Mapping Tunneling Nanotubes In Vitro & In Vivo: A Structural Approach

Keywords: Tunneling Nanotubes, Actin, Neuronal communication, Cerebellum, Connectomics

Inter-cellular communication has long been thought to be governed by juxta-, endo-, and paracrine signaling, tight junctions, and more recently, exosomes. However, large efforts from our and other groups revealed that Tunneling Nanotubes (TNTs), actin-rich membranous protrusions that connect the cytoplasm of distant cells and allow the dynamic inter-cellular transport of biological cargo, also provide the infrastructure and machinery for effective cell-tocell communication. Despite significant progress made to unveil TNT-mediated cell communication, the characterization of these novel organelles has been limited by unanswered questions that hail from the lack of both molecular and structural information. Exploring these gaps in the field using a series of state-of-the-art tools and novel approaches became the main focus of my dissertation. Specifically, I explored the specific role of actin-regulator complexes in the formation of TNTs connecting neuronal cells. My analyses show that molecular pathways known to be involved in the formation of other membranous protrusions behave differently in the generation of TNTs. By employing live imaging microscopy, cryo-correlative electron microscopy and tomography approaches, I also studied the nano-architecture of neuronal TNTs.

My findings demonstrated that TNTs of neuronal cells are comprised of multiple individual TNTs capable of transporting vesicles and mitochondria. Owing to the difficulties of identifying TNTs in vivo, my work also focused on the implementation of a structural Connectomic approach to detect TNTs in tissue without the need for a TNT-specific marker. My findings indicate that TNTlike structures connect migratory cerebellar granule cells of neonate mice, suggesting that intercellular communication during migratory events in the brain could be mediated by TNT-like processes. Skeletonization of the structures identified provide my findings with geometrical information that can be compared with observations made by corroborative dye-coupling experiments. Taken together, my dissertation work sheds light on the formation and structure of neuronal TNTs in vitro, and novel approaches for the identification of TNTs in vivo.

VII

III

Caractérisation des Tunneling Nanotubes In Vitro et In Vivo: Une approche structurelle

Mots clefs: Tunneling Nanotubes, Actine, communication neuronale, cervelet, Connectomics Resumé: On a longtemps pensé que la communication intercellulaire était essentiellement régie par les signalisations juxta-, endo-et paracrine, les gap junctions et, plus récemment, les exosomes. Cependant, les travaux de plusieurs groupes dont le nôtre ont révélé que les Tunneling Nanotubes (TNT), des protrusions membranaires riches en actine qui relient le cytoplasme de cellules distantes et permettent le transport intercellulaire dynamique de leur contenu biologique, fournissent également l'infrastructure et les machines pour une communication efficace entre cellules. Malgré des progrès significatifs, la caractérisation de ces nouveaux organites a été limitée par le manque d'informations moléculaires et structurelles. Combler ces lacunes à l'aide d'une série d'outils de pointe et d'approches novatrices est devenu l'objectif principal de ma thèse. Plus précisément, j'ai exploré le rôle des complexes régulateurs de l'actine dans la formation des TNT reliant les cellules neuronales. Mes analyses montrent que les voies moléculaires connues pour être impliquées dans la formation d'autres protrusions membranaires régulent différemment la génération des TNT. En utilisant la microscopie par imagerie en direct, la microscopie électronique cryocorrélative et la tomographie, j'ai également étudié la nanoarchitecture des TNT neuronaux. Mes découvertes ont démontré que les TNT des cellules neuronales sont composés de plusieurs TNT individuels permettant le passage de vésicules et de mitochondries. En raison des difficultés d'identification des TNT in vivo, mes travaux ont également porté sur la mise en oeuvre d'une approche « Connectomic » structurelle pour détecter les TNT dans les tissus sans avoir besoin d'un marqueur spécifique de TNT. Mes résultats indiquent que des structures de type TNT relient les cellules granulaires cérébelleuses migratrices des souris nouveau-nées, ce qui suggère que la communication intercellulaire pendant des événements migratoires dans le cerveau pourrait être médiée par des processus mettant en jeu des TNT. La squelettisation des structures identifiées fournit des informations géométriques qui corroborent les observations faites dans des expériences de couplage de colorants. L'ensemble de mes travaux de thèse fait la lumière sur la formation et la structure des TNT neuronaux in vitro et sur de nouvelles approches pour l'identification des TNT in vivo.

First, I'd like to thank Dr. Chiara Zurzolo for giving me the chance to carry out my thesis in her lab and build a project from the feet up, putting her full confidence on my practical and intellectual abilities. I trust that the lessons I learned in her lab will have a lasting impact on my future career.

I would also like to thank all the current and former members of the Traffic & Membrane Pathogenesis Unit for all the fun and engaging interactions we had inside and outside the laboratory over the last few years. To the members of the Ultrastructural BioImaging Unit and Image Analysis hub: thank you for all your help, it was a great pleasure working with you.

To the members of my dissertation committee: Dr. Thierry Galli, Dr. Pierre Marie Lledo, and Dr.

Isabelle Cloez-Tayarani, thank you for your time, feedback, and invigorating support.

To my scientific collaborators: Dr. Jeff Lichtman (and laboratory members), thank you for welcoming me in your lab on numerous occasions and sharing your technical expertise; to Dr. Laurence Cathala and Dr. David DiGregorio, thank you for believing in my scientific quest and committing your efforts and infrastructure to my project; to William Silversmith of the Seung lab, thank you for offering your expertise and sharing your computational tools with me.

To Susanna Celli and the rest of the Pasteur-Paris University steering committee: thank you for supporting my studies and my student organization -Graphics Visual Association (GVA).

To all the trainee students I had the pleasure of working with Santa Berzina, Ozlem Özen, Marie-Madeleine Trottmann, Owen Mulhern, Maximiliana Bogan, Neven Borak, Rio McLellan, Alexander Mancevski, Eric Sun, and Alex Atkin -thank you so much for your time and great work.

To my scientific and career mentor: Dr. Lida Katsimpardi, without whom I would have never made the bold 'move' to embark on a PhD in Europe, thank you for your great advice and support.

To my family -my mom, dad, and brother: gracias por el constante apoyo a través de una formación académica exhaustiva que ha durado mas de 20 años. El empuje que me dieron en todos los aspectos me ayudó a metamorfosearme en una persona altamente calificado para enfrentar desafíos del primer mundo.

XIII

List of Figures

Introduction to Cell to Cell Communication

From the discovery of the cell by English polymath, Robert Hooke, in 1665 [START_REF] Hooke | Micrographia : or some physiological descriptions of minute bodies made by magnifying glasses[END_REF], humans have embarked on the quest to better understand the building blocks that sustain life, and ultimately allow us to feel and think. This 354-year old ongoing voyage has revealed that cells interact in a vast universe in elegant harmony (Cajal, 1888;[START_REF] De Duve | A Guided Tour of the Living Cell[END_REF][START_REF] Golgi | Sulla Fina Anatomia degli Organi Centrali del Sistema Nervoso[END_REF]. Animal and plant life rely on this essential interaction; constant and effective cell-to-cell communication orchestrates all normal physiological functions but can give rise to diseases under certain conditions [START_REF] Virchow | Die cellularpathologie in ihrer begründung auf physiologische und pathologische gewebelehre[END_REF].

Cell-to-cell communication occurs through a variety of mechanisms that enable an extraordinarily diverse collection of biological messages to be shared between cells. Messages range from small molecules that maintain intra-and extra-cellular ion concentrations at normal physiological levels, such as potassium [START_REF] Kozoriz | Passing potassium with and without gap junctions[END_REF], to large energy-producing organelles, such as mitochondria [START_REF] Torralba | Mitochondria Know No Boundaries: Mechanisms and Functions of Intercellular Mitochondrial Transfer[END_REF]. In addition to size, two key factors that differentiate how these messages are transported inter-cellularly are (i) specificity and, (ii) the distance they need to reach their target.

Adjacent cells can communicate via nanoscopic water-filled channels called gap junctions (GJs) (Figure 1), which allow the inter-cellular transport of small (~1kDa) soluble molecules and inorganic ions (e.g. cyclic nucleotides and inositol phosphates) [START_REF] Bermudez-Fajardo | CD4+ T lymphocyte subsets express connexin 43 and establish gap junction channel communication with macrophages in vitro[END_REF].

GJs are formed by two hemichannel connexons (hexameric connexin complexes) that facilitate electrical and metabolic coupling [START_REF] Donahue | Joint diseases: from connexins to gap junctions[END_REF]. Connexin 43 (Cx43) is the primary component of GJs and it plays a wide variety of functions. For example, it can transport cellular death signals (e.g. Inositol trisphosphate, IP3) during the invasion of breast and lung tumorderived cancer [START_REF] Aasen | Connexins in cancer: bridging the gap to the clinic[END_REF]. Cx43 also plays a key role in development by contributing to the proper migration of pyramidal neurons in the cerebral cortex [START_REF] Liu | Connexin 43 controls the multipolar phase of neuronal migration to the cerebral cortex[END_REF].

Cells also use various forms of 'signal transmission' for inter-cellular communication: Paracrine signaling is used by cells in close proximity, where messages (paracrine ligand molecules) are secreted into the extracellular matrix (ECM) where they bind to a neighboring's cell receptor [START_REF] Saxen | Are morphogenetic tissue interactions mediated by transmissible signal substances or through cell contacts?[END_REF]. Endocrine signaling is a slow and passive cell-to-cell signaling process in which hormones (small, 5-20 kDa soluble factors) are secreted by stimulatory cells in endocrine tissue (e.g. adrenal glands), and travel through the bloodstream until they reach a specific receptor on a distant target cell in the body (Figure 1) [START_REF] Starling | On the chemical correlation of the functions of the body[END_REF]. Physiological examples of these processes include neurotransmitters that cross a synaptic cleft to reach the postsynaptic terminal, as a paracrine signal; and as endocrine signal, insulin, a hormone that controls glucose and lipid metabolism. Other forms of signaling modes of communication include autocrine and Juxtacrine signaling. During autocrine signaling, cells 'self-communicate' by secreting soluble ligands that bind to receptors on their own surface [START_REF] Doğaner | Autocrine Signaling and Quorum Sensing: Extreme Ends of a Common Spectrum[END_REF]. Depending on the type of signal, ligands can also bind to receptors within the cell. Common examples of autocrine signaling include interleukin-1, a soluble protein involved in immune response to antigens and mitogens [START_REF] Youk | Secreting and sensing the same molecule allows cells to achieve versatile social behaviors[END_REF]. Juxtacrine signaling is a contact-dependent class of signaling in which membrane ligands from one cell bind to cell surface receptors located on another cell, initiating an intracellular signaling cascade [START_REF] Zimmerman | Juxtacrine intercellular signaling: another way to do it[END_REF]. One of the best characterized examples of Juxtacrine signaling is the Notch signaling pathway, in which the ligand of the signaling cell, Delta, binds to the receptor Notch, activating cascades that affect a wide variety of functions that include cell fate, death, and proliferation [START_REF] Lai | Notch signaling: control of cell communication and cell fate[END_REF].

Other modes of cellular communication involve extracellular vesicles (EVs), which gained popularity as avenues for the inter-cellular exchange of genetic material, lipids, and proteins [START_REF] Van Niel | Shedding light on the cell biology of extracellular vesicles[END_REF]. EVs are classified into two categories, microvesicles (MVs) and exosomes (Figure 1). MVs are 100 -1,000 nm plasma membrane-derived particles that form from outward blebbing of the plasma membrane; exosomes are intraluminal vesicles (ILVs) of 30 -100 nm in diameter of endocytic origin that get exocytosed when the multivesicular endosomes (MVEs) containing them fuses with the plasma membrane [START_REF] Keller | Exosomes: from biogenesis and secretion to biological function[END_REF]. EVs are highly heterogeneous in size, composition, morphology and biogenesis. Although their precise biological role and presence in vivo remains to be defined, there is substantial evidence that links EVs to numerous physiological and pathological roles [START_REF] Buzas | Emerging role of extracellular vesicles in inflammatory diseases[END_REF].

Introduction to Tunneling Nanotubes

The quest to discover novel modes of cell-to-cell communication didn't stop with the identification of exosomes. 15 years ago, Hans-Hermann Gerdes and colleagues observed membranous tubes forming in cell cultures of rat kidney pheochromocytoma 12 (PC12), human embryonic kidney (HEK), and normal rat kidney (NRK) cells which they called Tunneling Nanotubes (TNTs) (Figure 2) (Rustom et al., 2004). Stained with fluorescently conjugated Wheat Germ Agglutinin (WGA), cells connected by TNTs created an inter-connected cellular network.

Using structural approaches (discussed in further detail in later sections of this chapter), the authors found that these 50 to 200 nm-thick, several μm-long actin-bearing membranous threads permitted the inter-cellular transport of synaptophysin-marked endosomes, small synaptic-like microvesicles, and lysotracker-labeled organelles. That same year, Davis and colleagues identified similar (30 to 140 μm-long) structures forming in various immune cells, including macrophages, B lymphocytes, and blood natural killer (NK) cells [START_REF] Onfelt | Cutting edge: Membrane nanotubes connect immune cells[END_REF]. In the same study, the authors discovered that TNTs could form upon disassembly of the immunological synapse (discussed in later sections of this chapter), and, like the connections described by Rustom et al., can also facilitate the trafficking of membrane components.

Further studies from our group and others shed light on TNTs connecting numerous other cell types, along with the detection of a long list of TNT-mediated cargo trafficking that includes ions, organelles, viruses, bacteria, apoptotic signals, genetic material, and proteins. TNTs were found to connect cells of the same-(homotypic) and different-(heterotypic) type and allow both uniand bi-directional cargo transport. Numerous review articles have described how these findings link TNTs to both physiological and pathological conditions, which range from tissue repair to tumor progression; bacterial to virus infection; and stem cell differentiation to progression of neurodegenerative diseases [START_REF] Abounit | Wiring through tunneling nanotubes--from electrical signals to organelle transfer[END_REF][START_REF] Ariazi | Tunneling Nanotubes and Gap Junctions-Their Role in Long-Range Intercellular Communication during Development, Health, and Disease Conditions[END_REF][START_REF] Austefjord | Tunneling nanotubes: Diversity in morphology and structure[END_REF][START_REF] Drab | Inception Mechanisms of Tunneling Nanotubes[END_REF][START_REF] Jash | Perspective on nanochannels as cellular mediators in different disease conditions[END_REF][START_REF] Marzo | Multifaceted roles of tunneling nanotubes in intercellular communication[END_REF][START_REF] Mittal | Cell communication by tunneling nanotubes: Implications in disease and therapeutic applications[END_REF][START_REF] Venkatesh | Tunnelling nanotubes: A bridge for heterogeneity in glioblastoma and a new therapeutic target?[END_REF][START_REF] Vignais | Cell Connections by Tunneling Nanotubes: Effects of Mitochondrial Trafficking on Target Cell Metabolism, Homeostasis, and Response to Therapy[END_REF][START_REF] Yamashita | Specialized Intercellular Communications via Cytonemes and Nanotubes[END_REF]. Particular morphological and functional differences (e.g. cell type, TNT length and thickness, open-vs. close-ended tip, nature of cargo transport, and relationship to a given disease), resulted in TNTs acquiring disparate names. For example, "nanoscale channels" connect metastatic cancer and endothelial cells [START_REF] Connor | Physical nanoscale conduit-mediated communication between tumour cells and the endothelium modulates endothelial phenotype[END_REF]; "T-cell nanotubes", connect Jurkat T cells (Sowinski et al., 2008); "membrane nanotubes" connect human NK cells [START_REF] Chauveau | Membrane nanotubes facilitate long-distance interactions between natural killer cells and target cells[END_REF]; "bridging conduits", or 'thick TNTs' contain actin and microtubules [START_REF] Kadiu | Macrophage bridging conduit trafficking of HIV-1 through the endoplasmic reticulum and Golgi network[END_REF];

"intercellular bridges", connect epiblast cells in the developing zebrafish [START_REF] Caneparo | Intercellular bridges in vertebrate gastrulation[END_REF];

"epithelial bridges" (EBs) connect primary human bronchial epithelial cells [START_REF] Zani | Tubular bridges for bronchial epithelial cell migration and communication[END_REF];

"tumor microtubes" (TMs) connect astrocytoma cells (Osswald et al., 2015a); and "membrane fronds" connect baby hamster kidney 21 (BHK-21) cells, 293 T cells, and NIH-3T3 fibroblasts [START_REF] Gill | A gamma-herpesvirus glycoprotein complex manipulates actin to promote viral spread[END_REF]; etc.

The heterogeneity of TNT-like structures justifies nomenclature diversity. After all, numerous studies describing similar organelles (e.g. thin filopodia during sea urchin gastrulation, [START_REF] Miller | Dynamics of thin filopodia during sea urchin gastrulation[END_REF]) were published prior to the work by Rustom et al., and it is conceivable that plenty others were also reported but never mentioned in any of our field's bibliography. However, the number of names increased for structures that could simply be one structure-an open-ended membranous connection that, through a contiguous cytoplasmic tunnel containing actin cytoskeletal railways, permit the transport of cellular cargo-or, entirely distinct structures which only share essential properties (e.g. enclosure by a membrane and extending between two distant cells), has been subject of much debate in our field [START_REF] Baker | How the Internet of cells has biologists buzzing[END_REF].

The lack of a TNT-specific marker and limitations in optical microscopy resolution have hampered our ability to catalog TNTs accurately and address the ambiguous heterogeneity described above. In order to better understand these structures and move the field forward, we will need the answers to pivotal questions, such as: how do TNTs form? How do TNTs function;

that is, what cytoskeletal filaments and motor proteins work together to enable cargo transport?

Are TNTs open-ended tunnels or tip-close-ended protrusions? Do TNTs exist in animals? Do TNTs play the essential roles they demonstrate in vitro, in living organisms? If so, how do they compete with other modes of cell-to-cell communication?

In the remaining sections of this chapter, I will present a synthesized review of the advances our group and others have made in the field of TNTs and point to specific biological questions that drove the direction of my doctoral dissertation, while also highlighting how my efforts have contributed to a better understanding of the unanswered points above. Specifically, I will describe the tools, methods, and criteria employed to study TNTs, as well as findings on TNT biogenesis, mechanisms of formation, structure, presence in vivo, pathological implications, and therapeutic applications. Throughout these sub-sections, I will also emphasize published and preliminary data from our group that served as the foundation for the projects I worked on during my doctoral work.

Methods and Criteria used for the study of Tunneling Nanotubes 1.3.1 Tunneling Nanotubes: Denotation In Vitro

Today, the properties that make up the criteria used by us and others to label TNTs as 'TNTs' are the following: (i) a cell protrusion with a surrounding plasma membrane that extends between two cells, (ii) the presence of of actin cytoskeletal filaments, and (iii) ability to transport cargo, which also serves as a strong indicator of (iiii) cytoplasmic continuity through open-ended cell-TNT-cell contact sites (Figure 3) [START_REF] Dupont | Tunneling Nanotubes: Intimate Communication between Myeloid Cells[END_REF]. Other protrusions, which share some or most of these features are often called TNT-like structures; however, for simplicity, I will use the term TNT for the remaining sections of this chapter unless stated otherwise.

Preservation of Tunneling Nanotubes

The long and thin topological features that characterize TNTs make them fragile and vulnerable to shearing forces. The preservation of TNTs through classical fixation methods has met obstacles along the way, as TNTs' susceptibility to low-concentrated chemical fixatives (e.g. paraformaldehyde) and long-light exposures often break or crook them. To circumvent this obstacle, we and others employ a two-step fixation protocol that leverages extensive crosslinking via glutaraldehyde prior to paraformaldehyde fixation [START_REF] Abounit | Identification and Characterization of Tunneling Nanotubes for Intercellular Trafficking[END_REF].

The fragility of TNTs impairs investigations involving membrane permeabilization experimentally cumbersome, as pores on the membrane weaken the scaffolding structure that builds TNTs. Presently, access to the inside of TNTs has been achieved with the use of milder membrane detergents (e.g. Saponin) at low concentrations (Costanzo et al., 2013;[START_REF] Rupp | Malaria parasites form filamentous cell-to-cell connections during reproduction in the mosquito midgut[END_REF]Zhu et al., 2018); however, the identification of components within TNTs by this method remains inefficient and unsatisfactory.

Labeling of Tunneling Nanotubes and Actin Backbone

A TNT-specific marker hasn't been identified yet. However, recent efforts to obtain molecular markers specific for labeling TNTs by using micro-proteomic approaches highlighted several unexpected candidate proteins, (e.g. Grk5 and Hist1h3b), which have yet to be investigated [START_REF] Gousset | A novel Microproteomic Approach Using Laser Capture Microdissection to Study Cellular Protrusions[END_REF]. At the present time, the method applied to label TNTs is to stain the entire plasma membrane of cells with fluorescently-conjugated proteins that bind to glycolipids and glycoproteins, such as WGA [START_REF] Abounit | Identification and Characterization of Tunneling Nanotubes for Intercellular Trafficking[END_REF], FM1-43 (McCoy-Simandle et al., 2016), and

MemBright [START_REF] Collot | MemBright: A Family of Fluorescent Membrane Probes for Advanced Cellular Imaging and Neuroscience[END_REF], or by genetic over-expressing proteins associated to the plasma membrane, such as H-Ras and CAAX [START_REF] Hanna | Tunneling nanotubes, a novel mode of tumor cell-macrophage communication in tumor cell invasion[END_REF][START_REF] Rainy | H-Ras transfers from B to T cells via tunneling nanotubes[END_REF].

The actin backbone composition of TNTs is labeled with fluorescently-conjugated Phalloidin [START_REF] Abounit | Identification and Characterization of Tunneling Nanotubes for Intercellular Trafficking[END_REF] or by genetic over-expressing the small F-actin peptide dye, Lifeact [START_REF] Riedl | Lifeact: a versatile marker to visualize F-actin[END_REF] fused to GFP or mCherry [START_REF] Sáenz-De-Santa-María | Control of long-distance cell-to-cell communication and autophagosome transfer in squamous cell carcinoma via tunneling nanotubes[END_REF]. A new report shed light on a novel approach to simultaneously label actin-, intermediate-, and microtubule-filaments of TNTs of urothelial cells [START_REF] Resnik | Triple labelling of actin filaments, intermediate filaments and microtubules for broad application in cell biology: uncovering the cytoskeletal composition in tunneling nanotubes[END_REF], with promising application for co-cytoskeletaldistribution studies. The reproducibility of this approach in TNTs of other cells has yet to be determined.

Detection by Microscopy

The detection of TNTs by microscopy requires high-magnification differential interference contrast (DIC) imaging and/or laser scanning confocal microscopy (LSCM), as TNTs, unlike peripheral filopodia (substrate-adherent filopodia), hover over the substrate and, in some cases, over other cells. To discriminate TNTs from dorsal filopodia, (filopodia located near or toward the upper surface of cells in culture), TNTs must not touch the substratum, extend towards the medium, and be sufficiently long and stable to connect at least two cells [START_REF] Abounit | Identification and Characterization of Tunneling Nanotubes for Intercellular Trafficking[END_REF].

The identification of TNTs in vitro is contingent upon adequate signal-to-noise ratio, which requires that cells in culture be seeded in a medium-confluent monolayer ecosystem that warrants sufficient physical space between cells to visualize TNTs. A recent study found that this paradigm could be avoided with treatment of proteases. In their study, Rustom and colleagues singularized cells by turning them spherical and cleared the way for TNTs to become visible. The authors confirmed this phenomena in various cell lines, including Rat Embryonic Fibroblasts 52 (REF52), NRK, Henrietta Lacks (HeLa), Madine-Darby canine kidney (MDCK), endothelial, adenocarcinoma, and HEK cells [START_REF] Staufer | Protease-resistant cell meshworks: An indication of membrane nanotube-based syncytia formation[END_REF]; however, it is currently unknown whether protease treatment could also be inducing the formation of TNT (via a stress response, which will be discussed later in this chapter), and if the observable connections are capable of inter-cellular cargo transport.

Quantitative Assessment

Unfortunately, a method to quantify individual TNTs does not exist but new artificial intelligence (AI) approaches and machine learning (ML) have been proposed [START_REF] Valdebenito | The Novel Roles of Connexin Channels and Tunneling Nanotubes in Cancer Pathogenesis[END_REF]. For now, our group and others extrapolate relevant biological information from experiments by quantifying the number of cells connected via TNTs using a computer-assisted manual approach in Fiji/ImageJ or ICY (TNT-Annotator plugin) (Delage et al., 2016).

These analyses are informative but in order to definitively identify TNTs with accuracy they must be accompanied by functional assays (i.e. transport of cargo). Cargo transfer is assessed by observing organelles (e.g. lysosomes), vesicles (or endosomes) labeled with fluorescent lipophilic cationic indocarbocyanine dyes (e.g. DiI and DiD) transfer between cells in a donor-and-acceptor cell co-culture system [START_REF] Abounit | Identification and Characterization of Tunneling Nanotubes for Intercellular Trafficking[END_REF][START_REF] Sharma | Rhes travels from cell to cell and transports Huntington disease protein via TNT-like protrusion[END_REF]. The amount of transfer is assessed by (i) live imaging microscopy or in fixed conditions by (ii) LSCM or Fluorescence Activated Cell Sorting (FACS) [START_REF] Abounit | Identification and Characterization of Tunneling Nanotubes for Intercellular Trafficking[END_REF]. In order to examine if the intercellular transport of vesicles in these functional assays is TNT-dependent and not, via long-range EV mediated mechanisms, experiments must be accompanied by three controls: (i) a transwell filter that reduces (or eliminates) cell-to-cell contact; (ii) assessment of supernatant transferred from donor-to acceptor cells; and (iii) a mixture of non-co-cultured cells to obtain background noise threshold (Gousset et al., 2009;[START_REF] Thayanithy | A transwell assay that excludes exosomes for assessment of tunneling nanotube-mediated intercellular communication[END_REF]. Integrating these assays into a controlled device where the position of cells and growth-direction of TNTs are confined to microchannels in a micro-fluidic chip (similarly to those routinely used for axonal growth studies [START_REF] Neto | Compartmentalized Microfluidic Platforms: The Unrivaled Breakthrough of In Vitro Tools for Neurobiological Research[END_REF]), could significantly increase the 'control', throughput, and versatility of our studies but have only been employed once, previously [START_REF] Yang | Biochip-based study of unidirectional mitochondrial transfer from stem cells to myocytes via tunneling nanotubes[END_REF].

The metrics described above hold a stringent enough criterion to identify TNTs in culture.

However, new challenges arise from cell type and model organism complexity. These technical and biological obstacles will continue to be discussed throughout this chapter in order to pinpoint the areas in which our field is lacking, and my dissertation work contributes to.

Tunneling Nanotubes: Biogenesis

TNTs emerge from two physico-mechanical origins: a 'cell dislodgement mechanism' and an 'actin-driven protrusion mechanism. In order to further understand these models, I will take a deeper look at each one of them over the next sub-sections.

Cell dislodgement mechanism

The cell dislodgement mechanism of TNT formation occurs when two adjacent cells fuse transiently and as they move apart, a thin actin-bearing membranous tube remains (Figure 4). It accounts for 100% of TNTs in squamous cell carcinomas (SCC), neuronal crest cells (NCCs), and NRK cells; 82% in HEK 293 cells [START_REF] Sáenz-De-Santa-María | Control of long-distance cell-to-cell communication and autophagosome transfer in squamous cell carcinoma via tunneling nanotubes[END_REF][START_REF] Wang | Animal cells connected by nanotubes can be electrically coupled through interposed gap-junction channels[END_REF], and the vast majority in human primary, hematopoietic progenitors, and leukemic KG1a cells [START_REF] Reichert | Tunneling nanotubes mediate the transfer of stem cell marker CD133 between hematopoietic progenitor cells[END_REF]. In neuronal cells such as PC12, only 7% of TNTs are formed by dislodgment [START_REF] Bukoreshtliev | Selective block of tunneling nanotube (TNT) formation inhibits intercellular organelle transfer between PC12 cells[END_REF], suggesting that this biogenesis mechanism is cell specific.

Interestingly, some of the TNTs derived from dislodgement were shown to be electrically coupled by Cx43 GJs [START_REF] Wang | Animal cells connected by nanotubes can be electrically coupled through interposed gap-junction channels[END_REF]. This observation agrees with the findings of several other groups, in which immune-cells, which are known to interact by immune synapses when they are in close proximity [START_REF] Finetti | Transcellular communication at the immunological synapse: a vesicular traffic-mediated mutual exchange[END_REF], form closed-tip TNT-like protrusions [START_REF] Davis | Membrane nanotubes: dynamic long-distance connections between animal cells[END_REF][START_REF] Dustin | Understanding the structure and function of the immunological synapse[END_REF][START_REF] Onfelt | Cutting edge: Membrane nanotubes connect immune cells[END_REF][START_REF] Onfelt | Structurally distinct membrane nanotubes between human macrophages support long-distance vesicular traffic or surfing of bacteria[END_REF][START_REF] Watkins | Functional connectivity between immune cells mediated by tunneling nanotubules[END_REF]. Whether TNTs in immune cells require adhesion molecules (e.g. Cadherins) to establish dislodged TNTs is currently unknown. The precise mechanism by which cells incorporate an actin backbone into TNTs formed by dislodgement is another unexplored question. However, recent studies provided new insights after finding that Fascin, an actin-bundling protein found in numerous membranous protrusions, is absent in the cell dislodgment-based formation of TNTs in epithelial lung cells [START_REF] Dubois | A role for RASSF1A in tunneling nanotube formation between cells through GEFH1/Rab11 pathway control[END_REF]. This finding is consistent with one of our previous reports In which we showed a decrease in TNT formation and increase in filopodial protrusions upon over-expression of Fascin in neuronal CAD cells (Gousset et al., 2013). Together, this data suggests significant variations amongst TNTs of distinct cell types formed by cell dislodgment and raises new directions for future studies.

Actin-driven protrusion mechanism

The actin-driven protrusion mechanism of TNT biogenesis occurs when the tip of a filopodiumlike structure extrudes from one cell and fuses with another, onto either the cell body or the tip of a filopodium emanating from it (Figure 4). Differently form dislodgement, the actin-driven protrusion mechanism is typical of relatively immobile cells (e.g. neurons and epithelial cells) (Gousset et al., 2013).

With the exception of a select group of "thick" (>0.7 μm), microtubule-bearing TNTs in macrophages and urothelial cells [START_REF] Onfelt | Structurally distinct membrane nanotubes between human macrophages support long-distance vesicular traffic or surfing of bacteria[END_REF][START_REF] Resnik | Helical organization of microtubules occurs in a minority of tunneling membrane nanotubes in normal and cancer urothelial cells[END_REF][START_REF] Veranic | Different types of cell-to-cell connections mediated by nanotubular structures[END_REF], all

TNTs contain only actin. This premise was demonstrated by two key findings: (i) microtubuledisrupting and microtubule-stabilizing agents (nocodazole and paclitaxel, respectively) do not alter induction of TNTs [START_REF] Wang | Tunneling-nanotube development in astrocytes depends on p53 activation[END_REF], and (ii) F-actin depolymerizing toxins, (latrunculin, cytochalasin B, and cytochalasin D) block TNT formation [START_REF] Bukoreshtliev | Selective block of tunneling nanotube (TNT) formation inhibits intercellular organelle transfer between PC12 cells[END_REF][START_REF] Dupont | Tunneling Nanotubes: Intimate Communication between Myeloid Cells[END_REF][START_REF] Dupont | Tunneling Nanotubes: Intimate Communication between Myeloid Cells[END_REF][START_REF] Schiller | LST1 promotes the assembly of a molecular machinery responsible for tunneling nanotube formation[END_REF][START_REF] Takahashi | Tunneling nanotube formation is essential for the regulation of osteoclastogenesis[END_REF]. These findings propose that TNTs form by the interaction of actin protein networks that also give rise to other actin-based protrusions such as filopodia, or filopodia-like structures. Live-imaging assays showing that 93%

of TNTs in HeLa cells originate from filopodia like protrusion interplay [START_REF] Bukoreshtliev | Selective block of tunneling nanotube (TNT) formation inhibits intercellular organelle transfer between PC12 cells[END_REF] support this notion, and underscore the importance to investigate the cellular and molecular players involved in the formation of filopodia, which I will describe in the sub-sections below.

Filopodia: Introduction

In 1907, Ross Granville Harrison discovered that upon isolating and culturing embryonic tissue from adult frogs, thin "hyaline protoplasm" filaments of 25 μm in length and 1.5 -3 μm in thickness, extended from the end of growing nerve fibers [START_REF] Harrison | Observations of the living developing nerve fiber[END_REF]. Over 100 years later, we now know that Harrison's "rhizopod"-resembling nerve protrusions drive several major neuronal events, which include axonal guidance, dendritic spine formation, and synaptic connectivity [START_REF] Gallo | Mechanisms underlying the initiation and dynamics of neuronal filopodia: from neurite formation to synaptogenesis[END_REF], and represent only one of various kinds of membranous protrusions classified under the umbrella of filopodia (Figure 5).

Today, filopodia are defined as thin (100 -300 nm), close-ended actin-rich finger-like protrusions at the leading edge of cells. Their 'antenna'-like ability to sense the extracellular environment has granted them roles in vital processes such as cell-to-cell adhesion and migration [START_REF] Gardel | Mechanical integration of actin and adhesion dynamics in cell migration[END_REF][START_REF] Jacquemet | Filopodia in cell adhesion, 3D migration and cancer cell invasion[END_REF]. As with TNTs, variations in length, thickness, dynamicity, and cellular localization make filopodia largely heterogenous. These variations could be due to organism-or cell type specific signaling proteins, actin-cross-linking proteins, nucleators, motor proteins, and actin-to-plasma membrane Bin/Amphiphysin/Rvs (I-BAR) linking proteins [START_REF] Mattila | Filopodia: molecular architecture and cellular functions[END_REF]. Studies by the group of Giorgio Scita and others have made seminal findings that will be described below, and which served as preliminary data for

Chapter 2 of my work.

Filopodia: Mechanisms of Formation

The formation of filopodia requires a highly dynamic actin cytoskeleton structure that forms by two models, a 'convergent elongation model' in which filopodia arise from the Lamellipodium dynamic actin filament meshwork assembled by the Actin-related protein 2/3 (Arp2/3), and a 'de novo filament nucleation model', in which the actin of filopodia is nucleated at the tips by the vasodilator-stimulated phosphoprotein (VASP) and the actin polymerizer, formin [START_REF] Mattila | Filopodia: molecular architecture and cellular functions[END_REF]) (Figure 6).

Small GTPases of the Ras and Rho superfamily lie at the heart of the 'convergent elongation model' [START_REF] Bishop | Rho GTPases and their effector proteins[END_REF][START_REF] Nobes | Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia[END_REF], where the Cell Division Control Protein 42 (CDC42) and Ras-related C3 botulinum toxin substrate (Rac1) play key roles in the activation of Arp2/3. Specifically, CDC42 acts on the Wiskott-Aldrich syndrome protein (WASP), or neural-WASP (N-WASP), to initiate the nucleation of new actin filaments through the activation of Arp2/3 [START_REF] Carlier | Signalling to actin: the Cdc42-N-WASP-Arp2/3 connection[END_REF], whereas Rac1 [START_REF] Heasman | Mammalian Rho GTPases: new insights into their functions from in vivo studies[END_REF] acts on its downstream interactor, WASP-family verprolin homologous (WAVE) protein, before activating Arp2/3 [START_REF] Kurisu | The WASP and WAVE family proteins[END_REF]. Importantly, CDC42 is involved in filopodia formation while Rac1 in lamellipodia formation [START_REF] Nobes | Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia[END_REF]. Schematic diagram depicting the molecular assembly of Arp2/3 (A) and Formins (B), (obtained from [START_REF] Mattila | Filopodia: molecular architecture and cellular functions[END_REF]. (A) The Arp2/3 complex is made up of seven subunits, two actin-related proteins and five other proteins. When Arp2/3 binds to pre-existing actin filaments, the actin-related subunits form a 'seed' that nucleates actin polymerization. The activation of the ARP2/3 complex is induced by binding to nucleation-promoting factors, such as WASP/WAVE-family proteins, which dissociate from the ARP2/3 complex after filament nucleation. (B) Formins are multi-domain proteins that nucleate polymerization of unbranched actin filaments. The formin homology-2 (FH2) domain initiates filament assembly and remains persistently associated with the fast-growing barbed end, enabling insertion of actin subunits while protecting the end from capping proteins. Formins can contain a profilin binding formin homology-1 (FH1) domain, which recruits profilin-G-actin complexes to the barbed ends of the growing filament.

An alternative mechanism to the CDC42-WASP-Arp2/3-dependent filopodia formation pathway involves the interaction of CDC42 and VASP with the I-BAR-domain, Insulin Receptor Tyrosine kinase substrate (IRSp53), an actin crosslinking protein involved in the formation of filopodia-like protrusions such as neurites [START_REF] Govind | Cdc42Hs facilitates cytoskeletal reorganization and neurite outgrowth by localizing the 58-kD insulin receptor substrate to filamentous actin[END_REF]. Using quantitative binding assays and Mouse Embryonic Fibroblasts (MEF) cells, Scita and colleagues demonstrated that filopodiainducing stimulus ensues the binding of CDC42 to IRSp53, and subsequently promotes the growth of a filopodial filament through the formation of a CDC42-IRSp53-VASP complex (Disanza et al., 2013). In the same study, the authors also observed a reduction in filopodia filament formation upon the interaction between IRSp53 with the Epidermal growth factor receptor kinase substrate 8 (Eps8), a GTPase which had previously been associated with actin dynamics through its barbedend capping activity (Di [START_REF] Fiore | Eps8 in the midst of GTPases[END_REF], and strong synergy with IRSp53 in mediating actin bundling [START_REF] Disanza | Regulation of cell shape by Cdc42 is mediated by the synergic actin-bundling activity of the Eps8-IRSp53 complex[END_REF]) (Figure 7).

Using a mouse neuronal cell line of catecholaminergic origin, Cath.a-differentiated (CAD) [START_REF] Qi | Characterization of a CNS cell line, CAD, in which morphological differentiation is initiated by serum deprivation[END_REF], which our group previously identified as a robust in vitro model for the study of TNTs in vitro [START_REF] Abounit | Identification and Characterization of Tunneling Nanotubes for Intercellular Trafficking[END_REF]Gousset et al., 2009Gousset et al., , 2013;;Zhu et al., 2018), we previously demonstrated that overexpression of VASP reduce TNT formation. Our more recent results

(detailed in Chapter 2) reinforce these findings and reveal that, contrary to their role in filopodia, (i) CDC42-VASP-IRSp53 network serve as negative regulators of TNTs and inter-cellular vesicle transport, while (ii) Eps8, induces TNT formation and increases cargo transfer (Delage et al., 2016). Cryo-electron microscopy and vitrification of specimens have revolutionized structural analyses of biological samples [START_REF] Murata | Cryo-electron microscopy for structural analysis of dynamic biological macromolecules[END_REF]. Cryo-transmission electron microscopy (cryo-TEM) and cryo-electron tomography (cryo-ET) of vitrified Dictyostelium discoideum amoeba revealed the intracellular ultrastructure of de novo filopodia with sufficient detail to elucidate several key topological features about their cytoskeletal architecture: actin filaments that make the shaft of the filopodia appear fragmented; at the close-ended tip, the "terminal cone", is built of short branched actin filaments that anchor to the tip of the filopodium on one end and flank on the other [START_REF] Medalia | Organization of actin networks in intact filopodia[END_REF].

Using similar imaging approaches, Yasunaga and colleagues observed over 30 filopodial filaments emanating from the neuronal cell line, NG108-15, and unveiled their cysoskeletal architecture by revealed that actin filaments (of 2.7 nm in thickness) are bundled by Fascin in a hexagonal shape [START_REF] Aramaki | Revealing the intracellular ultrastructure of filopodia with cryo-electron tomography[END_REF][START_REF] Aramaki | Filopodia formation by crosslinking of F-actin with fascin in two different binding manners[END_REF]. Fast Fourier transformation analyses confirmed that the bundles observed within the filopodial filament were in fact actin.

As part of one of my projects (described in detail in Chapter 3), I studied TNTs using cryo-TEM and cryo-ET in two neuronal cell models, CAD and SH-SY5Y cells [START_REF] Kovalevich | Considerations for the use of SH-SY5Y neuroblastoma cells in neurobiology[END_REF], in which we and others had observed functional TNTs [START_REF] Dieriks | α-synuclein transfer through tunneling nanotubes occurs in SH-SY5Y cells and primary brain pericytes from Parkinson's disease patients[END_REF]Dilsizoglu Senol et al., 2019;[START_REF] Epperla | Nanodiamond-Mediated Intercellular Transport of Proteins through Membrane Tunneling Nanotubes[END_REF]. In these cells, our observations indicate that TNTs are comprised of numerous individual TNTs (iTNTs), whereas filopodia are not; TNTs contain cargo (e.g. vesicles and organelles) within, filopodia do not (Sartori- Rupp et al., 2019). Electron micrographs showing the ultra-structure of filopodia, (obtained from [START_REF] Medalia | Organization of actin networks in intact filopodia[END_REF]Aramaki et al., 2016, respectively). (A) Four consecutive sections through the reconstructed volume of a wild-type Dictyostelium cell filopod shows clear branching of actin filaments that reaches the growth cone. (B) 3D structure of a filopodium and intracellular filaments of a (NG108-15) neuronal cell by cryo-ET. Segmentation shows filamentous structures, cell membrane, and a 10-nm gold marker in red, blue, and yellow, respectively. A highly ordered bundled filamentous structure can be clearly observed. (right) The periodicity of the bundled filamentous structure is shown using ">" and "<" with 36-nm helical repeats. The diameter of the filopodium is 100 nm at its thinnest region (**) and 250 nm at its thickest region (*).

Tunneling Nanotubes: Molecular Formation

In an attempt to identify the molecular pathways involved in the two biogenesis mechanisms described above, our group and others have focused on investigating a series of proteins and signaling pathways linked to actin networks that play a role in the formation and regulation of other common membranous protrusions, including those of filopodia.

Role of Actin Cytoskeleton and Actin Protein Regulators

CDC42 and Rac, whose roles in Arp2/3-dependent actin polymerization were mentioned above and previously studied in filopodia and lamellipodia formation [START_REF] Heasman | Mammalian Rho GTPases: new insights into their functions from in vivo studies[END_REF], were first observed at the base of TNTs connecting immune Jurkat T cells [START_REF] Lachambre | Preliminary characterisation of nanotubes connecting T-cells and their use by HIV-1[END_REF]). It's role in role in TNTs was revealed by the inhibition of CDC42 using Secramine A, which blocked TNT formation [START_REF] Arkwright | Fas stimulation of T lymphocytes promotes rapid intercellular exchange of death signals via membrane nanotubes[END_REF]. In the cervical cancer HeLa cell, inhibition of CDC42 by transfecting a dominant negative construct decreased the formation of TNTs.

Conversely, inhibition of Rac1 by overexpression of its dominant negative form had no significant effect (Hase et al., 2009). By treating macrophages with specific inhibitors for CDC42 and Rac1, ML-141 and 6-Thio-GTP, respectively, Hanna and colleagues confirmed that TNTs in immune cells require both CDC42 and Rac GTPases for their formation [START_REF] Hanna | The Role of Rho-GTPases and actin polymerization during Macrophage Tunneling Nanotube Biogenesis[END_REF].

A portion of my dissertation was devoted to studying the role of these GTPases on TNTs connecting neuronal CAD cells. Interestingly, as will be described in more detail in Section 2, our observations indicate that inhibition of CDC42 and Rac1 by transfecting CAD neuronal cells with dominant negative forms, significantly increases the number of cells connected by TNTs (Delage et al., 2016).

Downstream effectors of CDC42, WASP localize throughout macrophage TNTs. Silencing WASP by short hairpin (sh) RNA (shWASP) resulted in a 90% reduction of cells connected by TNTs and blocking of DiI-labeled cargo. Similar results were observed with macrophages depleted of Rac's downstream effector, WASP family verprolin-homologous 2 (WAVE2) by transient silencing [START_REF] Hanna | The Role of Rho-GTPases and actin polymerization during Macrophage Tunneling Nanotube Biogenesis[END_REF].

Direct inhibition of the common downstream effector of WASP and WAVE2, Arp2/3, with CK-666, that blocks movement of the Arp 2 and Arp 3 subunits into the activated filament-like conformation [START_REF] Hetrick | Small molecules CK-666 and CK-869 inhibit actin-related protein 2/3 complex by blocking an activating conformational change[END_REF], resulted in a decrease in the formation of TNTs in bone marrow-derived macrophage [START_REF] Hanna | The Role of Rho-GTPases and actin polymerization during Macrophage Tunneling Nanotube Biogenesis[END_REF]. On the contrary, as described in detail in my results, inhibition of the Arp2/3 complex in neuronal CAD cells induces the formation of TNTs and significantly increases inter-cellular DiD-labeled vesicle transfer (Sartori- Rupp et al., 2019).

Rab Cascades

Ras-related proteins in brain (Rabs), are peripheral membrane-anchored proteins best known for their essential roles in endo-and exocytic membrane trafficking, including vesicle formation and vesicle movement along cytoskeletal networks [START_REF] Stenmark | Rab GTPases as coordinators of vesicle traffic[END_REF]. Through various pathways, Rabs also regulate ciliary axoneme extension [START_REF] Blacque | Rab GTPases in cilium formation and function[END_REF], dendritic growth cones, and dendritic filopodia and spines [START_REF] Mori | Small GTPase Rab17 regulates dendritic morphogenesis and postsynaptic development of hippocampal neurons[END_REF].

Through a high-content screening in which 41 Rab GTPase subfamilies were studied in CAD neuronal cells, our group found that Rab8a and Rab11a increased TNT formation and promoted contact-dependent inter-cellular vesicle transfer (Zhu et al., 2018). On the contrary, the activation of Rabin8, a major Guanine nucleotide Exchange Factor (GEF) of Rab8 and important for ciliogenesis and filopodia elongation [START_REF] Homma | Rabin8 regulates neurite outgrowth in both GEF activity-dependent and -independent manners[END_REF][START_REF] Westlake | Primary cilia membrane assembly is initiated by Rab11 and transport protein particle II (TRAPPII) complex-dependent trafficking of Rabin8 to the centrosome[END_REF], increased the generation of filopodia but did not affect TNT formation. In the same study, the v-SNARE Vesicle-associated membrane protein 3 (VAMP3) was identified as an effector of Rab11aand Rab8-TNT formation, but not of filopodia. This study indicates that Rab11 and Rab8 act in a signaling cascade through VAMP3 to induce TNT formation. This study corroborates another finding in which knockdown of Rab8a or Rab11a in neuronal Schwann cells inhibited the formation of TNTs and reduced inter-cellular vesicle transfer (Zhu et al., 2016). By overexpressing Rab8 in HeLa cells, Gerdes and colleagues found an increase in TNT formation and transport of Transferrin (Tf) and Tf-receptor, suggesting that Rab8-dependent TNT formation mechanism plays a role in cancer cell lines as well [START_REF] Burtey | Intercellular transfer of transferrin receptor by a contact-, Rab8-dependent mechanism involving tunneling nanotubes[END_REF]. The authors confirmed this notion after observing that significantly less cargo transfer occurred between non-cancer NRK cells. In agreement with these observations, we found that blocking the Tf recycling pathway inhibits TNT formation (Zhu et al., 2018). Finally, in other cancer cells, bronchial epithelial and pleural mesothelial cells, recent reports by Levallet and colleagues identified the Ras association domain family 1 isoform A (RASSF1A) as a regulator of TNTs formation through induction of GEF-H1 and upregulation of Rab11 [START_REF] Dubois | A role for RASSF1A in tunneling nanotube formation between cells through GEFH1/Rab11 pathway control[END_REF]. Schematic diagram depicting neuronal TNT formation by Rab cascade, (obtained from Zhu et al., 2018). GTP-bound Rab11a acts upstream of Rab8a, while VAMP3 acts downstream of Rab8a in regulating TNT formation. Rab11a-induced activation of Rab8a is independent of Rabin8 and GRAB. One possibility is that Rab11a is released from the recycling endosome when Rab8a is activated. GTP-bound Rab8a interacts with VAMP3, which functions as a Rab8a effector and facilitates the fusion of recycling endosome-derived vesicle to supply lipids and specific proteins needed for TNT formation.

Together, these studies shed light on the formation of TNTs via Rab GTPases and supports our group's hypothesis that TNTs and filopodia harness distinct protein modulators underlying the regulation of their formation. However, it is currently unclear whether Rabs could affect TNT formation by modulating actin cytoskeletal dynamics or/in addition to regulating membrane supply and specific lipids and proteins through activation of the recycling pathway.

PI3K/AKT/mTOR Signaling Pathway

The Phosphatidylinositol-3-kinase (PI3K) and its downstream mediators Akt and the mammalian target of rapamycin (mTOR) constitute a crucial signaling pathway for the regulation of the cell cycle, cellular proliferation, metabolism, and survival [START_REF] Yu | Proliferation, survival and metabolism: the role of PI3K/AKT/mTOR signalling in pluripotency and cell fate determination[END_REF]. The pathway is also involved in the morphogenesis of filopodial structures such as dendrites [START_REF] Kumar | Regulation of dendritic morphogenesis by Ras-PI3K-Akt-mTOR and Ras-MAPK signaling pathways[END_REF].

Several studies have also revealed the importance of this signaling pathway in the formation of TNTs. Using a dominant negative mutant for Akt, Wang and colleagues found a significant reduction in TNT formation of PC12 cells. Similarly, inhibition of PI3K with specific inhibitors (ly294002 and wortmannin) also reduced TNT formation. Inhibition of mTOR via rapamycin had a similar effect [START_REF] Wang | Tunneling-nanotube development in astrocytes depends on p53 activation[END_REF]. Using the mTOR-specific inhibitors everolimus and metformin, studies in mesothelioma and ovarian cancer cells also observed a suppression of TNT formation [START_REF] Desir | Tunneling nanotube formation is stimulated by hypoxia in ovarian cancer cells[END_REF][START_REF] Lou | Tunneling nanotubes provide a unique conduit for intercellular transfer of cellular contents in human malignant pleural mesothelioma[END_REF].

Together, these studies indicate that the PI3K/AKT/mTOR signaling pathway plays a role in the formation of TNTs, and postulate the notion that TNTs might be inter-cellular mechanisms that aid cells under stress (Figure 10) [START_REF] Wang | Tunneling-nanotube development in astrocytes depends on p53 activation[END_REF].

M-Sec/TNFaip2/B94

Downstream of the PI3K/AKT/mTOR Signaling Pathway lies M-Sec, a cytosolic protein homolog of Sec6 that was first detected in specialized epithelial enterocytes called M cells. Originally known as 'TNF-a induced protein 2' (TNFaip2) (or B94) for exerting various proinflammatory and catabolic effects as a pleiotropic cytokine, M-Sec was one of the earliest markers found to be involved in the formation of TNTs (Hase et al., 2009). The depletion of M-Sec by RNA interference in Raw264.7 macrophages significantly decreases TNT formation and inter-cellular propagation of calcium flux. Conversely, its overexpression has the opposite effect, (i.e. it induces TNT formation). In HeLa, M-Sec's induction activity involves the interaction with the Ras-related A (RalA) GTPase and the Exocyst complex (Figure 10).

In other studies carried out in both HeLa and HEK-293 cells, M-Sec was shown to also depend on the transmembrane major histocompatibility complex (MHC) class III protein leucocyte specific transcript 1 (LST1) [START_REF] Schiller | LST1 promotes the assembly of a molecular machinery responsible for tunneling nanotube formation[END_REF]. The authors propose that LST1 recruits RalA to the plasma membrane, where RalA then interacts with the Exocist complex (Figure 10). This suggests that M-Sec activates a molecular assembly machinery that could be responsible for supplying the membrane required for TNT formation [START_REF] Hanna | The Role of Rho-GTPases and actin polymerization during Macrophage Tunneling Nanotube Biogenesis[END_REF].

A recent proteome interactomic analyses for M-Sec in human osteosarcoma-origin U2OS cells revealed an important upstream interactor of M-Sec, the endoplasmic reticulum chaperone ERp29 [START_REF] Pergu | The chaperone ERp29 is required for tunneling nanotube formation by stabilizing MSec[END_REF]. After depleting M-Sec by siRNA, while simultaneously over-expressing exogenous ERp29, the authors observed a significant reduction in TNT formation, indicating that ERp29 can only induce TNTs in the presence of M-Sec.

Interestingly, M-Sec does not appear to play the same role in all of immune cells. For example, M-Sec was not found, nor did it increase, TNT formation in immune Jurkat T cells [START_REF] Lachambre | Preliminary characterisation of nanotubes connecting T-cells and their use by HIV-1[END_REF]. Furthermore, M-Sec doesn't appear to be expressed in neuronal cells either (Gousset et al., 2013), which could suggest that its role in TNT formation is restricted to myeloid lineages only.

MyosinX

The myosins are a superfamily of actin-based motor proteins that "walk" on actin filaments.

Of all the myosins, MyoX is the most broadly distributed one in cells, inducing the formation of filopodia [START_REF] Sousa | Myosin-X: a molecular motor at the cell's fingertips[END_REF]. Studies from our group showed that MyoX is involved in TNT formation after findings revealed that its deletion inhibited TNT formation. Intriguingly, different domains of MyoX are essential for TNTs, and some for filopodia (Gousset et al., 2013). We demonstrated that neuronal TNT formation requires the motor and tail domains of MyoX, and, specifically, identified the F2 lobe of the tail protein-ezrin-radixin-moesin (FERM) domain. Given that MyoX increases the formation of adherent-and dorsal filopodia, we postulated that neuronal TNTs can arise from a subset of MyoX-driven dorsal filopodia, independent of integrins, transmembrane receptors required for the formation of adherent filopodia. Consistent with our hypothesis and subsequent data shown in Chapter 2 (Delage et al., 2016), MyoX acts downstream of CDC42 and is independent of VASP, another potent inducer of dorsal filopodia formation [START_REF] Bohil | Myosin-X is a molecular motor that functions in filopodia formation[END_REF]. Together, these findings demonstrate the existence of different mechanisms for dorsal filopodia formation.

In macrophages, recent studies showed that MyoX expression depends on the Immunodeficiency Virus Type 1 (HIV-1) Negative Regulatory factor (NEF) [START_REF] Uhl | Myosin-X is essential to the intercellular spread of HIV-1 Nef through tunneling nanotubes[END_REF], indicating, for the first time, that MyoX-dependent TNT formation is not restricted to neuronal cells.

Tunneling Nanotubes: Intrinsic and Extrinsic Induction

There are several physiological factors that can affect the molecular machineries described above [START_REF] Rustom | The missing link: does tunnelling nanotube-based supercellularity provide a new understanding of chronic and lifestyle diseases?[END_REF] and which indicate that stressful stimuli induce TNT formation. Some studies have postulated that TNTs arise from stress responses to conditions that favor metabolic dysregulation [START_REF] Lou | Tunneling nanotubes provide a unique conduit for intercellular transfer of cellular contents in human malignant pleural mesothelioma[END_REF][START_REF] Victoria | The spread of prion-like proteins by lysosomes and tunneling nanotubes: Implications for neurodegenerative diseases[END_REF][START_REF] Wang | Long-distance electrical coupling via tunneling nanotubes[END_REF]. The effectors known to date are described below.

Reactive Oxygen Species

Reactive Oxygen Species (ROS), whose free radicals have been long known to pose a threat to cellular integrity and viability [START_REF] Dixon | The role of iron and reactive oxygen species in cell death[END_REF], were found to be effective inducers of TNTs via the major form of endogenous ROS, hydrogen peroxide (H2O2) [START_REF] Liang | A Salutary Role of Reactive Oxygen Species in Intercellular Tunnel-Mediated Communication[END_REF]. In rat hippocampal astrocytes, neurons, and HEK293 cells, H2O2 significantly increased the number of (homotypic and heterotypic) cells connected by TNT-like protrusions compared to untreated cells [START_REF] Wang | Tunneling-nanotube development in astrocytes depends on p53 activation[END_REF]. Notably, upon co-culturing non-stressed and stressed cells, the authors observed that H2O2-treated cells were the initiators of TNT formation. Subsequent studies from our group showed that H2O2-induced stress increases TNT formation of neuronal CAD cells by 70% (Gousset et al., 2013). In one of our group's prior reviews [START_REF] Victoria | The spread of prion-like proteins by lysosomes and tunneling nanotubes: Implications for neurodegenerative diseases[END_REF], we proposed that, based on (i) the strict correlation between neurodegeneration-associated proteins and increased ROS production and oxidative stress (Kim et al., 2015), and (ii) our findings showing an increase in neuronal TNTs through intercellular aggregate buildup, ROS generated as a consequence of prion-like protein aggregation may stimulate TNT formation.

The mechanism leading to ROS-mediated TNT induction is not well understood; however, a study on TNTs in astrocytes indicates that H2O2 triggers the phosphorylation of the p38 mitogenactivated protein kinase (MAPK) [START_REF] Zhu | Hydrogen peroxide alters membrane and cytoskeleton properties and increases intercellular connections in astrocytes[END_REF], a kinase responsive to stress stimuli previously shown to induce actin cytoskeletal remodeling in fibroblasts [START_REF] Hoffman | Mechanical signals activate p38 MAPK pathway-dependent reinforcement of actin via mechanosensitive HspB1[END_REF]. Together, these observations support the established notion that H2O2 affects the actin cytoskeletal organization and induces the formation of actin-rich protrusions [START_REF] Huot | SAPK2/p38dependent F-actin reorganization regulates early membrane blebbing during stress-induced apoptosis[END_REF].

Hypoxia

Decreased oxygen availability (hypoxia) has been well acknowledged as a cellular state that severely affects the balance between ROS and antioxidant defenses, making cells vulnerable to oxidant damage that negatively impacts organ structure and function. Pathological conditions, such as tumor aggression and metastasis and cerebral and heart ischemia have been associated

to the deleterious side-effects of hypoxia [START_REF] Michiels | Physiological and pathological responses to hypoxia[END_REF]. In order to investigate if TNTs found in chemoresistant mammalian cancer cells were affected by hypoxia, Lou and colleagues cultured A2780, C200, and SKOV3 ovarian cancer cells in a chamber containing 2% oxygen and tested the expression of a factor known to maintain cellular homeostasis in low oxygen levels, hypoxia inducible factor-1α (HIF-1α). By 24 hrs, the authors observed an increase in HIF-1α expression and a 6-, 10-, and 11-fold increase in TNT formation compared to normoxic conditions, respectively [START_REF] Desir | Tunneling nanotube formation is stimulated by hypoxia in ovarian cancer cells[END_REF]. Similarly, in another study examining the prevalence of hypoxia in other carcinoma cells, Lou et al. discovered that hypoxia also induced TNT formation in three different colon cancer cell lines, SW480, HCT-116, and DLD-1 [START_REF] Lou | Cellular and Molecular Networking Within the Ecosystem of Cancer Cell Communication via Tunneling Nanotubes[END_REF]. Stress-induced TNT formation by hypoxia has also recently been observed in prostate cancer PC3 and LNCaP cells [START_REF] Kretschmer | Stress-induced tunneling nanotubes support treatment adaptation in prostate cancer[END_REF]. Taken together, these studies present convincing evidence that the tumor microenvironment, which is known to benefit from hypoxic conditions [START_REF] Petrova | The hypoxic tumour microenvironment[END_REF], benefits from an inter-cellular TNT-mediated network that can yield cancer spreading (TNTs and Cancers explored in more detail below).

Apoptosis Signaling

p53, Bax, and caspases play important roles in cell apoptosis. Wang and colleagues tested the role of each one and found that the overexpression of p53 increased the number of TNTconnected cells, while its depletion using a dominant negative mutant had the opposite effect.

In support of this finding, the authors silencing p53 (p53-1 & p53-2) by siRNA and observed similar results [START_REF] Wang | Tunneling-nanotube development in astrocytes depends on p53 activation[END_REF].

The apoptosis antigen 1, FAS receptor (also called APO-1 or CD95), is a type II transmembrane protein receptor that belongs to the TNF family that induces apoptosis when ligands bind to it [START_REF] Strasser | The many roles of FAS receptor signaling in the immune system[END_REF]. Esposti and colleagues observed that the activation of FAS in immune Jurkat T cells induced 20-fold increase in the number of TNT-connected cells [START_REF] Arkwright | Fas stimulation of T lymphocytes promotes rapid intercellular exchange of death signals via membrane nanotubes[END_REF]. Transfer of vesicles and fluorescent cytosolic proteins was also observed upon activation of Fas. Lastly, upon observing that Secramine A and actin Rho-GTPase inhibitor, toxin B, blocked FAS-stimulated TNT formation, the authors indicate that the role of FAS on TNT induction is mediated by Rho GTPases. Whether other immune cells, or cell types of other origins, behave similarly upon the activation of FAS is not currently known.

Stressful Environment

Low-serum, hyperglycemic, acidic growth medium stimulate TNT formation. In a study of TNTs connecting Mesothelioma cells, Lou and colleagues demonstrated that low Hyperglycemic lowserum medium (pH 6.6, 50mM glucose, 2.5% fetal calf serum) or hyperglycemic, low-serum medium with cytokines added to stimulate epithelial-to-mesynchymal transition (EMT), increased TNT formation in mesothelioma [START_REF] Lou | Tunneling nanotubes provide a unique conduit for intercellular transfer of cellular contents in human malignant pleural mesothelioma[END_REF]. Serum depletion in Schwann cells, described in more detail above, also increased TNT formation and TNT-mediated transport of GTPases, Rab8a and Rab11a (Zhu et al., 2016).

Tunneling Nanotube: Ultra-Structure

Specialized cellular membranous protrusions are defined by a strict structure and function relationship. For example, lamellipodia are membranous sheet projections that play a vital role in steering the migration of neuronal precursors during early brain cerebral and cerebellar development [START_REF] Krause | Steering cell migration: lamellipodium dynamics and the regulation of directional persistence[END_REF]. They owe this function to Arp2/3, which by promoting branched actin polymerization, it enables cells to propel across large distances [START_REF] Innocenti | New insights into the formation and the function of lamellipodia and ruffles in mesenchymal cell migration[END_REF].

Unfortunately, little is known about the structure of TNTs. In-depth ultrastructural analyses of Classic SEM analyses revealed that TNTs create a "seamless" membranous transition between the surface of PC12-connected cells (Rustom et al., 2004) (Figure 10) By serial sectioning-TEM (ssTEM) at 80nm/section, accurate diameter measurements were found in the range of 50 -200 nm (Figure 10). ssTEM micrographs also indicated cytoplasmic continuity between two cells connected. However, the example of a TNT provided by the authors is significantly shorter in length than the ones obtained by SEM or light microscopy, making comparisons between these two observations difficult. For example, the connection contained 'kinks' along its length, presumably from mechanical stress during sample preparation, as suggested by the authors.

Examples showing similar aberrations were shown in TNTs connecting SEP AS583 cells [START_REF] Miyazawa | Proliferative arrest of neural cells induces prion protein synthesis, nanotube formation, and cell-to-cell contacts[END_REF]. Whether these connections are representative of TNTs, or a sub-class of TNTs seen by light microscopy was not discussed in either report.

Another study using ssTEM at 60 nm found that HIV-1-transporting-TNTs (of 180-380 nm in diameter) in Jurkat lymphocyte T cells consisted of either a single protrusion invaginating into the opposing cell, or of two filopodia-like protrusions touching each other (Figure 10) [START_REF] Sowinski | Membrane nanotubes physically connect T cells over long distances presenting a novel route for HIV-1 transmission[END_REF]. Both observations explain how membrane dyes of two cell populations connected by TNTs do not mix and suggest that TNTs observed by light microscopy (at least in Jurkat T cells)

are derived solely from one of the cells that appeared connected. Moreover, the authors suggest that TNTs in T cells function through a receptor-dependent manner, indicating that TNTs in immune cells are unique compared to TNTs in other cells, such as neuronal cells (Rustom et al., 2004). Finally, to distinguish Jurkat T cell connections to other connections formed by homotypic cells, the authors also presented an example of an intercellular bridge derived from cytokinesis with dense microtubule bundles making up a midbody [START_REF] Skop | Dissection of the mammalian midbody proteome reveals conserved cytokinesis mechanisms[END_REF][START_REF] Sowinski | Membrane nanotubes physically connect T cells over long distances presenting a novel route for HIV-1 transmission[END_REF].

More recently, Eugenin and colleagues employed SEM and TEM on HIV-infected (and noninfected) human monocyte-derived macrophages and observed two distinct types of connections: open-ended structures that presumably allow cargo transport, and "non-fused", close-ended, synapse-like protrusions (Figure 10) [START_REF] Okafo | Tunneling nanotubes (TNT) mediate long-range gap junctional communication: Implications for HIV cell to cell spread[END_REF]. TEM revealed that the connection identified contained actin and numerous mitochondria within the connection but no viral particles in/outside. This finding is consistent with the cytoskeletal composition of TNT-like structures described by Sowinski et al., in Jurkat T cells-TNTs in immune cells are often closeended protrusions [START_REF] Sowinski | Membrane nanotubes physically connect T cells over long distances presenting a novel route for HIV-1 transmission[END_REF]. In MDCK and lung epithelial cells (A549), SEM analysis underscored the detrimental impact that sample preparation steps can have on the integrity of TNTs, most often affecting their 'straightness'. By SEM, the authors found that TNTs of A549 cells vary in diameter, from 250 nm -1.37 μm (Figure 10) [START_REF] Kumar | Influenza virus exploits tunneling nanotubes for cell-to-cell spread[END_REF]. TEM analysis showed an electron-dense bundle of fibers (presumably microtubules) running along the length of the inside of the expansion-processed connection, which wasn't expected based on the tubulinlacking TNTs previously reported for T lymphocytes [START_REF] Sowinski | Membrane nanotubes physically connect T cells over long distances presenting a novel route for HIV-1 transmission[END_REF]. The presence of mitochondria and ribosomes within the connections observed was also reported. Bladder cancer T24 and RT4 cells by SEM were measured at 100 -200 nm in diameter, with lengths that spanned from 20 μm to 1 mm. The electron micrographs showing TNTs connecting T24-to-RT4 appeared to be single connection with branches [START_REF] Lu | Tunneling nanotubes promote intercellular mitochondria transfer followed by increased invasiveness in bladder cancer cells[END_REF]. It is not clear whether the branches stemming from these TNTs were ever connections that snapped during sample preparation.

Other examples presented by the authors lacked a contiguous plasma membrane connecting both cells; that is, appeared as filopodia-like structures.

In another study, Corbeil and colleagues explored the ultra-structure of TNTs connecting human primary CD34+ hematopoietic stem and progenitor cells (HSPCs). Using SEM, the authors found that TNTs had a diameter of 100 nm, a length that reached > 100 μm, and importantly, appeared to create membrane continuity between the cells at both ends (Figure 10) [START_REF] Reichert | Tunneling nanotubes mediate the transfer of stem cell marker CD133 between hematopoietic progenitor cells[END_REF]. In contrast, KG1a cells, an immature hematopoietic cell line displayed a clear junction half-way along the length of their TNTs, indicating a close-ended connection, which the authors confirmed upon the observation of fluorescent membrane probes and covalently labeled longlived intracellular molecules not flowing seamlessly between cells.

In spite of the technical obstacles encountered, previous structural studies in neuronal, immune, and epithelial cells propose a variety of topologically different TNTs within and between cell types. Unfortunately, the resolution and sample quality based on the images provided does not provide sufficient detail to definitively distinguish whether the connections found contained actin, microtubules, or both. Moreover, only a couple of studies provide examples of cargobearing TNTs, and neither of these reports obtained any convincing evidence proving TNTs' openendedness. The lack of these pivotal pieces of information led to an ongoing debate regarding the structural identity and the mechanisms TNTs use to transport cargo [START_REF] Baker | How the Internet of cells has biologists buzzing[END_REF].

A significant portion of my dissertation dealt with the optimization and application of a correlative-cryo electron-microscopy-based pipeline to address these questions. Using these strategies, which will be described in detail in Chapter 3, we were able to observe TNTs connecting cells in culture under native conditions and discover that most TNTs, classically defined as single connections, were comprised of several individual TNTs. We also demonstrated that cargo (e.g. vesicles and mitochondria) get transported through the inside of the TNT, using actin. Our work also proves that TNTs, at least in neuronal cells, create cytoplasmic continuity.

Tunneling Nanotube-like structures In Vivo

Definitive evidence that TNTs exist in the complex biological contexts where in vitro observations point to is currently missing. However, numerous studies have reported the presence of membranous protrusions with signaling roles in several classes of tissues at embryonic and early stages of post-natal development.

During gastrulation of marine animals such as Sea Urchins, 200 -400 nm thick, and up to 80 µm-long actin-and microtubule-containing filopodial protrusions extend from ectodermal cells, and between primary and secondary mesenchyme cells (PMCs and SMCs, respectively) (Figure 12) [START_REF] Miller | Dynamics of thin filopodia during sea urchin gastrulation[END_REF]. Although sightings of thin filopodia were previously made in Sea Urchins and hypothesized to play a role in locomotion [START_REF] Gustafson | THE CELLULAR BASIS OF MORPHOGENESIS AND SEA URCHIN DEVELOPMENT[END_REF], McClay and colleagues suggest that the developmental timing, location, and rich cytoskeletal apparatus of filopodia formed by PMCs and SMCs point their function role towards signaling organelles that enable the communication of migratory cues (McClay, 1999).

During zebrafish gastrulation, long (up to 350 µm), actin-rich Intercellular Bridges (IBs) generate from dividing epiblast cells [START_REF] Caneparo | Intercellular bridges in vertebrate gastrulation[END_REF] that do not separate by abscission as daughter cells normally do during cytokinesis, (the final step of cell division) (Figure 12). The observation of membrane-linked Dendra2 moving along IBs at a rate that would only be possible through an active process led the authors to hypothesize that IBs could function as TNTs for intercellular signaling during embryonic patterning and development. However, recently published studies described similar protrusions as 'retraction fiber projections' that host Migrasomes (Figure 12) [START_REF] Jiang | Migrasomes provide regional cues for organ morphogenesis during zebrafish gastrulation[END_REF], vesicular structures at the tips of projections emanating from migrating cells that release contents into the environment through an EV-like mechanism called 'migracytosis' [START_REF] Huang | Migrasome formation is mediated by assembly of micron-scale tetraspanin macrodomains[END_REF][START_REF] Ma | Discovery of the migrasome, an organelle mediating release of cytoplasmic contents during cell migration[END_REF][START_REF] Tavano | Migrasomes take center stage[END_REF]. Although IBs and migrasome-carrying fibers resemble each other topologically, it is currently unclear whether migrasomes are derived from IBs, or whether these two structures coexist in the complex zebrafish gastrular micro-environment as two separate processes.

The formation of stable IBs formed by incomplete cytokinesis was also shown in various germline and somatic tissues (Haglund et al., 2011). For example, actin-rich germline bridges Intervascular bridges are actin-bearing fibrous strands that connect neighboring capillaries in the retina of humans and mice (Figure 12) [START_REF] Mendes-Jorge | Intercapillary bridging cells: immunocytochemical characteristics of cells that connect blood vessels in the retina[END_REF]. The expression of common pericyte markers (NG2 and PDGFR-b) led the authors to hypothesize that they are of pericytic nature. Interestingly, these bridges express Cx43, suggesting that these connections allow inter-cellular communication. However, the precise signals transported between cells via these bridges has not been explored yet.

Various malignant tumors, including those of lung cancer, ovarian cancer, laryngeal cancer, mesothelioma, and neuroblastoma were shown to form TNT-like connections [START_REF] Ady | Tunneling nanotubes: an alternate route for propagation of the bystander effect following oncolytic viral infection[END_REF]Antanavičiūtė et al., 2014a[START_REF] Antanavičiūtė | Exogenous connexin43-expressing autologous skeletal myoblasts ameliorate mechanical function and electrical activity of the rabbit heart after experimental infarction[END_REF][START_REF] Desir | Tunneling nanotube formation is stimulated by hypoxia in ovarian cancer cells[END_REF]. Mesothelioma cells forming TNTs of > 70 μm in length contained microvesicles labeled with MitoTracker [START_REF] Lou | Tunneling nanotubes provide a unique conduit for intercellular transfer of cellular contents in human malignant pleural mesothelioma[END_REF]. Human glioblastoma tumors labeled with an astrocytoma cell GFP reporter and implanted into a mouse brain form long (>500 μm) connections called Tumor Microtube (TMs) between glioblastoma cells (Figure 12) (Osswald et al., 2015a).

Stephanie Cherqui and colleagues identified human stem cells (HSCs) grafted to cystinotic kidneys also generated TNT-like, nanotubular extensions that crossed the dense basement membranes that delivered cystinosin into diseased proximal tubular cells (Figure 12) [START_REF] Cherqui | The renal Fanconi syndrome in cystinosis: pathogenic insights and therapeutic perspectives[END_REF][START_REF] Naphade | Brief reports: Lysosomal cross-correction by hematopoietic stem cell-derived macrophages via tunneling nanotubes[END_REF].

Collectively, these findings show that TNT-like structures exist in vivo under certain physiological and/or pathological conditions. Future studies will need to demonstrate whether these different structures feature the TNT criteria described above.

Roles in Disease

A growing body of literature suggests that TNTs play a role in a wide variety of diseases, including neurodegenerative diseases (NDs), viral diseases, bacterial infection, and cancer.

Neurodegenerative Diseases

Major NDs, such as Parkinson's disease (PD), Alzheimer's disease (AD), Hungtinton's disease (HD), and amytrophic lateral sclerosis (ALS) share the defining feature of proteinaceous aggregates that accumulate in the central nervous system (CNS) [START_REF] Jucker | Self-propagation of pathogenic protein aggregates in neurodegenerative diseases[END_REF]. A growing body of evidence suggests the notion that the progression of these diseases is the consequence of prion-like propagation of amyloid-like pathologies [START_REF] Zhang | Targeting prion-like protein spreading in neurodegenerative diseases[END_REF]. Our group demonstrated that the infectious form of prions (PrPsc) are able to spread to other cells by hijacking TNTs (Gousset et al., 2009). The fact that amyloid proteins behave like prions, in their ability to induce misfolding of normal proteins, led our group to propose TNTs as one of the avenues for the spreading of neurodegenerative diseases in the CNS [START_REF] Baker | How the Internet of cells has biologists buzzing[END_REF][START_REF] Gousset | Tunnelling nanotubes: a highway for prion spreading?[END_REF]Gousset et al., 2009). Over the last decade, our group and others confirmed this notion by demonstrating that in addition to prions (Gousset et al., 2009;[START_REF] Victoria | The spread of prion-like proteins by lysosomes and tunneling nanotubes: Implications for neurodegenerative diseases[END_REF][START_REF] Victoria | Astrocyte-to-neuron intercellular prion transfer is mediated by cell-cell contact[END_REF]Zhu et al., 2015), other aggregates of misfolded proteins get transported inter-cellularly via TNTs. These misfolded proteins include tau and amyloid beta (Aβ), the main components of amyloid plaques found in the brains of Alzheimer disease patients (Abounit et al., 2016a;[START_REF] Tardivel | Tunneling nanotube (TNT)-mediated neuron-to neuron transfer of pathological Tau protein assemblies[END_REF][START_REF] Wang | Tunneling-nanotube development in astrocytes depends on p53 activation[END_REF]. α-synuclein, the presynaptic neuronal protein genetically linked to Parkinson's disease, was also found transmitted across other cells via TNTs [START_REF] Abounit | Tunneling nanotubes spread fibrillar α-synuclein by intercellular trafficking of lysosomes[END_REF][START_REF] Dieriks | α-synuclein transfer through tunneling nanotubes occurs in SH-SY5Y cells and primary brain pericytes from Parkinson's disease patients[END_REF][START_REF] Rostami | Human Astrocytes Transfer Aggregated Alpha-Synuclein via Tunneling Nanotubes[END_REF]Vargas et al., 2019). Mutant Huntingtin (mHtt), the unstable protein associated to Huntington's disease was no exception. Our lab first observed aggregate spreading of polyglutamine and mHtt in TNTs connecting CAD cells (Costanzo et al., 2013). Recently published reports identified a brainenriched GTPase, Rhes, as a key factor in the biogenesis of TNTs and enhanced intercellular transfer of mHtt between donor and acceptor cell populations [START_REF] Sharma | Rhes travels from cell to cell and transports Huntington disease protein via TNT-like protrusion[END_REF]. Model of prion-like protein aggregate-induced formation of TNTs and intercellular spreading, (Obtained from [START_REF] Victoria | The spread of prion-like proteins by lysosomes and tunneling nanotubes: Implications for neurodegenerative diseases[END_REF]. The aggregation and accumulation of prions or prion-like proteins in an "infected" cell ( 1) induces an increase in TNT number via an unknown mechanism, (e.g. via ROS-induced stress pathway) ( 2). The prion/prion-like aggregates may then be propagated via TNTs from infected cells to naive cells ( 3), wherein they effect the conversion and seeding of naive self-protein molecules.

Viral Diseases

Viruses are inert pathogens that rely on host cellular mechanisms to transmit and propagate effectively to non-infected cells in order to strive. Numerous reports have shown that viral particles can also utilize TNTs to transport inter-cellularly [START_REF] Sattentau | Cell-to-Cell Spread of Retroviruses[END_REF].

The earliest detection of viral particles associated to TNT-like connections dates back to 2005, when Favoreel and colleagues found that a protein kinase of the pseudorabies virus induced the formation of "cell projections" in swine testicle and rabbit kidney cells. Viral particles used these projections to propagate onto non-infected cells [START_REF] Favoreel | Cytoskeletal rearrangements and cell extensions induced by the US3 kinase of an alphaherpesvirus are associated with enhanced spread[END_REF][START_REF] Boissière | Compartmentalization of VP16 in cells infected with recombinant herpes simplex virus expressing VP16-green fluorescent protein fusion proteins[END_REF].

Other members of the Herpes virus family, such as the Herpes Simplex Virus (HSV), NV1066 [START_REF] Ady | Tunneling nanotubes: an alternate route for propagation of the bystander effect following oncolytic viral infection[END_REF], and the Murine gamma-herpesvirus-68 (MHV-68), were also found in TNTs of distinct cells, including primary embryonic fibroblasts, MHV-68, NIH-3T3, BHK-21 cells, NMuMG cells and mesothelioma-infected cells [START_REF] Gill | A gamma-herpesvirus glycoprotein complex manipulates actin to promote viral spread[END_REF]. In bovine primary fibroblasts and oropharynx cells infected with the bovine herpesvirus 1 (BoHV-1), TNTs can transport mitochondria and the viral proteins glycoprotein E and capsid VP26 [START_REF] Panasiuk | Tunneling Nanotubes as a Novel Route of Cell-to-Cell Spread of Herpesviruses[END_REF]. Other studies have also linked TNTs to T cell leukemia virus type I (HTLV-1) [START_REF] Van Prooyen | Human T-cell leukemia virus type 1 p8 protein increases cellular conduits and virus transmission[END_REF], murine leukemia virus [START_REF] Sherer | Retroviruses can establish filopodial bridges for efficient cell-to-cell transmission[END_REF]; influenza A virus [START_REF] Kumar | Influenza virus exploits tunneling nanotubes for cell-to-cell spread[END_REF][START_REF] Roberts | Influenza A virus uses intercellular connections to spread to neighboring cells[END_REF]; and human immunodeficiency virus type 1 (HIV-1), the most studied TNT-mediated virus [START_REF] Aggarwal | Mobilization of HIV spread by diaphanous 2 dependent filopodia in infected dendritic cells[END_REF][START_REF] Eugenin | Tunneling nanotubes (TNT) are induced by HIVinfection of macrophages: a potential mechanism for intercellular HIV trafficking[END_REF][START_REF] Hashimoto | Potential Role of the Formation of Tunneling Nanotubes in HIV-1 Spread in Macrophages[END_REF][START_REF] Hashimoto | Potential Role of the Formation of Tunneling Nanotubes in HIV-1 Spread in Macrophages[END_REF][START_REF] Okafo | Tunneling nanotubes (TNT) mediate long-range gap junctional communication: Implications for HIV cell to cell spread[END_REF][START_REF] Shrivastava | Slit2N Inhibits Transmission of HIV-1 from Dendritic Cells to T-cells by Modulating Novel Cytoskeletal Elements[END_REF][START_REF] Sowinski | Membrane nanotubes physically connect T cells over long distances presenting a novel route for HIV-1 transmission[END_REF][START_REF] Xu | HIV-1 evades virus-specific IgG2 and IgA responses by targeting systemic and intestinal B cells via long-range intercellular conduits[END_REF].

In spite of the numerous findings describing TNTs in the transport of viruses, there is evidence that, as they do in filopodia, most viruses move on the outer surface of the protrusion and not through the inside [START_REF] Sherer | Retroviruses can establish filopodial bridges for efficient cell-to-cell transmission[END_REF]. This suggests that many of the TNTs described during viral infection may not be open-ended connections.

Bacterial Infection

Even though initial sightings of contact-dependent bacterial cell-to-cell transport in macrophages date back to 1989 [START_REF] Tilney | Actin filaments and the growth, movement, and spread of the intracellular bacterial parasite, Listeria monocytogenes[END_REF], bacteria spreading between macrophages derived from human monocytes along the membrane of TNTs was never shown prior to the study by Davis and colleagues [START_REF] Onfelt | Structurally distinct membrane nanotubes between human macrophages support long-distance vesicular traffic or surfing of bacteria[END_REF]. In their study, Onfelt et al., demonstrated that bacteria "surf" along the tubes from the outside until they reach the cell at the other end, where they get phagocytosed. It is likely that these structures exhibit distinct structural characteristics from classical TNTs, however that is not an avenue that has been pursuit.

Bacteria can also communicate amongst themselves via TNT-like connections. For example,

Bacillus subtilis form open-ended TNT-like connections that form in the course of minutes [START_REF] Dubey | Architecture and Characteristics of Bacterial Nanotubes[END_REF]. Other studies in the same bacteria indicate that TNT-like connections can form extensive networks known as biofilms [START_REF] Sretenovic | An early mechanical coupling of planktonic bacteria in dilute suspensions[END_REF]; however the function of these

tubes has yet to be defined.

Cancer

Cancer is a disease characterized by are a deleterious ability to proliferate, spread, and invade organisms. Inter-cellular transfer of chemical signals and materials responsible for stimulating cellular growth and proliferation, and the coordination of tumor invasion. The classical paradigm of inter-cellular communication between cancer cells is via diffusion of chemical signals, GJs, MVs, and exosomes. However, over the last decade, evidence from several groups have detected TNTs forming between cancer cells, as well as between cancer and non-cancer stromal cells.

It is hypothesized that heterotypic connections could contribute to the formation of cancer microenvironments and stem cell niches that support tumor growth. These studies include TNTs in mesothelioma cell lines and primary human mesothelioma cells capable of transporting mitochondria, vesicles, and proteins [START_REF] Lou | Tunneling nanotubes provide a unique conduit for intercellular transfer of cellular contents in human malignant pleural mesothelioma[END_REF]. In their study, Lou and colleagues also identified mitotracker-labeled structures in surgical specimens that resembled TNTs. The identification of TNTs capable of transporting mitochondria between primary human laryngeal SCC cells also linked TNTs to laryngeal cancer [START_REF] Antanavičiūtė | Long-distance communication between laryngeal carcinoma cells[END_REF]. Later studies reinforced these findings and added other TNT-mediated cargo transport (vesicles, lysosomes, and autophagosomes) to the list of SCC-derived TNTs [START_REF] Sáenz-De-Santa-María | Control of long-distance cell-to-cell communication and autophagosome transfer in squamous cell carcinoma via tunneling nanotubes[END_REF]. Other reports examining micro RNAs getting transported via TNTs in metastatic cancer cells and the endothelium linked TNTs to breast cancer [START_REF] Connor | Physical nanoscale conduit-mediated communication between tumour cells and the endothelium modulates endothelial phenotype[END_REF][START_REF] Parker | Lattice light sheet imaging of membrane nanotubes between human breast cancer cells in culture and in brain metastases[END_REF]. GJ-bearing thick TNTs, TM, that transport calcium between astrocytoma cells have also linked TNTs to Astrocytoma [START_REF] Osswald | Brain tumour cells interconnect to a functional and resistant network[END_REF]. Bladder cancer cell models can also form TNTs and share mitochondria [START_REF] Lu | Tunneling nanotubes promote intercellular mitochondria transfer followed by increased invasiveness in bladder cancer cells[END_REF]. MSC and Leukemic cell TNTs that transport autophagosomes and mitochondria have also linked TNTs to Leukemia [START_REF] Polak | B-cell precursor acute lymphoblastic leukemia cells use tunneling nanotubes to orchestrate their microenvironment[END_REF][START_REF] De Rooij | Tunneling nanotubes facilitate autophagosome transfer in the leukemic niche[END_REF]. A better understanding of how these in vitro findings could reproduce in real-life tumors are still in progress. The development of artificial 3D model systems (e.g. organoids) could be more representative of complex and heterogenous tumor microenvironments and are described in more detail in a review by Venkatesh and Lou [START_REF] Venkatesh | Tunnelling nanotubes: A bridge for heterogeneity in glioblastoma and a new therapeutic target?[END_REF]. In order to circumvent these limitations, I sought out to investigate the existence of TNTs in wild-type tissue using a "label everything, see everything" structural approach known as Connectomics, a method designed to study connectomes, (comprehensive maps of connections within the CNS).

In the remaining sections of this chapter, I will give an overview of the neurobiology field of Connectomics, with an emphasis on structural (nanoscale) connectomics. I will then discuss how this type of connectomic approach can be utilized for the identification of TNT-like structures in tissue, and specifically, between progenitor cells in postnatal mouse development. In my results, I will describe the connectomic datasets used, the identification of TNT-like structures in these datasets, and immunohistological and dye-coupling experiments that corroborate my structural findings. Thereafter, I will explain all of the experimental methods used throughout this project.

Finally, I will discuss the main results of this work and consider possible directions for future studies based on these results.

Connectomics

The first classical map of the human brain connectome dates back to 1909 [START_REF] Zilles | Brodmann: a pioneer of human brain mapping-his impact on concepts of cortical organization[END_REF], a map widely used today. Presently, the connectome is studied at several orders of magnitudes;

structural connections (physical wires and synaptic pathways), functional connections (macroscale interactions between brain regions), and in between the two, connections visible by optical microscopy. (A) Schematic diagram depicting an automated tape-collecting ultramicrotome (ATUM). Procedure as described by [START_REF] Kasthuri | Saturated Reconstruction of a Volume of Neocortex[END_REF] The bottom reel of the ATUM contains a plastic tape that is fed into the knife boat of a diamond knife mounted on a commercial ultramicrotome. The tape is collected on a take-up reel (top). (Red inset) Close-up view of the tape conveyor positioned in the knife boat. The diamond knife boat (dark blue) is filled with water (light blue). The diamond knife (green rectangle) is at the opposite end of the knife boat from the taping mechanism. It cuts serial ultrathin sections from tissue embedded in a plastic block. The sections then float on the surface of the water in the knife boat until they adhere to the moving tape. (B) 10 m of Kapton tape with 2,000 sections collected. Four of the 29-nm sections (red rectangle) are shown at a higher magnification at the bottom of the panel. (C) The reel of tape is then cut into individual strips and mounted on silicon wafers for post-staining and/or carbon coating. A low-power scanning electron microscopy image of part of a wafer containing 85 brain sections is shown. One of the sections (red rectangle) is shown at a higher magnification in the next panel. (D) One 29-nm section containing neocortex and hippocampus. The region that was studied at high resolution is the dark-looking box (red arrow). Scale bar, 1 mm. (E) Cortical neuronal somata reconstruction to aid in cortical layer boundaries (dotted lines) based on cell number and size. Large neurons are labeled red; intermediate ones are labeled yellow; and small ones are labeled blue. The site of the saturated segmentation is in layer V (pink arrow). These two layer VI pyramidal cell somata (red and green arrows) give rise to the apical dendrites that form the core of the saturated cylinders. (F) A single section of the manually saturated reconstruction of the high-resolution data. The borders of the cylinders encompassing the ''red'' and ''green'' apical dendrites are outlined in this section as red and green quadrilaterals. This section runs through the center of the ''green'' apical dendrite. (G) A single section of a fully automated saturated reconstruction of the high-resolution data. Higher magnification view (lower left inset) shows 2D merge and split errors.

From Connectomics to Tunneling Nanotubes: Paving the Road

Due to their small size, and the lack of a TNT-specific marker, nano-Connectomics via ssSEM is the best suited approach. Over the next sub-sections, I will describe the cells and developmental stage in which we hypothesize TNT-like structures can be found in vivo.

Neuronal Tunneling Nanotubes

TNTs have been identified and described in various neuronal cell lines (e.g. PC12 (Rustom et al., 2004), CAD (Gousset et al., 2009), and SH-SY5Y (Sartori-Rupp et al., 2019) cells) and primary Schwann cells (Zhu et al., 2016). A previous study from our group also showed that primary cerebellar granule neurons (CGNs) transferred mutant Htt aggregates onto other CGNs in a cellto-cell contact-dependent manner (Costanzo et al., 2013). A common factor that characterizes neuronal cells in which TNTs, or functional changes in which TNTs could be used as a mode of communication, is their undifferentiated and precursor state (see also [START_REF] Grudina | Human NPCs can degrade alpha-syn fibrils and transfer them preferentially in a cell contact-dependent manner possibly through TNT-like structures[END_REF] and (Vargas et al., 2019)). Combined, these features sparked our interest in progenitor cells.

In Vivo Tunneling Nanotube-like structures

As previously described in Chapter 1, in vivo TNT-like structures have previously been reported

during early stages of development, where cues necessary for migration and coordination could be shared inter-cellularly. These observations argue that TNTs could be used as a mechanism of inter-cellular communication between cells that haven't yet developed other modes of communication (e.g. synapses), and support our work showing undifferentiated or non-polarized neuronal cells and undifferentiated human neuronal precursor cells (hNPCs) [START_REF] Grudina | Human NPCs can degrade alpha-syn fibrils and transfer them preferentially in a cell contact-dependent manner possibly through TNT-like structures[END_REF] forming TNTs in culture.

Survey of the Developing Cerebellum for Tunneling Nanotubes

Cerebellum Anatomy and Ontogenesis

The cerebellum is a major feature of the hindbrain, playing vital functions in the control of gaze, gate, posture, motor learning, and prediction of moments [START_REF] Manto | Consensus paper: roles of the cerebellum in motor control--the diversity of ideas on cerebellar involvement in movement[END_REF].

Anatomically, the cerebellum comprises three layers that make up the cerebellar cortex: the outermost, 'molecular layer' (ML), where Golgi, stellate, and basket cells reside; the middle, purkinje cel layer' (PCL), where Purkinje cells sit side by side in a 1-cell-thick layer; and the Inner Glanular Layer (IGL), where granule cells (GCs) live.

During the first few weeks of postnatal development, the cortical layer of the cerebellum undergoes severe transformations. GCs, the most abundant cell in the cerebellum [START_REF] Fox | A quantitative study of the Purkinje cell dendritic branchlets and their relationship to afferent fibres[END_REF], reside in a transitory, mitotically and migratory active region called the external granule layer (EGL). The EGL forms at embryonic day 15 (E15) and GC progenitors (GCPs) continue to divide until the third week of postnatal development [START_REF] Espinosa | Timing neurogenesis and differentiation: insights from quantitative clonal analyses of cerebellar granule cells[END_REF]. After division, GCPs undergo two modes of migration, tangential, which takes place in the EGL, and radial, which takes place across the ML and PCL (Figure 17) [START_REF] Galas | Postnatal Migration of Cerebellar Interneurons[END_REF][START_REF] Komuro | Ca2+ transients control CNS neuronal migration[END_REF][START_REF] Komuro | Recent advances in cerebellar granule cell migration[END_REF].

External Granule Layer

The EGL comprises two sub-layers, the outer EGL (oEGL), where GCPs proliferate, and the inner EGL (iEGL), where GCPs migrate. After their final division in the oEGL, GCPs in the neonatal cerebellum orchestrate radial and tangential migrations from the EGL to the IGL without synapsemediated interactions, as first observed by Cajal in the late 1800s (Cajal, 1888). Improper migration of GCPs can have detrimental effects on the anatomy of the cerebellar cortex and lead to neurological disorders such as dystonia, ataxia, and psychiatric conditions such as autism spectrum disorders [START_REF] Lackey | Recent advances in understanding the mechanisms of cerebellar granule cell development and function and their contribution to behavior[END_REF]. We hypothesize that GCPs use TNT-like structures to communicate with each other during migratory movements, prior to synapse formation. To this end, I studied GCPs in the neonate developing cerebellum using ssSEM nanoscale Connectomics.

GCPs feature different morphologies depending on their location within the EGL. GCPs in, or close to the oEGL have spherical cell somas with few, if any, protruding neurites. Postmitotic GCPs in the iEGL feature typical morphology of migrating neurons, elongated somas, a leading process, and a thin trailing neurite oriented in the opposite direction (Figure 18).

Results

Datasets

To gain insight into the connectivity and cell-to-cell interactions made by GCPs in the EGL, we studied three large-scale ssSEM datasets of the cerebella of mice at P0, P3, and P7 postnatal stages of development, spanning 5.5 x 10 5 μm 3 , 1.1 x 10 6 μm 3 , and 1.7 x 10 6 μm 3 , respectively (Figure 11). All three volumes are columns of cerebellar tissue; in P0 reaching from the pia down to the ML, and in P3 and P7 reaching down to the white matter [START_REF] Wilson | The rich get richer: synaptic remodeling between climbing fibers and Purkinje cells in the developing cerebellum begins with positive feedback addition of synapses[END_REF].

Volumes were cut sagittal using an automatic tape-collecting ultramicrotome (ATUM) [START_REF] Kasthuri | Saturated Reconstruction of a Volume of Neocortex[END_REF] at 30 nm thickness. A total of 1450 sections for the P0 block, 2658 sections for the P3 block, and 2514 from the P7 block were used for imaging by SEM. The sections were placed on wafers and images were acquired at 4nm/px in lateral resolution (see methods).

GCPs form Tunneling Nanotube-like connections

For our study, we examined the existence of TNT-like structures between GCPs by identifying and performing a comprehensive screening of GCPs located in the EGL of our high-resolution volumes. Using accepted topological and positioning criteria [START_REF] Komuro | Recent advances in cerebellar granule cell migration[END_REF], we traced the cell soma and every emanating protrusion from 160 cells at P0, 70 cells at P3, and 60 cells at P7, and identified 14, 8, and 48 connected cells in pairs, respectively, which correspond to 7, 4, and 24 connections (Figure 12). These findings account for an 8.7%, 11%, and 80% of the total percentage of cells screened connected (Table 1).

Orientation of GCP-GCP vs. dividing GCPs

Due to the higher rate of cell division in the EGL, we needed a method to determine if connected GCPs were indeed evidence for the existence of TNT-like connections or simply remnants of cellular division such as mitotic intercellular bridges or stable intercellular bridges formed by incomplete cytokinesis (Haglund et al., 2011).

Due to existing classifications and studies with information on the spatial orientation of dividing GCPs through many early postnatal developmental stages [START_REF] Haldipur | Asymmetric cell division of granule neuron progenitors in the external granule layer of the mouse cerebellum[END_REF][START_REF] Miyashita | Dynamics of the cell division orientation of granule cell precursors during cerebellar development[END_REF], we decided to focus on creating a similar classification of our cells connected. Specifically, previous studies have described that finding whether the angle between the pia membrane that envelopes the brain and the plane of division between the cells is perpendicular or parallel is a very simple yet effective manner in providing a criterion for defining spatial orientation of cells. By using fluorescence data and this method of analysis, it has been shown that dividing GCPs in P0-P7 developmental stages are either non-discriminant in being either parallel or perpendicular in relation to the pia membrane or that they are predominantly parallel [START_REF] Haldipur | Asymmetric cell division of granule neuron progenitors in the external granule layer of the mouse cerebellum[END_REF][START_REF] Miyashita | Dynamics of the cell division orientation of granule cell precursors during cerebellar development[END_REF].

Overall these studies outline that an effective way to analyze spatial orientation is through measuring the difference in alignment between the pia membrane that envelops the brain and the plane that divides two cells, in our case bound by a TNT-like structure. Thus, spatial orientation of our connected cells was carried out through comparing their orientation in 3D in relation to the pia membrane. Cell pairs were then classified as either being parallel or perpendicular depending on the angle φ between the cell and pia plane (see methods). If the φ angle was <45°, cells were classified as parallel (Figure 22, C-G), while if the φ was between 45 -90°, cells were classified as perpendicular (Figure 22, H-L). Using this system of classification (Figure 22, A-E), we found that 100% of cell pairs were perpendicular in P0 (n=5), 75% in P3 cell (n=4), and 75% in P7 (n=12). Similarly, the average of all angles in P0 was 74.6°, 65.7° in P3, and 62.8° in P7, all of which are above the 45° perpendicular threshold. Next, through conducting a two-tailed, unpaired t-test, we found that all stages (P0-P7) had a significant (**, p=0.0048) tendency for connected cell pairs to be perpendicular to the pia in orientation (n=21). These results showing that our connected cells are predominantly perpendicular in P0-P7 is in contrast with the previous reports showing either no bias between a perpendicular or parallel axis of cell division or a predominantly parallel axe [START_REF] Haldipur | Asymmetric cell division of granule neuron progenitors in the external granule layer of the mouse cerebellum[END_REF][START_REF] Miyashita | Dynamics of the cell division orientation of granule cell precursors during cerebellar development[END_REF] (Figure 14,P). The discovery that our TNT-like connected cells are significantly more likely to be perpendicular at P0-P7 points in development argues that these connections are unlikely to be generated by dividing cells. Our observations were reinforced by the identification of cells in division, found through identifying midbodies and electron-dense cluster of microtubules (Figure 15). While the connected cells followed the pattern of having a tendency towards perpendicular, the dividing cells showed no tendency for either perpendicular or parallel, similar to the dividing cells described by [START_REF] Haldipur | Asymmetric cell division of granule neuron progenitors in the external granule layer of the mouse cerebellum[END_REF].

Assessment of dividing GCPs in the EGL

Given that by using our morphological criteria we only identified a handful of GCPs undergoing division throughout our connectome screenings, we wondered if GCPs in the EGL tend to divide with a frequency that is lower when compared to the frequency of TNT-like connection between cell pairs described above. To this end, we examined the proliferation of GCPs in tissue slices of mice from the same developmental stages as those used for our connectome analysis via Phosphohistone H3 (PH3) staining, (a marker that labels cell cycle stages S, G2, and M).

We employed two analysis techniques -one focused on the total percentage of dividing cells and another using a method closer to the one applied to identify connections in ssSEM volumes (see methods). Using our first approach, we found that the average percent pixel density of PH3/DAPI+ cells to be 3.9%, 4.5%, and 5.1% in P0, P3, and P7 stages, respectively. Averages per stage range from 2-5% in P0, 4-5% in P3, and 4-6% in P7. Using our second approach, we found that the average percent of PH3+ cells out of 50 randomly selected DAPI cells to be 2.9%, 4.7%, and 7.3% in P0, P3, and P7 stages, respectively. Averages per stage range from 1-4% in P0, 4-6% in P3, and 7-9% in P7.

These findings demonstrate that the number of dividing GCPs in the EGL within the same regions used for our structural analysis to be substantially lower compared to the number of connected cells. The values we obtained by our connectomic approach (see Table 1) demonstrate higher percentage values of connected cells, most noticeably in P7. These findings strengthen our hypothesis that the structures we identified could be TNT-like structures and not simply remnants of cell division or stable intercellular bridges. We also found that although some cells visibly connected by TNT-like structures, some GCPs appeared coupled with GCPs immediately adjacent to them (Figure 25C-H). During the acquisition of one of our examples (Figure 25G), we observed that the pipette displaced the patched GCP and presumably fractured a connection bridging the two cells, leaving behind a gap between the protrusion emanating from the patched GCP and the GCP beside it. Whether internal connections of small size, such as those shown by EM, lie squeezed in-between the pairs of cells coupled was not evident with our imaging setup. microtome with a reel-to-reel tape collecting system so that sections can be continuously cut and collected on the tape with minimal user intervention. The ATUM allows many consecutive sections to be cut, collected onto a reel of tape, and then imaged. Image acquisition, stitching, and alignment of the volume was carried out as previously described [START_REF] Wilson | The rich get richer: synaptic remodeling between climbing fibers and Purkinje cells in the developing cerebellum begins with positive feedback addition of synapses[END_REF].

Image Visualization

Autodesk 3D Studio Max (3Ds Max) is a powerful animation software used by game developers, animators, and other motion graphics purposes. It allows for not only the construction of complex 3D models like a traditional computer-aided design (CAD), but also provides the tools necessary for elaborate motion animation as well as the analysis of in-software structures. Our research relies upon the multiple facets of 3DS Max, not only utilizing it as a visualization method to reconstruct our EM segmentations, but also as a system to the collect measurements needed to characterize the spatial arrangement of GCPs within the cerebellum (angle measurements). By using 3DS Max as a tool to collect data on the spatial orientation of TNT-like structures in the cerebellum and specifically the outer EGL, it is possible to cross reference findings with other previous studies about dividing cells.

Skeletonization

Access to Kimimaro skeletonization pipeline code for Python can be accessed, (link: https://github.com/seung-lab/kimimaro).

Angle Measurements

The fully-segmented collection of 2D layers, produced by serial sectioning of the EGL in VAST, is fused together in 3DS Max by using custom MatLab script [START_REF] Berger | VAST (Volume Annotation and Segmentation Tool): Efficient Manual and Semi-Automatic Labeling of Large 3D Image Stacks[END_REF], in order to produce a full 3D model of a neuronal cell. The MatLab script turns the stack of colored-in (segmented) layers from VAST into .obj files that can then be opened in 3DS Max. These .obj files are already automatically fused into the full 3D model, while preserving features such as the different "children" found in VAST, as well as their global orientation and overall positioning within the EM stack in VAST. This allows manipulation of the different parts of the cells (provided they are colored as different children), such as adjusting translucency, color, and appearance individually, in order for the optimal animation. The preservation of position within the EM stack also allows for different .obj files to be imported while maintaining the same relative position to each other as in the cerebellum.

This characteristic thus makes it possible for spatial analysis of 3D models produced by .obj files in 3DS Max to accurately reflect that of their corresponding cells and segmentations in the EM stack in-vivo. Our research is novel as it adds in another factor of security into said analysis.

Instead of simply measuring the spatial orientation in 2D in-vivo using immunohistochemistry like prior works, we will also do spatial orientation in-vivo, but in 3D instead, since the .obj files represent 3D objects themselves and occupy a 3D space. This gives us the unique opportunity to be the first team to classify and characterize cell orientation in the cerebellum using this more accurate new method. Once the .obj files of each cell are loaded onto 3DS Max, we first need to define how we will represent the orientation of the connected cells and the pia. The orientation of the pia is represented by a single plane (pia plane) that is tangential to the pia membrane at the point of the membrane that is closest in proximity to the connected cells. The orientation of the connected cells is represented by a single plane (cell plane) that divides the two as accurately as possible. Precision of the plane between different cell pairs called for the adoption of a standard, which entails that a line is drawn between the center of the nucleus of one connected cell to the nucleus center of the other connected cell. This serves as the line normal to the cell plane, and the alignment tool in 3DS Max allows for the cell plane to snap into the correct angular orientation when the x, y, and z angles mirror that of said nucleus-to-nucleus line.

Processing of the data created by this series of cell planes and pia planes is done by using the 3DS Max alignment tool to move both planes so that each respective plane's center shares the same point in 3D space, while maintaining their original orientation. Lines of fixed length that are normal to the planes are then added, with each line originating at the center of their respective planes. Each of these normal lines of fixed length are now representative of the angular orientation of their plane, and the angle between them is thus used as a means to find whether the cell plane and pia plane are parallel or perpendicular. If the angle between the normal lines originating at the same point is small (<45°), then the planes are parallel, while if the angle of the normal lines is large (>45°), then the planes are perpendicular. The use of a single angle measurement to characterize objects in a 3D space is sufficient, because we only need to understand the position of the cell plane and the pia plane in relation to each other. Therefore, envisioning a spherical coordinate system (r, θ, φ) where the pia plane normal line represents φ=0 and the cell plane normal line represents a variable φ, we can use the single φ angle as a metric for being parallel or perpendicular. If the cell plane normal line φ is close to 0°, that means the two normal lines are similar in orientation and thus the planes are parallel, while a φ close to 90° would indicate the planes are perpendicular to each other. This method of angle measurement is used as it is efficient in condensing a lot of data from the 3D space and turning it into a single usable metric. This is evident as we only have to use φ, with θ not affecting "perpendicularity" and thus allowing us to not have to consider it.

This method of measurement of angles was conducted in P0, P3, and P7 samples. The P0 EM stack yielded 5 connections to analyze, while the P3 yielded 4 connections, and the P7 yielded 22 connections to analyze. The method for referencing the angle in P0 samples was completed by fully coloring and segmenting every layer. The complete segmentation gave clear insight into the curvature of the pia at every point, as well as provided a clear tilt along every slice. The presence and visibility of the pia in the majority of slices of the P0 EM stack allowed us the opportunity to do this one extremely thoroughly, and thus is shown in a supplementary video. The method for referencing the angle in P3 samples was similar to P0, however consisted of segmented and colored layers in only specific areas. Due to issues with the visibility of the pia membrane, segmentation was conducted in chunks every 200-300 slices in the EM stack. As some sections of slices didn't show the pia membrane, segmenting was done in concentrations of 50-100 slices in strategic locations that did show the pia. This still allowed for sufficient awareness of both the pia membrane's curvature and tilt. The method for referencing angle in P7 samples took a combination of methods, as the P7 pia was only visible in the lower third of the slices of the EM stack. Therefore, seeing its general curvature was clear, but there weren't enough slices to fully assess the tilt of the pia. This called for segmenting one of the EM stack's parallel fibers, as they span the entirety of all the slices and are parallel to the pia, allowing us to inference the tilt of the pia from said parallel fibers.

Immunohistochemistry

Brains were removed from P0 (n=3), P3 (n=3), and P7 (n=4) mice pups and postfixed in 4%

Microscopy

Brains were imaged using a Zeiss LSM 700 confocal microscope. P0 images were taken in middle of cerebellum to parallel P0 ROI in EM volume. P3 images were taken in Lobule 8 of cerebellum to parallel P3 ROI in EM volume. P7 images were taken in the fissure between lobules 7/8 to parallel P7 ROI in EM volume.

Image Analysis

Method 1: Images were first analyzed using a MACRO written on FIJI which quantifies an estimate for the amount of PH3+ cells. Because of the extremely high density of granule cells, DAPI cells could not be counted and compared with the number of PH3 positive cells. As most cells could not be properly dissociated from each other, we defined the cell density through the pixel density. The method used is based on utilizing an intensity thresholding and object filter using shape and size characteristic. For quantifying PH3+, we isolate the channel containing the signal PH3+ and we apply a maximum projection in order to get most of the cell signal. A light gaussian blur and background subtraction are applied before automatically thresholding the signal. All threshold objects that are small than a cell or have a too elongated shape are removed.

For quantification of DAPI, a similar approach is applied. The user selects the plan to process, otherwise we take the middle of the volume. A light gaussian and background are applied, followed by a threshold. Small threshold objects are then removed. The user can then provide a Region of Interest (RoI) to process. If no selection is provided, the entire image is used. A density value is compute with detected PH3+ pixels and the detected DAPI pixels on the RoI. We drew an ROI around a specific part of EGL in cerebella in all images and applied this approach to quantify an estimate for percent PH3+ cells.

Method 2: The second analysis approach was employed in order to better compare the data collected within this experiment with past experiments locating tunneling nanotubes in the EGL.

It involved randomly choosing 50 DAPI+ cells within the same ROI's of the cerebella and counting how many of these cells are PH3+ throughout the Z-Stack.

Dye Coupling

Discussion

Connectomics is the study of connectomes: comprehensive maps of connections within the CNS [START_REF] Lichtman | Ome sweet ome: what can the genome tell us about the connectome?[END_REF]. ssSEM emerged as an unparalleled tool to study connectomes at sufficient resolution to reconstruct entire neuronal diagrams at the synaptic vesicle level within a 'relatively' small region of the brain [START_REF] Kasthuri | Saturated Reconstruction of a Volume of Neocortex[END_REF]. On the premise that this method offers the advantage to preserve fine neuronal processes (e.g. dendritic spines) and circumvents the need for TNT-specific markers by labeling everything membranous [START_REF] Morgan | Digital tissue and what it may reveal about the brain[END_REF], we sought out to investigate whether connectomics could also reveal the existence of TNT-like structures in vivo, which would otherwise be inaccessible.

Based on our in vitro work showing that TNTs form and enable inter-cellular communication in the undifferentiated, neuroblast-like non-polarized CAD [START_REF] Abounit | Tunneling nanotubes: A possible highway in the spreading of tau and other prion-like proteins in neurodegenerative diseases[END_REF]Costanzo et al., 2013;Delage et al., 2016;Gousset et al., 2013;Gousset et al., 2009;Zhu et al., 2018;Zhu et al., 2015) and SH-SY5Y cells (Dilsizoglu Senol et al., 2019;Sartori-Rupp et al., 2019), and most recently, undifferentiated hNPCs [START_REF] Grudina | Human NPCs can degrade alpha-syn fibrils and transfer them preferentially in a cell contact-dependent manner possibly through TNT-like structures[END_REF] and primary cortical neurons (Vargas et al., 2019), we hypothesized that neuronal precursors in the brain with similar topological and genetic profiles could also generate and utilize these processes before establishing synaptic communication. Using this criterion, we focused our efforts on the EGL of the developing cerebellum, a transitory sub-region of the cerebellar cortex in which millions of GCs proliferate extensively before relocating to the IGL through elegantly orchestrated migratory events aided by BG [START_REF] Galas | Postnatal Migration of Cerebellar Interneurons[END_REF].

The aim of this work was to produce a mineable EM dataset obtained from the EGL of a P0 neonate mouse of sufficient scale to capture hundreds of GCPs in the process of division (oEGL) and migration (iEGL). To scale up our investigations in a manner that would substantially increase our likelihood of detecting these connections, we extended our study onto two other postnatal developmental stages, P3 and P7, using volumes that were previously produced but never used for these type of investigations [START_REF] Wilson | The rich get richer: synaptic remodeling between climbing fibers and Purkinje cells in the developing cerebellum begins with positive feedback addition of synapses[END_REF].

To this end, we investigated the presence of TNT-like structures by manually tracing the cell soma of GCPs and processes emanating from them in the EGL (both i-and o-EGL) of all three volumes. As hypothesized, we observed that TNT-like connections of various lengths and thicknesses formed between GCPs in the EGL across all of the developmental stages studied.

Surprisingly, we observed a surge in the formation of these structures over development, notably at P7. Although these connections were never previously described, one possibility that we considered is that they were remnants of cytokinesis. In this case, one possible explanation for the higher number of connections at P7 could be that GCPs tend to divide more at the base of the fissure than at the crown of the lobe. However, previous studies have shown that GCPs located in fissures express a higher mitotic index only until E18.5 [START_REF] Sudarov | Cerebellum morphogenesis: the foliation pattern is orchestrated by multi-cellular anchoring centers[END_REF], arguing against the likelihood that our connected GCPs are cells in division, tethered by a stable intercellular bridge or remnant of cytokinesis [START_REF] Mclean | Bridging the divide: illuminating the path of intercellular exchange through ring canals[END_REF].

In order to further investigate this matter, we decided to better characterize the proliferative profile of GCPs in cerebellar tissue slices of mice obtained from the same region and developmental stages as those used for our connectomic analyses by quantifying the percentage of GCPs that expressed PH3, a marker that labels cell cycle stages S, G2, and M. Our data indicates that the rate of proliferation between P0 and P7 has a slight tendency to increase, supporting previous published data [START_REF] Sudarov | Cerebellum morphogenesis: the foliation pattern is orchestrated by multi-cellular anchoring centers[END_REF]. These findings are consistent with the knowledge that fissures serve as anchor points for lobules to continue to grow past P7, until the second and third week of development. However, comparing our PH3 data with our connectomic analyses revealed substantial differences between the percentage of cells in division vs. the number of cells connected by ssSEM, whereby 2.9% divide vs. 8.7% connect in P0; 4.7% divide vs.

12% connect in P3; and 7.3% divide vs. 80% connect in P7. Although these results cannot prove that our connected GCPs by ssSEM are not dividing cells, they suggest that (at least the majority of) our connections do not derive from mitosis.

It is also conceivable that the increased number of cells connected in our datasets result from post-mitotic, non-PH3 expressing, GCPs 'holding on to' their intercellular bridges formed by incomplete cytokinesis [START_REF] Mclean | Bridging the divide: illuminating the path of intercellular exchange through ring canals[END_REF]. To the best of our knowledge, there isn't any published evidence to support intercellular bridges or ring canals forming between cerebellar GCPs, as these processes have been majorly described in germline and invertebrate somatic cells (e.g. during drosophila oogenesis) (Haglund et al., 2011). Based on the topological criteria employed to investigate these types of bridges, our connections differ in two significant ways: 1) intercellular bridges are short (0.4-1.8 μm) whereas our connection can exceed 4 μm in length;

2) intercellular bridges display electron-dense outer rims, our connections do not. In order to unequivocally assess the presence and longevity of stable intercellular bridges in the EGL, we plan to perform experiments and examine the expression of Aurora B kinase and Kif20b, markers previously used to effectively label midbodies in the embryonic mouse cerebral cortex [START_REF] Janisch | Imaging and quantitative analysis of cytokinesis in developing brains of Kinesin-6 mutant mice[END_REF].

Another consideration that we must take into account when interpreting our results is that although all three volumes were obtained from the same region in the cerebellum (vermis of lobule VIII), our analyses were carried out in three slightly distinct regions of one lobule, (crown of folia in P0; edge of the crown in P3; and base of the fissure in P7). Differences in the cytoarchitecture between these different regions might offer the potential to generate useful insights as to whether GCPs could form more/less TNT-like connections depending on where they locate in the lobule.

In an attempt to further characterize our TNT-like connections and test alternative methods to distinguish them from cell division remnants, we decided to classify our ssSEM connected cells according to their spatial orientation in 3D in relation to the pial membrane, using similar approaches used before to classify dividing cells [START_REF] Haldipur | Asymmetric cell division of granule neuron progenitors in the external granule layer of the mouse cerebellum[END_REF][START_REF] Miyashita | Dynamics of the cell division orientation of granule cell precursors during cerebellar development[END_REF].

Our preliminary data clearly indicate that our cells connected arrange mainly perpendicular to the pia, while the axis of cell division at the stages of development studied were found either non-biased or parallel. These observations were reinforced by the identification of very few cells in division in our ssSEM data, found through identifying midbodies with electron-dense cluster of microtubules. Furthermore, our preliminary data shows that the GCPs we classified as 'dividing', based on the morphological features of intercellular bridges, were oriented randomly in respect to the Pia, in agreement with the dividing cells previously described [START_REF] Haldipur | Asymmetric cell division of granule neuron progenitors in the external granule layer of the mouse cerebellum[END_REF].

Although our ssSEM data provided us with hints regarding the role these connections might play in inter-cellular communication and transport of cargo (shown by the presence of mitochondria and vesicles within our connections, we decided to study GCPs in living conditions and test whether these are functional; that is, capable of transporting or diffusing cargo, we patch-clamped GCPs in the EGL of P6-P8 cerebellar slices and filled them with a fluorescent dye.

Our preliminary results revealed dye-coupling between GCPs via TNT-like structures that proved to be open-ended. These connections shared similar thicknesses with TNTs in vitro (Gousset et al., 2009), and our connections by ssSEM; however, we observed two differences between our findings using these two different approaches: 1) live-TNTs measure long lengths and 2) form networks of more than two cells, our ssSEM connections do not. This discrepancy can be explained by several factors: (i) our ssSEM datasets span 45 -75 μm in which long connections would leave the volume and become untraceable; (ii) sample preparation deformation during specimen dehydration and embedding suggest that our (ssSEM) connections could have represented longer connections that shrank substantially; (iii) distortion during sample preparation could have also fractured long connections due to harsh shearing forces that are known to break, bend, and tear TNTs in vitro (Rustom et al., 2004;Sartori-Rupp et al., 2019).

Interestingly, our patch-clamping data indicates that GCPs that connect via TNT-like connections lie at the middle of the EGL, somewhere along the interface between the o-and iEGL. These observations are in agreement with our ssSEM findings, which also show that 54% of GCPs at P7 locate in the middle of the EGL. Altogether, these observations suggest that TNT-like structures could serve as inter-cellular avenues for GCPs to share information associated with changes in their microenvironment, specifically with respect to the transition between progenitor and migratory state (oEGL and iEGL, respectively). However, further experiments are required to sustain this hypothesis and support these preliminary findings.

In this study, we also attempted to extrapolate geometrical information such as the length and thickness from the 3D meshes obtained by our ssSEM reconstructions via TEASER-based Kimimaro skeletonization algorithms. We have successfully tested our skeletonization pipeline on a handful of TNT-like structures and obtained accurate length measurements with success; however, our current algorithm only computes the min, mean, and max diameter of our connections. After observing that the diameter of our connections shifts along their length, and when they contain cargo within, (as we also observed in vitro, (Sartori-Rupp et al., 2019)), we plan to implement an additional tool that can yield skeleton 'branches'. These branch-wires would sprout from the single-wire trunk every n-number of nm along the connection in order to provide us with a radii distribution description of the diameter of our connections.

We also plan to focus on strategies that can increase our throughput. For example, one of the obstacles we face is (i) the time it requires to screen our ssSEM volumes for TNT-like connections between GCPs and (ii) fully tracing/segmenting GCPs after they were found connected. Over the course of this study, we have manually segmented dozens of cells, nuclei, and TNT-like structures that serve as ground-truth for the adaption and implementation of robust automatic segmentation algorithms based on ML [START_REF] Plaza | Analyzing Image Segmentation for Connectomics[END_REF]. We are now currently working on applying these approaches on our volumes. Once completed, these algorithms will have the ability to automatically identify and segment TNT-like structures without human input. This approach will be tremendously useful to examining the presence of TNT-like structures in other ssSEM volumes where no connections of this type were ever described, including the fruit fly brain [START_REF] Zheng | A Complete Electron Microscopy Volume of the Brain of Adult Drosophila melanogaster[END_REF], and larval fish [START_REF] Hildebrand | Whole-brain serial-section electron microscopy in larval zebrafish[END_REF].

From the days of Cajal (Cajal, 1888), neuroscientists have been fascinated with understanding how the brain is wired. The advent of Connectomics has expanded our knowledge of these wires and provided unprecedented context to the remarkable drawings of Cajal. Taking advantage of the benefits offered by the tools used by Connectomics, specifically ultra-high resolution ssSEM and computational processing and reconstructions, we sought out to investigate the presence of TNT-like structures in the GCP subset of the developing cerebellum. Our findings shed light on processes that were never previously described in GCPs, cerebellar development, or brain tissue.

Presently, we cannot be certain that there isn't an underlying developmental significance that could explain these findings; therefore, extending this study to other brain area and developmental stages and testing whether these TNT-like connections are functional needs to be addressed. Nonetheless, the identification of these structures has the potential to shift the paradigm and change the way in which we think about brain connectivity.

  ............................................................................................................................................... Arp2/3 Actin-related protein 2/3 ................................................................................................................................ -B-BHK-21 Baby hamster kidney 21 ................................................................................................................................... BMP Bone morphogenetic protein ............................................................................................................................. -C-CAD Cath.a-differentiated ............................................................................................................................................. CDC42 Cell Division Control Protein 42 ....................................................................................................................... cryo-ET cryo-electron tomography ............................................................................................................................. cryo-TEM Cryo-transmission electron microscopy ..................................................................................................... Cx43 Connexin 43 ......................................................................................................................................................... -D-DIC Differential interference contrast ........................................................................................................................ -E-EB Epithelial bridges ..................................................................................................................................................... Eps8 Epidermal growth factor receptor kinase substrate 8 ........................................................................................ EV Extracellular vesicle ................................................................................................................................................. -F-FACS Fluorescence Activated Cell Sorting ................................................................................................................... Fgf Fibroblast growth factor ....................................................................................................................................... -G-GEF Guanine nucleotide Exchange Factor .................................................................................................................. GJ Gap Junction ........................................................................................................................................................... -H-HEK Human Embryonic Kidney ..................................................................................................................................... HeLa Henrietta Lacks .................................................................................................................................................. HIV-1 Immunodeficiency Virus Type 1 ........................................................................................................................ HSC Human Stem Cell ................................................................................................................................................. -I-I-BAR Bin/Amphiphysin/Rvs ........................................................................................................................................ IGL Inner Granule Layer ............................................................................................................................................ ILV Intraluminal vesicle ................................................................................................................................................. IP3 Inositol triphosphate ............................................................................................................................................... IRSp53 Insulin Receptor Tyrosine kinase substrate .................................................................................................... -L-LSCM Laser scanning confocal microscopy .................................................................................................................

Figure 12 .

 12 Figure 12. Examples of TNT-like structures in vivo. .....................................................................

Figure 13 .

 13 Figure 13. Model of prion-like protein aggregate-induced formation of TNTs. ..........................

Figure 16 .

 16 Figure 16. Connectomics by serial-sectioning scanning electron microscopy. ..........................

Figure 17 .

 17 Figure 17. Cerebellar granule cell migration during development. ...........................................

Figure 18 .

 18 Figure 18. Granule cell progenitor morphology characterization. .............................................

Figure 19 .

 19 Figure 19. Datasets produced by Serial-Sectioning Scanning Electron Microscopy. ..................

Figure 20 .

 20 Figure 20. ssSEM Reveals TNT-like connections between GCPs. ...............................................Figure 21. Skeletonization of TNT-like connections. ..................................................................Figure 22. Connected GCPs exhibit perpendicular orientation. .................................................

Figure 23 .

 23 Figure 23. Identification of GCPs undergoing division. ..............................................................

Figure 24 .

 24 Figure 24. Quantification of PH3+ cells in the Developing Cerebellum. ....................................

Figure 25 .

 25 Figure 25. Dye-coupling between GCPs in the EGL reveal TNT-like structures. .........................

Figure 1 .

 1 Figure 1. Modes of Inter-cellular Communication.Schematic diagram depicting: (A) Cells coupled by gap junctions, which allow various small (5-20 kDa) molecules, (e.g. ions, secondary messenger molecules, and electrical impulses) to directly pass through a regulated gate. (B) Paracrine signaling is mediated by the secretion of soluble signaling molecules that bind to specific receptors on neighboring cells. (C) Endocrine signaling is mediated by signaling molecules that travel through the blood stream before binding to a receptor on a distant cell; (D) Extracellular vesicles, which enable the transport of small particles (e.g. Proteins, RNA, and small molecules) though Microvesicles and Exosomes. Microvesicles (100 nm -1,000nm in diameter) arise from the budding of the plasma membrane. Exosomes (30 -100nm in diameter) originate from the exocytosis of intraluminal vesicle contained within a multivesicular body.

Figure 2 .

 2 Figure 2. Topological Architecture of Tunneling Nanotubes. (A) TNTs connecting Cath.a-differentiated (CAD) neuronal cells stained with Wheat Germ Agglutinin-Alexa Fluor (Orange), to label the membrane of cells, and DAPI (16-color Lookup table, Fiji), to label nuclei. Cells can connect by one or numerous TNTs, as seen in A. (B) Schematic diagram depicting Tunneling Nanotube-mediated communication between cells. Tunneling Nanotubes create direct conduits filled with actin filaments, which allow the transport of cargo (e.g. ions, proteins, RNA, viruses, and organelles). TNTs can form between cells of the same-(homotypic) and different-(heterotypic) type. As shown in A, cells can form one or numerous TNTs. Scale bar: A, 10 μm.

Figure 3 .

 3 Figure 3. Properties that define Tunneling Nanotubes. Schematic diagram depicting a TNT connecting two cells superimposed with labels that indicate the four underlying topological properties that make up TNTs: (i) a membranous extension of the plasma membrane from the cells connected; (ii) an actin cytoskeletal backbone; (iii) openended cell-TNT-cell contact sites, (iiii) functional ability to transfer cargo.

Figure 4 .

 4 Figure 4. Mechanisms of Tunneling Nanotube Biogenesis.Schematic diagram depicting the two physico-mechanical origins of TNTs and TNT-like connections (middle). The Actin-driven protrusion mechanism (left), in which one or both cells extend filopodia-like protrusions towards the target cell (or towards each other). Upon contact with the target cell, the tip of the protruding process fuses spontaneously or via fusion molecules leading to open-ended TNTs. The cell dislodgement mechanism (right) occurs when cells that are in sufficient proximity with each other fuse spontaneously and migrate away from each other, leaving behind membrane tethers that lead to TNTs. Cells interacting via adhesion molecules or electrically coupled with GJs can also lead to the formation of TNT-like structures (middle, bottom).

Figure 5 .

 5 Figure 5. Examples of filopodia. Schematic diagram depicting several examples of different types of filopodia, (obtained from Mattila and Lappalainen, 2008). (A) Filopodia localized at the leading edge of cells probe the microenvironment during cellular migration. (B) Filopodia of epithelial sheets, which protrude from opposing cells, help the sheets of cells to align and adhere together. (C) In neurons, filopodia participate in the guidance and migration of neuronal growth cones (top inset) and are precursors of dendritic spines (bottom inset).

Figure 6 .

 6 Figure 6. Arp2/3 and Formins: two major actin-nucleating machineries.

Figure 7 .

 7 Figure 7. Filopodia formation by CDC42, IRSp53, and VASP. Schematic diagram depicting how, during filopodia formation, CDC42 switches IRSp53 from inhibition of actin assembly to filament elongation by clustering of VASP (obtained from Disanza et al., 2013). (1) in the absence of stimuli, IRSp53 binds to the plasma membrane and slows down barbed-end elongation. (2) Upon stimuli, activated CDC42 binds to IRSp53, relieving IRSp53-mediated inhibition of filament growth. This promotes the formation of a CDC42-IRSp53-VASP complex and reduces the formation of the IRSp53-EPS8 complex. (3) Binding to CDC42 facilitates the formation of IRSp53 and VASP at the leading edge of the plasma membrane, in which the clustering of VASP allows processive actin filament elongation. (4) Membrane deforming activity of IRSp53 and processive filament elongation by VASP work together to extend actin filaments beyond the x, y plane of the membrane.

Figure 8 .

 8 Figure 8. Ultra-structure Architecture of filopodia.

Figure 9 .

 9 Figure 9. Regulation of Tunneling Nanotubes by Rab11a-Rab8a-VAMP3 cascade.

Figure 10 .

 10 Figure 10. TNT formation and induction.(A) Schematic diagram depicting the induction of TNT formation through the binding of M-Sec to the active form of Ral-A, an event dependent on the interaction between Ral-A and its downstream effector, the exocyst complex, whose function is to target secretory vesicles for membrane expansion. Through its interaction with Ral binding protein 1 (RalBP1), Ral-A is able to activate CDC42, which leads to actin remodeling. Given the role Ral-A plays in actin remodeling and the role of the exocyst complex, it is likely that M-Sec induces TNT formation by actin cytoskeleton remodeling and the supply of membrane. The association of Ral-A with filamin, an actin-filament crosslinking protein implicated in the regulation of actin remodeling in filopodia, may also be a candidate protein involved in TNT formation. (B) Stress conditions (H2O2 and serum starvation) induce TNT formation through the activation of the transcription factor p53, the EGF receptor (EGFR), and the Akt-PI3K-mTor pathway. M-Sec is also induced by the activation of p53. Black arrows indicate pathways previously described whereas dotted lines show hypothesized pathways. Blue colored arrows indicate interactions with the M-Sec-Ral-Aexocyst complex (blue square). Grey colored arrows show the exocyst-complex-mediated pathway. Single arrows indicate pathways, double arrows depict putative interactions. Both schematics and content were obtained from[START_REF] Abounit | Wiring through tunneling nanotubes--from electrical signals to organelle transfer[END_REF]

  TNTs have been scarce due to the transient and fragile nature of TNTs discussed at the beginning of this chapter. Only a small number of studies have been able to successfully examine the ultrastructure of TNTs using structural imaging approaches such as Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM). Unfortunately, the information provided by these studies lack (i) quantitative analysis of the number, prevalence, and significance of the connections presented, and (ii) answers to several key anatomical questions, such as: what does the cytoskeletal backbone of TNTs contain: actin, microtubules, or both? Does the cargo described as 'being transferred' by light microscopy get shuttled within TNTs, or on the outside of the membrane? Are TNTs open at both ends of the structure, or are they closed, filopodia-like protrusions that mediate cargo transport via trogocytosis-like event (in which an entire patch of plasma membrane gets engulfed by another cell)[START_REF] Dance | Core Concept: Cells nibble one another via the under-appreciated process of trogocytosis[END_REF]?

Figure 11 .

 11 Figure 11. Ultra-structural analyses of Tunneling Nanotubes. (A-B) TNTs connecting PC12 cells seen by Serial-sectioning TEM (A) and SEM (B), obtained from Rustom et al., 2004. Scale bar: Left (G), 2 μm, Middle (G1), Right (G2), 200 nm. (C-D) TNTs connecting Jurkat T cells seen by Serial-sectioning TEM (C) and SEM (D). Scale bar, C, D, 500 nm; insets (b´, b´´, d´, and d´´), 200 nm. Images were obtained from Sowinski et al., 2008. (E-G) Representative image of HIV infected cultures after 3 days post infection shown by SEM (E-F) and TEM (G). (F) High magnification electron micrograph of the end of the TNT process in HIV infected conditions. Images were obtained from Okafo et al., 2017. Scale bar: F, 50nm; G, 80nm. (H-I) Lung epithelial A549 cells seen by SEM (H) and TEM (I) show TNTs after expansion microscopy. Images obtained from Kumar et al., 2017. (J) TNT connecting Human primary CD34+ hematopoietic stem and progenitor cells (HSPC) as seen by SEM. Specific regions highlighted with green and blue boxes in are shown at high magnification as insets. Red circles indicate membrane bulges along TNTs. Dotted white lines and yellow arrowheads indicate the apparent membrane continuity of TNT ending points. Dotted white lines and yellow arrowheads indicate the apparent membrane continuity of TNT contact sites with HSPC body.Scale bar, 10 mm; Insets, 100 nm. Image obtained from[START_REF] Reichert | Tunneling nanotubes mediate the transfer of stem cell marker CD133 between hematopoietic progenitor cells[END_REF]

Figure 12 .

 12 Figure 12. Examples of TNT-like structures in vivo.Gallery of examples displaying Tunneling Nanotube-like structures In Vivo with the respective names given and model organisms in which they were identified. (A) Videomicrograph displaying a long, thin (200-400 nm in diameter) filopodium extending from a primary mesenchyme cell to the wall of the ectoderm ~80 µm away. The image also displays thin filopodia extending from ectoderm cells into the blastocoel (arrows). Scale bar, 10 µm. Image obtained fromMiller et al., 1995. (B) Confocal micrograph displaying an intercellular bridge (red arrows) formed between epiblast cells, acquired in a sub-region of a mosaic zebrafish embryo expressing mDendra2 at the onset of gastrulation. Scale bar, 20 µm. Image obtained from[START_REF] Caneparo | Intercellular bridges in vertebrate gastrulation[END_REF]. (C) Confocal image of retraction fibers containing migrasomes, acquired on a single blastomere of an eight-cell stage embryo injected with tspan4a-GFP mRNA. The arrow indicates an enlarged, process-connected vesicle. Scale bar, 10 μm. Image obtained from (Jiang et al., 2019). (D) Cells from the late third instar drosophila wing disks extending long processes containing GFP. (E) Confocal micrograph of Chimeric mouse corneal whole mount reveals a donor-derived (GFP+/green) MHC class II+ (red) and double positive (yellow) cell connecting via a fine membrane nanotube (arrows) to a resident MHC class II+ GFP+ cell (red only). Scale bar = 10 μm. Image obtained from Chinnery et al., 2008. (F) Differential chromatin distribution in mouse intervascular bridge cells (IBCs). Mouse IBCs were surrounded by a basement membrane stained by PAS reaction and containing collagen IV (left) and laminin (right). Image obtained from (Mendes-Jorge et al., 2012). (G) Lung adenocarcinoma tumor specimen with multiple mitotracker-labeled nanotubes of various lengths. (H) Example of a very stable T325 GBMSC tumor microtube (TM) (arrowheads), followed over 126 days in vivo. Scale bar, 50 µm. Image obtained from[START_REF] Osswald | Brain tumour cells interconnect to a functional and resistant network[END_REF] (I) Z-stack projections over 7 mm of confocal optical sections from an 8-month-old Ctns2/2 mouse kidney, transplanted at 2 months with eGFP-expressing wild-type hematopoietic stem cell (HSC). Green, immunolabeled eGFP; red, laminin immunolabeling provides a grazing view of basement lamina; blue, proximal tubular cell (PTC) brush border-specific sugars labeling by Lotus Tetragonolobus (LT)-lectin defines PTCs (lumen, #). Scale bar, 10 µm. Image obtained from[START_REF] Naphade | Brief reports: Lysosomal cross-correction by hematopoietic stem cell-derived macrophages via tunneling nanotubes[END_REF]

Figure 13 .

 13 Figure 13. Model of prion-like protein aggregate-induced formation of TNTs.

  complex organisms is one of the most outstanding challenges in the field of TNT-based cell-to-cell communication research. As described in Chapter 1, the lack of specific markers or antibodies that label TNTs and limitations in resolution of optical microscopy have hampered our ability to address this question, and therefore, to test whether the function of TNTs in vitro translate in vivo.

Figure 14 .

 14 Figure 14. Connectomics by serial-sectioning scanning electron microscopy.

  (H, left) The two pyramidal cells (red and green arrows) whose apical dendrites lie in the centers of the saturated reconstructions. Dendritic spines reconstructed in the high-resolution image stack only. (H, right) The saturated reconstruction volume. Images obtained from[START_REF] Kasthuri | Saturated Reconstruction of a Volume of Neocortex[END_REF]

Figure 15 .

 15 Figure 15. Cerebellar granule cell migration during development. Schematic diagram depicting a 3D representation of GCP migration from the EGL to the IGL. (1) Extension of two uneven horizontal processes near the top of the EGL. (2), tangential migration in the middle and bottom of the EGL. (3) Bergmann glia-(BG) associated radial migration in the ML (4) stationary state in the PCL. (5) glia-independent radial migration in the IGL. (6), completion of migration in the middle or the bottom of the IGL. Image obtained from[START_REF] Komuro | Recent advances in cerebellar granule cell migration[END_REF].

Figure 16 .

 16 Figure 16. Granule cell progenitor morphology characterization. Light microscopy micrographs showing the changes in the morphology of granule cells in a P10 mouse cerebellum revealed by Golgi-staining. (A-J), GCPs at the top of the EGL. (K-Z), tangentially migrating GCPs at the middle and bottom of the EGL. a1 and f1, Purkinje cells. b1, Bergmann glial cells. C1-E1 and G1-J1, radially migrating granule cells in the ML. K1 and R1-Y1, postmigratory granule cells in the IGL. l1-Q1, radially migrating granule cells in the IGL. Scale bar: A-Y1, 10 μm. Image obtained from (Kumada et al., 2009).

Figure 17 .

 17 Figure 17. Datasets produced by Serial-Sectioning Scanning Electron Microscopy. (A, D, & G) Low-resolution electron micrographs of a single section from P0, located in the crown of a folia; P3, located at the edge of the crown; and P7, located at the base of the fissure, respectively. The subregion that was imaged at high-resolution is superimposed onto each section. (B, E, and H) High-resolution electron micrographs of single superimposed images shown in A, D, and G, respectively. (C, F, and I) Electron micrograph volume collected in P0 (x, 135 μm; y, 90 μm; z, 45 μm), P3 (x, 120 μm; y, 190 μm; z, 50 μm), and P7 (x, 120 μm; y, 190 μm; z, 75 μm). Scale bars: A, D, and G, 100 μm; B, E, and H, 50 μm.

Figure 18 .

 18 Figure 18. ssSEM Reveals TNT-like connections between GCPs.Segmented EM images and corresponding 3D reconstructions of TNT-like connections identified at P0, P3, and P7. Raw EM images capture the points of fusion to each of the two connected cells and an intervening slice between both cells. 3D reconstructions illustrate the plasma membranes of the connected cells along with their nuclei and the connection (Cells), the position of the connection relative to the nuclei alone (Nuclei) and the connection alone in top, bottom, front and side orientations. The points of fusion of the tunnel with the connected cells are indicated by a different color inside the connection. Scale bar = 1 µm.

Figure 19 .

 19 Figure 19. Skeletonization of TNT-like connections. (A) 3D reconstruction of GCPs connected by a TNT-like protrusion (green) containing small vesicles (gray). (B) Crop of the connection shown in A (white, dashed box). (C and D) 3Ds Maxrendered polygon wireframe of reconstructions shown in A and B, respectively. (E) 3D representation of the reconstruction (red cloud) and single wire obtained by Kimimaro (solid red). Mean length = 4,025 nm; Mean radius = 189.6 nm; Min radius = 113.2 nm; Max radius = 265.9 nm.

Figure 20 .

 20 Figure 20. Connected GCPs exhibit perpendicular orientation.(A) 2D schematic diagram displaying an example of a perpendicularly aligned cell plane (dark purple) and pia plane (green). The cell plane is shown to divide and split the connected cell pair (black) into two parts and the pia plane is shown to be tangential to the pia membrane (pink) at the point closest to the connected cell pair. The dashed purple line shows that perpendicular alignment is characterized by plane φ angles in between 45° and 90°. (B) Shows an example of parallel aligned pia and cell planes (color code shown as in A). The dashed purple line shows that parallel alignment is characterized by plane φ angles in between 0° and 45°. (C) Gives the perspective view of an example of perpendicularly oriented pia planes (light blue dashed edges) and cell planes (yellow dashed edges), with the pia plane tangential to the P0 pia membrane and the cell plane dividing the two nuclei in the 3D space. The eye symbols represent the different types of viewpoints presented in figures E-G and J-L, with "1" being the Top View, "2" being the Side View, and "3" being the Front View. (D) Shows the pia (green) and cell (purple) planes overlaid in the same spot with perpendicular planes being over 45° from each other. (E-G) Shows assortment of top, side, and front views of the perspective view from C (colors preserved from C). (H) Perspective view of P3 parallel oriented pia and cell planes (colors preserved from C). (I) Shows pia and cell planes overlaid in the same spot with parallel planes being under 45° difference from each other (colors preserved from D). (J-L) Shows assortment of top, side, and front views of the perspective view from H (colors preserved from C). (M-O) Visual distribution and spread of all φ angle measurements of every pair of connected cells. Every pair of connected cells is represented by a single line in the 0-90° spread, respective to their unique φ angle. 0° represents planes that are perfectly parallel, while 90° represents perfectly perpendicular planes. Lines falling between the dashed 45° line and the 90° axis were classified as perpendicular, while lines falling between the dashed 45° line and the 0° axis were classified as parallel. (M-O) P0, P3, and P7 measurements were indicated by orange, cyan, and magenta lines, respectively, with colors from each developmental stage being preserved in (P) combined P0, P3, & P7 graphic. The percentages represent the proportion of parallel and perpendicular cell pairs there in respect to the total number of cell pairs found in each respective developmental stage.

Figure 21 .

 21 Figure 21. Identification of GCPs undergoing division. P7 volume segmented SEM micrographs (showing the contact points and middle of the connection) and corresponding 3D reconstructions of cells, nuclei, and intercellular mitotic bridge in a cell about to undergo cytokinesis. The 3D reconstructions show the segmented cells, the segmented nuclei and connection, and different side perspectives of the connection.

Figure 22 .

 22 Figure 22. Quantification of PH3+ cells in the Developing Cerebellum. (A) Representative micrograph of Phosphohistone H3 (PH3) immunohistochemistry (yellow) on P0 mouse cerebellum. Selected EGL region of interest for further imaging and analysis depicted within white box. (B) Magnified view of isolated DAPI channel of ROI depicted in A. (C) Isolated PH3 channel of selected ROI depicted in A and B. (D) Merged DAPI and PH3 channels of P0 cerebellar ROI. (E) Representative micrograph of Phosphohistone H3 (PH3) immunohistochemistry (yellow) on P3 mouse cerebellum. Selected EGL region of interest for further imaging and analysis depicted within white box. (F) Magnified view of isolated DAPI channel of ROI depicted in E. (G) Isolated PH3 channel of selected ROI depicted in E and F. (H) Merged DAPI and PH3 staining of P3 cerebellar ROI. (I) Representative micrograph of PH3 immunohistochemistry (yellow) on P7 mouse cerebellum. Selected EGL region of interest for further imaging and analysis depicted within white box. (J) Magnified view of isolated DAPI channel of ROI depicted in I. (K) Isolated PH3 channel of selected ROI depicted in I and J. (L) Merged DAPI and PH3 staining of P7 cerebellar ROI. (M) Mean and standard deviations of percentage PH3+/DAPI+ pixel density plotted for each age. (N) Average percentage PH3+/DAPI+ pixel density per brain (P0, n=3; P3, n=3; P7, n=4) plotted for each age. (O) Mean and standard deviations of percent PH3+ cells out of 50 randomly selected DAPI+ cells for each age. (P) Average percentage of PH3+ cells chosen out of 50 randomly selected DAPI+ cells per brain (P0 n=3; P3 n=3; P7 n=4) for each stage. Scale bar: A, E, I, 100μm; C, D, G, H, K, L, 20 μm.

Figure 23 .

 23 Figure 23. Dye-coupling between GCPs in the EGL reveal TNT-like structures.(A-B) Schematic diagram displaying the workflow: Harvesting of cerebellar tissue from P6-P8 mice (A), two-photon imaging of the slices in which we patched GCPs located in the EGL of the fissure between lobule VII and VIII. Patches where performed in GCPs located in the Outer, Middle, and Inner EGL, as shown by the diagram in B (x, y). In order to obtain complete GCPs and protrusions emanating from their soma, patches were performed in GCPs located deeper in the tissue (z, y), not on those at the surface. (C-H) 2-photon micrographs obtained from GCPs patched using the workflow shown in A and B. (C) GCP in the mEGL connected with GCP in the iEGL by a TNT-like structure of 10 μm in length and 600 nm in diameter. (D) GCP in the mEGL connected with GCP in the mEGL by a TNT-like structure of 68.9 μm in length and 900 nm in diameter. (E) GCP in the mEGL connected with GCP in the mEGL by a TNT-like structure of 30.6 (*), 32.5(**) and 400 nm in diameter. Yellow arrowhead indicates membrane bulge located along the TNT-like protrusion marked by '(*)', presumably cargo being transported along the connection as previously shown for TNTs in vitro. (F-H) Examples of patched GCPs appearing to be coupled with adjacent GCPs.

  Darby canine kidney ........................................................................................................................... MEF Mouse Embryonic Fibroblasts ............................................................................................................................. ML Machine learning .................................................................................................................................................. mTOR Mammalian target of rapamycin ..................................................................................................................... MV Microvesicle ........................................................................................................................................................... MVE Multivesicular endosome ..................................................................................................................................... -N-NEF Negative Regulatory factor .................................................................................................................................. NK Natural Killer ............................................................................................................................................................ NRK Normal Rat Kidney ................................................................................................................................................ N-WASP Neural-WASP ................................................................................................................................................

	-M-
	MDCK Madine-
	XII

-P-PC12 Pheochromocytoma 12 ........................................................................................................................................ PI3K Phosphatidylinositol-3-kinase ............................................................................................................................. PMC primary mesenchyme cell .................................................................................................................................. -R-Rab Ras-related proteins in brain ............................................................................................................................... Rac1 Ras-related C3 botulinum toxin substrate ......................................................................................................... RASSF1A Ras association domain family 1 isoform A ................................................................................................. RC Ring Canal .............................................................................................................................................................. REF52 Rat Embryonic Fibroblasts 52 ........................................................................................................................... -S-Shh Sonic hedgehog .................................................................................................................................................... SMC Secondary mesenchyme cell .............................................................................................................................. -T-TM Tumor microtube .................................................................................................................................................... TNF Tumor Necrosis Factor ........................................................................................................................................... TNFaip2 TNF-a induced protein 2 ............................................................................................................................... TNT Tunneling Nanotube .............................................................................................................................................. -V-VAMP3 v-SNARE Vesicle-associated membrane protein 3 ......................................................................................... VASP Vasodilator-stimulated phosphoprotein ........................................................................................................... -W-WASP Wiskott-Aldrich syndrome protein ................................................................................................................... WAVE WASP-family verprolin homologous protein ................................................................................................... WAVE2 WASP family verprolin-homologous 2 ........................................................................................................... WGA Wheat Germ Agglutinin ....................................................................................................................................... Wnt Wingless-related integration site ........................................................................................................................

  Figure 1. Modes of Inter-cellular Communication. ........................................................................Figure 2. Topological Architecture of Tunneling Nanotubes. .........................................................Figure 3. Properties that define Tunneling Nanotubes. ...............................................................Figure 4. Mechanisms of Tunneling Nanotube Biogenesis. .........................................................Figure 5. Examples of filopodia. ...................................................................................................Figure 6. Arp2/3 and Formins: two major actin-nucleating machineries. ....................................Figure 7. Filopodia formation by CDC42, IRSp53, and VASP. .......................................................Figure 8. Ultra-structure Architecture of filopodia. .....................................................................

Figure 9. Regulation of Tunneling Nanotubes by Rab11a-Rab8a-VAMP3 cascade. .....................

Figure 10. TNT formation and induction. .....................................................................................

Figure 11. Ultra-structural analyses of Tunneling Nanotubes. .....................................................

Table 1 . Cells connected by TNT-like structures over development.

 1

		P0	P3	P7
	# of cells screened	160	70	60
	# of connections	7	4	24
	% of cells connected	8.7	12	80

Table 3 . Properties of TNT-like structures.

 3

		Connection 1	Connection 2	Connection 3 & 4
	# of GCPs connected	2	2	3
	Length (μm)	10	68.9	30.6 (*), 32.5(**)
	Thickness (nm)	600	900	400

Acknowledgements

Preface

The work presented in this thesis consists of an introduction (Chapter 1), two published articles (Chapter 2 and 3), and significant progress towards unpublished research (Chapter 4). The papers are listed below (*co-author) followed with a description of my contributions to each project; my contributions are also stated in the Contributions section of each chapter (see Table of Contents).

Chapter 2 (Article 1): Delage, E., Cordero Cervantes, D.*, Pénard, E.*, Schmitt, C., Syan, S., Disanza, A., Scita, G., and Zurzolo, C. (2016). Differential identity of Filopodia and Tunneling Nanotubes revealed by the opposite functions of actin regulatory complexes. Scientific Reports 6, 39632.

In this project, in which I contributed equally as a second author when the study was welladvanced, we addressed the role of specific actin modifiers in the formation of TNTs in contrast to filopodia. I studied the role of Rac1 in the formation of neuronal TNTs by ectopic expression of the dominant negative-and constitutively active-form of the protein. Specifically, I performed the experiments, acquired the images, and manually analyzed the data collected to prepare Supplementary Figure 2. By Western Blotting, I also evaluated the expression of Eps8 in CAD and CHO cells, as well as brain (hippocampus and striatum) lysates, (see Supplementary Figure 6a). In addition, I examined the mRNA expression of Eps8 by RT-PCR in the cell lines above, (see Supplementary Figure 6b). I integrated my results and figures into the manuscript and replaced panels in Figure 1 and 5 with more representative, re-processed images. Finally, I updated several sections of the manuscript, including the Introduction, Results, Discussion, Methods, and Bibliography, and rephrased several key points throughout these sections. This work was carried out during the two-revision, peer-review process of the manuscript, which I handled allthroughout until publication of the article.

Chapter 3 (Article 2): Sartori-Rupp, A.*, Cordero Cervantes, D.*, Pepe, A.*, Gousset, K., Delage, E., Corroyer-Dulmont, S., Schmitt, C., Krijnse-Locker, J., and Zurzolo, C. (2019). Correlative cryoelectron microscopy reveals the structure of TNTs in neuronal cells. Nature Communications 10, 342.

V In this project, we designed and optimized a cryo-correlative electron microscopy (cryo-CLEM) approach to characterize the structure of TNTs at the nanometer resolution. I participated and contributed equally with the other two first authors in different aspects of the study. Specifically, I studied the role of Arp2/3 in the formation of neuronal TNTs by testing an Arp2/3 specific inhibitor (CK-666) in CAD cells. To this end, I performed the experiments, acquired the images, and manually analyzed the data collected to prepare Supplementary Figure 3c-3d. Setting up these experiments was fundamental for this project as CK-666 treatment of cells was used to increase the yield of TNTs on cryo-EM grids. I also labeled-, imaged-, and identified regions of interest (filopodia emanating from CAD cells) for the cryo-CLEM/cryo-electron tomography micrographs shown in Figure 5. I also obtained the cryo-TEM micrographs shown in Supplementary Figure 6, performed all the live-imaging experiments in the manuscript and implemented a plugin to analyze the speed of mitochondrial transport via TNTs (see Figure 6 and Supplementary Figure 5). I assisted with the manual analysis of cryo-ET images by computing plot profiles and fast Fourier transforms described in the manuscript and shown in Figure 4 and 5.

Moreover, I contributed to the manual segmentation of TNTs in FIB-SEM volumes and reprocessing electron micrographs throughout the manuscript for the preparation of figures (i.e. image stitching, alignment, enhancement, and addition of overlays and tracings). I designed and created the schematic diagrams found in Figure 1a and Figure 8. My role also included teaching one of the co-first authors (i) the protocols employed to study the effect of CK-666 in CAD cells' adherent filopodia and TNT function (see Supplementary Figure 3a-3b and 3e-3f); and (ii) the methods used to prepare samples for cryo-TEM and identification of TNTs on EM grids. Finally, I wrote the manuscript, assembled all the main and Supplementary Figures, as well as Supplementary Movies. I assembled, revised, submitted, and handled the manuscript throughout the three rounds of peer revision until publication of the article. VI called Ring Canals (RCs), are 500 nm -10 µm-thick rims that connect germ cells in cysts of Xenopus laevis [START_REF] Kloc | Formation, architecture and polarity of female germline cyst in Xenopus[END_REF], Drosophila [START_REF] Haglund | Cindr interacts with anillin to control cytokinesis in Drosophila melanogaster[END_REF], mice [START_REF] Pepling | Female mouse germ cells form synchronously dividing cysts[END_REF], and several others (Haglund et al., 2011). The function of these connections remains unknown; however, numerous reports have proposed that they promote the exchange of cytoplasm and organelles, in a similar fashion as TNTs, as TNTs do not form from cell division.

Whether ring canals or other IBs, such as those suggested by Caneparo et al., in zebrafish represent specialized TNT-like protrusions that enable inter-cellular communication and synchronization of cell division and differentiation requires future investigation.

During development of the Imaginal disc of Drosophila, 200 nm-thick and up to 700 μm in length, specialized filopodia called Cytonemes were identified, and are sometimes erroneously classified as the first demonstration of TNT-like structures in vivo (Figure 12) (Ramírez-Weber and [START_REF] Ramírez-Weber | Cytonemes: cellular processes that project to the principal signaling center in Drosophila imaginal discs[END_REF]. Differently from TNTs, cytonemes are close-ended, specialized filopodia [START_REF] Kornberg | Cytonemes as specialized signaling filopodia[END_REF], that play an important role in signaling of morphogens, (e.g. Bone morphogenetic protein, BMP; Sonic hedgehog, Shh; Wingless-related integration site, Wnt, and Fibroblast growth factor, Fgf), through ligands and receptors that are currently under investigation [START_REF] Huang | Cells must express components of the planar cell polarity system and extracellular matrix to support cytonemes[END_REF][START_REF] Zhang | Cytonemes in development[END_REF]. Interestingly, recent evidence from our group indicates that Wnt signaling is involved in the regulation of TNT formation of primary neurons (Vargas et al., EMBO in press). However, Wnt-dependent TNT formation is mediated by the activation of the downstream Calcium-Calmodulin signaling pathway, while the formation of cytonemes is mediated by the downstream activation of beta catenin [START_REF] Mattes | Wnt/PCP controls spreading of Wnt/β-catenin signals by cytonemes in vertebrates[END_REF]. Thus, while activation of the Wnt receptor is involved in both cytoneme and TNT formation, their downstream pathways differ, suggesting that cytonemes and TNTs are different structures with different functions during development.

In other studies, major histocompatibility complex (MHC) class II+ cells in the mouse cornea were observed to connect via TNT-like structures called membrane nanotubes (MNTs), of <800 nm in thickness and > 300 μm in length [START_REF] Chinnery | Cutting edge: Membrane nanotubes in vivo: a feature of MHC class II+ cells in the mouse cornea[END_REF][START_REF] Seyed-Razavi | Membrane nanotubes in myeloid cells in the adult mouse cornea represent a novel mode of immune cell interaction[END_REF]. The authors hypothesize that isolated (dendritic) cells might use TNTs to communicate and form an immunological syncytium, however, this hypothesis has yet to be explored.

Chapter 2 2 Role of Actin Regulatory Networks in

Neuronal Tunneling Nanotube Formation

Results

Summary

Filopodia are tip-closed membranous protrusions containing polymerized actin filaments, which have not been shown to transport cargo between cells. TNTs on the other hand share many similarities with filopodia but allow the long-range inter-cellular transport of cargo through openended contact sites. In this project, we studied different regulatory complexes of actin, which play a role in the formation of filopodia and asked whether they also played a role in TNT formation. To this end, we tested the role CDC42, IRsP5, and VASP play in the formation and function of neuronal TNTs through the ectopic expression of constitutively active-and dominant negative forms of each protein. Our analyses elucidated new insights on the cytoskeletal architecture and mechanisms underlying TNT formation and intercellular vesicle transfer in neuronal CAD cells. Specifically, our results show that the filopodia-promoting CDC42/IRSp53/VASP network negatively regulates TNT formation and impairs TNT-mediated intercellular vesicle transfer. We further found that these proteins act in concert to inhibit TNT formation. Conversely, elevation of Eps8, an actin regulatory protein that inhibits the extension of filopodia in neurons, increases TNT formation. Notably, Eps8-mediated TNT induction requires Eps8 bundling but not its capping activity. Thus, despite their structural similarities, filopodia and

TNTs form through distinct molecular mechanisms. Taken together, our results suggest that a switch in the molecular composition in common actin regulatory complexes is critical in driving the formation of either type of membrane protrusion.

Aims of the project

• Examine the molecular relationship between filopodia and TNTs.

• Test the role of the CDC42/IRSp53/VASP network in TNT formation and functionality.

• Examine the role of Eps8 in TNT formation and functionality.

• Test if Eps8-mediated TNT induction requires its bundling and/or capping activity.

Contribution

In this project, in which I contributed equally as a second author when the study was welladvanced, we addressed the role of specific actin modifiers in the formation of TNTs in contrast to filopodia. I studied the role of Rac1 in the formation of neuronal TNTs by ectopic expression of the dominant negative-and constitutively active-form of the protein. Specifically, I performed the experiments, acquired the images, and manually analyzed the data collected to prepare Supplementary Figure 2. By Western Blotting, I also evaluated the expression of Eps8 in CAD and CHO cells, as well as brain (hippocampus and striatum) lysates, (see Supplementary Figure 6a). In addition, I examined the mRNA expression of Eps8 by RT-PCR in the cell lines above, (see Supplementary Figure 6b). I integrated my results and figures into the manuscript and replaced panels in Figure 1 and 5 with more representative, re-processed images. Finally, I updated several sections of the manuscript, including the Introduction, Results, Discussion, Methods, and Bibliography, and rephrased several key points throughout these sections. This work was carried out during the two-revision, peer-review process of the manuscript, which I handled allthroughout until publication of the article.

Article 1: Differential identity of Filopodia and Tunneling Nanotubes revealed by the opposite functions of actin regulatory complexes

Article 1

Supplementary Material

Chapter 3

3 Tunneling Nanotubes: Ultra-Structural Identity Missing Links

Results

Summary

For over a decade, TNTs have gained substantial attention as an inter-cellular communication mechanism, allowing the transport of cargo between distant cells. Since their discovery, studies have focused on their functional role but information regarding their ultrastructure and the differences between them and other cellular protrusions such as filopodia is still lacking.

Here, we report on the structural characterization of TNTs in neuronal cells of different species, using correlative light-and cryo-electron tomography approaches. We contrast their structural identity with that of filopodia by showing that most TNTs -shown by fluorescence microscopy to be single connections -are in fact made up of multiple, smaller, individual tunneling nanotubes (iTNTs), all of which contain long, parallel actin filaments, presumably serving as railways for the transport of vesicles and organelles also observed within iTNTs. Our electron tomograms suggest the existence of thin filaments and coils that connect iTNTs, which could serve to increase the mechanical stability of the overall bundle. We demonstrated the functionality of these structures by showing that nanotubes transported mitochondria using live confocal imaging, which we validated by observations of mitochondria within iTNTs by cryoelectron tomography. Furthermore, we employed FIB-SEM to produce 3D electron micrograph volumes with sufficient resolution to clearly identify that TNTs are 'open' at both ends (contact sites) and thus form a continuous cytoplasmic bridge between two cells.

Collectively, our findings provide the first structural description of TNTs, support published fluorescence microscopy evidence suggesting bi-directional TNT-mediated transport of cargo, and demonstrate that TNTs are distinct from other actin-containing cellular protrusions such as filopodia. We also employ an imaging workflow that improves upon, and avoids, previous limitations of tools used to study the anatomy of TNTs, such as classic SEM and TEM. Our strategy better preserves these fragile structures in native conditions, which may be useful for studying the role TNTs play in other cell types and physio/pathological models This work is an essential step toward understanding TNT-mediated cell-to-cell communication and lays the groundwork for investigations into the spreading of pathogens such as viruses and misfolded proteins.

Aims of the project

• Design a strategy to study TNTs at the structural level by combining a series of EM approaches.

• Validate strategy in two neuronal cell lines, CAD (mouse) and SH-SY5Y (human) cells.

• Test the functionality of TNTs connecting SH-SY5Y cells by live-image analysis.

• Examine the F-actin cytoskeletal architecture of TNTs.

• Investigate whether cargo observed by light microscopy is present in/outside of TNTs.

• Identify similarities and differences in the structure of TNTs vs. filopodia.

• Examine whether TNTs are open-ended at each contact site via FIB-SEM.

Contribution

In this project, we designed and optimized a cryo-correlative electron microscopy (cryo-CLEM) approach to characterize the structure of TNTs at the nanometer resolution.

3D skeletonization of meshes reveals geometrical features.

In order to extract relevant geometrical information (e.g. length and diameter) from the TNTlike structures identified in our ssSEM datasets, we turned to simplifying our connections down to a single-wire curve-skeleton via Kimimaro (see methods), a derivation of the Tree-structure Extraction Algorithm delivering Skeletons that are Accurate and Robust (TEASER) [START_REF] Sato | TEASAR: Tree-structure Extraction Algorithm for Accurate and Robust Skeletons[END_REF]. Kimimaro smoothens the 3D connection mesh and contracts it into a 1-dimensional "wire", abstracting its shape and topology, all while maintaining connectivity. Through Kimimaro, we obtained the geometries of 3 TNT-like structures identified in our P3 volume with lengths that span 3 -4 μm and widths of 113 -265 nm (Example shown in Figure 21).

Dye-coupling reveals GCP TNT-like structure connectivity

In order to corroborate our findings with an alternative approach that would enable us to assess a larger number of samples, we investigated dye-coupling between GCPs in ex vivo cerebellar slices by 2-photon microscopy (see methods). To this end, we sectioned 250 μm acute parasagittal cerebellar slices from 6-to 8-day-old mice. We patch-loaded single GCPs in the EGL of distinct lobules with the fluorescence indicator Alexa 594 and imaged with two-photon laser scanning microscopy (2PLSM) in order to visualize the patched GC and potential connected cells.

(Figure 25A-B).

In three independent set of experiments we performed to date, we patch-loaded a total of 30 cells. In our first session, we patched 5 GCPs located in the top two-three rows of the EGL (oEGL) and didn't identify any coupling (Table 2). Next, we patched 11 GCPs located in the mEGL and identified 7 (63%) GCPs coupled by long, thin, membranous-bound protrusions (Figure 25C-E).

These structures spanned between 10 -68.9 μm in length and 400 -900 nm in diameter (Table 3). We observed that 2 out of the 3 GCPs patched coupled with cells in the iEGL using these connections that faced radial migration (Figure 25B), while only 1 coupled with a GCP found in the mEGL with a connection that faced tangential migration. Finally, we patched 14 GCPs in the lower 2-3 rows of the EGL (iEGL) and didn't identify any coupling (Table 2).

Assessing whether inter-cellular communication mediated by the exchange of cargo was not feasible by dye-coupling; however, in one of our examples (Figure 25E), we identified a bulge along one of the connections identified, resembling bumps previously indicative of cargo inside TNTs (Sartori- Rupp et al., 2019). Then the sample was transferred to the buffer solution for 5 2-hour changes and incubated in 1%

TCH in buffer that had been heated to 60 °C for 20 minutes. After washing the tissue again with buffer as described above, the tissue was incubated in 2% osmium tetroxide in buffer at 4 °C overnight. After staining and post-fixing with ROTO, we dehydrated the tissue by washing it in solutions of increasing ethanol content (5 minutes each in ice-cold solutions of 20%, 50%, 70%, 90%, 100%, and 100% ethanol in ddH2O) and finally by placing the tissue in anhydrous acetone left at room temperature for 10 minutes. We then embedded the tissue in Epon 812 resin mixed according to formula that would result in a medium-to-hard cured product (Electron Microscopy Sciences). We embedded each section in a block of resin by introducing solutions of propylene oxide (PO). Using mixtures of PO and resin thus allows the resin to better penetrate spaces in the tissue. We washed each section 2 times for 5 minutes each in pure PO, then left it in 3:1 PO/resin solution under constant agitation for 1 hour, followed by 1:1 PO/resin under agitation overnight, then 1:3 PO/resin under agitation for 1 hour, then in 100% resin overnight. Finally, we embedded each section in a tube of fresh resin and cured the block at 60 °C for 48 hours.

Next, from each block of embedded tissue we cut a series of ultrathin, serial sections for imaging using an automatic tape-collecting ultra-microtome (ATUM). The ATUM combines an ultra-5 Dissertation Perspectives

Conclusion and future work

During my dissertation work, I participated in three projects: (1) the role of actin regulatory networks in neuronal TNT formation, which resulted in an article entitled "Differential identity of Filopodia and Tunneling Nanotubes revealed by the opposite functions of actin regulatory complexes", of which I am a co-second author;

(2) the ultra-structural analyses of neuronal TNTs, which resulted in a second article entitled "Correlative cryo-electron microscopy reveals the structure of TNTs in neuronal cells", of which I am a co-first author; and (3) the search for TNTlike structures in vivo using a Connectomic approach, work that is currently in progress.

My first project was conceived and designed to investigate the underlying mechanism of neuronal cells' TNT formation in contrast to those engaged in filopodia formation. Our work elucidated the opposite role the CDC42/IRSp53/VASP and Eps8 actin regulatory complexes play in the formation of these two structures. In summary, when we overexpressed the CDC42/IRSp53/VASP complex in neuronal CAD cells, we observed a decrease in the number of TNT-connected cells (Delage et al., 2016), opposite from what others previously described for filopodia (Disanza et al., 2013). Similarly, overexpressed Eps8 increased the number of TNTconnected cells, once again, opposite from what the literature reported about the role of Eps8 in the formation of filopodia. These findings led us to hypothesize that TNTs and filopodia of neuronal cells form through distinct molecular pathways and with actin-regulatory proteins behaving in different manners.

My specific role in this project was to examine the role of Rac1 by testing whether its overexpression would affect the generation of TNTs. My results show that Rac1 negatively regulates TNT formation in a similar fashion as CDC42. These findings reinforced our previous data and our hypothesis that filopodia and TNTs harness distinct mechanisms of formation as well as the necessary evidence to recognize TNTs as bona fide organelles, and not specialized filopodia. Interestingly, our combined results disagree with those shown by [START_REF] Hanna | The Role of Rho-GTPases and actin polymerization during Macrophage Tunneling Nanotube Biogenesis[END_REF], which indicate that TNT formation in immune cells require CDC42 and Rac1 for their formation, and suggest a cell-specific underlying pathway activated to generate TNTs. This was supported by other studies, most notably as seen for M-Sec, which was shown to be involved in the formation of TNTs in immune cells (Hase et al., 2009) but not neuronal cells (Gousset et al., 2013). Furthermore, a ground-breaking study by [START_REF] Sowinski | Membrane nanotubes physically connect T cells over long distances presenting a novel route for HIV-1 transmission[END_REF] demonstrated that TNTs of immune T cells have a filopodia-like, close-ended tip. Therefore, it is conceivable that TNTs of immune and neuronal cells are equipped with distinct protein arsenals that yield two distinct structures: one close-and one open-ended structure, respectively. These observations led me to study the structure of TNTs in neuronal cells, whose anatomy had never been explored in detail.

To this end, my second project was devised to study the ultra-structure of TNTs in a correlative fashion and with sufficient resolution to compare the actin cytoskeletal topology of TNTs vs.

filopodia, assess whether TNTs transport cargo and organelles within its narrow constitution, and investigate whether TNTs connect the cytoplasmic lumen of two (or more) cells via an openended connection. To our surprise, our experiments not only revealed that the actin arrangement in TNTs looked notably straight compared to filopodia's, but also shed light on a TNT-specific arrangement we called iTNTs, (individual-TNTs), a bundle of several TNTs bundled together.

Given that no previous studies had employed the cryo-EM-based approaches that we used, it's currently beyond the bounds of possibility to ascertain that iTNTs are specific to neuronal cells, eventhough the work by Sowinski et al., [START_REF] Sowinski | Membrane nanotubes physically connect T cells over long distances presenting a novel route for HIV-1 transmission[END_REF] provide structural evidence of single TNTs. To ensure that our observations were not specific to cells of mouse origin, an important goal of this study was to analyze the structure of TNTs in different cell types. Immunogold labeling and cryo-EM tomography revealed that N-Cadherin served as links to support and presumably 'stabilize' iTNTs. Based on these observations, we now hypothesize that N-Cadherin interactions could be responsible for establishing (actin-driven mechanism-led) TNTs during their formation by growing filopodia that pull each other towards their cell somas and forming iTNTs upon fusion. Some of my lab peers are now testing this hypothesis. Our analyses also showed that vesicles and organelles squeeze between actin filaments and the membrane that make-up iTNTs, creating bulges along the connection that can also be detected by optical microscopy.

Furthermore, in our correlative study, we demonstrate that the actin-based motor protein, in tissue with a high-throughput, ultra-high-resolution, in vivo-optimized approach of significant size to obtain quantitative results. My studies on the cerebellar GCPs reveal TNT-like connections, uninterrupted by GJs or synaptic-like clefts. Many of the connections identified contain vesicles and mitochondria, suggesting that cargo can move along these connections and possibly get transported. The immunohistological and dye-coupling preliminary data presented in this thesis suggests that these connections are unlikely to derive from cytokinesis during cell division; however, future studies will have to examine and better test this possibility. Finally, streamlining the skeletonization methods to obtain accurate geometrical measurements will help us characterize and categorize our observations, and teach us about the heterogeneity of connections in the cerebellum. Although many questions remain open about the possible function these connections play during migratory events in the cerebellum, our findings provide new insights on GCP-to-GCP communication that did not exist prior to this study.

Taken together, my dissertation examined specific questions about TNT formation, structure, and presence in vivo. I consider these investigations to have been a worthwhile effort to understand how these organelles function. As a result of these studies, new avenues and projects have spurred in our group, demonstrating the usefulness of the studies described in this thesis.

Mapping Tunneling Nanotubes