Rodrigo Cáceres

Julie Plastino
email: julie.plastino@curie.fr

Majdouline Abou-Ghali

Cytoskeleton Dynamics: Actin in Cell Invasion

Keywords:

Actin filament dynamics have been studied for decades in pure protein solutions or in cell extracts, but a breakthrough in the field occurred at the turn of the century when it became possible to reconstitute networks of actin filaments, growing in a controlled but physiological manner on surfaces, mimicking the actin assembly that occurs at the plasma membrane during cell protrusion and cell shape changes. The story begins with the bacteria Listeria monocytogenes, the study of which led to the reconstitution of cellular actin polymerization on a variety of supports including plastic beads. These studies made possible the development of liposome-type substrates for filament assembly and micropatterning of actin polymerization nucleation. Based on the accumulated expertise of the last 15 years, many exciting approaches are being developed, including the addition of myosin to biomimetic actin networks to study the interplay between actin structure and contractility. The field is now poised to make artificial cells with a physiological and dynamic actin cytoskeleton, and subsequently to put these cells together to make in vitro tissues.

ABBREVIATIONS

Note: All C. elegans protein names, consisting of 3 letters and a number, are written first followed by a slash and the vertebrate or Drosophila equivalent. For example the WASP protein is designated WSP-1/WASP.

PREFACE

Cancer metastasis is the cause of 90% of human cancer deaths. During this process cells from a primary tumor escape and colonize other tissues. To do this the cells must cross extracellular matrix barriers called basement membranes (BM) in a process called invasion.

Although complex and multi-step, cell invasion is dependent, at least in part, on the dynamics of the acto-myosin cytoskeleton.

The goal of this thesis was to understand mechanistically how the acto-myosin cytoskeleton contributed to cell invasion. I used an experimental model known as anchor cell (AC) invasion in the nematode Caenorhabditis elegans. AC invasion occurs during the normal development of the vulva of C. elegans, and is the first step in the formation of an opening between the uterus and worm exterior. AC invasion resembles in many important respects cancer cell invasion, and all major BM and acto-myosin cytoskeleton components are conserved between worms and humans, making results relevant for understanding cancer cell invasion.

This thesis is organized into 6 chapters: the first 2 are introductory chapters, the third is a methods chapter and the last 3 detail the experiments I undertook over the course of my PhD.

My published review articles are in the annexes at the end of the document. In the first chapter, cell invasion is introduced, particularly cancer cell invasion, and the main actin structures, actin-binding proteins and their roles in cell shape changes and cancer cell invasion are explained. The second introductory chapter reviews the current state of the art about the experimental model system, AC invasion, used in the thesis. The last 3 chapters focus on the experiments done during the thesis, which are separated into 3 stories concerning 1) the importance of different actin components during AC invasion. 2) super-resolution study of the actin architecture in the AC and 3) the role of acto-myosin contractility during AC invasion in the absence of metalloproteases. Results from the first 2 studies will make up my first author publication, which should be submitted before the defense. I will be a contributing author in the third study that is nearing completion.

CHAPTER 1: CELL INVASION AND THE ACTIN CYTOSKELETON

OVERVIEW OF INVASION AND INTRODUCTION TO THE MODEL SYSTEM

Cell invasion across extracellular matrix (ECM) barriers such as basement membranes (BMs) occurs in many developmental and pathological contexts. BM is a two-dimensional layer of specialized ECM composed principally of laminin, type IV collagen polymers and glycoproteins that separate epithelia from the underlying tissue (Hohenester and Yurchenco, 2013). Owing to the small pore size of BM, non-permissive to cell passage, cells must enable specific cellular programs to move across BMs.

An example of this process is cancer cell invasion during metastasis. During metastasis, cancer cells leave the primary tumor, cross into the blood stream, are transported to new tissues, and migrate to form new colonies (Figure 1.1). In order to cross the physical barriers imposed by the intervening ECM including BMs, the cancer cell forms actin-rich protrusions called invadopodia, responsible for pulling the cell forward and for digesting and reorganizing the ECM to allow invasion (Rowe and Weiss, 2009). Cells leave the primary tumor by invading through the BM, cross the ECM stroma and invade through the BM of blood vessels. The circulatory system carries the tumor cells to new destinations where the cells invade out of the blood vessel and establish new tumor sites called metastases. From [START_REF] Nezhad | The cellular mechanics of an invasive lifestyle[END_REF].

Invasion also occurs in non-pathological contexts, one example being leucocyte transmigration. Leucocytes continually pass back and forth across the BMs of the blood and lymph vessels in order to mount the immune response (Vestweber, 2015). This BM is located beneath the endothelial cells that line the vessels. However a difference with cancer cell invasion is that this BM possesses low expression regions (LER) of collagen IV and laminins, forming weaker areas that are used by leukocytes to cross (Voisin et al., 2010) and (Figure 1.2). Experiments using microfluidic devices to mimic the transmigration of leucocytes through small gaps show the importance of actin polymerization for leucocyte transmigration [START_REF] Thiam | Perinuclear Arp2/3-driven actin polymerization enables nuclear deformation to facilitate cell migration through complex environments[END_REF]Vargas et al., 2016). [START_REF] Kelley | Traversing the basement membrane in vivo: A diversity of strategies[END_REF].

Angiogenesis, the sprouting and growth of new blood vessels from pre-existing ones, is another non-pathological process involving cell invasion. In response to proangiogenesis signals such as vascular endothelial growth factor (VEGF), endothelial cells lining the vessels become motile, lose contact with their neighbors and invade across the underlying BM (Eilken and Adams, 2010) and (Figure 1.3). This process is led by cells displaying actin-filled protrusions. (Eilken and Adams, 2010).

Cell invasion also occurs during organ development. One example, which is the focus of this PhD, is anchor cell (AC) invasion during vulval development in C. elegans. In immature worms, the uterine and vulva cells are separated by BM, which must be pierced to make a hole that will later become the vulva. A single cell, the AC, is responsible for forming the hole in the BM, and this process is reminiscent of cancer cell invasion through BMs (Figure 1.4). Cáceres, unpublished, 2016). Bottom: cancer cell invasion of BM--human colon carcinoma cells (actin in green, DNA in blue) invading rat peritoneal BM immunostained for laminin (red). Degradation of the BM and formation of invasive protrusions (invadopodia) allow the infiltration of the cell to the other side of the membrane. Stars indicate sites of penetration. Adapted from (Schoumacher et al., 2010). Scale bars 5 μm.

In the present project AC invasion was used as a model to better understand cell invasion with the goal of determining the combination of actin cytoskeleton components that was necessary for invasion as well as the potential role of myosin in this process. AC invasion allows for the study of invasion in a native 3D environment, without the caveats associated with current 2D and 3D cell culture where the mechanical properties of the native microenvironment are not preserved (Paszek et al., 2005). An additional advantage of working with a C. elegans is the ease of imaging in a 3D environment and tractable genetics, while sharing the same highly conserved components of the BM and acto-myosin cytoskeleton that are important for cancer cell invasion.

ACTIN CYTOSKELETON IN CELL PROTRUSION AND CELL SHAPE CHANGES

Shape changes of cells, including protrusions, are driven in large part by the actin cytoskeleton, a network of actin filaments that continuously elongate (polymerize) and disassemble (depolymerize) inside the cell beneath the membrane. Actin filaments are initially made by a "nucleation" process whereby 3 actin monomers come together to make a nascent filament [START_REF] Sept | Thermodynamics and Kinetics of Actin Filament Nucleation[END_REF]. Once the trimer is formed, assembly of actin monomers into filaments occurs in a directional manner due to the polarity of actin filaments that have dynamic (barbed) ends and less dynamic (pointed) ends (Pollard, 1986). In vivo, further polarity of assembly is conferred by the actin monomer-binding protein profilin that inhibits monomer addition to the pointed ends of filaments [START_REF] Pring | Profilin-actin complexes directly elongate actin filaments at the barbed end[END_REF]. Actin monomers carry a molecule of ATP that is hydrolyzed to ADP once in the filament. This also contributes to filament polarity by generating an actin-ATP cap at the barbed end while the rest of the filament contains actin-ADP. To enable cell protrusion and movement, actin filaments form different types of structures at different sites in the cell by interacting with different types of actin-binding proteins (Figure 1.5).

Figure 1.5 Actin structures in a protruding cell: In the cell there are 3 main actin networks, the filopodium, the lamellipodium and the acto-myosin cortex. Actin is represented by gray lines and the cell nucleus in blue. Adapted from (Blanchoin et al., 2014).

In the following sections, I will describe these different networks: branched structures that make up lamellipodia that push out the front of the cell, unbranched filaments that are found in filament bundles like filopodia likewise at the front of the cell and the acto-myosin cortex, a mix of branched and unbranched filaments along with the molecular motor myosin that underlies the cell membrane at the cell rear and can contract to squeeze forward the back of the cell during cell motility and other cell shape changes. I will also devote a section to actin cross-linking proteins since they play roles in the formation and stabilization of actin organization in all 3 architectures in the cell. The actin structures in a protruding cell and their biochemistries are similar to those found in invading cells, allowing them to enact cell shape changes to cross ECM barriers.

BRANCHED ACTIN NETWORKS

Cellular lamellipodia are composed principally of branched actin networks produced by the nucleating activity of the Arp2/3 complex (Pollard and Borisy, 2003;[START_REF] Svitkina | Arp2/3 Complex and Actin Depolymerizing Factor/Cofilin in Dendritic Organization and Treadmilling of Actin Filament Array in Lamellipodia[END_REF] and (Figure 1.6). This complex catalyzes the polymerization of a new actin filament on the side of a preexisting filament, creating a branch at a 70° angle from the side of the mother filament. The daughter filament grows by the barbed end while the pointed end remains attached to the Arp2/3 complex at the branch point [START_REF] Mullins | The interaction of Arp2/3 complex with actin: Nucleation, high affinity pointed end capping, and formation of branching networks of filaments[END_REF] and(Figure 1.6). [START_REF] Svitkina | Arp2/3 Complex and Actin Depolymerizing Factor/Cofilin in Dendritic Organization and Treadmilling of Actin Filament Array in Lamellipodia[END_REF]) b) Single-molecule time-lapse TIRFM of actin polymerization by the Arp2/3 complex (indicated by a red triangle), forming a branch off the side of a mother filament. Scale bar 1 μm. Adapted from [START_REF] Smith | Three-color single molecule imaging shows WASP detachment from Arp2/3 complex triggers actin filament branch formation[END_REF].

The Arp2/3 complex on its own has very low nucleating capacity and must be activated by a nucleation promoting factor from the WASP (Wiskott-Aldrich Syndrome protein)/WAVE family of proteins [START_REF] Kurisu | The WASP and WAVE family proteins[END_REF]. WASP proteins are composed of several domains, which give it its multifunctional properties. At the N-terminus, WASP has a WH1 (WASP homology) domain, the binding site for WASP-interacting protein (WIP), an adaptor protein involved in endocytosis [START_REF] Miki | Regulation of actin dynamics by WASP family proteins[END_REF]. This domain is followed by a basic region able to interact with the lipid phosphatidylinositol- [START_REF] Vogel | Minimal systems to study membrane-cytoskeleton interactions[END_REF][START_REF] Mullins | In vitro studies of actin filament and network dynamics[END_REF]-bisphosphate (PIP2), and a GTPase-binding domain "CRIB/GBD". The C-terminal half of the WASP molecule is composed of a proline rich region that can bind profilin-actin, followed by a verprolin homology domain (V), a cofilin-like region (C) and an acidic domain (A). Collectively, these three domains form what is known as the VCA region. The VCA region binds actin monomers and the Arp2/3 complex via its V and CA domain respectively, thus activating the Arp2/3 complex to nucleate a new filament [START_REF] Machesky | Scar, a WASp-related protein, activates nucleation of actin filaments by the Arp2/3 complex[END_REF][START_REF] Marchand | Interaction of WASP/Scar proteins with actin and vertebrate Arp2/3 complex[END_REF]. In its resting state, WASP is folded into an auto-inhibited conformation by interaction of the C-terminus with the "B" and "CRIB" domains, masking the VCA domain. This conformation can be released by interaction of WASP at the cell membrane with PIP2 and the GTPase Cdc42 [START_REF] Higgs | Activation by Cdc42 and Pip(2) of Wiskott-Aldrich Syndrome Protein (Wasp) Stimulates Actin Nucleation by Arp2/3 Complex[END_REF][START_REF] Rohatgi | The interaction between N-Wasp and the Arp2/3 complex links Cdc42dependent signals to actin assembly[END_REF] and (Figure 1

.7).

Another member of the WASP/WAVE family of proteins is WAVE, also known as Scar.

WAVE is very similar to WASP, with the difference that its interaction with GTPases is indirect because it lacks a CRIB domain, and it preferentially binds phosphatidylinositol- [START_REF] Brüggemann | Model systems for studying cell adhesion and biomimetic actin networks[END_REF][START_REF] Vogel | Minimal systems to study membrane-cytoskeleton interactions[END_REF][START_REF] Mullins | In vitro studies of actin filament and network dynamics[END_REF]-bisphosphate (PIP3) instead of PIP2 [START_REF] Oikawa | PtdIns(3,4,5)P3 binding is necessary for WAVE2-induced formation of lamellipodia[END_REF]. As compared to WASP, WAVE is a less efficient activator of the Arp2/3 complex due to its less acidic A domain even though both molecules bind the Arp2/3 complex with similar affinities (Zalevsky et al., 2001a;Zalevsky et al., 2001b). Like WASP, WAVE exists in an inhibited form, but in the case of WAVE, this is because of an inhibitory complex called the WAVE complex [START_REF] Eden | Mechanism of regulation of WAVE1-induced actin nucleation by Rac1 and Nck[END_REF]. The WAVE complex is composed of Abi1 (Abelson interacting protein 1), Nap1 (NCK-associated protein 1), Sra1 (specifically Rac-associated protein 1) and HSPC300. The hetero-complex is formed by binding the N-and C-terminal parts in a complex, thus hiding the VCA domain [START_REF] Ismail | The WAVE Regulatory Complex is Inhibited[END_REF]. This conformation is released by action of the GTPase Rac along with PIP3 [START_REF] Miki | WAVE, a novel WASP-family protein involved in actin reorganization induced by Rac[END_REF] and (Figure 1.7).

Figure 1.7 Activation mechanism of WASP and WAVE. N-WASP, one form of the WASP protein, has an N-terminal WASP-homology 1 (WH1) domain where WIP can bind. WAVE2, one form of the WAVE protein, has an N-terminal WAVE-/Scar-homology domain (WHD/SHD) that mediates protein complex formation with, Abi1, Nap1, and Sra1. The basic region (B) is common to both N-WASP and WAVE2, where phosphoinositides (PIP2 or PIP3) bind for protein localization or activation of the Arp2/3 complex. N-WASP has the Cdc42-Rac interactive binding region (CRIB) for Cdc42 GTPase binding. WAVE2 binds to Rac GTPase through Sra1. Adapted from [START_REF] Takenawa | Phosphoinositide-binding interface proteins involved in shaping cell membranes[END_REF].

Capping protein is another protein that is important for the dynamics of branched networks.

Capping protein binds to the barbed ends of actin filaments, stopping their elongation. In so doing capping proteins favor branching over barbed end elongation and thus play a role in the architecture of the branched network by generating short filaments with a high density of branches [START_REF] Akin | Capping Protein Increases the Rate of Actin-based Motility by Promoting Filament Nucleation by the Arp2/3 Complex[END_REF]Pollard and Borisy, 2003). This provides mechanical stiffness to generate force via barbed end growth towards the cell membrane (Figure 1.8). [START_REF] Akin | Capping Protein Increases the Rate of Actin-based Motility by Promoting Filament Nucleation by the Arp2/3 Complex[END_REF].

Branched network dynamics are also regulated by the actin filament severing protein ADF/cofilin. This protein has highest affinity for ADP actin subunits in the filament, which accumulate in the older regions, leading to uneven decoration of the filament with ADF/cofilin. ADF/cofilin decoration renders the filament less stiff, and fluctuations create breaking points between stiffer, undecorated and flexible ADF/cofilin-coated parts of the filament [START_REF] Mcgough | The gelsolin family of actin regulatory proteins: modular structures, versatile functions[END_REF] and (Figure 1.9). Figure 1.9 Actin filament severing by ADF/cofilin. Cofilin (grey symbols) binds to ADP actin regions of the actin filament (in red) as opposed to ATP or ADP-Pi portions (yellow and orange). This creates a discontinuity in filament stiffness, and breakage (scissor symbol) occurs at frontier regions between decorated and undecorated filament. Adapted from (Blanchoin et al., 2014).

Lamellipodium dynamics can also be regulated by numerous other proteins. Two examples are lamellipodin and Ena/VASP, which both localize to the leading edge of moving cells.

Ena/VASP is a barbed end elongation enhancement molecule that is associated with enhanced cell protrusion [START_REF] Breitsprecher | Clustering of VASP actively drives processive, WH2 domain-mediated actin filament elongation[END_REF][START_REF] Breitsprecher | Molecular mechanism of Ena/VASP-mediated actin-filament elongation[END_REF][START_REF] Rottner | VASP dynamics during lamellipodia protrusion[END_REF].

Ena/VASP also competes with capping protein for barbed end binding and crosslinks filaments together into parallel bundles [START_REF] Barzik | Ena/VASP Proteins Enhance Actin Polymerization in the Presence of Barbed End Capping Proteins[END_REF][START_REF] Laurent | Role of proteins of the Ena-VASP family in actinbased motility of Listeria monocytogenes[END_REF][START_REF] Suei | Fascin and VASP synergistically increase the Young's modulus of actin comet tails[END_REF]. Furthermore Ena/VASP may also bind directly to the WAVE molecule or to the Abi1 subunit of the WAVE regulatory complex to enhance WAVE-dependent actin polymerization [START_REF] Chen | Ena/VASP Proteins Cooperate with the WAVE Complex to Regulate the Actin Cytoskeleton[END_REF][START_REF] Havrylenko | WAVE binds Ena/VASP for enhanced Arp2/3 complex-based actin assembly[END_REF]. Lamellipodin is also associated with enhanced motility and binds Ena/VASP. Lamellipodin acts by tethering Ena/VASP to filaments, thus increasing its elongation efficiency [START_REF] Hansen | Lamellipodin promotes actin assembly by clustering Ena/VASP proteins and tethering them to actin filaments[END_REF], and by enhancing the ability of Rac to activate the WAVE complex for Arp2/3-mediated actin polymerization [START_REF] Carmona | Lamellipodin promotes invasive 3D cancer cell migration via regulated interactions with Ena/VASP and SCAR/WAVE[END_REF][START_REF] Law | Lamellipodin and the Scar/WAVE complex cooperate to promote cell migration in vivo[END_REF].

Branched actin network dynamics in a moving cell is summarized in (Figure 1.10).

Extracellular signals activate GTPases in the PIP2 and PIP3-rich plasma membrane. This recruits and activates WASP/WAVE/Scar, which in turn activates the Arp2/3 complex to polymerize branched actin filaments (Pollard and Borisy, 2003). These growing ends are capped by capping protein, preventing their further growth. Hydrolysis of ATP to ADP in the actin filament encourages ADF/cofilin binding and severing. Filaments disassemble from the pointed end, and the released monomers are bound with profilin to enhance exchange of ADP to ATP and to prevent pointed end polymerization, thus funneling growth toward the new barbed ends at the leading edge. (Pollard and Borisy, 2003).

UNBRANCHED FILAMENTS

Although principally composed of branched filaments, the leading edge of protruding cells also contains unbranched filaments. There are several factors that can nucleate unbranched filaments in the cell, the most important being a family of proteins called formins. Formins are dimeric proteins typically composed of a C-terminal region containing a diaphanous autoregulatory domain (DAD) and two formin homology domains (FH1 and FH2) with a diaphanous inhibitory domain (DID) and GTPase-binding (GBD) at the N-terminus [START_REF] Chesarone | Unleashing formins to remodel the actin and microtubule cytoskeletons[END_REF] and (Figure 1.11). Formins are autoinhibited via an intermolecular interaction between DAD and DIA. GTPase binding disrupts this conformation and opens it up to expose the FH1-FH2 domain, which is the minimal domain for nucleation of actin polymerization [START_REF] Alberts | Identification of a Carboxyl-terminal Diaphanous-related Formin Homology Protein Autoregulatory Domain[END_REF][START_REF] Watanabe | Cooperation between mDia1 and ROCK in Rho-induced actin reorganization[END_REF]. The FH1-FH2 domain acts as a dimer, with the FH2 domains tracking the barbed end as the filament grows, increasing the association of monomeric actin with the barbed end and protecting it from capping proteins [START_REF] Kovar | Control of the Assembly of ATP-and ADP-Actin by Formins and Profilin[END_REF][START_REF] Romero | Formin is a processive motor that requires profilin to accelerate actin assembly and associated ATP hydrolysis[END_REF][START_REF] Shemesh | A novel mechanism of actin filament processive capping by formin: solution of the rotation paradox[END_REF]. The FH1 domains have multiple sites for binding of profilin-actin, delivering it to the FH2 domains at the growing barbed end [START_REF] Kovar | Control of the Assembly of ATP-and ADP-Actin by Formins and Profilin[END_REF] and (Figure 1.11). The FH1 domains are thought to pull in monomers via their interaction with profilin-actin, while the FH2 domains are responsible for creating doughnut shaped homodimers in which actin nucleation can occur. Actin monomers are shown in green, profilin in blue, the FH1 domain in yellow and the FH2 domain in purple. From [START_REF] Randall | A formin-g role during development and disease[END_REF].

One of the main unbranched actin structures present at the leading edge of a protruding cell is filopodia. There are two models to explain filopodia formation, but both depend on the action of formins to make unbranched filaments. The "convergent elongation model" is based on a reorganization of branched networks at the edge of the lamellipodium via clustering of barbed ends into a "tip complex" composed of formins and anticapping proteins like Ena/VASP, which then elongates [START_REF] Svitkina | Mechanism of filopodia initiation by reorganization of a dendritic network[END_REF] and (Figure 1.12). The second model is known as "tip nucleation" and involves direct actin nucleation at the membrane by clustered formins without need of a previous branched network [START_REF] Yang | Filopodia initiation[END_REF]. In both models actin filament cross-linkers are necessary to bundle filaments and give the structure rigidity [START_REF] Vignjevic | Role of fascin in filopodial protrusion[END_REF]. These two models of filopodia initiation are both possible, and it is probable that they co-exist, as has been shown in vitro via reconstitution of filopodia assembly on lipid bilayers [START_REF] Lee | Self-assembly of filopodia-like structures on supported lipid bilayers[END_REF]. [START_REF] Svitkina | Mechanism of filopodia initiation by reorganization of a dendritic network[END_REF]. B) Mechanistic models of filopodia initiation. Convergent elongation model (top) and tip nucleation model (bottom). See text for details. Adapted from [START_REF] Yang | Filopodia initiation[END_REF].

THE ACTO-MYOSIN CORTEX

Another actin structure important for cell shape changes is the cell cortex, a layer of actin beneath the cell membrane composed of both branched and unbranched filaments [START_REF] Morone | Three-dimensional reconstruction of the membrane skeleton at the plasma membrane interface by electron tomography[END_REF] and (Figure 1.13). Cortical actin is nucleated by both the Arp2/3 complex and formins, thus accounting for its mixed structure (Bovellan et al., 2014). The actin layer also contains minifilaments of the molecular motor non muscle myosin II [START_REF] Charras | Reassembly of contractile actin cortex in cell blebs[END_REF].

Myosin recognizes the polarity of actin filaments and moves directionally. In the case where actin filaments are in an antiparallel arrangement, the bipolar nature of the myosin minifilament causes the filaments to slide past each other and contract [START_REF] Aguilar-Cuenca | Myosin II in mechanotransduction: master and commander of cell migration, morphogenesis, and cancer[END_REF][START_REF] Levayer | Biomechanical regulation of contractility: spatial control and dynamics[END_REF] [START_REF] Levayer | Biomechanical regulation of contractility: spatial control and dynamics[END_REF].

The contractility of the acto-myosin cortex plays an important role in cell rounding for mitosis, contraction of the cytokinetic furrow and squeezing forward the back of the cell during cell crawling [START_REF] Levayer | Biomechanical regulation of contractility: spatial control and dynamics[END_REF]. Acto-myosin tension is also what drives the formation of the cellular bleb. A bleb is a bulge of membrane, initially devoid of cytoskeleton, which forms when the link between the cortex and the membrane ruptures [START_REF] Charras | Reassembly of contractile actin cortex in cell blebs[END_REF] or when a hole appears in the cortex itself [START_REF] Paluch | Cortical Actomyosin Breakage Triggers Shape Oscillations in Cells and Cell Fragments[END_REF] and (Figure 1.15). Once the bleb is initiated, its rapid expansion and round shape suggest that the increase in bleb volume is mainly due to hydrostatic pressure created by myosin contractility rather than vesicle trafficking or local unwrinkling of the cell membrane (Charras et al., 2008). These observations also fit theoretical and experimental data that demonstrate the existence of a critical tension for bleb formation, and an increase in bleb size with increasing tension [START_REF] Tinevez | Role of cortical tension in bleb growth[END_REF]. Once the bleb has achieved its maximum size, the cortex reforms in the bleb, either leading to stabilization of the protrusion or to retraction of the bleb and reabsorption (Charras et al., 2008).

CROSSLINKED ACTIN STRUCTURES

For all three architectures discussed in the preceding sections (branched structures in lamellipodia, unbranched structures in bundles like filopodia and mixed branched and unbranched networks in the cell cortex), actin cross-linking proteins play key roles in the formation and properties of the different actin arrangements [START_REF] Revenu | The co-workers of actin filaments: from cell structures to signals[END_REF]Stevenson et al., 2012). There are many actin crosslinkers in cells, but the best-studied ones are fascin, -actinin, plastin (also called fimbrin) and filamin [START_REF] Adams | Roles of fascin in cell adhesion and motility[END_REF][START_REF] Bartles | Parallel Actin Bundles and Their Multiple Actin-bundling Proteins[END_REF][START_REF] Otey | α-actinin revisited: A fresh look at an old player[END_REF][START_REF] Stossel | Filamins as integrators of cell mechanics and signalling[END_REF]. Fascin is a monomeric protein able to form small bridges of 8 nm between parallel actin filaments [START_REF] Jansen | Mechanism of Actin Filament Bundling by Fascin[END_REF] and (Figure 1.16), whileactinin is a dimeric protein able to form longer filament crosslinks of around 17-24 nm [START_REF] Goldstein | The Z lattice in canine cardiac muscle[END_REF][START_REF] Luther | Three-Dimensional Structure of a Vertebrate Muscle Z-band: Implications for Titin and α-Actinin Binding[END_REF] and (Figure 1.16). Due to its flexible amino-terminal Factin binding domains that rotate freely with respect to each other, -actinin is able to bind non-aligned actin filament networks, antiparallel arrangements or parallel bundles [START_REF] Courson | Actin Cross-link Assembly and Disassembly Mechanics for α-Actinin and Fascin[END_REF]. Both fascin and -actinin are implicated in filopodial structure and function [START_REF] Fukumoto | α-Actinin-4 Enhances Colorectal Cancer Cell Invasion by Suppressing Focal Adhesion Maturation[END_REF][START_REF] Huang | Targeted inhibition of fascin function blocks tumour invasion and metastatic colonization[END_REF][START_REF] Shao | α-Actinin-4 Is Required for Amoeboidtype Invasiveness of Melanoma Cells[END_REF] and have been shown to work synergistically in increasing actin network stiffness [START_REF] Tseng | How actin crosslinking and bundling proteins cooperate to generate an enhanced cell mechanical response[END_REF].

Plastin, like fascin, is a monomeric protein with two actin binding domains able to bundle parallel filaments, but with a distance of about 12 nm, giving a looser bundle than fascin [START_REF] De Arruda | Fimbrin is a homologue of the cytoplasmic phosphoprotein plastin and has domains homologous with calmodulin and actin gelation proteins[END_REF] and(Figure 1.16). Plastin is also found in filopodia, and additionally in filopodial-like structures called microvilli in the intestines [START_REF] Bretscher | Fimbrin, a new microfilament-associated protein present in microvilli and other cell surface structures[END_REF][START_REF] Volkmann | An Atomic Model of Actin Filaments Cross-Linked by Fimbrin and Its Implications for Bundle Assembly and Function[END_REF]. Another type of crossslinker is filamin, a dimeric protein with two actin binding domains at the N-terminus, two rigid rod domains, and two flexible hinges, thus conferring the mix of flexibility and stiffness required to tether actin filaments and hold them into an orthogonal arrangement [START_REF] Gorlin | Human endothelial actin-binding protein (ABP-280, nonmuscle filamin): a molecular leaf spring[END_REF][START_REF] Hartwig | Structure of macrophage actin-binding protein molecules in solution and interacting with actin filaments[END_REF] and (Figure 1.16). Before the discovery of the Arp2/3 complex, it was believed that filamin was responsible for the X, T and Y-shaped junctions visible in the lamellipodium [START_REF] Flanagan | Filamin A, the Arp2/3 complex, and the morphology and function of cortical actin filaments in human melanoma cells[END_REF][START_REF] Hartwig | Association of gelsolin with actin filaments and cell membranes of macrophages and platelets[END_REF][START_REF] Schliwa | Structural interaction of cytoskeletal components[END_REF]. As well as its role in crosslinking, filamins have a role in signaling via interactions with integrin adhesion proteins, and small GTPases, RhoA, Rac1, and Cdc42 [START_REF] Kiema | The Molecular Basis of Filamin Binding to Integrins and Competition with Talin[END_REF][START_REF] Ohta | The small GTPase RalA targets filamin to induce filopodia[END_REF]. Fascin, -actinin, plastin and filamin are all present in lamellipodial networks [START_REF] Small | The lamellipodium: where motility begins[END_REF][START_REF] Stossel | Filamins as integrators of cell mechanics and signalling[END_REF]. All but fascin are also present in the acto-myosin cortex where they presumably crosslink filaments formed by both Arp2/3 complex and formin nucleation [START_REF] Charras | Reassembly of contractile actin cortex in cell blebs[END_REF], and play a role in mediating cortex contractility as has been shown with crosslinked acto-myosin networks in vitro and in vivo via filamin [START_REF] Alvarado | Molecular motors robustly drive active gels to a critically connected state[END_REF][START_REF] Ding | Plastin increases cortical connectivity to facilitate robust polarization and timely cytokinesis[END_REF].

PUTTING IT ALL TOGETHER: ACTIN NETWORKS IN MOTILITY

The branched, unbranched and cortical networks described in the preceding sections work together in many cellular processes including cell motility. The overall picture of cell motility involves the front of the cell being pushed out by the formation of branched lamellipodial networks and bundled filopodial structures (called mesenchymal migration) or by the formation of blebs due to cortical contraction (called bleb-based migration).

During mesenchymal migration, lamellipodia or filopodia can generate adhesions with the substrate/ECM via integrins, transmembrane proteins able to link the ECM with the cytoskeleton [START_REF] Hanein | The structure of cell-matrix adhesions: the new frontier[END_REF][START_REF] Vicente-Manzanares | Adhesion dynamics at a glance[END_REF] Figure 1.17 Mesenchymal migration. Actin polymerization at the leading edge via Rac GTPase extends the cell membrane to create new adhesion points while the back of the cell contracts via Rho GTPase detaching the integrins from the focal adhesion. Adapted from [START_REF] Matsuoka | Rho/ROCK signaling in motility and metastasis of gastric cancer[END_REF].

On the other hand, in bleb-based migration, cells are minimally adherent. Blebs can intercalate amongst ECM fibers and Rho dependent myosin contraction can force the cell forward (Lämmermann and Sixt, 2009) and (Figure 1.18). Figure 1.18 Bleb-based migration. Blebs expand amongst ECM fibers, anchoring the cell in place and allowing the back of the cell to be squeezed forward by myosin contractility (myosin in red, actin in green). From (Lämmermann and Sixt, 2009).

Switches between mesenchymal and bleb-based motility are observed in 2D situations, where lamellipodia inhibition by reducing the Arp2/3 complex or reducing cell-substrate adhesion triggers bleb-based motility, while increasing lamellipodial dynamics by activating Rac1 suppresses bleb formation (Bergert et al., 2012). Similar behavior is observed when cancer cells are confined in channels passivated to decrease the level of cell adhesion to the substrate (Liu et al., 2015). Under these conditions, slow-moving mesynchymal-type cells show fast bleb-based locomotion.

ACTIN CYTOSKELETON IN CANCER CELL INVASION

Cancer cell invasion is a special type of cell motility that involves the movement of cells through the layers of ECM that separate different kinds of tissue. Cancer cell invasion has been observed to proceed by both mesenchymal and bleb-based mechanisms.

MESENCHYMAL-TYPE INVASION

This type of invasion resembles the moving cell described in the previous section. Movement is preceded by a change in the cell properties, termed epithelial-mesenchymal transition whereby epithelial cells lose their apical/baso-lateral polarity and their cell-cell adhesion attributes that make them part of a cohesive tissue and acquire mesenchymal markers including cell-matrix adhesions (Thiery, 2002). They form a protrusion that allows them to move, but also to cross ECM barriers, and in this case the protrusion is called an invadopodium. Invadopodia are actin-filled structures, but they also are equipped with the ability to proteolytically degrade ECM fibers by secreting matrix metalloproteases (MMPs) [START_REF] Linder | Degrading Devices: Invadosomes in Proteolytic Cell Invasion[END_REF][START_REF] Murphy | The 'ins' and 'outs' of podosomes and invadopodia: characteristics, formation and function[END_REF][START_REF] Revach | The interplay between the proteolytic, invasive, and adhesive domains of invadopodia and their roles in cancer invasion[END_REF] and (Figure 1.19). The actin network in invadopodia is a mix of a branched network and unbranched bundled filaments, and at later stages, additional cytoskeletal elements like microtubules and intermediate filaments are also observed (Schoumacher et al., 2010) and (Figure 1.19). [START_REF] Friedl | Tumour-cell invasion and migration: diversity and escape mechanisms[END_REF]. Right panel: electron microscopy image of an invadopodium. Microtubules and intermediate filaments make up the core of the mature invadopodium, while the sides and tip are composed of a dense array of actin filaments, difficult to resolve because of tight packing. Scale bar 0.5 µm. Adapted from (Schoumacher et al., 2010).

The Arp2/3 complex is necessary for invadopodia formation, and the Arp2/3 complex activator N-WASP has been clearly visualized in invadopodia, implying that WASP proteins may be the activators for invadopodial actin nucleation (Nürnberg et al., 2011;[START_REF] Oser | Cortactin regulates cofilin and N-WASp activities to control the stages of invadopodium assembly and maturation[END_REF]Yamaguchi et al., 2005). However WASP has also been reported to play a role in vesicle trafficking [START_REF] Benesch | Phosphatidylinositol 4,5-Biphosphate (PIP2)-induced Vesicle Movement Depends on N-WASP and Involves Nck, WIP, and Grb2[END_REF][START_REF] Co | Mechanism of Actin Network Attachment to Moving Membranes: Barbed End Capture by N-WASP WH2 Domains[END_REF][START_REF] Kovacs | Tuba stimulates intracellular N-WASPdependent actin assembly[END_REF], coordinating protease delivery at invadopodia [START_REF] Nusblat | The non-redundant role of N-WASP in podosome-mediated matrix degradation in macrophages[END_REF][START_REF] Yu | N-WASP coordinates the delivery and F-actin-mediated capture of MT1-MMP at invasive pseudopods[END_REF], so it is possible that WASP's role in invasion relates to trafficking of components to the protrusion and not to actin polymerization to push out the invasive protrusion. The other Arp2/3 complex activator, WAVE, is dim or absent from invadopodia in classical invasion assays (Yamaguchi et al., 2005). However in 3D environments, both WAVE and N-WASP appear to promote invasion [START_REF] Giri | The Arp2/3 complex mediates multigeneration dendritic protrusions for efficient 3dimensional cancer cell migration[END_REF][START_REF] Takahashi | WAVE2, N-WASP, and mena facilitate cell invasion via phosphatidylinositol 3-kinase-dependent local accumulation of actin filaments[END_REF]. Other studies show that inhibition of different forms of WAVE can both upregulate and downregulate cancer cell invasion, depending on the context (Kurisu and Takenawa, 2010). Overall the role of WAVE in invasion is not clear.

Given the mixed architecture of actin in invadopodia, it is not surprising that formins are also linked to cancer invasion and metastasis. Indeed Arp2/3 complex independent invasion, dependent on the formin FHOD3, has been reported [START_REF] Paul | α5β1 integrin recycling promotes Arp2/3-independent cancer cell invasion via the formin FHOD3[END_REF]. Upregulation of the formin FMNL2 was found in metastatic colorectal cancer cell [START_REF] Zhu | Overexpression of FMNL2 is closely related to metastasis of colorectal cancer[END_REF]. However the same formin was found at lower levels in hepatocellular carcinoma lines [START_REF] Liang | Down-regulation of formin-like 2 predicts poor prognosis in hepatocellular carcinoma[END_REF]. Similarly for the formin mDia2, some studies show a positive correlation with invasion and others the opposite [START_REF] Vizio | Oncosome Formation in Prostate Cancer: Association with a Region of Frequent Chromosomal Deletion in Metastatic Disease[END_REF][START_REF] Lizárraga | Diaphanous-Related Formins Are Required for Invadopodia Formation and Invasion of Breast Tumor Cells[END_REF]. These observations suggest that the correlation between invasive behavior and the presence of formins depends on the tissue and the type of formin.

Actin crosslinkers have also been implicated in invadopodia formation. High expression levels of fascin are correlated with tumor cell migration and invasion in vitro, and metastasis in vivo [START_REF] Huang | Targeted inhibition of fascin function blocks tumour invasion and metastatic colonization[END_REF][START_REF] Vignjevic | Fascin, a Novel Target of β-Catenin-TCF Signaling, Is Expressed at the Invasive Front of Human Colon Cancer[END_REF]. Conversely other studies report that loss of filamin enhances ECM degradation, but this seems to be due to filamin's role in regulating proteolytic activity, not its actin crosslinking activity [START_REF] Baldassarre | Filamin A controls matrix metalloproteinase activity and regulates cell invasion in human fibrosarcoma cells[END_REF]. Other studies link both filamin upregulation and downregulation with breast cancer progression [START_REF] Caruso | Proteomic profiling of lipid rafts in a human breast cancer model of tumorigenic progression[END_REF][START_REF] Xu | Filamin A regulates focal adhesion disassembly and suppresses breast cancer cell migration and invasion[END_REF]. So like formins, it appears that the role of crosslinkers in invasion depends on the cell type and how invasion is evaluated.

BLEB-DRIVEN INVASION

Invasive cancers cells can also display blebbing motility. In particular this is observed when cancer cells migrate in 3D environments in the absence of MMP activity. Because the ECM cannot be digested, cells squeeze through matrix gaps by using the contractile activity of the acto-myosin cortex [START_REF] Sabeh | Protease-dependent versus -independent cancer cell invasion programs: three-dimensional amoeboid movement revisited[END_REF]Wolf et al., 2003) and (Figure 1.20). Bleb-based invasion in the absence of MMPs is mainly regulated by the phosphorylation of myosin light chain (MLC) by ROCK (Rho kinase) which promotes the interactions between myosin and Factin to generate contractile force [START_REF] Wolf | Physical limits of cell migration: Control by ECM space and nuclear deformation and tuning by proteolysis and traction force[END_REF]. Adapted from [START_REF] Pinner | Imaging amoeboid cancer cell motility in vivo[END_REF].

As for healthy motile cells, invading cancer cells can switch between mesenchymal and blebbased modes of invasion depending on the mesh size of the 3D environment, cell-substrate adhesion levels, the presence of proteases and the balance between actin protrusivity via RacGTPase and actomyosin contractility via RhoGTPase-ROCK (Lämmermann and Sixt, 2009;Petrie and Yamada, 2012) and(Figure 1 In early stages, C. elegans larvae do not possess a vulva and the uterus is a closed organ, unconnected to the outside of the worm. At the site of the future vulva, the uterine and prevulval tissue are separated by a double BM (Morrissey et al., 2014). The role of AC invasion is to pierce the double BM at a specific larval stage, making a hole that will later become the vulva. As briefly introduced in Chapter 1, AC invasion looks morphologically similar to the invading cancer cell, since both are dependent on a dynamic actin-filled protrusion to make a hole in a BM. AC invasion generally starts with a unique small hole in the BM at a certain time in development, colocalizing in the AC with actin and with signaling molecules such as PIP2 (Hagedorn et al., 2013) and (Figure 2.1). The protrusion grows and the hole in the BM expands until it reaches the AC border. In later stages, as the vulval tissue begins to invaginate, the hole expands beyond the edges of the AC due to mechanisms that are distinct from the initial invasive process, termed BM sliding [START_REF] Ihara | Basement membrane sliding and targeted adhesion remodels tissue boundaries during uterine vulval attachment in Caenorhabditis elegans[END_REF][START_REF] Matus | Cell Division and Targeted Cell Cycle Arrest Opens and Stabilizes Basement Membrane Gaps[END_REF][START_REF] Mcclatchey | Boundary cells restrict dystroglycan trafficking to control basement membrane sliding during tissue remodeling[END_REF]. (Hagedorn et al., 2013).

It is important to note that, at the time of invasion, the AC is expressing 3 MMPs, the zinc matrix proteases ZMP-1, -3 and -6 [START_REF] Matus | Invasive Cell Fate Requires G1 Cell-Cycle Arrest and Histone Deacetylase-Mediated Changes in Gene Expression[END_REF]. However there is evidence that BM hole formation and expansion is not all due to matrix digestion. BM accumulates at the perimeter of the gap (Figure 2.1) and a photo-conversion experiment using photoconvertible laminin showed that not all of the BM beneath the AC is digested; some is pushed aside (Hagedorn et al., 2013) and (Figure 2.2). The goal during my PhD was to find the source of these pushing forces, with the long-term objective of relating these results to understanding cancer cell invasion. The BM just beneath the AC and a control flanking region are photocoverted just before invasion and imaged after invasion. The control region remains intact, and while much of the photoconverted BM under the AC has disappeared, some remains, pushed to the sides of the AC. Bar 5 m. AC and BM in green; BM photoconverted to purple. Adapted from (Hagedorn et al., 2013).

WHY STUDY AC INVASION?

Due to the difficulty of studying cancer cell invasion in living animals, most of what is known about the role of the actin cytoskeleton in cell invasion comes from the study of cultured cancer cells invading artificial BM mimics in 2D assays [START_REF] Bowden | Co-localization of cortactin and phosphotyrosine identifies active invadopodia in human breast cancer cells[END_REF][START_REF] Linder | Degrading Devices: Invadosomes in Proteolytic Cell Invasion[END_REF]. However reconstituted extracellular matrices, such as Matrigel, do not retain all of the biochemical properties of in vivo matrices, and the thin 2D geometry of native BM sheets is not respected. Even more importantly, mechanical properties of reconstituted BM do not reflect native membranes: estimates of native BM Young's modulus are in the kPa-100 kPa range, whereas Matrigel has a 10 to 1000-fold lower elasticity, in the 100 Pa range (Paszek et al., 2005;[START_REF] Wood | Biophysical Cueing and Vascular Endothelial Cell Behavior[END_REF]. Since actin dynamics and organization responds to mechanical cues in the environment [START_REF] Fletcher | Cell mechanics and the cytoskeleton[END_REF], it is important to approximate in vivo mechanics as closely as possible in order to acquire biologically relevant information on the actin cytoskeleton in cell invasion. In this context, it is useful to study cell invasion using developmental models where non-pathologic invasion occurs at a specified place and time during organ development, making it easier to detect and image in the living animal. An example of such a process is AC invasion.

RELEVANCE OF AC INVASION TO CANCER CELL INVASION

Transcriptionally AC invasion is mainly regulated by fos-1a, the C. elegans ortholog of the transcription factor Fos, which in vertebrates is upregulated in many types of metastatic cancers and regulates MMPs via the AP-1 transcription factor complex [START_REF] Aoyagi | Specific transcription factors prognostic for prostate cancer progression[END_REF]Hagedorn and Sherwood, 2011;[START_REF] Saez | c-fos is required for malignant progression of skin tumors[END_REF]. Adding to this, a whole-genome RNA interference (RNAi) screen identified 99 genes that promote AC invasion, and most of these genes have human orthologs, many of which have been implicated in cell invasion [START_REF] Matus | In Vivo Identification of Regulators of Cell Invasion Across Basement Membranes[END_REF].

The molecular organization of BMs in C. elegans is highly conserved with vertebrates and contains orthologs of the major structural components, including type IV collagen and laminin. The acto-myosin cytoskeleton is also well-conserved in worms, which contain counterparts of the Arp2/3 complex subunits, named ARX1-7 [START_REF] Sawa | Essential role of the C.elegans Arp2/3 complex in cell migration during ventral enclosure[END_REF], homologs of the activators WASP and WAVE (WSP-1 and WVE-1, respectively) [START_REF] Miki | Regulation of actin dynamics by WASP family proteins[END_REF][START_REF] Patel | The WAVE/SCAR complex promotes polarized cell movements and actin enrichment in epithelia during C. elegans embryogenesis[END_REF] and a 6-member family of formin proteins: FHOD-1, CYK-1, FRL-1, INFT-2, DAAM-1 and EXC-6 (Mi-Mi et al., 2012;Schönichen and Geyer, 2010). Concerning upstream signaling to the actin cytoskeleton, C. elegans has one real Cdc-42, CDC-42, and one true Rac, CED-10, and another Rac, MIG-2, which contains Rac-like and Cdc-42-like motifs [START_REF] Lundquist | Three C. elegans Rac proteins and several alternative Rac regulators control axon guidance, cell migration and apoptotic cell phagocytosis[END_REF][START_REF] Reiner | Small GTPases[END_REF][START_REF] Shakir | The Arp2/3 activators WAVE and WASP have distinct genetic interactions with Rac GTPases in Caenorhabditis elegans axon guidance[END_REF]. As for mammals, CDC-42 is the Rac that is responsible for activating WSP-1 to bind and activate the Arp2/3 complex, with MIG-2 also playing this role, while CED-10 is the upstream activator of WVE-1 [START_REF] Shakir | The Arp2/3 activators WAVE and WASP have distinct genetic interactions with Rac GTPases in Caenorhabditis elegans axon guidance[END_REF][START_REF] Walck-Shannon | CDC-42 orients cell migration during epithelial intercalation in the Caenorhabditis elegans epidermis[END_REF]. C. elegans also has most major actin binding regulatory proteins: 2 forms of ADF/cofilin UNC-60A and B (Ono et al., 2003), Ena/VASP, UNC-34 [START_REF] Withee | Caenorhabditis elegans WASP and Ena/VASP protiens play compensatory roles in morphogenesis and neuronal cell migration[END_REF]), 3 profilin isoforms PFN1-3 (Polet et al., 2006) and 2 non-muscle myosin heavy chains NMY-1 and -2 (Piekny et al., 2003). Like in vertebrates, non-muscle myosin activity in C. elegans is controlled by kinases and phosphatases: NMYs associate with light chains, for example MLC-4, which need to be phosphorylated by the Rhobinding kinase LET-502/ROCK or MRCK-1 in order to be active [START_REF] Gally | Myosin II regulation during C. elegans embryonic elongation: LET-502/ROCK, MRCK-1 and PAK-1, three kinases with different roles[END_REF][START_REF] Piekny | Embryonic Morphogenesis in Caenorhabditis elegans Integrates the Activity of LET-502 Rho-Binding Kinase, MEL-11 Myosin Phosphatase, DAF-2 Insulin Receptor and FEM-2 PP2c Phosphatase[END_REF][START_REF] Shelton | The Nonmuscle Myosin Regulatory Light Chain Gene mlc-4 Is Required for Cytokinesis, Anterior-Posterior Polarity, and Body Morphology during Caenorhabditis elegans Embryogenesis[END_REF]. In addition MLC-4 is negatively regulated by the phosphatase MEL-11, inhibiting actomyosin contraction [START_REF] Wissmann | TheCaenorhabditis elegans mel-11Myosin Phosphatase Regulatory Subunit Affects Tissue Contraction in the Somatic Gonad and the Embryonic Epidermis and Genetically Interacts with the Rac Signaling Pathway[END_REF]. With the notable exception of fascin, the major classes of actin bundling proteins are also present in C. elegans: α-actinin, ATN-1 [START_REF] Francis | Muscle organization in Caenorhabditis elegans: localization of proteins implicated in thin filament attachment and I-band organization[END_REF][START_REF] Moulder | α-actinin is Required for Proper Assembly of Z-disk / Focal Adhesion-Like Structures and for Efficient Locomotion in Caenorhabditis elegans[END_REF]; filamin, FLN-1 (DeMaso et al., 2011) and plastin/fimbrin, PLST-1 [START_REF] Skop | Dissection of the Mammalian Midbody Proteome Reveals Conserved Cytokinesis Mechanisms[END_REF].

Overall acto-myosin biochemistry, BM composition and transcriptional control are similar in the AC as compared to an invading cancer cell, making AC invasion a relevant model for studying the role of actin dynamics during invasion.

THE PHYSIOLOGICAL CONTEXT OF AC INVASION

Before invasion occurs, the AC communicates with the VPCs so they assume the correct fate for subsequent vulval development, for review [START_REF] Schmid | Signal transduction during C. elegans vulval development: a NeverEnding story[END_REF]. Briefly in the early third larval stage, there are 6 VPCs called P3.p-P8.p arranged along the ventral surface of the worm, with P6.p centered on the AC (Figure 2 [START_REF] Schmid | Signal transduction during C. elegans vulval development: a NeverEnding story[END_REF] Leading up to AC invasion, the 1° and 2° VPCs undergo three rounds of division with precise timing. The progression of AC invasion can be staged by examining whether the central VPC P6.p is at the 2, 4 or 8 cell stage (Figure 2.4). In wild type worms, the AC breaches the BM between the 2-cell and 4-cell stage. AC invasion is not a cell-autonomous process. In vulvaless lin-3 mutant animals or when P6.p is ablated, invasion does not occur [START_REF] Sherwood | Anchor Cell Invasion into the Vulval Epithelium in C. elegans[END_REF]. In addition AC invasion is blocked in mutants where all VPCs adopt 2° fates [START_REF] Sherwood | Anchor Cell Invasion into the Vulval Epithelium in C. elegans[END_REF]. Conversely when P8.p assumes a 1° fate after ablation of P3.p-P7.p, the AC protrusion breaches the BM and elongates in order to reach it, so AC invasion appears to be driven by the 1° VPC independently of its location [START_REF] Sherwood | Anchor Cell Invasion into the Vulval Epithelium in C. elegans[END_REF]. Moreover studies using mutant animals with multiple 1° VPCs, but only one AC, show breaches in the BM only where the AC is located, confirming that the VPCs do not stimulate AC invasion by removing the BM themselves [START_REF] Sherwood | Anchor Cell Invasion into the Vulval Epithelium in C. elegans[END_REF]. On the uterine side of the BM, the gonadal cells adjacent to the AC can be ablated without affecting invasion, so the direct neighbors of the AC do not appear to play a role in helping the AC to pierce the BM [START_REF] Sherwood | Anchor Cell Invasion into the Vulval Epithelium in C. elegans[END_REF]. Notably although the VPCs divide over the course of AC invasion, the AC itself does not. This is because the AC is arrested at the G1 phase of the cell cycle via transcriptional control [START_REF] Matus | Invasive Cell Fate Requires G1 Cell-Cycle Arrest and Histone Deacetylase-Mediated Changes in Gene Expression[END_REF] and Section 2.6. When arrest is interfered with and cell division and proliferation of the AC continues, invasive behavior is lost [START_REF] Matus | Invasive Cell Fate Requires G1 Cell-Cycle Arrest and Histone Deacetylase-Mediated Changes in Gene Expression[END_REF].

AC INVASION REGULATION BY SIGNALING

AC invasion is regulated by two main external signals, a diffusible vulva cue from the 1° VPC and another emitted by the ventral nerve cord (VNC) that is adjacent to the vulval cells [START_REF] Sherwood | Anchor Cell Invasion into the Vulval Epithelium in C. elegans[END_REF]Ziel et al., 2009). The cue from the 1° VPC is unidentified, but it is known that it is diffusible because, as mentioned in the previous section, when P8.p acquires a 1° fate, the AC extends a protrusion to it over a long distance [START_REF] Sherwood | Anchor Cell Invasion into the Vulval Epithelium in C. elegans[END_REF]. The signal from the 1° VPC doesn't completely control the timing of AC invasion however, since in mutant animals where the 1° VPC forms precociously, the AC still invades at or near the normal time of invasion [START_REF] Sherwood | Anchor Cell Invasion into the Vulval Epithelium in C. elegans[END_REF].

In vulvaless animals there is still 20% successful invasion, and this is believed to be dependent on a UNC-6/netrin signal produced by the VNC [START_REF] Sherwood | Anchor Cell Invasion into the Vulval Epithelium in C. elegans[END_REF]Ziel et al., 2009). UNC-6/netrin signal is high in the VNC and accumulates in the BM under the AC where is acts via the netrin receptor UNC-40/DCC located at the AC invasive membrane (Ziel et al., 2009). In neurons, netrin/DCC signaling is known to act on actin regulators, including RacGTPase [START_REF] Gitai | The Netrin Receptor UNC-40/DCC Stimulates Axon Attraction and Outgrowth through Enabled and, in Parallel, Rac and UNC-115[END_REF]. When UNC-6/netrin is not present, UNC-40/DCC remains active in assembling actin patches, however the actin clusters are mislocalized and dynamic, undergoing an oscillatory behavior of assembly-disassembly (Wang et al., 2014c). UNC-6/netrin is crucial for clustering and stabilizing UNC-40/DCC to form a proper protrusion (Hagedorn et al., 2013;Wang et al., 2014c) and(Figure 2.5). Loss of UNC-40/DCC and/or UNC-6/netrin does not inhibit the ability of the AC to create the initial holes in the BM, a process that is apparently not linked to netrin signaling (Hagedorn et al., 2013;Ziel et al., 2009). Adapted from (Wang et al., 2014c) Instead the formation of initial actin-rich foci that occur before invasion is dependent on integrins (Hagedorn et al., 2013). When integrin is knocked down, foci don't form and furthermore, components of the mature protrusion, UNC-6/netrin, UNC-40/DCC and other actin regulatory proteins are not localized normally at the invasive membrane (Hagedorn et al., 2009;Wang et al., 2014a;Wang et al., 2014c). These results suggest that the integrin and netrin pathways work together to signal to the invasive program [START_REF] Hagedorn | Integrin Acts Upstream of Netrin Signaling to Regulate Formation of the Anchor Cell's Invasive Membrane in C. elegans[END_REF].

AC INVASION REGULATION BY GENE EXPRESSION

The invasive behavior of the AC is conferred by the specific expression of certain genes in the AC (Figure 2.6). As mentioned briefly above, the transcription factor fos-1, the sole C. elegans ortholog of the fos bZIP transcription factor family, is specifically expressed in the AC and controls the expression of important target genes [START_REF] Sherwood | FOS-1 Promotes Basement-Membrane Removal during Anchor-Cell Invasion in C. elegans[END_REF]. These include zmp-1, an MMP, him-4, an ECM component involved in adhesion of adjacent BMs and cdh-3, a cadherin family protein involved in cell-cell adhesions (Morrissey et al., 2014;[START_REF] Sherwood | FOS-1 Promotes Basement-Membrane Removal during Anchor-Cell Invasion in C. elegans[END_REF]. Individually mutation of these 3 genes slightly affects AC invasion, however the triple knock-out has a more severe effect, only 75% invasion at the 4-cell stage where wild-type shows 100% [START_REF] Sherwood | FOS-1 Promotes Basement-Membrane Removal during Anchor-Cell Invasion in C. elegans[END_REF]. fos-1a also positively regulates MIG-10/lamellipodin expression (Wang et al., 2014b).

Another transcription factor found in the AC is egl-43, the C. elegans ortholog of vertebrate proto oncogene EVI1 [START_REF] Hwang | C. elegans EVI1 proto-oncogene, EGL-43, is necessary for Notch-mediated cell fate specification and regulates cell invasion[END_REF][START_REF] Rimann | Regulation of anchor cell invasion and uterine cell fates by the egl-43 Evi-1 proto-oncogene in Caenorhabditis elegans[END_REF]. egl-43 acts downstream fos-1 [START_REF] Hwang | C. elegans EVI1 proto-oncogene, EGL-43, is necessary for Notch-mediated cell fate specification and regulates cell invasion[END_REF][START_REF] Matus | In Vivo Identification of Regulators of Cell Invasion Across Basement Membranes[END_REF] and represses the expression of MIG-10 (Wang et al., 2014a;Wang et al., 2014b). The contradictory effects on MIG-10 expression, positively regulated by fos-1 but negatively regulated by egl-43, are believed to tightly control MIG-10 availability since too much MIG-10 is deleterious to invasion (Wang et al., 2014a). EGL-43 is also required for cell fate specification to convert a uterine cell (VU) into the AC [START_REF] Rimann | Regulation of anchor cell invasion and uterine cell fates by the egl-43 Evi-1 proto-oncogene in Caenorhabditis elegans[END_REF]).

An RNAi screen for genes that perturb AC invasion yielded numerous candidates from many protein families, such as chaperones, kinases and transcription factors including a histone deacetylase, hda-1 [START_REF] Matus | In Vivo Identification of Regulators of Cell Invasion Across Basement Membranes[END_REF]. The gene hda-1 plays a key role in transducing G1 arrest to protrusion formation, downstream of the nuclear hormone receptor nhr-67 and cki-1, a cyclin-dependent kinase inhibitor. In brief, G1 arrest upregulates hda-1, which controls the expression of the proinvasive genes zmp-1, cdh-3 and him-4 both directly and via upregulation of fos-1, in addition to directly affecting the expression of the actin regulatory genes exc-6 (a formin) and unc-34 (an Ena/VASP protein) [START_REF] Matus | In Vivo Identification of Regulators of Cell Invasion Across Basement Membranes[END_REF][START_REF] Matus | Invasive Cell Fate Requires G1 Cell-Cycle Arrest and Histone Deacetylase-Mediated Changes in Gene Expression[END_REF]. 1, cdh-3, him-4, unc-34 and exc-6. Modified from [START_REF] Matus | In Vivo Identification of Regulators of Cell Invasion Across Basement Membranes[END_REF][START_REF] Matus | Invasive Cell Fate Requires G1 Cell-Cycle Arrest and Histone Deacetylase-Mediated Changes in Gene Expression[END_REF][START_REF] Sherwood | FOS-1 Promotes Basement-Membrane Removal during Anchor-Cell Invasion in C. elegans[END_REF]Wang et al., 2014a)

ACTIN AND MEMBRANE DYNAMICS FOR AC INVASION

As seen from the preceding sections, the physiology, signaling and transcriptional control of AC invasion is fairly well described. It is equally clear that actin cytoskeleton and membrane components are important for this process. However the molecular players involved in actin and membrane dynamics in the AC are less well understood. UNC-6/netrin localizes the RacGTPases MIG-2 and CED-10 to the invasive leading edge and their combined loss results in invasion defects (Wang et al., 2014b;Ziel et al., 2009). Since MIG-2 signals to WSP-1 and CED-10 to WVE-1, this suggests the participation of both WSP-1/WASP and WVE-1/WAVE in AC invasion. In addition loss of another GTPase, CDC-42, also upstream of WSP-1/WASP, results in a delay in AC invasion (Lohmer et al., 2016).

Active CDC-42 is localized in the F-actin puncta that form prior to the growth of the invasive protrusion, and not in the invasive protrusion itself, indicating that WASP may be important for initial but not later stages of AC invasion (Lohmer et al., 2016).

The implication of both WSP-1/WASP and WVE-1/WAVE in AC invasion indicates that the process may depend on actin polymerization nucleation via the Arp2/3 complex. Nevertheless a formin, EXC-6, is specifically expressed in the AC under control of hda-1, but the role of formins during AC invasion is not known [START_REF] Matus | Invasive Cell Fate Requires G1 Cell-Cycle Arrest and Histone Deacetylase-Mediated Changes in Gene Expression[END_REF]. Two other actin assembly factors important for actin dynamics in lamellipodia, UNC-34/Ena/VASP and MIG-10/lamellipodin, are also known to contribute to AC invasion, and they do so independently of the UNC-6/netrin pathway (Wang et al., 2014b).

As for lamellipodia, actin disassembly proteins are also important for AC invasion. In animals mutant for the C. elegans orthologue of ADF/cofilin, UNC-60, AC invasion is blocked (Hagedorn et al., 2014). Excessive actin accumulates at the invasive membrane, but experiments using a photoconvertible fluorophore coupled to actin show that this actin is nondynamic, indicating that ADF/UNC-60 is important for actin turnover (Hagedorn et al., 2014). UNC-40/DCC and HIM-4/hemicentin are correctly localized in unc-60 mutants, but the phospholipid PI(4,5)P2, MIG-2/Rac and CED-10/Rac are not normally polarized when ADF/UNC-60 is disrupted (Hagedorn et al., 2014).

Moreover mispolarization and ectopic accumulation of lysosomal markers in internal compartments after loss of UNC-60 support the idea that UNC-60/ADF promotes membrane recycling from the protrusion via endolysosomes, although it is not clear if there is a direct link or if it's a steric effect created by excess polymerized actin that impedes proper membrane trafficking in the cytosol (Hagedorn et al., 2014). In keeping with a role for membrane dynamics in AC invasion, knocking down GDI-1, a Rab GDP-dissociation inhibitor that is related with vesicle trafficking, reduces AC invasion (Lohmer et al., 2016).

The scheme in (Figure 2.7) shows what we knew about membrane and actin dynamics in the AC during invasion at the point when I started my PhD studies. To summarize, in response to an unknown signal from the vulval cells, actin foci form in the AC via the activity of CDC-42 activating WSP-1/WASP, presumably upstream of Arp2/3 complex actin nucleation. The vulval signal also appears to communicate to other assembly factors such as UNC-34/Ena and MIG-10/lamellipodin. Only one or two of these foci will make a hole in the BM and transition into an invasive protrusion, dependent on Racs and presumably WAVE downstream of UNC-6/netrin signaling from the VNC to UNC-40/DCC. As for most force-producing actin structures, actin turnover via UNC-60/ADF is also important and may participate, along with GDI-1, in the trafficking of membrane to the protrusion.

CHAPTER 3: EXPERIMENTAL METHODS

PROTOCOLS

One of the main techniques of the PhD was RNAi by feeding. Sections 3.1.1-Section 3.1.3 explain the steps of the procedure.

WORM SYNCHRONIZATION

Worms were synchronized prior to most RNAi treatments so that large numbers of worms in the right stage of invasion could be evaluated.

1. Collect worms from 4 plates full of adult worms (use water) and transfer to a 15 ml Falcon tube.

2. Fill the tube with water and centrifuge at 200 g for 3min.

3. With a Pasteur pipette and a pump, suck off the supernatant (be careful not to touch the pellet). Repeat step 2 and 3 until the liquid is transparent (usually 2 times).

4. Add 100 -150ul of bleach solution (see below), and shake by hand (do not invert the tube or vortex, this makes the worms attach to the walls and not be in contact with the bleach solution). 5. Verify using the stereo microscope that all the worms are disintegrated (around 4-5 minutes after addition of the bleach solution), at this point the solution will have turned yellow.

6. Immediately fill the tube with PBS and mix by inversion 8 times.

7. Centrifuge at 600g at 3 min at room temperature (4°C will kill the eggs).

8. With a Pasteur pipette and a pump, suck off the supernatant (be careful not to touch the pellet). 9. Repeat steps 6-8. After the second wash, leave around 1ml of solution.

10. Verify with a pH strip that the pH is around 7. If it's not, perform another wash.

11. Shake the tube until all the eggs are floating, and pour the solution into a 50 ml Falcon tube (do not use a pipette, all the eggs will attach to the walls).

12. Put the 50 ml Falcon tube on a rotating wheel at room temperature.. 13. After 36 hrs most of the worms will be hatched and arrested in L1 larval stage due to lack of food.

Bleach solution in a 15 ml Falcon tube

Bleach* (2.6% active hypochlorite) 3ml NaOH (35% solution =12.1 M) 333ul H20

Fill to 5ml * Do not use bottles that have been opened for more than 2 months.

PREPARING BACTERIA WITH PROBES FOR FEEDING

1. Put 100 ul of HT115 competent cells into a 1.5ml tube.

2. Add 150 ng of DNA, mix very gently by hand (2 taps on the tube). Be sure there are no drops on the walls. DNA is the RNAi probe in the L4440 vector.

3. Incubate 30 minutes on ice.

4. Heat shock at 42°C for 2 minutes in a water bath.

5. Add 1 ml of LB media, mix by inversion and incubate 1h at room temperature.

6. Apply 20 ul and 100 ul to the middle of ampicillin agar plates with tetracycline (see below) and spread with a glass spatula.

7. Centrifuge the rest of the bacteria at 20000g for 2 minutes at room temperature.

8.

Remove almost all the supernatant (leave around 100 ul). 9. Resuspend the bacteria and spread it on an Amp/Tet plate as above.

10. Incubate the plates overnight at 37°C. 11. Only work with the big colonies that grow close to the center of the plate.

Ampicillin plates with tetracycline

Add 55 ul of tetracycline (100 mg/ml) to the middle of an ampicillin plate and homogenize with a glass spatula.

RNAI BY FEEDING

1. Pick a single colony with a 10 ul sterile pipette and grow it in 3ml LB medium + 3µl 100 mg/ml ampicillin in a 14ml culture tube at 37°C, 225 rpm agitation.

2. After 3 hours, check the OD using a 100ul of culture+900 LB, after blanking the machine on 900ul LB. If 20 min after spotting, the plates are not yet dry, put the plates in the hood with the lids ajar. Keep the windows of the lab closed to avoid contamination at all times.) 5. After induction, apply around 80 arrested L1s (see above, synchronization) to each plate.

Evaluate AC invasion after 32-35hrs.

NGM-IPTG plates Make 1.5 L batch and then divide into 3, 500 ml bottles after autoclaving and before adding the salts and the antibiotics. 500 ml bottles can be heated up individually in the microwave, supplemented with salts and antibiotics and poured. 500 ml makes about 50 plates. Mix, autoclave and Leave it at 55°C on a water bath, After add following the order: 1M CaCl2

1.5 ml (0.5 ml for 500 ml batch)

1 M MgSO4 1.5 ml (0.5 ml for 500 ml batch)

1 M KPO4 pH 6.0 37.5 ml (12.5 ml for 500 ml batch)

mg/mL cholesterol

This solution is in ethanol Avoid the flame.

1.5 ml (0.5 ml for 500 ml batch) Add IPTG and carbenicillin to 1mM and 25 ug/ml, respectively (1.5ml of IPTG at 1M and 375 uL of carbenicillin at 100 mg/ml for a 1.5 L batch; 500ul and 125 ul for a 500 mL batch).

The plates can be stored at 4°C for up to 3 weeks.

LIVE WORM IMAGING

Sample mounting

1. Put 100 ul of levamisole (0.02%) into the well of a watch glass.

2. Transfer 30-40 worms (minimize bacteria) and incubate until the worms stop moving (15 min approx).

3. Transfer the worms with a mouth pipette onto a 4.5% noble agar pad.

4. Cover the sample with a coverslip and add more drug at the edges of the agar pad to keep it humid.

5. Use VALAP to seal the sample, leaving 2 small windows for oxygen exchange.

6. Use a humid chamber to transport the sample to the microscope room.

Image acquisition (Spinning disk)

Samples are imaged at 20°C with an inverted confocal spinning disk microscope from Nikon or an upright spinning disc from Zeiss, using a 100x NA1.4 OIL DIC N2 PL APO VC objective and a CoolSNAP HQ2 (Photometrics) camera controlled by Metamorph software.

1. Find and center the worms using the 10x objective.

2. Change to the 100X objective and with low laser power (15-20%) and 50 ms of exposure, find the fluorescent AC and select the top and bottom planes.

Start time-lapse acquisition using:

-491nn laser at 30% 150ms exposure time -561nn laser at 35% 250ms exposure time (increase over time if necessary, see below*) -Transmitted light at max power 350 ms exposure time-only use to check worm morphology, do not use during stack acquisition. -0.5 um Z-step -Acquire a stack every 45s 4. The worm moves in the z plane because the agar pad dries. Check that the top and bottom plane of the stack still covers the whole AC by changing the stage position by hand when necessary. * Keep the laser at 0% power during acquisition. Just turn it on at 35% when the AC actin protrusion start to form, when the protrusion expands and at the end (not during the whole acquisition).

RESOLFT microscopy

Point scanning RESOLFT imaging was carried out using a custom setup built by the laboratory of Ilaria Testa. The setup was built around a Leica DMI 6000 microscope with a 63x STEDwhite glycerol objective lens NA 1.4. The correction index collar was used in order to match the refraction index from the worm with the mounting medium. The collar was adjusted for each worm in order to collect the most photons to minimize the expansion of the focal spot along the optical z-axis. RESOLFT requires 3 separate controllable light sources, one for each phase in the RESOLFT scheme: activation (405 nm), off-switching (488 nm) and read-out (488 nm). The hardware was controlled using a custom designed software based on LabVIEW and National Instrument's FPGA. This allowed us, through a single interface, to control the XYZ scanner, the sequential pulsing scheme of the lasers, and ensured we only collected the photons emitted during the read-out phase.

Image acquisition

1. Once the anchor cell is centered, reduce the scanning window to match as much as possible in the X and Y axes. On the Z axis only take the planes that contain the protrusion (0,5µm steps).

2. Select on the acquisition software the following parameters:

"Act Time": 20 ms "wait1Time":50 ms "donutTime":50 ms "wait2Time":500 ms "readoutTime":100 ms * "minDwellTime":0 ms "decisionTime From":0 ms "decisionTime To":70 ms Note: The system is not able to take a time-lapse. It acquires images continuously.

3. The worm moves in the z plane because the agar pad dries. Check that the top and bottom plane of the stack still covers the whole AC by changing the stage position by hand when necessary.

MICROINJECTION

The VCA-BFP, Lifeact-BFP and Lifeact-Dronpa strains were prepared by microinjection, followed by integration (see below) for the VCA and Dronpa strains.

1. Put a drop of halocarbon oil on a dry pad of noble agar 4.5% on a 60x50 mm coverslip.

2. Pick an adult worm into the oil and with a flat platinum filament, smooth the worm down from the middle body to the tail and head to immobilize the worm on the agar.

INTEGRATING TRANSGENIC LINES

1. Take an injected line that transmits the transgene well, that is, ones that rescue well for the unc-119 phenotype (DP38 was the injected strain). Take the best transmitters.

2.

Put a drop of water onto an NGM plate without food.

3.

Pick 40 young adult DP38 (unc-119) worms into the drop to remove bacteria from their bodies.

4.

Re-pick the 40 clean worms onto a new NGM plate without food.

5.

Irradiate the worms (lid off) in a UV Stratalinker with the "300" energy setting of the machine (corresponds to 30000 microjoules).

6.

Transfer the worms onto NGM plates with food (2 worms per plate).

7.

After starvation transfer a chunk with approx. 100 worms to a new plate with food.

8.

After starvation transfer 8 worms per plate onto new NGM plates (1 worm per plate = 160 NGM plates total). 9.

After 3-5 days keep only the plates that have 100% of the selected phenotype, in this case mover, non-uncs.

10. From each selected plate, transfer 3 worms onto separate NGM plates.

11. After 3-5 days confirm all 3 plates still have 100% of the selected phenotype.

12. Check the expression levels of your protein of interest and verify normal development and keep the best ones.

SINGLE WORM PCR

This technique was used to verify transgenic worms and to detect alleles during crosses.

1.

Put 15 ul of lysis buffer (see below) into each PCR tube of a strip and put on ice.

2.

Pick a L4 /young adult into a drop of water on a clean NGM plate.

3.

Move the worm around in the drop of water to remove the bacteria.

4.

Pick the worm into the PCR tube, confirm the worm is in the lysis buffer and put back on ice (avoid touching the walls of the PCR tube).

5.

Centrifuge for 4 s (use the strip centrifuge).

6.

Put the PCR strip into the -80 o C for 10 min (until the solution is totally frozen).

7.

Put the PCR tube/strip into the thermocycler (verify the tubes are well closed).

8.

Once the program is finished, flick the tubes to mix (make sure to mix the condensation from the walls into the rest of the solution) and recentrifuge before using for PCR. The cdh-3 promoter was amplified as in (Ziel et al., 2009) and introduced into Gateway cloning vector pDONR[P4-P1R]. Lifeact::GFP was from [START_REF] Havrylenko | WAVE binds Ena/VASP for enhanced Arp2/3 complex-based actin assembly[END_REF]. The fragments were recombined as above with unc-54 UTR and pCFJ150. Lifeact and linker as in [START_REF] Havrylenko | WAVE binds Ena/VASP for enhanced Arp2/3 complex-based actin assembly[END_REF] was synthesized in frame with DronpaM159T. The Dronpa sequence was obtained by taking the mammalian sequence and adapting it for C. elegans codon usage and by putting in syntrons using the website http://worm-srv3.mpi-cbg.de/codons/cgi-bin/optimize.py. The gene was the synthesized by Eurofins Genomics.

Lysis

CHAPTER 4: THE ROLE OF ACTIN POLYMERIZATION NUCLEATORS AND CROSSLINKERS IN ANCHOR CELL INVASION

GOAL OF THE STUDY

As described in the previous chapters, most studies on cell invasion are performed in vitro using cancer cells plated on or seeded in BM mimics where native mechanical and geometrical properties of the BM are not entirely respected. These are important factors for most cell biology questions: for example a 10-fold difference in substrate elasticity is enough to change the fate of a mesenchymal stem cell from a brain cell to a muscle cell [START_REF] Engler | Matrix Elasticity Directs Stem Cell Lineage Specification[END_REF]. However lack of preservation of the mechanical microenvironment is particularly problematic for actin cytoskeleton studies where changes in ECM properties and geometry have been shown to change the dynamics and biochemistry of actin-based structures [START_REF] Fraley | A distinctive role for focal adhesion proteins in threedimensional cell motility[END_REF][START_REF] Geraldo | Do cancer cells have distinct adhesions in 3D collagen matrices and in vivo?[END_REF]Petrie et al., 2012). Using AC invasion as a model allows us to study the role of the actin cytoskeleton in an invasive process in a native 3D environment.

Specifically during this part of my PhD, I sought to address the role of the Arp2/3 complex and the relative contributions of its two activators, WAVE and WASP, in an invasive process, if formins played a role and if the invasive protrusion required actin cross-linkers to increase its mechanical stability. In all this my overall hypothesis was that actin polymerization creates forces that help break open the BM and push it aside, and does not just play a structural or trafficking role as has been suggested by some studies [START_REF] Yu | N-WASP coordinates the delivery and F-actin-mediated capture of MT1-MMP at invasive pseudopods[END_REF].

THE ARP2/3 COMPLEX AND ITS ACTIVATORS DURING AC INVASION

As mentioned in Chapter 2, WSP-1/WASP plays a role during AC invasion, along with its upstream effector CDC-42/Cdc-42 (Lohmer et al., 2016). Also the Rac GTPases CED-10 and MIG-2 are found at the invasive front of the AC (Ziel et al., 2009), suggesting the involvement of WAV-1/WAVE and WSP-1/WASP. The involvement of its activators pointed toward the Arp2/3 complex as a nucleator for actin polymerization in AC invasion, but this had not been proven. Likewise how the loss of WSP-1/WASP and WAV-1/WAVE affected the formation of the invasive protrusion had not been quantified.

THE EFFECT OF REDUCTION OF ARP2/3 COMPLEX AND WASP ON AC INVASION

The components of the Arp2/3 complex are essential genes, so worms completely lacking Arp2/3 complex function are not available. I therefore used RNAi knockdown followed by scoring of invasion to evaluate the role of the Arp2/3 complex in AC invasion. This is an approach that I used extensively during this and other sections, and involves performing RNAi by feeding (see Chapter 3) and then evaluating invasion at the 4-cell stage (see Chapter 2, Section 2.4). At this stage in WT worms, 100% of ACs had cleared a large opening in the BM. At the 4-cell stage, I grouped my observations into 3 categories: 1) the hole in the BM was as wide as the AC, this was "full invasion"; 2) a defect in the BM was detectable, but the hole was not as wide as the AC, this was "partial invasion"; 3) there was no break in the BM, this was "no invasion". When arx-2, the equivalent of the Arp2 subunit of the Arp2/3 complex, was knocked down, 30% of the worms showed a complete lack of a hole in the BM under the AC at the 4-cell stage (Table 4.1). This indicated a role for the Arp2/3 complex in AC invasion, despite the known resistance of the Arp2/3 complex to RNAi due to its long-term stability [START_REF] Wu | Arp2/3 is critical for lamellipodia and response to extracellular matrix cues but is dispensable for chemotaxis[END_REF][START_REF] Zhu | Functional Coordination of WAVE and WASP in C. elegans Neuroblast Migration[END_REF]. This was in keeping with the reported role for WASP in AC invasion, a result that I reproduced, finding that only 23% of ACs had invaded at the 4-cell stage (Table 4.1). For this and in the following, I used a mutant worm that is a functional null for the sole copy of C. elegans WASP [START_REF] Withee | Caenorhabditis elegans WASP and Ena/VASP protiens play compensatory roles in morphogenesis and neuronal cell migration[END_REF], strain NG324, genotype wsp-1(gm324). This allele will be referred to as "WASP-deleted". WASP-deleted worms have low brood size, but are otherwise viable and fertile.

ACTIN DYNAMICS IN THE AC IN THE ABSENCE OF WASP

In order to characterize the effect of WASP deletion on actin dynamics during AC invasion, a worm strain was constructed that carried the wsp-1(gm324) and additionally expressed

AC INVASION IN THE ABSENCE OF WAVE

In the case of WASP deletion, the protrusion was not entirely absent. Indeed WASP-deleted In order to probe further the role of WVE-1/WAVE in AC invasion, invasion was scored in different mutant and RNAi backgrounds at the 4-cell stage by DIC microscopy as described above. First a WAVE-deleted worm was evaluated, strain OX308, genotype wve-1(ne350),

which contains an early stop codon giving non-functional protein [START_REF] Patel | The WAVE/SCAR complex promotes polarized cell movements and actin enrichment in epithelia during C. elegans embryogenesis[END_REF]. The WVE-1/WAVE deletion is embryonic lethal so this worm strain was maintained as a heterozygote. Homozygotic WAVE-deleted larval worms were isolated for the analysis and probably due to low RNAi efficiency against the Arp2/3 complex as mentioned above.

DOMINANT NEGATIVE INHIBITION OF ARP2/3 COMPLEX ACTIVITY

To get around this limitation, we employed a dominant negative approach to inhibit the Arp2/3 complex that had been shown to be effective in mammalian cells [START_REF] Machesky | Scar1 and the related Wiskott-Aldrich syndrome protein, WASP, regulate the actin cytoskeleton through the Arp2/3 complex[END_REF]) (Figure 4.8). This consisted in expressing under control of an AC-specific promoter the VCA domain of WASP, the part of WASP that binds and activates the Arp2/3 complex. The cytosolic expression of VCA sequesters the Arp2/3 complex in the cytosol away from its normal membrane localization and thus acts as a dominant negative without disturbing other known pathways for actin polymerization that are Arp2/3 complexindependent [START_REF] Koestler | Arp2/3 complex is essential for actin network treadmilling as well as for targeting of capping protein and cofilin[END_REF]. Indeed even at later stages of vulval morphogenesis, the BM remained intact in 90% of the VCA-expressing worms (Figure 4.10), the vulva did not form properly, and worms "bagged", that is eggs hatched inside the mothers instead of being laid. We concluded from these results that AC invasion is very strongly dependent on Arp2/3 complex-based actin nucleation, and except for rare events, AC invasion does not occur when the Arp2/3 complex at the invasive leading edge is completely turned off.

INTERPLAY OF WASP AND WAVE FOR AC INVASION

As mentioned above, invasion driven by Arp2/3 complex-based polymerization still happened in WASP-deleted worms seemingly due to the presence of WVE-1/WAVE. In order to provide evidence for this, I examined where endogenous WSP-1/WASP and WVE-1/WAVE were localized during AC invasion, using a worm strain carrying GFP-labeled WSP-1/WASP and RFP-labeled WVE-1/WAVE, produced via genome modification by CRISPR (courtesy of the Guangshuo Ou laboratory, alleles cas723 and cas762, respectively [START_REF] Zhu | Functional Coordination of WAVE and WASP in C. elegans Neuroblast Migration[END_REF]). In this background, we introduced Lifeact::tagBFP to visualize where WVE-1/WAVE and WSP-1/WASP were in relation to the actin cytoskeleton during AC invasion.

We observed that WSP-1/WASP was present in the AC before ( 2

RELATIVE ROLES OF WASP AND WAVE IN AC INVASION

WVE-1/WAVE played a minor role in AC invasion as compared to WSP-1/WAVE despite their similar function of activating the Arp2/3 complex for branched actin polymerization nucleation. The upstream activators of WVE-1/WAVE have been shown to be present at the invasive front (Ziel et al., 2009), in keeping with my observed localization analysis above, so it was not clear why there was such an unequal contribution to invasion of the two Arp2/3 complex activators. One possibility was a reduced efficiency of WVE-1/WVE to activate the Arp2/3 complex in comparison to WSP-1/WAVE to form a branched protrusion able to push aside the BM. Mammalian WAVEs are known to be less active than mammalian WASP molecules due to differences in acidity of the C-terminal, Arp2/3 complex-binding domain (Zalevsky et al., 2001b). To see if this was likewise the case with C. elegans WASP and WAVE, a pyrene-actin assay was performed using the purified VCA domains from C. elegans WSP-1/WASP and WVE-1/WAVE in presence of the Arp2/3 complex. A pyrene assay is a spectroscopic method to evaluate the speed of formation of actin filaments over time, and more efficient Arp2/3 complex activation gives steeper polymerization curves [START_REF] Doolittle | Measurement and Analysis of in vitro Actin Polymerization[END_REF].

Representative polymerization curves for WSP-1/WASP and WVE-1/WAVE are shown in

LOCALIZATION OF INVASIVE COMPONENTS UPON WASP DELETION

Given the known role of WASP in endocytosis and in protease delivery in invadopodia (Campellone and Welch, 2010;[START_REF] Yu | N-WASP coordinates the delivery and F-actin-mediated capture of MT1-MMP at invasive pseudopods[END_REF], it was possible that the effect of WASP deletion on invasion was related to a defect in trafficking of invasive components to the leading edge.

In This data confirmed that WASP deletion was not affecting localization, and presumably trafficking, of essential components to the invasive edge. This supported the idea that the branched actin network made by WASP was not playing a passive scaffolding role, but was actively driving the invasive process by applying forces on the BM.

ROLES OF UPSTREAM REGULATORS OF WASP AND WAVE IN AC INVASION

Despite its reduced activity, WVE-1/WAVE appeared to "stand in" for WSP-1/WASP to ensure AC invasion, albeit with a smaller, less dynamic protrusion and a corresponding delay in BM invasion. If WSP-1/WASP and WVE-1/WAVE were working together in this way, this should be reflected in the upstream regulatory pathways involving GTPases. In C.

elegans, WASP is known to be regulated by both CDC-42/Cdc-42 and MIG-2/Rac, while WVE-1/WAVE is activated by the CED-10/Rac [START_REF] Walck-Shannon | Polarized Rac-dependent protrusions drive epithelial intercalation in the embryonic epidermis of C. elegans[END_REF]. We therefore set out to probe the role of these upstream regulators, in order to lend evidence to the idea that there were two independent pathways for activating the Arp2/3 complex for AC invasion.

Interfering with the GTPase regulators individually gave little or no invasion defect: RNAi against cdc-42 gave a slight defect (14% no invasion) (Lohmer et al., 2016), mig-2 mutant worms had 100% normal invasion (Ziel et al., 2009) and ced-10 mutant worms with ced-10

RNAi to eliminate maternal contribution (it was a balanced strain) had 97% full invasion at

ROLE OF FORMINS IN AC INVASION

From the results of the dominant negative Arp2/3 complex activator study, it seemed unlikely that formins played a role in AC invasion. Indeed in studies on cells, when the Arp2/3 complex is inhibited by the VCA dominant negative approach or other methods, lamellipodia collapse, but actin structures that are dependent on other nucleators such as formins become more pronounced [START_REF] Koestler | Arp2/3 complex is essential for actin network treadmilling as well as for targeting of capping protein and cofilin[END_REF][START_REF] Suraneni | The Arp2/3 complex is required for lamellipodia extension and directional fibroblast cell migration[END_REF][START_REF] Wu | Arp2/3 is critical for lamellipodia and response to extracellular matrix cues but is dispensable for chemotaxis[END_REF]. The fact that the VCA-expressing AC did not display actin spikes or other structures was an indication that other nucleators, including formins, were not active in the AC, see Figure 4.9 and 4.10.

In order to confirm that formins were not playing a role, I evaluate the invasion for all 6 known C. elegans formin mutants separately, and also evaluated a triple formin mutant that included EXC-6, a formin strongly and specifically expressed in the AC [START_REF] Matus | Invasive Cell Fate Requires G1 Cell-Cycle Arrest and Histone Deacetylase-Mediated Changes in Gene Expression[END_REF].

All of these animals showed WT invasion at the 4-cell stage (Table 4.2). Perhaps EXC-6 is strongly expressed in the AC because it is needed in later stages of vulval morphogenesis, during lumen formation for example [START_REF] Estes | The anchor cell initiates dorsal lumen formation during C. elegans vulval tubulogenesis[END_REF]. EXC-6 is known to be important for lumen formation during secretory canal development [START_REF] Shaye | The Disease-Associated Formin INF2/EXC-6 Organizes Lumen and Cell Outgrowth during Tubulogenesis by Regulating F-Actin and Microtubule Cytoskeletons[END_REF]. These RNAi results, together with the unspikey appearance of ACs treated with the VCA dominant negative, suggested that formins did not nucleate actin for AC invasion.

AC Invasion

FORMINS Complete Invasion partial Invasion No Invasion

Control

ROLE OF ACTIN FILAMENT CROSSLINKERS IN AC INVASION

As mentioned previously, actin crosslinkers play important roles in the mechanics of actin structures in general, and the actin bundler fascin is key for invadopodia formation in particular (Schoumacher et al., 2010). In order to know if the actin crosslinkers were playing a role in AC cell invasion, I performed RNAi and evaluated mutants of the 3 main actin crosslinkers in the C. elegans genome: ATN-1/-actinin, PLST-1/plastin (fimbrin) and FLN-1/filamin. Mutations knocking out the activity of individual cross-linkers did not affect AC invasion (Table 4.3). To bypass possible redundancy amongst crosslinkers, I constructed a strain that was mutant in ATN-1/-actinin and PLST-1/plastin (fimbrin) and I further knocked down fln-1 by RNAi in this background. These worms also displayed near WT levels of invasion (Table 4.3). From this I concluded that crosslinkers were not essential for AC invasion. It has been shown that entanglement due to a high degree of branching can provide cohesion in an actin network despite the absence of crosslinkers [START_REF] Achard | A "Primer"-Based Mechanism Underlies Branched Actin Filament Network Formation and Motility[END_REF]Dayel et al., 2009). Experiments using super-resolution microscopy (Chapter 5) indicated that the actin network in the AC protrusion was very dense, suggesting that additional crosslinking was unnecessary.

AC Invasion

CROSSLINLERKS Complete Invasion partial Invasion

No Invasion

atn-1(ok84) 100% 0% 0% plst-1(tm4255) 100% 0% 0% fln-1 (tm545) 91% 7% 2%
atn-1(ok84) ; plst-1(tm4255)+fln-1 (RNAi) 96% 4% 0% were reported for cell movements in the C. elegans embryo during a process known as dorsal intercalation [START_REF] Walck-Shannon | Polarized Rac-dependent protrusions drive epithelial intercalation in the embryonic epidermis of C. elegans[END_REF] and during axon migration [START_REF] Shakir | The Arp2/3 activators WAVE and WASP have distinct genetic interactions with Rac GTPases in Caenorhabditis elegans axon guidance[END_REF].

On the other hand an analysis of WSP-1/WASP and WVE-1/WAVE during Q neuroblast migration, a process where a single cell migrates through the body cavity in C. elegans, showed that WVE-1/WAVE is the main Arp2/3 complex activator for motility although WSP-1/WASP can relocalize upon WVE-1/WAVE depletion to compensate [START_REF] Zhu | Functional Coordination of WAVE and WASP in C. elegans Neuroblast Migration[END_REF]. In other lamellipodial-type protrusions, like that of the ventral epidermal cells during ventral enclosure, WSP-1/WASP plays no role in actin assembly dynamics, which are entirely dependent on WVE-1/WAVE [START_REF] Havrylenko | WAVE binds Ena/VASP for enhanced Arp2/3 complex-based actin assembly[END_REF][START_REF] Patel | The WAVE/SCAR complex promotes polarized cell movements and actin enrichment in epithelia during C. elegans embryogenesis[END_REF]. Likewise WAVE elimination in mammalian cells abolishes lamellipodia formation and WASP is not localized at the leading edge [START_REF] Leithner | Diversified actin protrusions promote environmental exploration but are dispensable for locomotion of leukocytes[END_REF], and it is generally accepted that Rac signaling to WAVE is what forms lamellipodia while Cdc42 signaling to WASP has other functions (Campellone and Welch, 2010;[START_REF] Rotty | New insights into the regulation and cellular functions of the Arp2/3 complex[END_REF].

In AC invasion, this is not the case, and the lamellipodial-like protrusion of the AC is mostly WSP-1/WASP driven with some contribution from WVE-1/WAVE. This is similar to cancer cell invadopodia, as presented in Chapter 2, where there is strong evidence for WASP activity in invadopodia while the role of WAVE in invadopodia is less clear. My results point clearly to a role for WAVE as a back-up player to WASP in invasive protrusion formation, and adds to a growing list of recent studies showing that the two molecules play more similar roles than previously thought [START_REF] Fritz-Laylin | WASP and SCAR are evolutionarily conserved in actin-filled pseudopod-based motility[END_REF][START_REF] Zhu | Functional Coordination of WAVE and WASP in C. elegans Neuroblast Migration[END_REF]. I also show that the strong dependence of AC invasion on actin polymerization via WASP is not due to its trafficking or scaffolding role: 3 main players in AC invasion, including a protease, are correctly localized in the absence of WASP. Despite the presence of the key components, WASP-deleted ACs do not invade with normal timing, presumably because insufficient actin is produced to breach and push aside the BM.

The results of this chapter and some elements from the next chapter will be included in a publication that is currently in preparation: Cáceres R., Bojanala N., Kelley L.C., Dreier J., Manzi J., Di Federico F., Chi Q., Risler T., Testa I., Sherwood D.R. and Plastino J "WASP and WAVE activate the Arp2/3 complex for actin-based force production during basement membrane invasion".

introduce super-resolution in general before describing RESOLFT and its predecessor STED, and the results I obtained with the former.

SUPER-RESOLUTION MICROSCOPY

OVERVIEW OF SUPER-RESOLUTION TECHNIQUES RELATED TO RESOLFT

In order to increase the resolution of the light microscope, and be able visualize two adjacent filopodia for example, a decrease in the wavelength or an increase in the numerical aperture is needed, something that in practice is difficult to do with conventional confocal, bright field, and spinning disk microscopy. These techniques are limited by the wavelengths of available probes and the NA of the objectives. However in 1994 Hell and Wichmann broke the diffraction limited using an approach based on spatial minimization of the focal spot created by the laser, increasing the resolution of confocal fluorescence microscopy [START_REF] Hell | Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy[END_REF]. This technique is called Stimulated Emission Depletion Fluorescence Microscopy (STED) and it can achieve a resolution of around 70 nm (Willig et al., 2006a). I will come back in more detail to this technique in Section 5.

REversible Saturable Optical

Linear Fluorescence (RESOLFT) relies on the same idea as STED, but additionally using reversible photoswitching between a fluorescent "on" state and a dark "off" state to decrease the focal spot [START_REF] Hell | Imaging and writing at the nanoscale with focused visible light through saturable optical transitions[END_REF][START_REF] Testa | Nanoscopy of Living Brain Slices with Low Light Levels[END_REF]. I will describe RESOLFT more fully in Section 5.2.3.

At about the same time as STED, other super-resolution techniques were being developed, including Photo-Activated Localization Microscopy (PALM) (Betzig, 1995;Betzig et al., 2006). PALM uses a different approach to break the diffraction limit, based on repeated cycles of photoactivation and photobleaching: fluorophores such as photoactivatable GFP are stochastically activated, an image is taken and then the fluorophores are bleached, allowing for the pinpointing of individual fluorescent molecules. Once all the single fluorophore positions are obtained, a reconstruction produces a final super-resolution image. A similar strategy can be used with photoswitchable fluorophores, such as Eos and Dendra, in order to turn "on" and "off" different sub-sets of fluorophores in the same region. This approach is known as stochastic optical reconstruction microscopy (STORM) [START_REF] Rust | Stochastic optical reconstruction microscopy (STORM) provides sub-diffraction-limit image resolution[END_REF]. PALM, STORM and STED super-resolution techniques use long acquisition times (5-30 minutes) [START_REF] Shroff | Photoactivated Localization Microscopy (PALM) of Adhesion Complexes[END_REF] and high intensity light of MW.cm -2 [START_REF] Li | Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics[END_REF], and are thus suited to studies of fixed samples but not to following the dynamics of a cellular process. This is not the case with RESOLFT, which was one of the reasons we chose this technique to image Factin in the AC.

STIMULATED EMISSION DEPLETION FLUORESCENCE MICROSCOPY (STED)

I will describe STED before moving on to RESOLFT, since pedagogically and historically, RESOLFT was inspired by STED. STED consists in the overlapping of 2 synchronized lasers that illuminate the same position. One is responsible for activating the fluorophore (excitation laser) while the second one turns off the excitation (depletion laser). The depletion laser has a doughnut-shaped pattern and thus sharpens the excitation laser and decreases the focal spot.

This doughnut-shaped depletion is accomplished using a phase mask in the path of the depletion laser (Figure 5.1). The depletion laser works by de-exciting fluorophores from the excited state to the ground state without photon emission. The probability that the fluorophore will remain in a high energy state decreases in an exponential manner with increasing intensity of the depletion laser (Figure 5.2). In practice,

STED is performed at a laser intensity where almost all the exited fluorophores are turned off,

Is. This intensity is called a "saturated intensity", but if a higher intensity is used (I in Figure 5.2), this will not only increase the probability of turning off the exited fluorophores, but also increase the resolution in an inversely proportional manner (Figure 5.2). When I equals Is, the equation is reduced to the Abbé diffraction limit, but if I is larger than Is, the focal spot becomes very narrow achieving super-resolution. To achieve this gain in resolution, the depletion laser must be high enough in energy to send all the exciting fluorophores to the ground state before they release fluorescence which happens in less than 2 ns. For this reason high depletion laser energies, on the order of MW.cm -2 , are required.

= √ +

REVERSIBLE SATURABLE OPTICAL LINEAR FLUORESCENCE (RESOLFT)

The concept of RESOLFT was developed in order to reduce the intensity levels required in STED [START_REF] Hell | Imaging and writing at the nanoscale with focused visible light through saturable optical transitions[END_REF]. This technique uses on/off switching as in STED but with reversible photoswitchable fluorophores (rsFPs) such as the molecule Dronpa. On/off states reflect cistrans molecular isomerization within the fluorophore of the fluorescent protein label [START_REF] Grotjohann | Diffraction-unlimited all-optical imaging and writing with a photochromic GFP[END_REF]. These states have long lifetimes (µs-ms), reducing the light intensity needed to perform depletion to W.cm -2 to kW.cm -2 , around 10 5 lower than STED [START_REF] Stefan | The 2015 super-resolution microscopy roadmap[END_REF]. As for STED, the gain in spatial resolution is due to use of a doughnutshaped light pattern to transiently silence the florescence of rsFPs, and allow only the fluorophores from the center of the doughnut to contribute to the fluorescence signal (Figure 5.3). In order to achieve nano-resolution, the rsFPs need to survive several "on" and "off"

cycles. Nevertheless due to the low energies used, studies on living neurons show that after 2 hours of continuous scanning there is no obvious photodamage [START_REF] Testa | Nanoscopy of Living Brain Slices with Low Light Levels[END_REF].

SUPER-RESOLUTION VISUALIZATION OF F-ACTIN STRUCTURES DURING AC INVASION

Results described in Chapter 4 strongly suggest that AC protrusion is mainly driven by a branched actin network that indents the BM before invasion and pushes it aside during invasion. However the actin architecture in the invasive protrusion is not resolvable with conventional microscopy techniques, as seen from the images presented in Chapter 4. In order to overcome this obstacle, and after having tried other techniques such as structured illumination and light sheet microscopy without success, I decided to do RESOLFT imaging in collaboration with Ilaria Testa, one of the authors of the first RESOLFT publication. I constructed a worm strain expressing an rsFP, DronpaM159T, fused to Lifeact specifically in the AC as an integrated transgenic line. The M159T variant is a faster switcher than the original Dronpa [START_REF] Kaucikas | Photoisomerization and Proton Transfer in the Forward and Reverse Photoswitching of the Fast-Switching M159T Mutant of the Dronpa Fluorescent Protein[END_REF]. I took these worms to Sweden, and spent about 3 weeks in the Testa laboratory to perform RESOLFT imaging.

Experiments were performed on live anesthetized worms in view to doing super-resolution of the invasive protrusion over time. However with the point scanning acquisition mode of the RESOLFT microscope, acquiring a stack that covered the entire AC took around 3 minutes, a window of time much too long considering that the whole invasion process only lasted about 30 minutes. In order to remedy this problem, the scanning time was reduced to around 1.5

minutes by reducing the number of z-planes, and only acquiring those planes where the AC actin protrusion was most visible. Image acquisition of 1.5 minutes was still very long, especially considering that even with an interval of 45 seconds on the spinning disk, consecutive frames appeared discontinuous, indicating that structures formed and collapsed faster than the stacks could be acquired. However with the 3D nature of the AC and the slow scan speed imposed by the point-scanning set-up and RESOLFT technology, 1.5 minute image acquisition was the best that could be achieved. Reducing the number of z-planes also helped reduce photodamage, but even with this restricted z-stack mode, we were only able to image AC invasion for around 15 minutes before fluorophore bleaching, not enough time to acquire the entire AC invasion process. So in the end, acquiring RESOLFT images of AC invasion over time turned out not to be practicable, due mostly to insufficient time resolution. The dense unresolvable core was also visible in post-invasion stages, where the BM hole started to expand beyond the AC edges and the protrusion was bigger (Figure 5.5). In addition to short branch-type structures at the edges of the BM hole as observed pre-invasion (above), a few elongated structures were also visualized on the shaft of the AC protrusion, emanating from the dense core (Figure 5.5). These structures resembled filopodia protruding from a lamellipodia network. In this regard it should be remembered that some formins such as EXC-6 are highly expressed in the AC, and although they don't appear to play a role in invasion (Chapter 4, Section 4.4), they could participate in the formation of post-invasive structures such as these filopodia-like protrusions.

CONCLUSION: AC PROTRUSION IS A HIGHLY DENSE ACTIN NETWORK

On a technological level, the study of the AC protrusion is a proof-of-concept as to the potential of RESOLFT microscopy for following cellular events on live samples. In addition it shows that complex thick tissues can be successfully imaged with RESOLFT. Since RESOLFT is a relatively new technique, this is useful information for the Testa laboratory.

As to understanding more about the architecture of the invasive protrusion, the RESOLFT study was not as informative as I had hoped. Partly due to the long acquisition time and the dynamic movements of the sample, the amount of additional detail observable by RESOLFT as compared to spinning disk was not dramatic. In the AC, RESOLFT microscopy distinguishes actin structures around 100 nm thick on the edges of the protrusion. However the actin core of the protrusion can't be resolved by RESOLFT. This is a piece of information in itself, indicating that the protrusion is very densely packed with actin filaments. This supports my results in Chapter 4, showing that strong filament branching via both WASP and WAVE activation of the Arp2/3 complex is occurring during normal invasion.

CHAPTER 6: ROLE OF ACTO-MYOSIN CONTRACTION DURING AC INVASION IN THE ABSENCE OF PROTEASES

CONTEXT OF THE STUDY: AC INVASION AND MMPS

Up until recently the role of proteases in AC invasion was not clear. As discussed in Chapter 2, an MMP, ZMP-1, was known to be expressed in the AC under control of the transcription factors responsible for AC identity. However knocking out zmp-1 on its own had no effect on AC invasion [START_REF] Sherwood | FOS-1 Promotes Basement-Membrane Removal during Anchor-Cell Invasion in C. elegans[END_REF]. Furthermore RNAi knock-down of 90% of the C.

elegans genes that possessed a predicted protease or protease inhibitor domain gave no perturbations of AC invasion [START_REF] Ihara | Basement membrane sliding and targeted adhesion remodels tissue boundaries during uterine vulval attachment in Caenorhabditis elegans[END_REF]. This lack of effect may be due to protease redundancy, so a study was undertaken by our collaborators to remove all MMPs in the C.

elegans genome. As opposed to the mammalian genome that has 20+ MMPs, C. elegans only possesses 6, one of which is not expressed in the AC at the relevant stage (Hagedorn and Sherwood, 2011) and (L. Kelley and D. Sherwood, unpublished). Based on this, L. Kelley and coworkers from the Sherwood lab created a worm strain that was mutated in all 5 relevant MMPs, hereafter referred to as "MMP-" conditions. Somewhat surprisingly AC invasion in these worms was only slightly affected, with 84% of the ACs invading by the 4-cell stage.

Given what was known about protease-independent invasion of cancer cells (Chapter 1, Section 1.3.2), I undertook the study of the role of myosin in AC invasion in MMP-worms, with the hypothesis that, in analogy to cancer cells, enhanced contractility might compensate for reduced proteolysis of ECM barriers.

ACTO-MYOSIN MACHINERY DURING AC INVASION IN ABSENCE OF MMPS

In order to test for compensation, I compared the effects of RNAi in WT versus in MMP-, looking for knock-downs that affected MMP-invasion while having no effect in WT worms. I knocked down by RNAi the genes for myosin heavy chain nmy-2, mlc-4, a regulatory light chain for non-muscle myosin, mel-11, a myosin phosphatase and negative regulator of myosin activity, and two other main regulators of myosin contraction, the Rho kinase let-502 and the myosin light chain kinase mrck-1 (see also Chapter 2, Section 2.3). All of these components are implicated in myosin-based processes like embryonic elongation in C. elegans [START_REF] Gally | Myosin II regulation during C. elegans embryonic elongation: LET-502/ROCK, MRCK-1 and PAK-1, three kinases with different roles[END_REF]. Scoring was performed at the 4-cell stage as either invaded or not.

As shown in Table 6.1, none of the treatments had an impact on WT worms, indicating that myosin contractility was not important for AC invasion. The effectiveness of the RNAi treatment was confirmed by the presence of vulval malformations and a reduction in egglaying for mlc-4 and mell-11 probes. From this it appeared that myosin activity was necessary for later stages of vulval development in keeping with other studies [START_REF] Farooqui | Coordinated Lumen Contraction and Expansion during Vulval Tube Morphogenesis in Caenorhabditis elegans[END_REF],

but not for AC invasion in itself. The same RNAi treatments were applied to MMP-worms, and as compared to control RNAi treatment containing empty vector, there were no differences in the % of ACs invaded at the 4-cell stage (Table 6 In keeping with this, a GFP fusion of MRCK-1 expressed under the endogenous promotor as a transgene was not expressed in the AC (Figure 6.1), nor was its substrate MLC-4, although MEL-11, LET-502 and NMY-2 were all present in the AC at the 4-cell stage (L. Kelley unpublished results). The fact that some of the essential myosin machinery was missing from the AC at the critical time was further evidence that myosin activity was not important for invasion. Of note MRCK-1::GFP was observed in the VPCs (Figure 6.1), indicating that the transgene was active. This localization is probably related to the importance of myosin contraction for normal vulva development during lumen formation that starts in the stage just after AC invasion [START_REF] Farooqui | Coordinated Lumen Contraction and Expansion during Vulval Tube Morphogenesis in Caenorhabditis elegans[END_REF].

ACTO-MYOSIN CORTEX DURING AC INVASION

In parallel with this RNAi study, I approached the question from another angle, examining WT and MMP-ACs during invasion to see if the AC exhibited a cortex. Since myosin contributes to invasion and motility by contracting the cell cortex at the back of the cell (Chapter 1, Section 1.2.3 and 1.2.5), it was expected that a cortex would be visible in the AC if myosin contractility was playing a role. In order to visualize the actin cytoskeleton in MMP-worms, I induced the expression of Lifeact::BFP in the AC and did the same in WT as a control. Imaging the AC at the 4-cell stage revealed the presence of both cortical and protrusive actin in WT worms (Figure 6.2). Surprisingly in MMP-worms, F-actin was concentrated in the AC protrusion and barely detectable in the cortex (Figure 6.2), as if actin polymerization was enhanced in the protrusion to compensate for the absence of MMPs. This difference was quantified by calculating the actin ratio: the mean intensity of the protrusion (maximum intensity projection) was divided by the mean intensity of the cortex (single cortical plane) using the AC membrane (mCherry::PH) as a marker to detect the plane where the cortex was most in focus. As shown in Figure 6.2, the ratio protrusion/cortex in MMPwas double that of WT, meaning that F-actin was much more polarized toward the invasive edge of the cell in MMP-worms as compared to WT. In fact, as evident in Figure 6.2, cortical actin was so sparse that it appeared to be absent in many cases. This data added to the RNAi data in confirming that myosin contraction of the cortex was not helping the cell squeeze through the BM.

BLEB VISUALIZATION DURING AC INVASION

The worms strains that I constructed for the previous section allowed me to visualize F-actin, AC membrane and BM in both WT and MMP-backgrounds. In MMP-only, I sometimes observed membrane bulges coming off the front of the protrusion (Figure 6.3). These could be called blebs since they contained no F-actin, however I was never able to obtain any dynamics, so it was not clear how these bulges formed. Some appeared to have no connection to the AC body, implying that they were severed membrane fragments. Although these structures were observed at low frequency (5% of worms examined), they were consistently seen in MMP-as opposed to WT. MMP-ACs appeared to be more constricted by the BM during invasion, giving rise to "onion"-type shapes (Figure 6.2 and Figure 6.3). Perhaps membrane bulges originated from rupture events as the protrusion attempted to squeeze through small openings in the BM. Given the lack of certain myosin components in the AC, although I couldn't rule out that they were just at undetectable levels, it seemed unlikely that these were real blebs created by contraction and cortex detachment as described in Chapter 1, Section 1.2.3.

CONCLUSION: MYOSIN IS NOT ESSENTIAL FOR AC INVASION

The conclusion of this chapter is that myosin contractility is not important for AC invasion even in the absence of proteases. So unlike cancer cells in in vitro 3D environments (Wolf et al., 2003), the AC does not switch to a bleb-based mode of invasion powered by myosin contractility when MMPs are inhibited. In this context it should be mentioned that the Wolf et al. study was somewhat controversial due to the uncrosslinked nature of the reconstituted ECM used. Other laboratories found that cancer cells were unable to migrate without MMPs in ECM mimics that were physiologically crosslinked [START_REF] Sabeh | Tumor cell traffic through the extracellular matrix is controlled by the membrane-anchored collagenase MT1-MMP[END_REF]. Indeed in our AC study, native BM was used, so this might explain why invasion in the absence of MMPs was not powered by myosin contractility. Another reason is that the physical context of the AC is very different from a solitary invading cancer cell. The AC is part of a tissue, adhering to adjacent cells at its back and sides as well as to the BM that it is invading. Indeed AC invasion is not a real transmigration event in that the back of the cell does not move forward and cross over the BM. AC invasion is a lamellipodia-type protrusion event, and myosin is not known to play a role in the formation of this kind of protrusion.

Our collaborators have continued their studies of the MMP-strain, looking for components that are responsible for invasion in this background. They find that mitochondrial proteins are necessary, implying that energy consumption is increased in the MMP-case as compared to WT. The hypothesis is that the cell needs more ATP, probably to power actin polymerization, in order to break through a BM in the absence of proteases. These results and the results in this chapter will make up a publication that is currently in preparation: Kelley L.C, Hastie E.L., Cáceres R., Matus D.Q., Chi Q., Plastino J. and Sherwood D.R. "Localized ATP production and F-actin polymerization drive basement membrane invasion in the absence of MMPs".

Overall this study of MMP-independent invasion emphasizes the plasticity of the invading cell to adapt its machinery in order to accomplish its invasive program. This is a crucial point that needs to be better understood in order to develop new treatments targeting cell invasion in disease.

GENERAL CONCLUSION

In this thesis I analyzed the acto-myosin cytoskeleton during a cell invasion event that occurs during C. elegans development, with the long-term perspective of better understanding the similar process of cancer cell invasion. Although relatively well-characterized as far as signaling and genetics were concerned, AC invasion had not been much thought of in terms of physical mechanisms. The ease of genetics and imaging in the worm made quantitative measurements possible while respecting the native microenvironment.

In Chapter 4 taking advantage of C. elegans genetic and imaging tools, as well as using the actin biochemistry strengths of the host lab (the dominant negative approach, the in vitro analysis…), I provided evidence for actin polymerization-based forces in the invading cell, produced by teamwork between WASP and WAVE. In Chapter 5 I used RESOLFT to visualize in more detail the actin cytoskeleton. This study confirmed that the actin network in the AC during invasion was unresolvably dense, but some dendritic-like structures were visible. During this study in Ilaria Testa's lab, I was lucky to work with an expert in optics, Jess Dreier. I experienced first-hand the power of super-resolution techniques, but also the constraints of working with opticians. In Chapter 6 I participated in the characterization of a new invasion strategy in the absence of proteases, independent of myosin contraction, but relying on increased ATP production in the invading cell. Here again I was lucky to collaborate with Laura Kelley and David Sherwood, experts in the field of AC development.

The results of this study are not contradictory to previous studies with human cancer cells, but it shows the importance of studying cellular processes in their native environment.

Overall the main contribution of this PhD was to provide evidence that mechanical forces generated by the cytoskeleton are important during cell invasion, an idea that is not universally accepted in the invasion field. AC invasion and other cell movements in the worm could be powerful models for studying the cell biology and biophysics of cell motility, but they need to be better exploited at the mechanical and structural level.

Introduction

Basement membrane (BM) is a two-dimensional layer of specialised extracellular matrix (ECM) composed principally of laminin, type IV collagen polymers and glycoproteins that separate epithelia from the underlying tissue (Hohenester and Yurchenco, 2013). Owing to the small pore size of BM, nonpermissive to cell passage, cells must enable speciic cellular programs to invade across BMs. Cell invasion occurs during tissue development and also in pathologies such as cancer progression and is controlled by genetics and signalling, as well as by the physical properties and mechanical activity of invasive cells [START_REF] Wirtz | The physics of cancer: the role of physical interactions and mechanical forces in metastasis[END_REF]. The mechanical properties of cells are determined in large part by the actin cytoskeleton, a network of biopolymers that continuously assemble and disassemble inside the cell in a directional manner due to the polarity of actin ilaments that have dynamic (barbed) ends and less dynamic (pointed) ends (Blanchoin et al., 2014). Filaments also slide past each other by the action of the molecular motor myosin, which can recognise the polarity of the actin ilament and move directionally. One of the main structures of the moving cell is the actin-rich protrusion, the lamellipodium, formed by polarised polymerisation of the actin network beneath the plasma membrane, which creates enough force to deform the membrane and pull the cell forward via adhesions to the substrate (Pollard and Borisy, 2003). Filopodia, inger-like actin protrusions, are also observed at the leading edge of cells [START_REF] Faix | The making of ilopodia[END_REF]. At the back of the moving cell is the cell cortex, an actomyosin network that lies just underneath the plasma membrane. Myosin contraction of actin ilaments serves to squeeze forward the back of the cell during cell motility and also drives other aspects of cell motility and cell shape changes [START_REF] Salbreux | Actin cortex mechanics and cellular morphogenesis[END_REF]. Cycles of contraction and detachment from the substrate at the back of the cell, coupled with protrusion and the formation of new cell-matrix adhesions at the front, allow the cell to move forward.

Depending on the environment, different strategies are employed by the cell in order to cross BM barriers. In most invasion events, the breach in the BM is mediated by a lamellipodia-type protrusion, generally known as an invadopodium. Actin assembly dynamics in invadopodia are essential for invasion (Nürnberg et al., 2011). Making holes in BMs is also known to depend on matrix metalloproteases (MMPs) that are displayed on the surface of invadopodia and digest the matrix ibres (Rowe and Weiss, 2009). However, protease-independent mechanisms of invasion have also been reported, involving the cell squeezing through matrix gaps due to actomyosin contractility (Wolf et al., 2003).The objective of this article is to summarise the roles of cytoskeleton dynamics and actin-binding proteins in different modes of cell invasion. Studies on cancer cell invasion, immune cell invasion of endothelia and developmental models such as angiogenesis and anchor cell (AC) invasion in Caenorhabditis elegans will be used to illustrate the different strategies used by cells to breach BMs.

The Actin Cytoskeleton

To better understand the actin structures that participate in cell invasion, we will briely introduce the main concepts of actin architecture and the role of actin-binding proteins in different structures. For more details, see also Blanchoin et al. (2014)and Actin and Actin Filaments.

Branched networks

Cellular lamellipodia are composed principally of branched actin networks produced by the activity of the Arp2/3 complex (Pollard and Borisy, 2003). This complex catalyses the polymerisation of a new actin ilament on the side of a preexisting ilament and is thus called 'a nucleator', creating a Y-branched actin structure. The Arp2/3 complex on its own has a very low nucleating capacity and must be activated by a nucleation-promoting factor from the WASP (Wiskott-Aldrich Syndrome protein)/WAVE family of proteins. The activity of WASP/WAVE proteins, which are constitutively inactive due to inhibitory interactions, is in turn controlled by Rho family GTPases (Rho, Cdc42 and Rac) and by the lipids phosphatidylinositol-(4,5)-bisphosphate (PIP2) and phosphatidylinositol- [START_REF] Brüggemann | Model systems for studying cell adhesion and biomimetic actin networks[END_REF][START_REF] Vogel | Minimal systems to study membrane-cytoskeleton interactions[END_REF][START_REF] Mullins | In vitro studies of actin filament and network dynamics[END_REF]-bisphosphate (PIP3) [for review, see Campellone and Welch, (2010)]. For WASP activation, in response to extra-or intracellular signals, the autoinhibited conformation of WASP is recruited to the plasma membrane through binding to membrane-bound Cdc42-GTP which, in cooperation with PIP2, switches WASP into an active conformation. On the other hand, WAVE, which exists in an inhibited complex with other components, is activated by binding PIP3 and via an indirect interaction with Rac-GTP. Both mechanisms expose the binding site for the Arp2/3 complex on the WASP/WAVE molecule and thus trigger Arp2/3 complex-based actin assembly. Another important component of actin networks nucleated by the Arp2/3 complex is the protein cortactin. Cortactin synergises with WASP to enhance Arp2/3 complex activation by triggering the release of WASP from nascent branch points, permitting new ilaments to elongate from the branching point [ (Blanchoin et al., 2014) and references therein].

Two other important components for branched actin network dynamics are capping proteins and ADF/coilin. Capping protein binds growing ilament ends, stopping their growth, and is believed to promote branched network growth by helping to maintain the pool of monomeric actin for branch formation and elongation. Similarly, ADF/coilin, in association with other cellular factors such as Aip1 in the cell, severs ilaments, thus contributing to actin dynamics by creating more ilament ends that can disassemble to provide monomers for subsequent rounds of polymerisation [ (Blanchoin et al., 2014) and references therein].

Unbranched filament arrangements

The Arp2/3 complex is not the only actin polymerisation nucleator in the cell. There exists another class of nucleators that form unbranched ilaments, including a family of proteins called formins. Formins exist in an inactive, autoinhibited conformation that can be switched to an active conformation by interaction with Rho-GTP or Cdc42-GTP. In the active conformation, the formin homology domains FH1 and FH2 work together to nucleate and elongate unbranched ilaments, with FH2 attaching to the growing ilament end and FH1 recruiting proilin-actin to fuel barbed end elongation [START_REF] Goode | Mechanism and function of formins in the control of actin assembly[END_REF]. The dynamics of growing ilaments is also controlled by Ena/VASP proteins, which enhance barbed end elongation, compete with capping protein and crosslink ilaments together [START_REF] Bear | Ena/VASP: towards resolving a pointed controversy at the barbed end[END_REF]. Unbranched ilaments formed by formins and Ena/VASP proteins can organise into parallel bundles, with barbed ends pointing in the same direction, and such structures are preferentially stabilised and rigidiied by actin crosslinking proteins such as fascin. In vivo such structures are observed in ilopodia. Unbranched ilaments can also adopt an antiparallel orientation in bundles stabilised by -actinin, while crosslinkers such as plastin/imbrin bundle both parallel and antiparallel ilaments. Plastin/imbrin and -actinin, as well as ilamin, a T-crosslinker, confer mechanical stability to unorganised networks formed by both formins and the Arp2/3 complex (Blanchoin et al., 2014)

Actomyosin cortex

Another actin structure in the cell, important for cell shape changes, is the cell cortex. The cortex is a layer of actin attached to the inner lealet of the plasma membrane by the ERM proteins (ezrin, radixin and moesin), which have the ability to interact with both the plasma membrane and actin ilaments [START_REF] Mcclatchey | Merlin and the ERM proteins -regulators of receptor distribution and signaling at the cell cortex[END_REF]. The actin layer also contains miniilaments of myosin that can contract the actin network due to their bipolar nature. The actin network in the cell cortex is generated by both Arp2/3 complex-and formin-based nucleation, making a mix of branched and unbranched ilaments (Bovellan et al., 2014). The balance between branched and unbranched ilaments, their interaction with myosin mini ilaments and the actin crosslinkers -actinin and ilamin act together to tune the properties of the cortical actomyosin network.

The actomyosin cortex dynamically polymerises, depolymerises and contracts, deforming the plasma membrane. However, if the attachment between the cortex and the membrane ruptures or if a rupture occurs in the cortex itself, a phenomenon known as blebbing occurs (Charras and Paluch, 2008). A bleb is a bubble of naked membrane that is forced out by myosin contraction of the cortex of the cell, downstream of Rho/ROCK signalling. This membrane bulge will be either retracted or stabilised by subsequent actin polymerisation to make a protrusion. In the latter case, cellular blebs can drive migration, in what is commonly called amoeboid migration based on its resemblance to the bleb-based motility of amoeba.

Actin Cytoskeleton in Cancer Cell Invasion

One of the irst steps of cancer metastasis from epithelial tissues, the origin of many cancers, is the invasion of tumour cells across the BM that underlies the epithelia. They subsequently invade the stroma and migrate towards the blood stream, from which they are disseminated to new sites to develop metastasis. Two main modes of cancer cell invasion have been described: mesenchymal and amoeboid.

Mesenchymal-type invasion

The epithelial-mesenchymal transition is a process by which epithelial cells lose their polarity and their cell-cell adhesion attributes that make them part of a cohesive tissue (Thiery, 2002). Once the cell is no longer part of the epithelial tissue, it can acquire characteristics that allow it to move and invade, including increased adhesion to the substrate and the production of actin-rich protrusions capable of remodelling the ECM, invadopodia (Figure 1).

There is very little structural information concerning the actin network of invadopodia, but one electron microscopy study indicates that invadopodia are composed of a mixture of a dendritic, branched network and unbranched, bundled ilaments (Schoumacher et al., 2010). In keeping with this, both formins and the Arp2/3 complex are necessary for invadopodia formation (Nürnberg et al., 2011). The dynamics of Arp2/3 complex-based networks in invadopodia have been shown to be controlled by an interplay between the actin-binding proteins ADF/coilin and cortactin, modiied by phosphorylation (Sibony-Benyamini and Gil-Henn, 2012). However, it is not entirely clear what the upstream activator of the Arp2/3 complex is for invadopodia formation. As mentioned before, both WASP and WAVE can activate the Arp2/3 complex; however, only N-WASP has been clearly visualised in invadopodia, with WAVE localisation very dim or absent (Yamaguchi et al., 2005). Other studies show that the inhibition of different forms of WAVE can upregulate or downregulate cancer cell invasion, depending on the context (Kurisu and Takenawa, 2010).Other actin-binding proteins associated with invadopodia and enhanced invasion eficiency are the Ena/VASP proteins and various ilament bundling proteins, including -actinin, fascin, ilamin and plastin/imbrin (Stevenson et al., 2012). How all of these proteins come together temporally to construct an invadopodium is not clear. One study presents at least a partial picture involving some of the previously mentioned components [START_REF] Sharma | Tks5 and SHIP2 regulate invadopodium maturation, but not initiation, in breast carcinoma cells[END_REF]. Invadopodia precursors are initiated by the association of coilin, cortactin, actin and N-WASP, which is stabilised by the adaptor protein Tks5 that also binds PIP2 at the future invadopodial membrane. The maturation phase involves intense actin polymerisation, augmented by the recruitment of SHIP2, a 5 ′ -inositol phosphatase, that locally produces more PIP2, thus retaining the Tks5 complex at the invading membrane and maintaining strong polymerisation (Figure 1). Other studies indicate that there is a subsequent elongation phase involving ilopodia-type machinery, including fascin (Schoumacher et al., 2010). A recent study shows that the distal ends of invadopodia extend into the cytosol and abut the nucleus, indenting it [START_REF] Revach | Mechanical interplay between invadopodia and the nucleus in cultured cancer cells[END_REF]. This implies that mature invadopodia are mechanically supported for invasion by bracing against the nucleus, 'pushing off' from the relatively rigid nuclear surface to help drive the protrusion through the ECM.

Amoeboid-type invasion

Cancer cells have also been reported to undergo bleb-based invasion, independent of invadopodia. For example, cancer cells migrating in three-dimensional matrices via invadopodia switch to amoeboid motion upon inhibition of MMPs (Wolf et al., 2003). From this study, it was concluded that when BM holes cannot be made by digestion and actin pushing, cells use actomyosin contractility to squeeze through existing BM gaps. Switches in motility mode can also be observed in two-dimensional situations, where inhibiting lamellipodia by reducing the Arp2/3 complex or reducing cell-substrate adhesion triggers bleb-based motility, while increasing lamellipodial dynamics by activating Rac suppresses bleb formation (Bergert et al., 2012). For some cancer cell types in three-dimensional environments, a rounded migration mode is observed with bleb-like protrusions at the back of the cell [START_REF] Poincloux | Contractility of the cell rear drives invasion of breast tumor cells in 3D matrigel[END_REF]. This motility mode is independent of the Arp2/3 complex but dependent on actomyosin contractility components that are believed to squeeze the cell through the ECM. Similar behaviour is observed when cancer cells are conined in channels passivated to decrease the level of cell adhesion to the substrate (Liu et al., 2015). Under these conditions, slow-moving mesynchymal-type cells show fast bleb-based locomotion. Overall, it is clear that invading cancer cells can exhibit both mesenchymal and amoeboid modes of invasion and can switch between the two depending on the three-dimensional environment and the balance among actin protrusivity, actomyosin contractility and adhesion (Lämmermann and Sixt, 2009).

Actin Cytoskeleton in Invasion in Nonpathological Contexts

Actin in anchor cell invasion in C. elegans

Most of what is known about the role of actin cytoskeleton in cell invasion comes from the study of cultured cancer cells invading artiicial BM mimics due to the dificulty of studying cancer cell invasion in living animals. BM mimics do not relect the stiff microenvironment of the tumour (Paszek et al., 2005), and the invadopodia observed in such conditions may therefore not be representative of what happens in vivo. In this context, it is useful to study cell invasion in near native conditions, using developmental models where nonpathologic invasion occurs at speciied times during organ development. An example of such a process is AC invasion in the development of the vulva of C. elegans. This process starts at an early larval stage of C. elegans, where the uterine and vulva cells are separated by a double BM, one laid down by the vulval epithelial cells and the other by the gonadal tissue. The double BM is held together by a tight adhesion known as BM-LINKage (B-LINK), consisting of a hemicentin linker between the adjacent BMs that is regulated by signalling from the AC (Morrissey et al., 2014). During the third larval stage, the AC pierces the BM by extending a protrusion, making a hole that will later become the vulva (Figure 2a).

The AC protrusion resembles cancer cell invadopodia visually and also in some important biochemical respects (Figure 2b). The AC protrusion is full of actin ilaments, and a mutant for ADF/coilin (UNC-60), which accumulates large amounts of nondynamic ilamentous actin in its AC, shows a drastically reduced invasion eficiency (Hagedorn et al., 2014). From this study, it appears that dynamic actin ilaments must be present at the invading membrane to actuate invasion, as is the case in invasive cancer cells. Indeed, much of the signalling that happens in the AC could potentially feed into actin dynamics, including the netrin-DCC pathway. Netrin (UNC-6) is produced by the ventral nerve cord, diffuses to the AC and binds to the DCC receptor (UNC-40) on the AC membrane (Hagedorn et al., 2013;W a n get al., 2014;Ziel et al., 2009). This receptor polarises PIP2 to the AC invading membrane (Ziel et al., 2009), which could in turn participate in the activation of actin polymerisation as discussed earlier. Indeed, the same study shows that a double mutant of two C. elegans RacGT-Pases, MIG-2 and CED-10, displays a signiicant reduction in invasion, suggesting the participation of downstream effectors such as WAVE and the Arp2/3 complex (Ziel et al., 2009). Loss of another GTPase, Cdc-42, as well as its downstream effector WASP (WSP-1), also results in delayed BM breaching (Lohmer et al., 2016). Two other actin-binding proteins important for actin dynamics in lamellipodia, Ena/VASP (UNC-34) and lamellipodin (MIG-10), are also known to contribute to AC invasion (Wang et al., 2014).

Future work will address what exact combination of actin polymerisation nucleators and actin ilament bundlers is necessary for AC invasion as well as the potential role of myosin in this process. It is interesting to note that certain components known to be key for invasion of cancer cells, such as fascin and cortactin, are lacking in the C. elegans genome, implying that other molecules fulil these roles in AC invasion.

Actin in sprouting angiogenesis

The term 'angiogenesis' commonly refers to the process of vessel growth, including vessel sprouting from pre-existing ones. In stable vessels, endothelial cells typically form a monolayer of polarised cells that make up the blood vessel wall. However, in the presence of proangiogenesis signals such as vascular endothelial growth factor (VEGF), the endothelial cells initiate motile and invasive behaviour. During this process, leading cells, called 'tip cells', elongate, principally by employing ilopodia, whereas the following cells, 'stalk cells', are less protrusive and play a role in establishing a lumen and proliferating to support sprout elongation (Eilken and Adams, 2010).

An in vivo imaging study in zebraish shows that the ilopodia of the tip cells are promoted by Cdc42, which binds a formin, FMNL3, to nucleate actin polymerisation [START_REF] Wakayama | Cdc42 mediates Bmp-induced sprouting angiogenesis through Fmnl3-driven assembly of endothelial ilopodia in zebraish[END_REF]. A similar role forCdc42 in tip cells is reported using human dermal microvascular endothelial cells [START_REF] Fantin | NRP1 regulates CDC42 activation to promote ilopodia formation in endothelial tip cells[END_REF]. However, lamellipodia are also observed on protruding tip cells, and in keeping with this, proteins such as WAVE and Ena/VASP are required for vascular formation and remodelling [START_REF] Furman | Ena/VASP is required for endothelial barrier function in vivo[END_REF][START_REF] Yamazaki | WAVE2 is required for directed cell migration and cardiovascular development[END_REF].

In addition to ilopodia and lamellipodia, structures called podosomes have recently been implicated in sprouting angiogenesis [for review, see [START_REF] Seano | Podosomes and invadopodia: tools to breach vascular basement membrane[END_REF]]. Similar to invadopodia, podosomes are actin-rich structures that can degrade the ECM; however, one main difference is their life time. Podosomes are relatively short lived (2-20 min), whereas invadopodia can persist for several hours [START_REF] Murphy | The 'ins' and 'outs' of podosomes and invadopodia: characteristics, formation and function[END_REF]. Another main difference is their structure. Podosomes consist of a ring of cell-substrate adhesion molecules called integrins with an actin core that can further organise into clusters called rosettes [START_REF] Juin | Extracellular matrix rigidity controls podosome induction in microvascular endothelial cells[END_REF].

The actin core of the podosome is known to contain the actin nucleating machinery Cdc42, WASP and the Arp2/3 complex, as well as associated proteins such as cortactin, -actinin, imbrin and the ilament capping/severing protein gelsolin [START_REF] Linder | Podosomes: adhesion hot-spots of invasive cells[END_REF]. In another physiological context, podosome assembly in osteoclasts is observed to proceed through steps involving irst assembly and then core consolidation by -actinin [START_REF] Luxenburg | Involvement of actin polymerization in podosome dynamics[END_REF]. Actomyosin contraction does not appear to play a role during podosome actin core enlargement; however, it plays a role in clustering of adjacent podosomes [START_REF] Meddens | Actomyosin-dependent dynamic spatial patterns of cytoskeletal components drive mesoscale podosome organization[END_REF].

Overall from what is known concerning actin dynamics in invasion during angiogenesis, invasion of endothelial cells shares many features with cancer cell invasion and AC invasion.

Actin in leukocyte transmigration

In the preceding sections, invasion has been described in speciic pathological or developmental contexts, but in fact, BM invasion is a common occurrence in the blood and lymphatic systems. Immune cells, the leukocytes, must continually pass back and forth across the BMs of the vasculature in order to mount the immune response, in a process known as transmigration. The vasculature barrier that leukocytes cross is made up of a uniform layer of endothelial cells and a discontinuous layer of pericytes that sandwich a BM which presents uneven expression of collagen IV and laminins because of the discontinuous nature of the pericytes (Voisin et al., 2010). This must be kept in mind as BM in other invasion events is generally uniform. Indeed, a recent study shows that leukocytes preferentially cross the BM in patchy regions and that gaps can further be enlarged by pericyte shape change [START_REF] Proebstl | Pericytes support neutrophil subendothelial cell crawling and breaching of venular walls in vivo[END_REF].

Although much is known about the rearrangement and dynamics of actin in underlying endothelial cells during leukocyte transmigration (Vestweber, 2015), relatively little is known about how actin is reorganised in an invading leukocyte to penetrate not only the endothelial cell layer but also the supporting BM. In fact, most studies on the actin cytoskeleton in immune cell migration have been performed in conining devices that mimic the geometry of invasion in the absence of BM. Using such devices, it appears that formin-based nucleation of actin polymerisation at the back of the cell is the driving force for migration through small openings, while Arp2/3 complex-dependent polymerisation is necessary for other immune cell functions (Vargas et al., 2016). Another report indicates that Arp2/3 complex-based networks around the nucleus are necessary for the nuclear deformation that accompanies cell passage through constrictions [START_REF] Thiam | Perinuclear Arp2/3-driven actin polymerization enables nuclear deformation to facilitate cell migration through complex environments[END_REF].

Conclusion

Overall, the molecular signature of the actin cytoskeleton is similar in different types of invasive protrusions, and many of the same actin polymerisation nucleators, adaptors and structuring proteins are found in different contexts. On the other hand, modes of invasion can be very clearly distinguished based on the biochemistry of the actin cytoskeleton, with myosin driving bleb-based invasion and the Arp2/3 complex playing the predominant role in invadopodia-type invasion. Despite distinct modes, the system is inherently plastic, and many invasive cell types are capable of switching modes in order to adapt to the microenvironment and realise their migratory program. This point needs to be taken into account when developing therapies targeting invasive cell motility.

Introduction

Actin is a protein that exists in a globular soluble form and in an assembled filamentous form, echoing a common theme observed in other types of cytoskeleton like microtubules and intermediate filaments.

Cell shape changes in general, including cell motility, cell division and cancer cell invasion, are due in part to the controlled assembly of actin into filamentous networks that can push membranes or contract in the presence of the molecular motor myosin thus leading to cell shape changes. The fact that actin filaments are polar, with a dynamic barbed end that grows and shrinks more quickly than the pointed end, is important for the directionality of network growth and for myosin motor activity.

Actin has been studied since the 1940s when it was first isolated from muscle. By the time the last century was drawing to a close, the dynamics of individual actin filaments had been well characterized in vitro [START_REF] Pollard | Rate constants for the reactions of ATP-and ADP-actin with the ends of actin filaments[END_REF] and much had been discovered about other factors that interacted with both the globular and filamentous forms of actin [START_REF] Pollard | Molecular mechanisms controlling actin filament dynamics in nonmuscle cells[END_REF]. The great step forward at the turn of the century was the successful recreation of dynamic actin networks growing at surfaces in a controlled fashion using cellular components, a departure from previous single filament studies where polymerization was generally occurring in the bulk solution. This review will be about the progress over the last 15 years in the field of reconstitution of dynamic actin and acto-myosin networks at surfaces or under confinement, and how technological advances have been used to further our understanding of cellular actin dynamics. Other excellent reviews on reconstitution have been published over the last 5 years concentrating on actin and adhesion, membranebound actin and single filament dynamics [START_REF] Brüggemann | Model systems for studying cell adhesion and biomimetic actin networks[END_REF][START_REF] Vogel | Minimal systems to study membrane-cytoskeleton interactions[END_REF][START_REF] Mullins | In vitro studies of actin filament and network dynamics[END_REF][6][START_REF] Loose | Biomimetic membrane systems to study cellular organization[END_REF]. The focus here is actin and acto-myosin networks at or near surfaces in vitro, to mimic cellular confinement and geometry.

The beginnings of actin network reconstitution

Listeria in cells

Somewhat surprisingly, most modern approaches to studying actin networks in vitro can trace their inspiration back to the food-borne pathogen Listeria monocytogenes (Fig. 1). This bacterium propels itself in the host cell cytosol not by swimming with a flagellum, but by building a network of filamentous actin behind itself, dubbed an actin tail or actin comet due to its appearance by electron and light microscopy (reviewed in [START_REF] Tilney | The wily ways of a parasite: induction of actin assembly by Listeria[END_REF]). What made this motility mode interesting to the general cell biology community was the discovery that the bacteria produced a single factor necessary for its motility, the ActA protein, which was displayed on its surface and was responsible for forming the actin comet from host cell components (reviewed in [START_REF] Cossart | Actin-based bacterial motility[END_REF]). In addition landmarking experiments in the actin network of moving cells and in Listeria tails showed that both processes involved insertion of newly polymerized actin at the cell membrane or bacterial surface, and this was hypothesized to be the driving force for propulsion in both cases [START_REF] Theriot | Actin microfilament dynamics in locomoting cells[END_REF][START_REF] Theriot | The rate of actin-based motility of intracellular Listeria monocytogenes equals the rate of actin polymerization[END_REF]. It was quickly realized by pioneers in the field that the Listeria actin network could be a powerful tool to study the biochemical basis of mammalian actin assembly, in isolation from cell signaling and adhesion. This discovery also opened up new avenues for studying how actin assembly created movement from a physical perspective since bacterial movement was a more tractable object to manipulate and model than an entire cell [START_REF] Peskin | Cellular motions and thermal fluctuations: the Brownian ratchet[END_REF][START_REF] Mogilner | Cell motility driven by actin polymerization[END_REF]. We will discuss here Listeria motility, but other pathogens with similar motility mechanisms have also been useful in the study of actin-based motility [START_REF] Gouin | A comparative study of the actin-based motilities of the pathogenic bacteria Listeria monocytogenes, Shigella flexneri and Rickettsia conorii[END_REF].

Initial experiments involved observation of Listeria movement in living cells. Such studies revealed that many host cell actin-binding proteins were present in the Listeria comet tail ( [START_REF] Pistor | The bacterial actin nucleator protein ActA of Listeria monocytogenes contains multiple binding sites for host microfilament proteins[END_REF] and references therein). Further this type of experiment led to more unexpected results, such as the fact that the actin tail composition changed depending on the intracellular location: in the cell body, comets contained α-actinin, while in cell protrusions, comets shed α-actinin concomitant with an evolution of the comet structure toward an aligned unbranched array of long filaments [START_REF] Sechi | The isolated comet tail pseudopodium of Listeria monocytogenes: a tail of two actin filament populations, long and axial and short and random[END_REF]. Information about how the actin network was constructed was also gleaned from altering the ActA protein itself and observing how this changed Listeria motility in cells, notably identifying the Arp2/3 complex and Ena/VASP binding domains as important motility motifs [START_REF] Skoble | Three regions within ActA promote Arp2/3 complex-mediated actin nucleation and Listeria monocytogenes motility[END_REF][START_REF] Skoble | Pivotal role of VASP in Arp2/3 complex-mediated actin nucleation, actin branch-formation, and Listeria monocytogenes motility[END_REF]. However the limitations of this approach quickly became apparent. For example, a back-to-back study of Listeria motility in cells expressing different forms of Ena/VASP proteins as compared to the movement of the cells themselves showed that cell movement and Listeria movement required different domains of Ena/VASP [START_REF] Loureiro | Critical roles of phosphorylation and actin binding motifs, but not the central prolinerich region, for Ena/Vasodilator-stimulated Phosphoprotein (VASP) function during cell migration[END_REF][START_REF] Geese | Contribution of Ena/VASP proteins to intracellular motility of Listeria requires phosphorylation and proline-rich core but not F-actin binding or multimerization[END_REF].This perplexing result could have resulted from off-target effects, including mislocalization of the mutant proteins in the host cells, and changes in the internal structure of the host cell that could have decreased or enhanced Listeria motility. Indeed other studies showed that the mechanical inhomogeneity of the cell interior altered the motile behavior of Listeria [START_REF] Giardini | Effects of intermediate filaments on actin-based motility of Listeria monocytogenes[END_REF].

Listeria in cell extracts and pure protein mixes

The cell interior was too complex of a place to conduct controlled biochemical motility assays, and physical manipulations were rendered difficult. The solution to the confounding effects of the biochemical and mechanical heterogeneity of the cell interior was the use of cell extracts, homogenous cytosolic preparations lacking organelles and cell membrane. Although not without its own challenges, mostly associated with obtaining cell extracts sufficiently concentrated in cytoskeleton factors that were not even entirely known at the time, cell extracts were successfully used to perform some first quantitative physical and biochemical characterizations. For example Listeria actin tail elasticity was measured using optical tweezers, and the roles of profilin and Ena/VASP proteins in Listeria movement were examined [START_REF] Gerbal | Measurement of the elasticity of the actin tail of Listeria monocytogenes,E u r .B i o p h y s[END_REF][START_REF] Theriot | Involvement of profilin in the actin-based motility of L. monocytogenes in cells and cell-free extracts[END_REF][START_REF] Laurent | Role of proteins of the Ena-VASP family in actin-based motility of Listeria monocytogenes[END_REF].At about the same time, great advances were being made in the understanding of how actin assembly was catalyzed in cells. A major step was the discovery of the Arp2/3 complex as a weak catalyzer or "nucleator" of actin assembly that made branches from the sides of existing filaments, and the subsequent finding that the Listeria ActA protein and the mammalian nucleation promoting factors (NPFs) WASp and Scar activated the activity of the Arp2/3 complex [START_REF] Mullins | The interaction of Arp2/3 complex with actin: nucleation, high affinity pointed end capping, and formation of branching networks of filaments[END_REF][START_REF] Welch | Interaction of human Arp2/3 complex and the Listeria monocytogenes ActA protein in actin filament nucleation[END_REF][START_REF] Machesky | Scar, a WASp-related protein, activates nucleation of actin filaments by the Arp2/3 complex[END_REF]. All together these findings paved the way for the next great advance: the reconstitution of Listeria motility in a mix of pure proteins [START_REF] Loisel | Reconstitution of actin-based motility of Listeria and Shigella using pure proteins[END_REF].T h e purified protein mix provided tight control of biochemical parameters, and is still today the method of choice for studying actin-based motility, especially for attaining the reproducibility needed for quantitative measurements.

However cell extracts should not be neglected. The study of a pure protein can reveal its mechanism in isolation, but not necessarily its mode of action in vivo in association with other proteins. A case in point is ADF/cofilin, an actin filament fragmenting protein. When pure ADF/cofilin was mixed with pure actin filaments in conditions where ADF/cofilin fully decorated the filaments, ADF/cofilin lost its ability to , 192-198 (2009), with permission from Elsevier.

sever [START_REF] Andrianantoandro | Mechanism of actin filament turnover by severing and nucleation at different concentrations of ADF/Cofilin[END_REF]. This was perplexing since high ratios of ADF/cofilin to actin are in fact physiological in some cell types. Recent results using cell extracts showed that an additional factor, Aip1, was present in cytosol that permitted ADF/cofilin to efficiently sever and disassemble actin at high ratios [START_REF] Nadkarni | Aip1 destabilizes cofilin-saturated actin filaments by severing and accelerating monomer dissociation from ends[END_REF], although the exact mode of action of Aip1 is the subject of some controversy [START_REF] Chen | Aip1 promotes actin filament severing by cofilin and regulates constriction of the cytokinetic contractile ring[END_REF][START_REF] Gressin | Architecture dependence of actin filament network disassembly[END_REF][START_REF] Jansen | Single-molecule imaging of a three-component ordered actin disassembly mechanism[END_REF]. The use of cell extracts also permitted other exciting developments such as the reconstruction of complex actin structures like the cleavage furrow in cytokinesis [START_REF] Nguyen | Spatial organization of cytokinesis signaling reconstituted in a cell-free system[END_REF]. Recent advances make possible the production of mutant extracts to study individual proteins while retaining the complexity of the cell cytosol and the preparation of staged extracts to examine how actin assembly varies with the cell cycle [START_REF] Miao | Cell-cycle regulation of formin-mediated actin cable assembly[END_REF][START_REF] Michelot | Actin filament elongation in Arp2/3-derived networks is controlled by three distinct mechanisms[END_REF].

The next generation

Replacing Listeria with beads

The first reconstituted motility systems using Listeria set the stage for the next generation of in vitro systems where the pathogen was replaced by a bead or other particle coated with the ActA protein (Fig. 1). This allowed for control of the size and properties of the cargo and the density and nature of the activating protein on the surface, including, importantly, the use of mammalian factors (next section).

The first successful bead systems were performed with ActA-coated particles in cell extracts [START_REF] Cameron | Motility of ActA protein-coated microspheres driven by actin polymerization[END_REF]. This study brought to light one of the stumbling blocks of working with particles in the place of Listeria: homogenous distribution of the ActA protein on the bead surface led to homogenous actin growth, which had to undergo "symmetry breaking" to form a polarized actin network and directional motility. Symmetry breaking was shown to depend on particle size, coating density and the concentration of the cell extract, and could be circumvented by preparing artificially asymmetric beads via silicon monoxide shadowing [START_REF] Cameron | Motility of ActA protein-coated microspheres driven by actin polymerization[END_REF][START_REF] Cameron | Dendritic organization of actin comet tails[END_REF]. Studies of such comets allowed for the important demonstration that actin comet tails observed by electron microscopy had a similar dendritic organization to that found in the lamellipodia of moving cells, thus further validating the use of the bead system as a minilamellipodium mimic [START_REF] Svitkina | Arp2/3 complex and actin depolymerizing factor/cofilin in dendritic organization and treadmilling of actin filament array in lamellipodia[END_REF].

Although an impediment to forming actin comets, symmetry breaking was an interesting topic in and of itself, and much was learned about actin network mechanics by observing the growth and rupture of actin networks on spherical beads. In particular it was demonstrated that the network had elastic properties, due to its entangled nature, and stresses could develop in the network and affect growth dynamics [START_REF] Noireaux | Growing an actin gel on spherical surfaces[END_REF][START_REF] Plastino | The effect of diffusion, depolymerization and nucleation promoting factors on actin gel growth[END_REF].Later with the purified protein mix, symmetry breaking on beads was thoroughly characterized and it was shown that stress build-up drove the polarization of the actin network and that stress development depended in predictable ways on the biochemical components of the protein mixture and the balance between nucleation of new filaments, capping and crosslinking [START_REF] Van Der Gucht | Stress release drives symmetry breaking for actin-based movement[END_REF][START_REF] Dayel | In silico reconstitution of actin-based symmetry breaking and motility[END_REF][START_REF] Kawska | How actin network dynamics control the onset of actin-based motility[END_REF].

What to coat the beads with?

ActA-coated beads are less employed today, but these original studies opened the door to grafting beads with the mammalian equivalent of ActA, the WASP/WAVE/Scar proteins. Reconstitution of actin comet tails and motility of beads coated with the NPF WASP in bovine brain extracts was the first entirely mammalian reconstitution of actinbased motility [START_REF] Yarar | The Wiskott-Aldrich syndrome protein directs actin-based motility by stimulating actin nucleation with the Arp2/3 complex[END_REF]. Subsequently the WASP proteins and the related Scar/WAVE molecules were picked apart by absorbing different protein fragments to bead surfaces and observing which domains gave optimal actin network growth and optimal motility in cell extracts and pure protein mixes [START_REF] Yarar | Motility determinants in WASP family proteins[END_REF][START_REF] Castellano | A WASP-VASP complex regulates actin polymerization at the plasma membrane[END_REF][START_REF] Fradelizi | ActA and human zyxin harbour Arp2/3-independent actinpolymerization activity[END_REF]. Different domains from different actin-binding proteins were also absorbed simultaneously and in different proportions to bead surfaces, for example to recruit and activate the Arp2/3 complex in varying proportions with Ena/VASP proteins [START_REF] Plastino | Actin filaments align into hollow comets for rapid VASP-mediated propulsion[END_REF]. When formin proteins were identified as actin polymerization nucleators that produced unbranched networks, in contrast to the Arp2/3 complex-based branched networks, formin-based actin assembly and movement were also reproduced on bead surfaces [START_REF] Romero | Formin is a processive motor that requires profilin to accelerate actin assembly and associated ATP hydrolysis[END_REF][START_REF] Michelot | Actin-filament stochastic dynamics mediated by ADF/Cofilin[END_REF].G i v e n this history, it is remarkable that no one has yet recreated Arp2/3 complex-based and formin-based nucleation together on a bead surface, despite the biological relevance to the lamellipodium where both nucleation systems co-exist and actin networks are generally mixes of branched and unbranched filaments [START_REF] Bovellan | Cellular control of cortical actin nucleation[END_REF][START_REF] Morone | Three-dimensional reconstruction of the membrane skeleton at the plasma membrane interface by electron tomography[END_REF]. This is particularly pertinent given a recent study that showed that the Arp2/3 complex and formin worked together in a mechanism where the new filament ends created by the Arp2/3 complex were captured and elongated by the formin FMNL2 [START_REF] Block | FMNL2 drives actin-based protrusion and migration downstream of Cdc42[END_REF]. However other studies showed that formin and the Arp2/3 complex compete for actin monomers in cells [START_REF] Burke | Homeostatic actin cytoskeleton networks are regulated by assembly factor competition for monomers[END_REF], and are not favored by the same conditions in profilin in vitro [START_REF] Suarez | Profilin regulates F-actin network homeostasis by favoring formin over Arp2/3 complex[END_REF],soreconstitution of the two activities together may be a challenge.

In general exotic surface coatings remain rare in the biomimetic field, and the predominant activating proteins used today in in vitro systems are human WASP protein fragments, in particular the VCA domain that binds and activates the Arp2/3 complex or its variant pVCA that additionally encompasses the proline-rich portion of WASP that binds profilin actin. VCA is also called WA, due to vocabulary created simultaneously by different labs [START_REF] Rohatgi | The interaction between N-Wasp and the Arp2/3 complex links Cdc42-dependent signals to actin assembly[END_REF][START_REF] Machesky | Scar1 and the related Wiskott-Aldrich syndrome protein, WASP, regulate the actin cytoskeleton through the Arp2/3 complex[END_REF][START_REF] Miki | WAVE, a novel WASP-family protein involved in actin reorganization induced by Rac[END_REF]. The pVCA construct is more effective for Arp2/3 complex activation than VCA when monomeric actin is bound with profilin [START_REF] Machesky | Scar, a WASp-related protein, activates nucleation of actin filaments by the Arp2/3 complex[END_REF]. Indeed most modern reconstitution studies use high concentrations of globular actin bound with profilin to prevent spontaneous nucleation, a closer mimic of actual conditions in cell cytosol and a departure from the original pure protein reconstitution system which used a reservoir of prepolymerized filamentous actin to maintain a low but stable concentration of actin monomers via depolymerization [START_REF] Loisel | Reconstitution of actin-based motility of Listeria and Shigella using pure proteins[END_REF][START_REF] Blanchoin | Actin dynamics, architecture and mechanics in cell motility[END_REF].

The choice of pVCA from WASP as the most-used NPF is more motivated by history than by physiology. WASP is in fact a protein that is only found in hematopoietic cells, while the closely-related N-WASP protein is ubiquitous, but was discovered later (reviewed in [START_REF] Miki | Regulation of actin dynamics by WASP family proteins[END_REF]). N-WASP-coated beads were used in some studies [START_REF] Marcy | Forces generated during actin-based propulsion: a direct measurement by micromanipulation[END_REF][START_REF] Wiesner | A biomimetic motility assay provides insight into the mechanism of actin-based motility[END_REF], and it is the VCA domain of human N-WASP that is currently commercially available. N-WASP is a more effective Arp2/3 complex activator than either WASP or WAVE/Scar due to the enhanced acidity of the A domain in the case of N-WASP, not as originally believed due to the extra V domain that N-WASP proteins contain [START_REF] Zalevsky | Different WASP family proteins stimulate different Arp2/3 complex-dependent actin-nucleating activities[END_REF]. WAVE/Scar-derived bead coatings have been used for some studies, but less extensively than the other NPFs [START_REF] Yarar | Motility determinants in WASP family proteins[END_REF][START_REF] Havrylenko | WAVE binds Ena/VASP for enhanced Arp2/3 complex-based actin assembly[END_REF]. WAVE proteins exist in regulatory complexes, which are impossible to mimic in pure protein mixtures although the WAVE regulatory complex has been successfully recruited to membrane-coated glass beads to form actin comets in cell extracts [START_REF] Koronakis | WAVE regulatory complex activation by cooperating GTPases Arf and Rac1[END_REF]. In the cell, NPFs have very different roles downstream of signaling cascades: WAVE/ Scar proteins are involved in lamellopodial protrusion, while WASP proteins are implicated in filopodia formation and endocytosis (for review [START_REF] Campellone | A nucleator arms race: cellular control of actin assembly[END_REF]). However, as far as biomimetics are concerned, where the regulatory portions of the NPFs are removed, the different NPFs can be used interchangeably since the VCA portion of the different NPFs give the same end product: an Arp2/3 complex-branched network.

The power of the bead system in the pure protein mix

The combination of the bead system with the pure protein mix changed the face of how actin polymerization was studied. Most importantly it made possible a type of biophysical experiment that had been impossible before, namely varying biochemical and physical parameters and observing how that changed actin assembly and motility. For example it was observed that simply changing particle size or beadcoating density could completely change how the actin comet created movement, switching between continuous and periodic, even though biochemical conditions were identical [START_REF] Bernheim-Groswasser | The dynamics of actin-based motility depend on surface parameters[END_REF]. Controlled force measurements also became possible in a variety of different experimental set-ups [START_REF] Marcy | Forces generated during actin-based propulsion: a direct measurement by micromanipulation[END_REF][START_REF] Pujol | Impact of branching on the elasticity of actin networks[END_REF]. Bead/pure protein mixes were also used to study the role of the important actin factor, capping protein, showing that capping protein restricted polymerization to the surface via promotion of Arp2/3 complex activity [START_REF] Akin | Capping protein increases the rate of actin-based motility by promoting filament nucleation by the Arp2/3 complex[END_REF][START_REF] Achard | A "primer"-based mechanism underlies branched actin filament network formation and motility[END_REF].

Bead speeds were a particularly easy parameter to measure while changing the biochemistry of the mix. As one example, this approach was used to resolve the confusion concerning Ena/VASP proteins and Listeria motility mentioned previously. When recruited to the bead surface, Ena/VASP proteins were shown to indeed increase bead speed and different mutants of Ena/VASP showed concordant effects on beads and on an in vivo cell motility event [START_REF] Plastino | Actin filaments align into hollow comets for rapid VASP-mediated propulsion[END_REF][START_REF] Havrylenko | WAVE binds Ena/VASP for enhanced Arp2/3 complex-based actin assembly[END_REF][START_REF] Samarin | How VASP enhances actin-based motility[END_REF]. However the relation between actin polymerization and particle speed is a complex one. It has been observed since the conception of the pure protein mix that movement velocity has a bell-curve dependence on the concentration of polymerization factors: both too much and too little of a given component can reduce speed [START_REF] Loisel | Reconstitution of actin-based motility of Listeria and Shigella using pure proteins[END_REF]. In the case of Ena/VASP for example, under different conditions than the study cited above, it was observed that a bead that was already moving very efficiently displayed drastically reduced motility when treated with Ena/VASP, concomitant with the production of a much denser comet tail (Fig. 2). So it seems that when motility is optimal, adding factors that increase polymerization (like Ena/VASP or even the Arp2/3 complex) can slow bead motility and this is something to keep in mind when using bead velocity as a readout of protein function.

Polymerization from soft, fluid and deformable substrates

The work on beads spawned a whole other branch of the reconstitution family (Fig. 1) involving polymerization on an assortment of fluid and sometimes deformable substrates like oil droplets, liposomes, lipid-coated beads or supported bilayers, moving one step closer to the real conditions for actin polymerization at a cell membrane bilayer.

The first of such studies involved the absorption of a His-tagged form of ActA to liposomes containing nickel lipids and incubation in cell extracts or cell extracts supplemented in the Arp2/3 complex to form actin comets [START_REF] Upadhyaya | Probing polymerization forces by using actin-propelled lipid vesicles[END_REF][START_REF] Giardini | Compression forces generated by actin comet tails on lipid vesicles[END_REF]. Several interesting observations came out of these studies, observations that were corroborated subsequently under different conditions: using the mammalian NPFs VCA-WASP and N-WASP absorbed to liposomes or non-specifically to oil droplets and incubated in either cell extracts or purified protein mixes [START_REF] Boukellal | Soft Listeria: actin-based propulsion of liquid drops[END_REF][START_REF] Delatour | Arp2/3 controls the motile behavior of N-WASP-functionalized GUVs and modulates N-WASP surface distribution by mediating transient links with actin filaments[END_REF][START_REF] Trichet | VASP governs actin dynamics by modulating filament anchoring[END_REF]. Although liposomes were more physiological, the advantage of oil droplets was that the surface tension was known so the curvature of the droplet surface could be used to calculate stresses exerted by the growing actin cytoskeleton. One of the main findings from such studies was, first of all, a direct visual proof of the elastic squeezing effect evoked to explain symmetry breaking, mentioned previously. The growth of an actin gel on a convex surface created compressive or squeezing stresses, and this could be clearly seen with both liposomes and oil droplets as a deformation from spherical shape (Fig. 3a,b). Furthermore it was shown that the actin comet exerted retarding or pulling forces on its substrate, presumably due to transient attachments between the actin network and the surface-bound NPFs mediated by the Arp2/3 complex. As a result, the NPFs on the fluid surface were convected under the comet (Fig. 3c). In line with this, another study using the bead system showed that cortactin enhanced motility by releasing NPF molecules from new branches [START_REF] Siton | Cortactin releases the brakesinactin-basedmotilitybyenhancing WASP-VCA detachment from Arp2/3 branches[END_REF]. Another proposed mechanism for transient network-surface attachment was the binding of the WH2 (or V) domain of NPFs to filament barbed ends, an interaction that was mediated by monomeric actin, giving convection of NPFs on lipid-coated glass beads [START_REF] Co | Mechanism of actin network attachment to moving membranes: barbed end capture by N-WASP WH2 domains[END_REF]. WASP/WAVE WH2 domains do not bind profilin-actin [START_REF] Ferron | Structural basis for the recruitment of profilin-actin complexes during filament elongation by Ena/VASP[END_REF], the predominant form of actin in vivo so, in the cell, a combination of attachment via the Arp2/3 complex and WH2 domains may be occurring. From all this, it is clear that actin growth exerts both protrusive and braking forces on the objects it acts upon.

However much was also gleaned from biomimetic membrane systems in conjunction with actin polymerization in the absence of motility (for review [START_REF] Loose | Biomimetic membrane systems to study cellular organization[END_REF]). For example actin polymerization was shown to induce phase separation of lipids in giant vesicles grafted with N-WASP, incubated in actin and the Arp2/3 complex [START_REF] Liu | Actin polymerization serves as a membrane domain switch in model lipid bilayers[END_REF]. In a similar experiment, the branched actin network produced by Arp2/3 complex-based polymerization was observed to be reorganized into bundled filopodiatype structures by the deformable lipid bilayer [START_REF] Liu | Membrane-induced bundling of actin filaments[END_REF].Evensimpler,and in a continuum with approaches using lipid-coated glass beads, actin polymerization was reproduced on supported lipid bilayers. In particular filopodia formation was recreated on such bilayers, showing that recruitment of biochemical factors from the cell extract gave spontaneous self-assembly of the bundled structure in the absence of membrane deformation [START_REF] Lee | Self-assembly of filopodia-like structures on supported lipid bilayers[END_REF].

Overall the actin network-on-liposome/droplet systems were a great advance in the field because they brought information as to the interplay between actin assembly and lipid bilayer properties and also opened the door to looking at actin-based deformations. Supported bilayers as a subset of this family have the advantage that they are easier to manipulate physically and image by techniques such as Total Internal Reflection Microscopy (TIRF), but give up the deformability of the liposome system and reduce the mobility of factors in the membrane via friction with the support [START_REF] Loose | Biomimetic membrane systems to study cellular organization[END_REF].

Expanding the biomimetic repertoire

Confining physiologically nucleated dynamic actin networks

There is nothing new about encapsulating actin polymerization. For decades people have been incorporating monomeric actin into liposomes, triggering polymerization and then observing shape changes. A non-exhaustive list of such studies includes [START_REF] Cortese | Actin polymerization induces a shape change in actin-containing vesicles[END_REF][START_REF] Limozin | On the organization of self-assembled actin networks in giant vesicles[END_REF][START_REF] Limozin | Polymorphism of cross-linked actin networks in giant vesicles[END_REF][START_REF] Miyata | Morphological changes in liposomes caused by polymerization of encapsulated actin and spontaneous formation of actin bundles[END_REF][START_REF] Miyata | Protrusive growth from giant liposomes driven by actin polymerization[END_REF].S o m es t u d i e s included non-physiological bonds between the encapsulated actin network and the liposome inner leaflet, such as the linking of biotin actin to biotin lipids via streptavidin [START_REF] Tsai | Encapsulation of active cytoskeletal protein networks in cell-sized liposomes[END_REF]. Similar experiments have been performed with pure actin and actin-binding proteins or with cell extracts confined in stabilized aqueous-in-oil emulsions, two examples of which are [START_REF] Pinot | Confinement induces actin flow in a meiotic cytoplasm[END_REF][START_REF] Claessens | Actin-binding proteins sensitively mediate F-actin bundle stiffness[END_REF]. More recently actin polymerization has been confined in Fig. 2. Enhancing polymerization does not always increase bead motility. a) When motility is very fast (2-3 μm/min), the addition of VASP b) slows the beads down (below 1 μm/ min) even though the comet is denser. So the effect of VASP on motility seems to depend on the initial state of the system, and when speed is already optimal, adding an enhancing molecule like VASP does not have the expected effect. Images taken at about 10-15 min reaction time of PRD-VCA-WAVE-coated beads in reconstituted motility mix as described in [START_REF] Havrylenko | WAVE binds Ena/VASP for enhanced Arp2/3 complex-based actin assembly[END_REF], but with commercial Arp2/3 complex. Phase contrast microscopy. Comet appears as a dark streak behind the white bead. Since there is no depolymerization in this system, comet length is proportional to bead velocity. Images M. Abou-Ghali, 2014. microchambers [START_REF] Alvarado | Alignment of nematic and bundled semiflexible polymers in cell-sized confinement[END_REF]. In all cases restricting actin polymerization led to interesting phenomena including self-organization, which were not seen in unconfined solutions. This can be understood in the larger framework of how confinement changes biological processes, including cytoskeleton dynamics [START_REF] Vahey | The biology of boundary conditions: cellular reconstitution in one, two, and three dimensions[END_REF].

A new development concerning confined actin polymerization builds on these experiments, but with several additional characteristics that were previously absent. Namely, to truly reproduce cellular dynamics, the actin network should be growing from the surface via localized actin polymerization nucleation. This means that there are transient attachments between the network and the surface, and the barbed ends are growing mostly toward or near the surface. The actin network should also be depolymerizing, and monomers continually recharging with ATP and repolymerizing to make a dynamic network. These aspects are important for mimicking not only lamellipodia-type protrusions, but also for reconstituting other cytoskeletal organelles as we will see in the next section.

Advances have been made in this direction over the last few years. Liposomes were made from native membranes and swelled in the presence of actin, with or without the membrane-actin crosslinking proteins ankyrin/spectrin. In the presence of ankyrin/spectrin, polymerized actin was anchored and bundled at the membrane [START_REF] Merkle | Reconstitution and anchoring of cytoskeleton inside giant unilamellar vesicles[END_REF]. This was a physiological link, however the filaments were not dynamic. At about the same time, liposomes were made by a different technique, the inverted emulsion technique, whereby the reconstituted motility mix of pure proteins described earlier was encapsulated in low salt conditions that prevented polymerization and then polymerization was triggered by inserting pores in the membrane to allow passage of salts [START_REF] Pontani | Reconstitution of an actin cortex inside a liposome[END_REF].Importantly polymerization occurred preferentially at the membrane because a VCA protein was specifically bound there by interaction of its histidine tag with nickel lipids in the membrane, and additionally this actin layer was shown to be actively turning over due to the presence of actin depolymerizing and recycling factors in the liposome interior. This study produced for the first time a dynamic membrane-associated actin structure in a liposome, polymerized in a physiological manner. Subsequently the inverted emulsion technique was used for actin/ actin-binding protein encapsulation and micropipette aspiration experiments to show that the membrane-associated actin layer was determinant for the mechanical properties of the liposome [START_REF] Luo | Mimicking the mechanical properties of the cell cortex by the self-assembly of an actin cortex in vesicles[END_REF][START_REF] Campillo | Unexpected membrane dynamics unveiled by membrane nanotube extrusion[END_REF]. Additionally membrane-bound actin layers have since been formed in aqueous-in-oil emulsions, using interface-targeted ActA protein and cell extracts [START_REF] Shah | Symmetry breaking in reconstituted actin cortices[END_REF]. These actin networks were shown to not only be actively turning over, but also were capable of auto-organization to break symmetry. An added motivation to use liposome-type biomimetic systems is to study proteins that recognize or impose membrane curvature and also interface with the actin cytoskeleton, such as BAR domain proteins [START_REF] Suetsugu | Dynamic shaping of cellular membranes by phospholipids and membrane-deforming proteins[END_REF].

Patterning actin assembly

Another innovation in the actin biomimetics field is that of making defined actin structures via micropatterning of nucleation sites [START_REF] Reymann | Geometrical control of actin assembly and contractility[END_REF]. In some ways this is similar to the previous challenge, but the confinement is imposed by the filament source instead of being created by the envelope. A pioneering study showed that the angle and distance between nucleation sites for actin assembly determined the proportion of parallel bundles versus anti-parallel structures within a given actin network although the biochemistry of the networks was identical [START_REF] Reymann | Nucleation geometry governs ordered actin networks structures[END_REF]. This showed that the geometry of filament growth could determine macroscopic structure formation, something that had previously been ascribed to actin-binding proteins. However in cells there is surely a mixture of both geometrical and biochemical control, for when the anti-parallel actin bundler α-actinin was added in high concentrations into the actin polymerization mix, antiparallel filament structures were favored even though the geometry dictated predominant parallel bundle formation [START_REF] Reymann | Nucleation geometry governs ordered actin networks structures[END_REF].

Reconstituting acto-myosin contractility in vitro in cell-like systems

The stage is now set for one of the next big challenges in actin biomimetics: reproducing the acto-myosin contractile structure found in non-muscle cells juxtaposed to the plasma membrane, an organelle commonly called the cell cortex. This mixed network of actin filaments and myosin motors dynamically polymerizes, depolymerizes and contracts, while at the same time being transiently linked to the plasma membrane that it deforms to produce cell shape changes. In the wellstudied contractile system of the muscle sarcomere, unbranched actin filaments are arranged in an anti-parallel manner so as to enable myosin-based contraction. In non-muscle cells, the actin network in the cell cortex is a random array of branched and unbranched actin filaments, not organized like in a muscle sarcomere [START_REF] Bovellan | Cellular control of cortical actin nucleation[END_REF][START_REF] Morone | Three-dimensional reconstruction of the membrane skeleton at the plasma membrane interface by electron tomography[END_REF].Thequestion then is: how does the cortex contract efficiently? To answer this, the previously-described techniques are being used to produce cell-like dynamic actin networks, but now containing myosin.

Interplay between actin organization and myosin contractility

As would be predicted from consideration of how myosin functions, it has been shown experimentally that the overall actin architecture can modify where and how effectively myosin contracts the actin network. The micropatterning approach described above was used to create different network geometries, mixed parallel bundles and anti-parallel structures. When myosin was added to this network, it preferentially contracted anti-parallel structures although it decorated parallel When the oil droplet is grafted with VCA a), motility is slow, comets are uniform and dense and the droplet is deformed in a pear shape. When the droplet is coated with a mix of VCA and PRO b), a fragment of the ActA protein that recruits VASP, movement is rapid, the comet is partially hollow and the droplet is therefore deformed differently than in a) into a kiwi shape. See also [START_REF] Trichet | VASP governs actin dynamics by modulating filament anchoring[END_REF]. Phase contrast microscopy. c) On the fluid surface of the oil droplet, VCA (green) is enriched under the comet (actin in red), as observed by the dimmer intensity of VCA at the front of the droplet. The droplet is undergoing jumping movement. For more details see [START_REF] Trichet | VASP governs actin dynamics by modulating filament anchoring[END_REF]. Confocal fluorescence microscopy. All images Léa Trichet, 2004-2005. bundles as well [START_REF] Reymann | Actin network architecture can determine myosin motor activity[END_REF]. Myosin was capable of contracting entangled branched networks, albeit much more slowly. However this appeared to be due to the spontaneous occurrence of anti-parallel structures within such networks that were the real substrate for myosin function [START_REF] Reymann | Actin network architecture can determine myosin motor activity[END_REF]. A very different experimental approach involving acto-myosin layers near but not attached to supported lipid bilayers also showed that a disordered actin network was efficiently contracted by myosin, but only above a critical myosin concentration [START_REF] Murrell | Actomyosin sliding is attenuated in contractile biomimetic cortices[END_REF].

When a static disordered acto-myosin network was attached to the outside or the inside of a lipsome, the outcome of contraction was modulated by the attachment to the bilayer [START_REF] Carvalho | Cell-sized liposomes reveal how actomyosin cortical tension drives shape change[END_REF].Inthe"outside geometry", the balance between contraction and membrane attachment determined whether the acto-myosin network compacted and peeled off the exterior of the lipsome or whether the network contracted and crushed the liposome. In the "inside geometry", contraction either occurred on the bilayer or pulled off the bilayer depending on attachment strength. Taking this experiment one step further, actin was polymerized in the outside geometry with a physiological attachment to the bilayer via a membrane-bound VCA molecules, with the Arp2/3 complex, capping protein and profilin to mimic cellular actin polymerization [START_REF] Carvalho | Actin polymerization or myosin contraction: two ways to build up cortical tension for symmetry breaking[END_REF]. It was observed that both myosin contraction and actin polymerization contributed to stress build-up in this system, and importantly, that the cocktail of actin-binding proteins determined the window where myosin produced contraction. All together, these results emphasize the importance of the geometry of the network, its attachment to the bilayer and the biochemistry of network formation for determining myosin contractility. This is why there is much to be learned by performing biomimetic experiments, which could give very different behavior from pure acto-myosin networks in the absence of constraints, attachments and physiological polymerization.

Another aspect of actin architecture that could affect myosin contractility efficiency is the presence of crosslinkers. The contraction of the anti-parallel regions of the actin network grown from micropatterns was slower in the presence of the anti-parallel cross-linking protein α-actinin, presumably due to resistance to filament sliding imposed by the cross-links [START_REF] Reymann | Actin network architecture can determine myosin motor activity[END_REF]. However a macroscopic contraction assay using suspended actin layers showed that the connectivity conferred by actin cross-linking proteins was necessary for a global contraction [START_REF] Alvarado | Molecular motors robustly drive active gels to a critically connected state[END_REF]. These biomimetic studies show that cross-linking may play a role in controlling how the network contracts. Indeed cross-linking proteins are abundant in the acto-myosin cell cortex [START_REF] Charras | Reassembly of contractile actin cortex in cell blebs[END_REF], and myosinregulatory roles for the actin-binding proteins fascin and ADF/cofilin, sometimes contradictory in the latter case, have been recently reported in cells [START_REF] Chen | Aip1 promotes actin filament severing by cofilin and regulates constriction of the cytokinetic contractile ring[END_REF][START_REF] Elkhatib | Fascin plays a role in stress fiber organization and focal adhesion disassembly[END_REF][START_REF] Wiggan | ADF/Cofilin regulates actomyosin assembly through competitive inhibition of Myosin II binding to F-actin[END_REF][START_REF] Pinto | Actin depolymerization drives actomyosin ring contraction during budding yeast cytokinesis[END_REF]. These issues will be one of the many questions to address in the future with biomimetics.

Myosin contractility as a disassembly agent

Contraction was expected to change the organization of the actin network by compacting it. What was somewhat unexpected was the observation that motor activity also severed and dismantled the network. This had been observed with actin bundles in bulk assays [START_REF] Haviv | A cytoskeletal demolition worker: myosin II acts as an actin depolymerization agent[END_REF]. However as concerns biomimetic networks, this depolymerization effect was most clearly demonstrated with the micropatterning experiments where contraction of the anti-parallel portions of the network led to their disappearance, and seemingly liberated monomeric actin, Fig. 4. The ideal artificial acto-myosin in vitro system. The main characteristics include: 1) the system has a cell-like geometry confined by a lipid bilayer to mimic the cell membrane, 2) actin filament nucleation occurs at the membrane by physiological factors such as the Arp2/3 complex or another nucleator such as formin, 3) attachment to the membrane is ensured by transient links via the Arp2/3 complex and physiological actin filament-membrane linkers such as ezrin, 4) non-muscle myosins are included in the artificial cell interior, 5) actin filaments disassemble either due to the activity of proteins such as ADF/cofilin or to the buckling/severing action that results from myosin contraction and 6) the actin monomers thus liberated are recycled to the cell membrane for subsequent rounds of nucleation. Like in cells, spontaneous formation of filaments in the "cell" interior is inhibited by maintaining free actin in its profilin-bound form. In gray are depicted the future of such systems where, in addition to all the characteristics listed above, the artificial cell is also capable of adhering to its substrate and to its neighboring "cells" via its cytoskeleton and transmembrane proteins, thus mimicking epithelial tissues.

as evidenced by an enhanced growth of the parallel bundles in the assay [START_REF] Reymann | Actin network architecture can determine myosin motor activity[END_REF]. This macroscopic effect reflected what was happening on the single filament level, where myosin activity was observed to buckle and fragment filaments that were attached to a lipid bilayer [START_REF] Murrell | F-actin buckling coordinates contractility and severing in a biomimetic actomyosin cortex[END_REF][START_REF] Vogel | Myosin motors fragment and compact membrane-bound actin filaments[END_REF].

Conclusion

One of the next challenges for biomimetics is to put together all that we have learned over the last 15 years in order to produce the ideal artificial acto-myosin in vitro system (Fig. 4). The goal is to reconstitute inside a cell-like confinement the acto-myosin network, while preserving the architecture of the network as found in living cells, its attachment to the bilayer and the biochemistry of network formation, all of which appear to be important for determining myosin contractility. Such systems should allow for the in vitro study of shape changes and spontaneous oscillations. Down the road, the next step will be to include adhesion to the substrate to make motile biomimetic cells, and adhesion to adjacent "cells" to build up artificial tissues in order to mimic and study collective shape changes.

Transparency document

The Transparency document associated with this article can be found, in the online version.

AC

Figure 1 . 1

 11 Figure 1.1 Cancer cell invasion.Cells leave the primary tumor by invading through the BM, cross the ECM stroma and invade through the BM of blood vessels. The circulatory system carries the tumor cells to new destinations where the cells invade out of the blood vessel and establish new tumor sites called metastases. From[START_REF] Nezhad | The cellular mechanics of an invasive lifestyle[END_REF].

Figure 1 . 2

 12 Figure 1.2 Leukocyte transmigration. Representation of a cross-section of an artery lined with endothelial cells on top of a patchy BM showing low expression regions (LERs). Leukocytes invade through the LERs. Adapted from (Kelley et al., 2014).

Figure 1 . 3

 13 Figure 1.3 Angiogenesis: VEGF induces endothelial cells to breach and remodel the BM, forming a new blood vessel bud. Adapted from (Eilken and Adams, 2010).

Figure 1 . 4

 14 Figure 1.4 Cell invasion through BMs. Top: AC invasion during vulval development: The AC (actin in green) starts out on top of the BM (red), which disintegrates over the course of development due to the invasive activity of the AC to allow the uterine-vulval connection. (R. Cáceres, unpublished, 2016). Bottom: cancer cell invasion of BM--human colon carcinoma cells (actin in green, DNA in blue) invading rat peritoneal BM immunostained for laminin (red). Degradation of the BM and formation of invasive protrusions (invadopodia) allow the infiltration of the cell to the other side of the membrane. Stars indicate sites of penetration. Adapted from (Schoumacher et al., 2010). Scale bars 5 μm.

Figure 1 . 6

 16 Figure 1.6 Branched filaments created by the Arp2/3 complex. a) Electron microscopy images of a cell lamellipodium. Zoom images show branched structures in various locations in the lamellipodium. Scale bar 0.5 μm. Adapted from (Svitkina and Borisy, 1999) b) Single-molecule time-lapse TIRFM of actin polymerization by the Arp2/3 complex (indicated by a red triangle), forming a branch off the side of a mother filament. Scale bar 1 μm. Adapted from[START_REF] Smith | Three-color single molecule imaging shows WASP detachment from Arp2/3 complex triggers actin filament branch formation[END_REF].

Figure 1 . 8

 18 Figure 1.8 How capping protein increases the density of a branched actin network. Capping favors actin monomer participation in branching (right panel) by preventing barbed end elongation (left panel). CP: capping protein. NPF: WASP or WAVE. Adapted from (Akin and Mullins, 2008).

Figure 1 .

 1 Figure 1.10 Actin dynamics in lamellipodia. Transduction of extracellular signals to nucleation of new actin filaments by the Arp2/3 complex, followed by disassembly via the action of capping protein, ADF/cofilin and ATP hydrolysis to ADP in the actin filament. See text for details. Adapted from(Pollard and Borisy, 2003).

Figure 1 .

 1 Figure 1.11 Schematic representation of a formin molecule and its mediation of actin filament assembly. (A) The domain arrangement that characterizes a typical formin. GBD, GTPase binding domain; DID, diaphanous inhibitory domain; FH1, formin homology domain 1; FH2, formin homology domain 2; DAD, diaphanous autoregulatory domain. (B)The FH1 domains are thought to pull in monomers via their interaction with profilin-actin, while the FH2 domains are responsible for creating doughnut shaped homodimers in which actin nucleation can occur. Actin monomers are shown in green, profilin in blue, the FH1 domain in yellow and the FH2 domain in purple. From[START_REF] Randall | A formin-g role during development and disease[END_REF].

Figure 1 .

 1 Figure 1.12 A) Filopodia. A) Electron microscopy image of a filopodia formed by bundle of unbranched actin filaments. Scale bar 0.2 μm. Adapted from (Svitkina et al., 2003). B) Mechanistic models of filopodia initiation. Convergent elongation model (top) and tip nucleation model (bottom).See text for details. Adapted from[START_REF] Yang | Filopodia initiation[END_REF].

  and (Figure 1.14). Myosin minifilaments are able to interact with unbranched or branched actin filaments, generating high and low contractility respectively.

Figure 1 .

 1 Figure 1.13 Actin cortex. Tomography from a EM of a cell cortex showing the mix of a branched and unbranched network. From (Morone et al., 2006).

Figure 1 .

 1 Figure 1.14 Actomyosin contraction. Top: bipolar myosin minifilaments slide antiparallel actin filaments. Bottom panels: myosin minifilaments are also able to generate low or high contraction depending on the geometry of the actin network. Adapted from[START_REF] Levayer | Biomechanical regulation of contractility: spatial control and dynamics[END_REF].

Figure 1 .

 1 Figure 1.15 Blebbing. The bleb starts by detachment or rupture of the cortex. Cytosol flows into the bleb, expanding it until it achieves a maximum size. Then the cortex reforms to stabilize the protrusion or to generate retraction and reabsorb it. From (Charras and Paluch, 2008).

Figure 1 .

 1 Figure 1.16 Actin crosslinkers. A representation of different actin crosslinkers. Yellow represents the actin binding domains, green the rest of the protein and blue are the actin filaments. Barbed and pointed ends are visible. Adapted from https://www.mechanobio.info/.

  and (Figure 1.17). The adhesion points stabilize the protrusion and exert traction forces to move the whole cell forward. Turnover of adhesions induces detachment and release of the rear part of the cell. Attachment/detachment is regulated by Rho and Rac GTPases. While Rac proteins activate actin polymerization and promote cell adhesion at the leading edge, Rho produces the detaching of integrins (focal adhesions) from ECM proteins and cortex contraction at the back of the cell (Vicente-Manzanares and Horwitz, 2011) and (Figure 1.17).

Figure 1 .

 1 Figure 1.19 Mesenchymal cancer cell invasion. Left panel: representation of a cell invading through a 3D ECM (yellow and orange lines) Arrow indicates the direction of invasion. Adapted from[START_REF] Friedl | Tumour-cell invasion and migration: diversity and escape mechanisms[END_REF]. Right panel: electron microscopy image of an invadopodium. Microtubules and intermediate filaments make up the core of the mature invadopodium, while the sides and tip are composed of a dense array of actin filaments, difficult to resolve because of tight packing. Scale bar 0.5 µm. Adapted from(Schoumacher et al., 2010).

Figure 1 .

 1 Figure 1.20 Bleb-based cancer cell invasion in a 3D environment. Cells move via membrane blebs that are subsequently stabilized by actin polymerization. In this type of invasion, adhesion is low and ROCK mediates contractile forces to increase the hydrostatic pressure to squeeze the cell forward.Adapted from[START_REF] Pinner | Imaging amoeboid cancer cell motility in vivo[END_REF].

Figure 1 .

 1 Figure 1.21 Switch between mesenchymal and bleb-based amoeboid cancer migration. Cancer cells switch to the low adhesion, high contractility mode of bleb-based cancer cell migration upon inhibition of protease activity or upon modulation of Rho GTPase crosstalk. Adapted from (Petrie and Yamada, 2012).

CHAPTER 2 :

 2 ANCHOR CELL INVASION IN CAENORHABDITIS ELEGANS 2.1 HOW THE AC OPENS THE HOLE IN THE BM

Figure 2 . 1

 21 Figure 2.1 AC protrusion growth and BM hole expansion. A) Side view of an AC breaching the BM (magenta) with a PIP2-rich membrane protrusion (blue). B) Ventral view of BM hole expansion to the width of AC. Bar 5 m. Adapted from(Hagedorn et al., 2013).

Figure 2 . 2

 22 Figure 2.2 BM is displaced as well as being digested.The BM just beneath the AC and a control flanking region are photocoverted just before invasion and imaged after invasion. The control region remains intact, and while much of the photoconverted BM under the AC has disappeared, some remains, pushed to the sides of the AC. Bar 5 m. AC and BM in green; BM photoconverted to purple. Adapted from(Hagedorn et al., 2013).

. 3 )

 3 . The AC secretes a LIN-3/Epidermal Growth Factor (EGF) signal, which is received by the VPCs via the receptor tyrosine kinase LET-23/EGFR. P6.p receives the most signal and is induced to adopt the 1° VPC fate, while the two flanking VPCs are induced to the 2° fate. Fate determination is further reinforced by lateral signaling via LIN-12/Notch that confirms the secondary fate of P5.p and P7.p. The other cells adopt non-vulval fates and contribute to the hypodermis.

Figure 2 . 3

 23 Figure 2.3 VPC arrangement. A representation of a third larval stage worm (L3). AC induces cell fate specification of the VPCs P3.p-P8.p. Adapted from[START_REF] Schmid | Signal transduction during C. elegans vulval development: a NeverEnding story[END_REF]

Figure 2 . 4

 24 Figure 2.4 VPC division is coordinated with AC invasion. From top to bottom, 1-cell, 2-cell and 4cell stage, named according to the division of the central VPC P6.p. The AC is indicated with a purple arrow. The BM is observed by differential interference microscopy (DIC) microscopy as a line under the AC which becomes discontinuous upon invasion, which happens between the 2 and 4-cell stage.

Figure 2 . 5

 25 Figure 2.5 Netrins role on actin polarity in the AC. Top panel: in WT animals UNC-6/netrin polarizes the receptor DCC/UNC-40 at the invasive membrane where actin starts to polymerize via DCC/UNC-40 partners. Bottom panel: in the absence of netrin/UNC-6, DCC/UNC-40 is mislocalized at the apical and lateral AC membrane and actin polymerization is not focused at the invasive edge.Adapted from(Wang et al., 2014c)

Figure 2 . 6 A

 26 Figure 2.6 A gene expression model of AC invasive differentiation. Summary of how nhr-67 and hda-1 affect genes related with AC invasion behavior, including zmp-1, cdh-3, him-4, unc-34 and exc- 6. Modified from[START_REF] Matus | In Vivo Identification of Regulators of Cell Invasion Across Basement Membranes[END_REF][START_REF] Matus | Invasive Cell Fate Requires G1 Cell-Cycle Arrest and Histone Deacetylase-Mediated Changes in Gene Expression[END_REF][START_REF] Sherwood | FOS-1 Promotes Basement-Membrane Removal during Anchor-Cell Invasion in C. elegans[END_REF] Wang et al., 2014a)

Figure 2 . 7

 27 Figure 2.7 Actin-binding proteins and membrane dynamics in AC invasion. Scheme of what was known when I started my PhD. Actin filaments are represented in orange. All other molecules are labeled. Black arrows represent interactions and pink arrows represent membrane recycling. Adapted from (Caceres and Plastino, 2017).

3 .

 3 When the OD is between 0.6-0.75, spot 100 ul onto 4 NGM-IPTG plates (see below) for each condition. (If the OD is around 0,4 check it again after 20-25 minutes.) 4. Once the bacteria soaks in, invert the plates and incubate at room temperature overnight to induce expression. (For better induction the bacteria need to dry quickly.

For

3 .

 3 Transfer the sample to the microinjector and center the worm with the 10X objective.4. Change to the 100X objective and insert the microinjection needle in the gonad arm.5.Inject the microinjection mix by using the "Injection" button.6. Remove the needle and transfer the worm to the stereoscope. 7. Put 10 ul of M9 solution onto an NGM plate with food.8. Transfer the injected worm into the drop and remove all the oil from the worm Note: This process must be done as quickly and as gently as possible to avoid worm damage.DNA MIX for injectingDNA 1Kb ladder up to 100 ng/ul Plasmid of interest 30 ng/ul Co-injection marker (use only one) rol-6 same concentration as the plasmid of interest (plasmid pRF4) 30 ng/ul Pmyo2>mCherry (plasmid pCFJ90) 2.5 ng/ul Final Volume 50 ul Centrifuge the DNA MIX at 20000g for 15minutes at 4°C. Take 15 ul from the top of the solution into a new tube and throw the rest away.

  Buffer-for one reaction of 15 ul, mix in order H20 12.75 ul Worm PCR buffer 10X 1.5 ul (made in 2007-recipe unknown) Proteinase K (NEB)* 0.75 ul *put the enzyme at the bottom of the solution and clean the tip by pipetting worm lysis *Only use basic Taq Polymerase. Fancy polymerases don't work. Clean the tip by pipetting. Mix the solution by flicking. Verify the tubes are well closed. Centrifuge for 4 s (use the strip centrifuge) before putting into the thermocycler. crossed with NK1073-both from David Sherwood, Duke University) unc-119(ed4) III; qyIs127 [Plam-1::lam-1::mCherry + unc-119(+)]; qyIs242 [Pcdh-3::Lifeact::GFP + unc-119(+)] WASP-DELETED JUP75 (NG324 crossed with JUP60. NG324 was from the CGC.) wsp-1(gm324) IV; unc-119(ed4) III; qyIs127 [Plam-1::lam-1::mCherry + unc-119(+)]; qyIs242 [Pcdh-3::Lifeact::GFP + unc-119(+)] WAVE-DELETED wve-1(ne350); I/hT2[bli-4(e937) let-?(q782) qIs48](I;III) (from Martha Soto, Rutgers University) --DP38 unc-119(ed3) III (from the CGC) This strain was injected to make integrated lines with VCA-BFP and Lifeact-Dronpa. VCA-BFP JUP64 unc-119(ed3) III; curIs20 [Pzmp-1-pes10::TagBFP:: VCA(WASP) + unc-119(+)] VCA+ Lifeact-GFP JUP84 (JUP64 injected with Lifeact-GFP) unc-119(ed3) III; curIs20 [Pzmp-1-pes10::TagBFP:: VCA(WASP) + unc-119(+)]; curEx21 [Pcdh-3 ::Lifeact::GFP + Pmyo2::mCherry] Endogenous WSP-1-GFP; WVE-1-RFP GOU2062 (from Guangshuo Ou) cas762[TagRFP::wve-1a knock-in] I; cas723[GFP::wsp-1a knock-in] IV Endogenous WSP-1-GFP and WVE-1-RFP + Lifeact-BFP GOU2062 injected with Lifeact-BFP cas762[TagRFP::wve-1a knock-in] I; cas723[GFP::wsp-1a knock-in] IV; curEx22 [Pcdh-3::Lifeact::tagBFP] + Pmyo-2::mCherry] mig-2 CF162 (from the CGC) mig-2(mu28) X ced-10 LE1012 (from the CGC) ced-10(tm597)/dpy-13(e184) IV cdc-42 VC898 (from the CGC) cdc-42(gk388)/mIn1 [mIs14 dpy-10(e128)] II frl-1 RB696 (from the CGC) Y48G9A.4(ok460) III fhod-1 fhod-1(tm2363) (from National BioResource Project) cyk-1 VC1895 (from the CGC) cyk-1(ok2300)/mT1 [dpy-10(e128)] III daam-1 daam-1(tm2133) (from National BioResource Project) inft-2 RB1280 (from the CGC) F15B9.4(ok1296) V/nT1 [qIs51] (IV;V) exc-6 (a.k.a. inft-1) NJ833 (from the CGC) exc-6(rh103) III Triple formin mutant cyk-1, inft-2, exc-6 GS7960 (from Daniel Shaye, Univeristy of Illinois) cyk-1(or596ts) exc-6(gk386); inft-2(ok1296); arIs198 -actinin mutant RB1812 (from the CGC) atn-1(ok84) V plastin mutant plst-1(tm4255) IV (from National BioResource Project) filamin mutant fln-1 (tm545) IV (from National BioResource Project) double mutant JUP80 (RB1812 crossed with plst-1 (tm4255)) atn-1(ok84) V ; plst-1(tm4255) IV UNC-40-GFP NK389 (from David Sherwood, Duke University) qyIs67 [Pcdh-3::UNC-40::GFP + unc-119(+)] PIP2-mCherry NK1588 (from David Sherwood, Duke University) qyIs108 [Plam-1::lam-1::dendra + unc-119(+)]; qyIs23[Pcdh-3::mCherry∷ PLCδ PH + unc-119(+)] ZMP-1-GFP NK1123 (from David Sherwood, Duke University) qyEx260 [Pzmp-1::GFP::ZMP-1-GPI] CHAPTER 5 Lifeact-DRONPA JUP72 unc-119(ed3) III; curIs23 [Pcdh-3::Lifeact::Dronpa-M159T + unc-119(+)] CHAPTER 6 MMP-strain used for RNAi NK1267 (from David Sherwood, Duke University) zmp-1(cg115); zmp-3(tm3482); zmp-4(tm3078); zmp-6(tm3073); zmp-5(tm3209); qyls108 [Plam-1::lam-1::dendra + unc-119(+)]; qyIs23[Pcdh-3 ::mCherry∷ PLCδ PH + unc-119(+)] WT control for MMP-RNAi experiments NK1588 (from David Sherwood, Duke University) qyls108 [Plam-1::lam-1::dendra + unc-119(+)]; qyIs23[Pcdh-3 ::mCherry∷ PLCδ PH + unc-119(+)] (MMP-) + LifeACT-BFP, cortex measurements JUP63 (NK1267 injected with Lifeact-BFP) zmp-1(cg115); zmp-3(tm3482); zmp-4(tm3078); zmp-6(tm3073); zmp-5(tm3209); qyls108 [Plam-1::lam-1::dendra + unc-119(+)]; qyIs23[Pcdh-3 ::mCherry∷ PLCδ PH + unc-119(+)]; curEx24 [Pcdh-3::Lifeact::tagBFP + Pmyo2::mCherry] WT control for MMP-cortex measurements JUP62 (NK1588 injected with Lifeact-BFP) qyls108 [Plam-1::lam-1::dendra + unc-119(+)]; qyIs23[Pcdh-3 ::mCherry∷ PLCδ PH + unc-119(+)]; curEx25 [Pcdh-3::Lifeact::tagBFP + rol-6(su1006) + Pmyo2::mCherry] MRCK-GFP ML1602 (from Michel Labouesse, UPMC) mrck-1(ok586) unc-42(e270); mcEx551[Pmrck::MRCK-1::GFP + pCFJ90 (Pmyo-2-mCherry) 3.3 CONSTRUCTS pCFJ150-Pzmp-1-pes10::tagBFP::VCA(WASP) tagBFP was from plasmid pBS Plam-1::BFP-3'UTR; unc-119 from David Sherwood.VCA was amplified from a cDNA clone of C. elegans WASP starting at Gly1477 and going to the stop codon. BFP was fused with VCA as an N-terminal tag and then recombined into Gateway cloning vector pDONR221. The promoter sequence Pzmp-1 and the enchancer element pes-10 were amplified from pBS-zmp-1p-pes-10-spGFP1-10 from David Sherwood as done in[START_REF] Hagedorn | Integrin Acts Upstream of Netrin Signaling to Regulate Formation of the Anchor Cell's Invasive Membrane in C. elegans[END_REF], and introduced into Gateway cloning vector pDONR[P4-P1R]. The entry vectors were recombined along with the unc-54 3′UTR (gift of G. Seydoux; Addgene plasmid #17253: pCM5.37) into the destination vector pCFJ150 -pDESTttTi5605[R4-R3] (gift from Erik Jorgensen Addgene plasmid # 19329). pCFJ150-Pcdh-3::Lifeact::GFP

  pCFJ150-Pcdh-3::Lifeact::tagBFP As above except Lifeact was fused to tagBFP. pCFJ150-Pcdh-3::Lifeact::Dronpa-M159T

Figure 4 . 1

 41 gives an example of the 3 categories with coupled DIC and fluorescent images of BM and AC actin. This shows that BM openings can be seen by DIC microscopy, and in fact, all of the scoring after RNAi treatments in this and other sections was performed using DIC images.

Figure 4 . 1

 41 Figure 4.1 Images of complete, partial and no invasion at the 4-cell stage. The 4 nuclei are visible in the cells directly beneath the AC, indicated by a white arrow. Invasion was evaluated by examining DIC images (left), where a break in the contrasted line of the BM indicated invasion (marked by white arrowheads). Corresponding epifluorescent images are of laminin::mCherry under the native promoter to visualize BM in the entire worm and Lifeact::GFP under an AC-specific promoter to visualize actin filaments in the AC only. Alleles qyIs242 and qyIs127; see Chapter 3. Fluorescent images are shown for illustration, but scoring was done only on DIC images. Scale bar 5 µm.

  LifeAct::GFP under an AC-specific promoter and laminin::mCherry to visualize BM, as for (Figure4.1). Typical still images of F-actin and BM in WT and WASP-deleted worms at the 2-cell, 4-cell and invagination stages are shown in Figure 4.2. The defect in invasion quantified in Table4.1 by DIC is clearly visible by fluorescence: at the 4-cell stage where the WT AC has invaded, the WASP-deleted AC has not. This is not a complete block, however, and by the invagination stage, WT and WASP-deleted ACs look fairly similar.

Figure 4 . 2

 42 Figure 4.2 WT and WASP-deleted AC invasion. Representative epifluorescence images of the AC (Lifeact::GFP) and BM (laminin::mCherry) before, during and after invasion in WT and WASPdeleted worm. Alleles wsp-1(gm324), qyIs242 and qyIs127; see Chapter 3. Scale bar 5 µm.

Figure 4 . 3

 43 Figure 4.3 Protrusion growth. Representative maximum intensity projections from spinning disk movies of the AC (Lifeact::GFP) over about 1 hour for WT (top panels) and WASP-deleted (bottom panels). WT images are from before invasion, just at the moment of BM breach, during and after invasion, and WASP-deleted images are taken over about an hour from the 4-cell stage when the AC should have invaded. Alleles wsp-1(gm324) and qyIs242; see Chapter 3. Scale bar 5 µm.

Figure 4 . 4

 44 Figure 4.4 Analysis of the size of the AC protrusion over the time course of invasion. Left: plot of the area of the actin-filled protrusion in WT worms (red, N=6) and WASP-deleted worms (blue, N=6). All WT traces were aligned so that invasion started at about 30 minutes, and WASP-deleted traces were placed at 30 minutes as well. The cloud of blue points is lower than the cloud of red points. Right: plot of the time course of a single representative cell taken from the previous plot. The red and blue lines are guides to the eyes.

Figure 4 . 5

 45 Figure 4.5 AC protrusion dynamics during invasion. Left: the area of unshared regions between consecutive time points was plotted for WT worms (red N=6) and WASP-deleted worms (blue N=6). The cloud of red points is clustered around 3 µm 2 , while the blue points cluster around 1 µm 2 . The traces are shorter than in Figure 4.4 because only post-BM breach points were considered for WT. Right: plot of shape changes of a single representative invasive event taken from the previous plot.

  worms were still able to invade, but with a 40 minute delay as compared to the normal developmental program, suggesting that another component assured invasion in the absence of WSP-1/WASP. A possible candidate was the other Arp2/3 complex activator, WVE-1/WAVE.To evaluate the contribution of WVE-1/WAVE to AC invasion, I measured the size of the Factin protrusion of the AC of worms treated with wve-1 RNAi. The protrusion was slightly, but significantly, smaller than WT, about 8 µm 2 as opposed to 10 µm 2 at the 4-cell stage of invasion (Figure4.6).

Figure 4 . 6

 46 Figure 4.6 Area of the actin-rich protrusion from maximum intensity projections. WT animals and worms treated with RNAi against wve-1. N ≥ 6. The difference is small but significant (p < 0.098). The dotted line shows the perimeter of the AC. Alleles wsp-1(gm324) and qyIs127; see Chapter 3. Scale bar 5 µm.

  further treated with RNAi targeting wve-1 to eliminate maternally contributed WVE-1/WAVE as much as possible. As compared to an RNAi control, WAVE-deleted worms treated with wve-1 RNAi showed no significant reduction in invasion efficiency and almost 100% of the animals showed full invasion at the 4-cell stage (Figure 4.7).

Figure 4 . 7

 47 Figure 4.7 AC invasion upon WVE-1/WAVE and WSP-1/WASP disruption. Left: representative DIC images of the major phenotype observed in the indicated conditions. Right: score of AC invasion in different mutant and RNAi backgrounds. WASP-deleted and WAVE-deleted, alleles wsp-1(gm324) and wve-1(ne350); see Chapter 3. N>40. Scale bar 5µm. *Same data as shown inTable 4.1, represented here for comparison.

Figure 4 . 8 :

 48 Figure 4.8: Effect of VCA treatment in mammalian cells. Left: untreated cell. Right: the same cell microinjected with purified VCA. Lamellipodial actin networks are diminished by the treatment, but non-Arp2/3 complex-dependent structures like stress fibers and filopodia increase. NIH3T3 cells; actin structures are labeled with fluorescent Lifeact. Scale bar 3 µm. Adapted from[START_REF] Koestler | Arp2/3 complex is essential for actin network treadmilling as well as for targeting of capping protein and cofilin[END_REF]

Figure 4 . 9

 49 Figure 4.9 Effect of VCA expression on AC invasion, 4-cell stage. Left: representative images of a VCA-expressing AC. The boxed region in the DIC is shown in the green and blue channels, single plane spinning disk images. F-actin is visualized with Lifeact::GFP and VCA is labeled with tagBFP. Middle and right: actin distribution line scan for WT (blue curve) and VCA strain (orange curve) for the images shown on the right. Alleles curIs20 and curEx21; see Chapter 3. Spinning disk microscopy addition projection image. Scale bars 5µm.

Figure 4 .

 4 Figure 4.10 Effect of VCA expression on AC invasion, late stage. Left: the unbroken line of the BM in DIC indicates a full block of invasion. Middle and right: the boxed region in the DIC is shown in the green channel (LifeACT::GFP) and blue channel (VCA::tagBFP). Spinning disk microscopy. Maximum intensity projection. Alleles curIs20 and curEx21; see Chapter 3. Scale bars 5µm.

  -cell stage) and after (4-cell stage) invasion, and also present at cell junctions in the vulval and uterine tissue, while WVE-1/WAVE, although faint in the vulva and uterine tissue, was more expressed at the leading edge of the invading AC (Figure4.11). To measure the co-localization of WSP-1/WASP, WVE-1/WAVE and actin in the AC protrusion, linescans were drawn along the leading edge of the protrusion and the fluorescence of actin, WVE-1/WAVE and WSP-1/WASP were measured. This analysis was performed before and after invasion, and showed that WVE-1/WAVE was co-localized with WSP-1/WASP in the actin-rich protrusion at both stages (Figure4.11). This analysis showed that WVE-1/WAVE was present in the right place and at the right time to contribute to the formation of the invasive protrusion, and explained how the protrusion could still form in the absence of WSP-1/WASP.

Figure 4 .

 4 Figure 4.11 Localization of WASP and WAVE in relation to actin in the invading AC. Left: images of the AC before (top panels) and after (bottom panels) invasion. Actin in the AC labeled by LifeAct::tagBFP and WVE-1::RFP and WSP-1::GFP are shown. Right: linescans were drawn along the protruding edge of the AC. The borders of the AC are marked with a bracket on the linescans. Alleles cas762, cas723, curEx22; see Chapter 3. Scale bar 5µm.

Figure 4 . 12 .

 412 Figure 4.12.Both drastically increased the amount of filamentous actin formed over the no addition control, but WSP-1/WASP was 40% faster reaching the plateau than WVE-1/WAVE at identical concentration. Applying some assumptions, the number of filament barbed ends can be roughly calculated using the slopes at half-maximum of such polymerization curves[START_REF] Higgs | Influence of the C terminus of Wiskott-Aldrich syndrome protein (WASp) and the Arp2/3 complex on actin polymerization[END_REF]. The number of barbed ends formed by each activator in the presence of Arp2/3 complex as a function of concentration showed that WSP-1/WASP created 40% more barbed ends (Figure4.12). This difference could explain why WVE-1/WAVE plays a less important role in AC invasion as compared to WSP-1/WASP.

Figure 4 .

 4 Figure 4.12 Pyrene assay comparison of WSP-1/WASP and WVE-1/WAVE activity. Left: pyrene actin curves 4 M actin, 12 M profilin, 50 nM Arp2/3 complex. Red is no VCA addition, blue is 100 nM VCA(WVE-1) addition, and black is 100 nM VCA(WSP-1) addition. WSP-1 gets to the plateau faster than WVE-1. Right: amount of barbed ends (nM) formed by VCA activation of Arp2/3 complex at different VCA concentrations. The data was fit to a saturation curve (Michaelis-Menten) to get the plateau. Plateau for WSP-1 is 3.4 ± 0.1 nM and for WVE-1 2.4 ± 0.2 nM.

  order to test this idea I knocked down wsp-1 by RNAi and measured the localization of fluorescently labeled UNC-40/DCC, the metalloprotease ZMP-1 and a marker for PI[START_REF] Vogel | Minimal systems to study membrane-cytoskeleton interactions[END_REF][START_REF] Mullins | In vitro studies of actin filament and network dynamics[END_REF]P2, components known to be involved in AC invasion and enriched at the invasive membrane (see Chapter 2). Enrichment was quantified by dividing the mean intensity of the invasive membrane by the mean intensity of the apical and lateral membranes (the non-invasive membranes) using a single plane where the lateral, apical and invasive membranes were most visible. As shown in Figure4.13, in WT worms, all 3 components were enriched by a factor of 2-3 in the invasive membrane as compared to the non-invasive membrane, and there was no significant difference (p < 0.9) in polarization in WT worms as compared to those blocked by wsp-1 RNAi.

Figure 4 .

 4 Figure 4.13 wsp-1 RNAi effect on the polarity of key invasive components ZMP-1, UNC-40 and PI(4,5)P2. Left: representative images of a single plane by DIC and fluorescence in WT and wsp-1RNAi treated animals. Right: quantification of enrichment/polarity as described in the text. N>12. Alleles qyEx260, qyIs67 and qyIs108; see Chapter 3. Scale bar 5µm.

the 4 -

 4 cell stage. RNAi against cdc-42 and mig-2 in the WASP-deleted strain gave no additional effect above the 20% invasion already observed for WASP-deleted alone, confirming that CDC-42 and MIG-2 were in the same pathway as WSP-1/WASP (Figure 4.14A and B). However when ced-10 was knocked down in the WASP-deleted strain, full invasion decreased to 7%, very similar to what was obtained with RNAi against WVE-1/WAVE in the WASP-deleted background (Figure 4.14A and see Figure 4.7).

Figure 4 .

 4 Figure 4.14 A) Evaluation of the role of RhoGTPases in relation to WSP-1/WASP and WVE-1/WAVE in AC invasion. Score of AC invasion after RNAi knock-down of mig-2, cdc-42 and ced-10 in WASP-deleted and WAVE-deleted worms, and of cdc-42 and ced-10 knock-down in MIG-2deleted worms. Green is full invasion, yellow is partial and red is no invasion. N>40 for each condition. Alleles wsp-1(gm324), wve-1(ne350) and mig-2(mu28); see Chapter 3. B) Regulatory pathways involving the RhoGTPases.

Figure 4 .

 4 Figure 4.15 Summary of the main results of this study. The main regulatory pathways of the Arp2/3 complex are shown, and their relative abilities to generate branched filaments in order to expand the protrusion during invasion are presented as green filaments issuing from WSP-1/WASP activation of the Arp2/3 complex and red filaments from WVE-1/WAVE.

Figure 5 . 1

 51 Figure 5.1 Principle of STED microscopy. Left: the excitation beam (blue) and the depletion doughnut-shaped laser (orange) with a phase mask. The overlap of the lasers decreases the focal spot achieving a resolution around 70 nm. Adapted from(Willig et al., 2006b). Right: energy diagram of fluorescence (FI, green arrow) which occurs at the focal spot and depletion (de-excitation of fluorophores) by the depletion laser of the doughnut. Adapted from[START_REF] Donnert | Macromolecular-scale resolution in biological fluorescence microscopy[END_REF].

Figure 5 . 2

 52 Figure 5.2 Laser power and resolution for STED microscopy. Left: representation of fluorophore intensity vs depletion laser intensity. Is saturated intensity, I depletion laser intensity used during STED microscopy. Right: the modified Abbé equation showing how altering depletion laser intensity (I) and the saturated intensity (Is) can increase resolution. Adapted from[START_REF] Christian | Exploring single-molecule dynamics with fluorescence nanoscopy[END_REF].

Figure 5 . 3

 53 Figure 5.3 Principle of RESOLFT microscopy. Schematic representation of the light patterns used during RESOLFT microscopy. Adapted from[START_REF] Stefan | The 2015 super-resolution microscopy roadmap[END_REF].

Figure 5 . 4

 54 Figure 5.4 RESOLFT image of an AC during invasion. Left: arrows indicate the dense actin regions, the white dotted line represents the BM, red box indicates the presence of a branched structure in close proximity to the BM. Scale bar 5µm. Right: zoom of the red box shown on the left. Scale 0.2 µm. Allele curIs23; see Chapter 3.

Figure 5 . 5

 55 Figure 5.5 RESOLFT image of an AC protrusion at a later stage when tissue invagination begins. Red arrows point to elongated structures coming from an actin dense region indicated by a yellow arrow. BM is represented by the white dotted line. Allele curIs23; see Chapter 3. Scale bar 5µm.

Figure 6 . 1

 61 Figure 6.1 MRCK-1 expression at the 4-cell stage. MRCK::GFP is not expressed in the AC but is present in the VPCs. Allele mcEx551; see Chapter 3. Scale bar 5 µm.

Figure 6 . 2

 62 Figure 6.2 Cortex analysis in WT and MMP-worms. Left: actin isosurface renderings made from z-stacks of WT and MMP-worms expressing Lifeact::BFP in the AC. Dashed blue line represents the AC membrane, labeled by AC-specific expression of mCherry::PH. Right: N>13. Alleles for WT were qyls108, qyIs23 and curEx24; MMP-additionally carried cg115, tm3482, tm3078, tm3073 and tm3209; see Chapter 3. Scale bar 5 m.

Figure 6 . 3

 63 Figure 6.3 Membranous structures observed during AC invasion in MMP-worms. Membrane bulges that lack filamentous actin were observed that could be blebs or membrane fragments (indicated by arrows). Maximum intensity projections of AC membrane and F-actin in the AC. For BM, a single plane shows the small gap (arrow) that constricts the AC. Scale bars 5µm.

  cells from the underlying tissue. The penetration of cells through BM barriers, called 'invasion', is an important process during normal tissue development and in cancer metastasis. To enable invasion, the cell adopts different shapes and creates different protrusive structures powered mainly by actin cytoskeleton dynamics. However, the exact cytoskeletal strategy that the cell uses to cross the physical BM barrier depends on the physiological context and the physical environment, as observed by examining actin structures in invading cancer and immune cells, and in cells that invade during developmental processes such as angiogenesis and anchor cell invasion in Caenorhabditis elegans.

  eLS subject area: Cell Biology How to cite: Cáceres, Rodrigo and Plastino, Julie (April 2017) Cytoskeleton Dynamics: Actin in Cell Invasion. In: eLS. John Wiley & Sons, Ltd: Chichester. DOI: 10.1002/9780470015902.a0001254.pub2

Figure 1

 1 Figure 1Actin biochemistry in invadopodia. Scheme of a hypothetical invadopodium, summarising all actin-binding proteins reported to be present in invasive structures in different contexts. The lipid bilayer of the cell membrane is represented by a thick grey line. Actin filaments are represented in orange, with barbed ends labelled 'b.e.' and pointed ends labelled 'p.e.'.

Figure 2

 2 Figure 2 Anchor cell invasion in C. elegans. (a) DIC and epifluorescence images of AC invasion in an early stage (before BM penetration) and in a late stage (BM effaced), showing the accumulation of filamentous actin at the AC/BM interface just before BM breaching and the actin-rich protrusion that forms during invasion. Actin in the AC is labelled with Life act-GFP and BM with mCherry-laminin. Bar 5 μm. Rodrigo Cáceres, unpublished, 2016. (b) Scheme of what is known about actin dynamics in the AC. Actin filaments are represented in orange.

Fig. 1 .

 1 Fig. 1. The family tree of biomimetic systems of actin motility and dynamics. The original inspiration came from Listeria motility in cells a), which led to studies of Listeria in cell extracts and pure protein mixes b). The next generation of in vitro systems can be split into two groups, one involving reconstitution on solid supports such as beads c) and the other involving the use of fluid, deformable substrates such as liposomes d). ActA from Listeria was used to coat the beads and liposomes, but also mammalian nucleation promoting factors (NPFs) of the WASP/WAVE/Scar family. The recent innovations in each branch of the family consist of reconstitution of actin dynamics on micropatterns on one hand e), and reconstitution of actin cortices inside liposomes on the other hand f). The lateral double-headed arrows indicate cross-talk between the different systems. a) Reprinted from [114]: Cell,vol.68,C. Kocks, E. Gouin, M. Tabouret, P. Berche, H. Ohayon, P. Cossart, L. monocytogenes-induced actin assembly requires the actA gene product, a surface protein, 521-531 (1992), with permission from Elsevier. b) Adapted by permission from Macmillan Publishers Ltd: Nature [28], 1999. c) Adapted by permission from Macmillan Publishers Ltd: Nature [68], 2002. d) Adapted by permission from the National Academy of Sciences: PNAS [74], 2003. e) Adapted by permission from Macmillan Publishers Ltd: Nature Materials [101], 2010. f) Reprinted from [95]: Biophysical Journal, vol. 96, L.-L. Pontani, J. Van der Gucht, G. Salbreaux, J. Heuvingh, J.-F. Joanny, C. Sykes, Reconstitution of an actin cortex inside a liposome, 192-198 (2009), with permission from Elsevier.

Fig. 3 .

 3 Fig.3. Actin polymerization on deformable, fluid supports. a) and b) Oil droplets are deformed by the actin comet, depending on how the comet is organized. When the oil droplet is grafted with VCA a), motility is slow, comets are uniform and dense and the droplet is deformed in a pear shape. When the droplet is coated with a mix of VCA and PRO b), a fragment of the ActA protein that recruits VASP, movement is rapid, the comet is partially hollow and the droplet is therefore deformed differently than in a) into a kiwi shape. See also[START_REF] Trichet | VASP governs actin dynamics by modulating filament anchoring[END_REF]. Phase contrast microscopy. c) On the fluid surface of the oil droplet, VCA (green) is enriched under the comet (actin in red), as observed by the dimmer intensity of VCA at the front of the droplet. The droplet is undergoing jumping movement. For more details see[START_REF] Trichet | VASP governs actin dynamics by modulating filament anchoring[END_REF]. Confocal fluorescence microscopy. All imagesLéa Trichet, 2004-2005.

Table 4 .1 The effect of Arp2/3 complex knock-down and WASP deletion on AC invasion.

 4

		No invasion	Partial invasion	Full invasion
	arx-2 RNAi a	30%	23%	47%
	WASP-deleted worm a	64%	13%	23%
	WASP-deleted worm (Lohmer et al., 2016)	54%	28%	18%

a N>30 worms were observed for each condition.

Table 4 .2 Evaluation of AC invasion in formin mutants.

 4 Formins were reduced by a combination of mutants (as specified) or RNAi (when indicated). CYK-1 is the equivalent of mammalian Dia, while EXC-6 and INFT-2 are similar to mammalian Inverted Formins INF proteins. FHOD and DAAM carry the same names as their mammalian counterparts. Score of AC invasion at the 4-cell stage by DIC microscopy. N>40 for each condition. Alleles as indicated in the table; see also Chapter 3.

Table 4 .3 Evaluation of AC invasion in the absence of crosslinkers

 4

. Crosslinkers were reduced by a combination of mutants (as specified) or RNAi (when indicated). ATN-1 is α-actinin, PLST-1 is plastin/fimbrin and FLN-1 is filamin. Score of AC invasion at the 4-cell stage by DIC microscopy. N>40 for each condition. Alleles as indicated in the table; see also Chapter

3. 4.6 CONCLUSION: WASP AND WAVE ACTIVATE THE ARP2/3 COMPLEX FOR BM INVASION

I show that AC invasion is almost exclusively driven by the Arp2/3 complex, which is activated at the invasive membrane by both WSP-1/WASP and WVE-1/WAVE working together. WSP-1/WASP is the main contributor since it is a more efficient activator of the Arp2/3 complex than WVE-1/WAVE (Figure

4

.15).

Table 6 .1 Evaluation of myosin participation in invasion

 6

. Percentage of full invasion at the 4-cell stage after RNAi knock down. Control is empty vector. N>40 for each condition. WT were carrying the alleles qyIs108 and qyIs23 for BM and AC membrane label, respectively. MMP-worms were additionally carrying alleles for the 5 MMPs: cg115, tm3482, tm3078, tm3073 and tm3209; see Chapter 3.

eLS © 2017, John Wiley & Sons, Ltd. www.els.net

R.Cáceres et al. / Biochimica et Biophysica Acta 1853 (2015) 3006-3014

Acknowledgements R.C. was funded by a PhD fellowship from ITMO Cancer. J.P. acknowledges inancial support from the Fondation pour la Recherche Médicale (Grant Number DEQ20120323737) and the Fondation ARC (Grant Number PJA 20151203487). Additionally R.C. and J.P. have received support under the program ≪Investissements d'Avenir≫ launched by the French Government and implemented by ANR with the references ANR-10-LABX-0038 and ANR-10-IDEX-0001-02 PSL.

Acknowledgments

We thank Cécile Sykes for discussions and reading of the manuscript. This work was funded by the Fondation pour la Recherche Médicale (Grant DEQ20120323737). R.C. was funded by a PhD fellowship from ITMO Cancer.

Actin filaments have a diameter of 7-10 nm depending on where along the double helix the measurement is taken [START_REF] Aebi | The Three-Dimensional Structure of the Actin Filament Revisiteda[END_REF]. Filaments can pack close together in structures such as crosslinked bundles with individual filaments only 8-24 nm apart [START_REF] Goldstein | The Z lattice in canine cardiac muscle[END_REF][START_REF] Jansen | Mechanism of Actin Filament Bundling by Fascin[END_REF][START_REF] Volkmann | An Atomic Model of Actin Filaments Cross-Linked by Fimbrin and Its Implications for Bundle Assembly and Function[END_REF] or cell cortices that have a mesh size of only 50-200 nm (Morone et al., 2006). In addition to being tightly packed, actin structures in the cell can sometimes be quite small such as filopodia that can be under 100 nm in diameter [START_REF] Lewis | Nerve growth cone lamellipodia contain two populations of actin filaments that differ in organization and polarity[END_REF][START_REF] Svitkina | Mechanism of filopodia initiation by reorganization of a dendritic network[END_REF]. The small size and tight packing of actin structures limits the structural information that can be obtained by conventional light microscopy techniques. Indeed the limit of fluorescence microscopy, the diffraction limitation, was described by Ernst Abbé in 1873: = 2 , where the resolution of the image d is equal to the wavelength of incident light  divided by 2 times the numerical aperture NA.

With numerical apertures of around 1 and illumination with green light for example, this means that the resolution of the image is approximately 250 nm, so structures that are closer together than 250 nm will be seen as a single object. Because of these issues, almost all information concerning actin architecture in protrusions has been obtained via electron microscopy, a technique that is incompatible with live-cell imaging.

The visualization of actin structures in invadopodia is a particularly difficult problem even for electron microscopy due to the 3D nature of invasive protrusions. The only structural information available for an invasive protrusion comes from electron microscopy of thin sections of cells invading into BM mimic-coated pores (images shown Chapter 1, Section 1.3.1) (Schoumacher et al., 2010). The goal of this part of my PhD was to apply superresolution microscopy to AC invasion, in view of obtaining for the first time structural information on the actin cytoskeleton during BM invasion in vivo. The choice of the technique, RESOLFT, was a result of discussions with Ilaria Testa (KTH, Sweden) who gave a seminar at Institut Curie in 2016. This technique also offered the exciting possibility of doing super-resolution dynamics (time-lapse), since the samples are not fixed. I will first ANNEXE 1: PUBLISHED REVIEW ARTICLE 2017

Summary:

Basement membrane (BM) is a dense sheet of specialized extracellular matrix that separates epithelia from underlying tissue. The penetration of cells through BM barriers, called "invasion", is an important process during normal tissue development and in cancer metastasis. Much has been understood concerning the genetics and signaling of how holes are formed in the BM during invasion. However less is clear about the physical forces involved:

how myosin contractility participates in BM removal and how different actin polymerization factors and crosslinkers contribute to the invasive process. To address these questions, we studied an invasion event in a developmental process, anchor cell (AC) invasion in Caenorhabditis elegans. AC breaching of the BM is known to depend on an actin-rich protrusion and the activity of matrix metalloproteases (MMPs), similar to cancer cell invasion.

RNAi knockdown of different actin polymerization activators and nucleators, and expression of a dominant negative form of an Arp2/3 complex activator specifically in the AC showed that AC invasion depended strongly on branched filaments formed via WASP/WSP-1 activation of the Arp2/3 complex. Super-resolution microscopy indicated that the AC invasive protrusion was densely packed with filaments, in keeping with the idea that the invasive protrusion was highly branched. We further showed that another Arp2/3 complex activator, WAVE/WVE-1, could enable invasion when WASP/WSP-1 was absent. Formins appeared not to play a major role and actin cross-linking proteins were likewise dispensable for AC invasion.

In wild type worms, we observed that myosin activity was not needed for invasion.

However it has been reported that cancer cells upregulate myosin contractility to invade in the absence of proteases, so we used a worm deleted for the five main MMPs of the worm genome to test the role of myosin in this context. AC invasion took place in MMP-worms, but with a time delay. RNAi knockdown of different components of the myosin machinery gave no enhancement of the invasion defect. In addition visualization of the actin cytoskeleton in MMP-worms revealed that actin was concentrated in the AC protrusion and barely detectable in the cortex, making it unlikely that myosin contraction of the cortex was helping the cell squeeze through the BM as reported in cancer cells in the absence of proteases.

All together these results showed that the invasive cell adapted its branched actin filament polymerization to maintain invasion in different biochemical and environmental contexts. This plasticity is a crucial point that needs to be better understood in order to develop future treatments targeting cancer cell invasion.