Yassine Cherrak

Nicolas Flaugnatti

Eric Durand

Laure Journet

Eric Cascales

Structure and Activity of the Type VI Secretion System

The Type VI secretion system (T6SS) is a multiprotein machine that uses a spring-like mechanism to inject effectors into target cells. The injection apparatus is composed of a baseplate on which is built a contractile tail tube/sheath complex. The inner tube, topped by the spike complex, is propelled outside of the cell by the contraction of the sheath. The injection system is anchored to the cell envelope and oriented towards the cell exterior by a trans-envelope complex. Effectors delivered by the T6SS are loaded within the inner tube or on the spike complex, and can target prokaryotic and/or eukaryotic cells. Here, we summarize the structure, assembly and mechanism of action of the T6SS. We also review the function of effectors and their mode of recruitment and delivery.

INTRODUCTION

The Type VI secretion system (T6SS) is a multiprotein machine that belongs to the versatile family of contractile injection systems (CISs) [START_REF] Bönemann | Tubules and donuts: a type VI secretion story[END_REF][START_REF] Zoued | Architecture and assembly of the type VI secretion system[END_REF][START_REF] Cascales | Microbiology: and Amoebophilus invented the machine gun![END_REF][START_REF] Taylor | Contractile injection systems of bacteriophages and related systems[END_REF]. CISs deliver effectors into target cells using a spring-like mechanism [START_REF] Taylor | Contractile injection systems of bacteriophages and related systems[END_REF][START_REF] Brackmann | Using force to punch holes: mechanics of contractile nanomachines[END_REF][START_REF] Cianfanelli | Aim, load, fire: the type VI secretion system, a bacterial nanoweapon[END_REF]. Briefly, CISs assemble a needle-like structure, loaded with effectors, wrapped into a sheath built in an extended, metastable, conformation (Fig. 1).

Contraction of the sheath propels the needle toward the competitor cell. Genomes of Gramnegative bacteria usually encode one or several T6SSs, with an overrepresentation in Proteobacteria and Bacteroidetes (for a review on the role of T6SS in gut-associated Bacteroidales, see chapter by Coyne and Comstock ( 7)) [START_REF] Bingle | Type VI secretion: a beginner's guide[END_REF][START_REF] Cascales | The type VI secretion toolkit[END_REF][START_REF] Boyer | Dissecting the bacterial type VI secretion system by a genome wide in silico analysis: what can be learned from available microbial genomic resources?[END_REF]. The broad arsenal of effectors delivered by T6SS includes antibacterial-specific proteins such as peptidoglycan hydrolases, eukaryotic-specific effectors that act on cell cytoskeleton, and toxins that can target all cell types such as DNases, phospholipases, or NAD + hydrolases [START_REF] Russell | Type VI secretion system effectors: poisons with a purpose[END_REF][START_REF] Durand | Tle, and beyond: the versatile arsenal of type VI secretion effectors[END_REF][START_REF] Diniz | Molecular weaponry: diverse effectors delivered by the Type VI secretion system[END_REF][START_REF] Hachani | Type VI secretion and anti-host effectors[END_REF]. As such, the T6SS plays a critical role in reshaping bacterial communities, and directly, or indirectly, in pathogenesis [START_REF] Fu | Tn-Seq analysis of Vibrio cholerae intestinal colonization reveals a role for T6SS-mediated antibacterial activity in the host[END_REF][START_REF] Sana | Salmonella Typhimurium utilizes a T6SS-mediated antibacterial weapon to establish in the host gut[END_REF][START_REF] Sana | T6SS: The bacterial "fight club" in the host gut[END_REF][START_REF] Chassaing | Antibacterial weapons: targeted destruction in the microbiota[END_REF][START_REF] García-Bayona | Bacterial antagonism in host-associated microbial communities[END_REF]. Destroying bacterial competitors also provides exogenous DNA that can be acquired in naturally competent bacteria and that serves as reservoir for antibiotic resistance gene spreading [START_REF] Veening | Interbacterial predation as a strategy for DNA acquisition in naturally competent bacteria[END_REF]. This chapter lists the major effector families, and summarizes the current knowledge on the assembly and mode of action of the T6SS.

TYPE VI SECRETION SYSTEM EFFECTORS

Several T6SSs have been shown to target eukaryotic cells [START_REF] Pukatzki | Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system[END_REF][START_REF] Sana | Internalization of Pseudomonas aeruginosa strain PAO1 into epithelial cells is promoted by interaction of a T6SS effector with the microtubule network[END_REF][START_REF] Schwarz | VgrG-5 is a Burkholderia type VI secretion system-exported protein required for multinucleated giant cell formation and virulence[END_REF]. By promoting or preventing cytoskeleton re-arrangements through the action of specific effectors that target actin or tubulin, the T6SSs of Vibrio cholerae, Aeromonas hydrophila, and Pseudomonas aeruginosa disable phagocytic cells or stimulate internalization into non-phagocytic cells [START_REF] Pukatzki | Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system[END_REF][START_REF] Sana | Internalization of Pseudomonas aeruginosa strain PAO1 into epithelial cells is promoted by interaction of a T6SS effector with the microtubule network[END_REF][START_REF] Pukatzki | Type VI secretion system translocates a phage tail spike-like protein into target cells where it cross-links actin[END_REF][START_REF] Suarez | A type VI secretion system effector protein, VgrG1, from Aeromonas hydrophila that induces host cell toxicity by ADP ribosylation of actin[END_REF][START_REF] Durand | Crystal structure of the VgrG1 actin cross-linking domain of the Vibrio cholerae type VI secretion system[END_REF]. Other T6SSs have been demonstrated to manipulate host cells, although the molecular determinants are not yet entirely understood [START_REF] Aubert | A Burkholderia Type VI effector deamidates Rho GTPases to activate the pyrin inflammasome and trigger inflammation[END_REF][START_REF] Eshraghi | Secreted effectors encoded within and outside of the Francisella pathogenicity island promote intramacrophage growth[END_REF][START_REF] Ledvina | A phosphatidylinositol 3-kinase effector alters phagosomal maturation to promote intracellular growth of Francisella[END_REF][START_REF] Lennings | The Burkholderia Type VI secretion system 5: composition, regulation and role in virulence[END_REF]. However, T6SS gene clusters are widespread in Gram-negative bacterial genomes, and not restricted to pathogens [START_REF] Boyer | Dissecting the bacterial type VI secretion system by a genome wide in silico analysis: what can be learned from available microbial genomic resources?[END_REF].

Most of them encode proteins with potent antibacterial activities such as enzymes that cleave essential macromolecules such as DNA, phospholipids or the peptidoglycan mesh, or essential metabolites such as NAD + /NADP + [START_REF] Hood | A type VI secretion system of Pseudomonas aeruginosa targets a toxin to bacteria[END_REF][START_REF] Russell | Type VI secretion delivers bacteriolytic effectors to target cells[END_REF][START_REF] Russell | Diverse type VI secretion phospholipases are functionally plastic antibacterial effectors[END_REF][START_REF] Ma | Agrobacterium tumefaciens deploys a superfamily of type VI secretion DNase effectors as weapons for interbacterial competition in planta[END_REF][START_REF] Whitney | An[END_REF](36). Additional T6SS antibacterial effectors include ADP-ribosyltransferases that specifically target the Z-ring and hence inhibit cell division (37). Antibacterial effectors are active in the periplasm or cytoplasm of the target cell, and are co-produced with immunity proteins that remain in the producing cell and act as antitoxins to prevent autointoxication during dueling between sister cells [START_REF] Russell | Type VI secretion system effectors: poisons with a purpose[END_REF][START_REF] Durand | Tle, and beyond: the versatile arsenal of type VI secretion effectors[END_REF][START_REF] Diniz | Molecular weaponry: diverse effectors delivered by the Type VI secretion system[END_REF]. More recently, T6SS effectors that collect manganese or zinc in the environment to provide metals to the cell have been described (38)(39)(40). By deploying antibacterial effectors or scavenging metals, T6SSs play an important role in bacterial communities, and hence T6SS gene clusters are usually highly represented in species present in multispecies microbiota such as the human gut [START_REF] Coyne | Type VI secretion systems and the gut microbiota[END_REF][START_REF] Sana | Salmonella Typhimurium utilizes a T6SS-mediated antibacterial weapon to establish in the host gut[END_REF][START_REF] Sana | T6SS: The bacterial "fight club" in the host gut[END_REF][START_REF] Chassaing | Antibacterial weapons: targeted destruction in the microbiota[END_REF]41). In general the regulatory mechanisms and signals underlying expression of T6SS genes, production of T6SS subunits or post-translational activation of the secretion apparatus are tightly linked to environmental cues in the niche in which the T6SS is required destroy competitors (42-45).

TYPE VI SECRETION MECHANISM OF ACTION

T6SSs use a contractile mechanism to inject effectors (Fig. 2). This mechanism is shared with all CISs: a sheath, assembled in an extended conformation, wraps a needle. Contraction of the sheath into a stable state propels the needle [START_REF] Bönemann | Tubules and donuts: a type VI secretion story[END_REF][START_REF] Cascales | Microbiology: and Amoebophilus invented the machine gun![END_REF][START_REF] Taylor | Contractile injection systems of bacteriophages and related systems[END_REF][START_REF] Brackmann | Using force to punch holes: mechanics of contractile nanomachines[END_REF]. The needle is composed of an inner tube capped by the spike complex that pierces the membrane of the target cell (Fig. 1). The tail tube/sheath complex (TTC) is built on an assembly platform named baseplate (BP) (Fig. 1).

TTC and BP are collectively called tail, a structure that is conserved among all CISs. In addition to this common theme to all CISs, T6SSs have evolved (i) a membrane complex (MC), which docks the tail to the cell envelope and serves as channel for the passage of the needle upon sheath contraction, and (ii) a specialized BP component to properly orient the needle toward the cell exterior, by recognizing and binding the MC (2-5, 46-48) (Fig. 1). T6SS biogenesis starts with the assembly of the MC in the cell envelope, and that of the BP in the cytoplasm (49-51) (Fig. 2). Once the BP is docked to the MC, the inner tube and sheath are coordinately assembled (49-52) (Fig. 2).

ARCHITECTURE OF THE TYPE VI SECRETION SYSTEM The Membrane Complex

The vast majority of T6SS gene clusters of Proteobacterial species encode three membrane proteins: TssJ, TssL, and TssM (8-10, 53) (Fig. 1). TssJ is an outer membrane-associated lipoprotein that protrudes in the periplasm (54). TssL and TssM are anchored in the inner membrane (55-57). The structures of several TssJ homologues have been reported: they all share a classical transthyretin fold with an additional loop, of variable length and composition, located between β-strands 1 and 2 (58-60). TssL bears a single C-terminal membrane-spanning segment (56) and a cytoplasmic domain that comprises two bundles of α-helices (61-63). TssM possesses three transmembrane helices followed by a large periplasmic region (55, 57). The periplasmic region of TssM comprises three domains, including the C-terminal domain that engages in interaction with the TssJ extra-loop (49, 58), TssL and TssM interact through their transmembrane segments (55, 64, 65). The cytoplasmic domains of TssL and TssM mediate contacts with the baseplate (50, 57, 64, 66, 67).

The electron microscopy structure of the fully assembled 1.7-MDa TssJLM MC from enteroaggregative Escherichia coli has been reported (49,[START_REF] Rapisarda | In situ and high-resolution cryo-EM structure of the type VI secretion membrane complex[END_REF][START_REF] Yin | Architecture of type VI secretion system membrane core complex[END_REF]. The complex has a rocketlike structure: a large base, that contains the cytoplasmic and membrane domains of TssL and TssM, is followed by arches and pillars which correspond to the TssM periplasmic domains and TssJ [START_REF] Rapisarda | In situ and high-resolution cryo-EM structure of the type VI secretion membrane complex[END_REF]. The TssJLM complex, which has five-fold symmetry in vivo and after purification, comprises 15 copies of TssJ, and 10 copies of TssL and of TssM (49, 58). The MC delimits an internal lumen with a diameter insufficient for the passage of the tail tube. In addition, this lumen is partly occluded by a periplasmic constriction gate, suggesting that large conformational changes occur upon BP docking or sheath contraction (49, 58).

The MC can be accessorized by additional subunits, such as peptidoglycan-binding proteins (53, [START_REF] Aschtgen | Anchoring the type VI secretion system to the peptidoglycan: TssL, TagL, TagP... what else?[END_REF][START_REF] Santin | Role and recruitment of the TagL peptidoglycan-binding protein during Type VI secretion system biogenesis[END_REF]. MC anchorage to the cell wall likely stabilizes the MC to resist the forces generated during sheath contraction [START_REF] Aschtgen | Anchoring the type VI secretion system to the peptidoglycan: TssL, TagL, TagP... what else?[END_REF]. Finally, recent studies have shown that proper assembly of the MC requires the activity of peptidoglycan-degrading enzymes [START_REF] Weber | Genetic dissection of the type VI secretion system in Acinetobacter and identification of a novel peptidoglycan hydrolase, TagX, required for its biogenesis[END_REF][START_REF] Santin | Domestication of a housekeeping transglycosylase for assembly of a Type VI secretion system[END_REF]. Interestingly, while the tail complex is evolutionarily related to contractile injection machines, the evolution history of the MC is less clear. TssL and TssM present significant homologies with two accessory subunits associated with Type IVb secretion systems, DotU and IcmF, respectively [START_REF] Bingle | Type VI secretion: a beginner's guide[END_REF][START_REF] Cascales | The type VI secretion toolkit[END_REF]. No homologue of TssJ is found associated with DotU/IcmF complexes, suggesting that TssJ is from a different ancestry. Indeed, while essential when present, TssJ is lacking in some T6SSs such as that of Agrobacterium and Acinetobacter.

Further studies are required to understand whether other proteins can substitute for the absence of TssJ in these species. The fact that the MC has a distinct history compared to the tail is also exemplified by the observation that no TssJLM complex is present in Bacteroidales T6SSs [START_REF] Russell | A type VI secretion-related pathway in Bacteroidetes mediates interbacterial antagonism[END_REF][START_REF] Coyne | Type VI secretion systems of human gut Bacteroidales segregate into three genetic architectures, two of which are contained on mobile genetic elements[END_REF]. However, putative uncharacterized membrane proteins are encoded within these T6SS gene clusters suggesting that a different transenvelope complex has been domesticated to anchor the tail [START_REF] Russell | A type VI secretion-related pathway in Bacteroidetes mediates interbacterial antagonism[END_REF][START_REF] Coyne | Type VI secretion systems of human gut Bacteroidales segregate into three genetic architectures, two of which are contained on mobile genetic elements[END_REF].

The Tail

The baseplate

The baseplate (BP) (Fig. 1) is a large complex of 2.7 MDa comprising > 60 polypeptides of at least six different proteins (50). The role of the baseplate is to initiate the polymerization of the tail tube/sheath complex. While it has not been formally shown yet, the T6SS baseplate is believed to trigger sheath contraction, as demonstrated in other CISs. A specific role of the T6SS baseplate is to anchor the TTC to the MC. The BP is composed of six wedge subcomplexes organized around the central hub, i.e., the N-terminal domain of the VgrG spike (76, 77) (Fig. 1). VgrG hence belongs to two tail sub-complexes: it constitutes the tip of the needle, and the hub for the baseplate. The wedge complex is composed of 4 proteins: TssE, -F, -G, and -K. These four proteins assemble a structure of 1:2:1:6 stoichiometry, the TssG peptide being the central core [START_REF] Cherrak | Biogenesis and structure of a type VI secretion baseplate[END_REF][START_REF] English | Biochemical analysis of TssK, a core component of the bacterial type VI secretion system, reveals distinct oligomeric states of TssK and identifies a TssK-TssFG subcomplex[END_REF][START_REF] Taylor | Structure of the T4 baseplate and its function in triggering sheath contraction[END_REF]. Two TssF subunits wrap TssG to form a triangular shape called trifurcation unit, whereas two extensions of TssG make contacts with two TssK trimers [START_REF] Cherrak | Biogenesis and structure of a type VI secretion baseplate[END_REF]. TssE, -F, -G are respectively homologues of phage T4 gp25, gp6 and gp7 and phage Mu Mup46, Mup47 and Mup48 (50, [START_REF] Cherrak | Biogenesis and structure of a type VI secretion baseplate[END_REF][START_REF] Taylor | Structure of the T4 baseplate and its function in triggering sheath contraction[END_REF][START_REF] Büttner | Baseplate assembly of phage Mu: Defining the conserved core components of contractile-tailed phages and related bacterial systems[END_REF], that also constitute the inner part of phage baseplates [START_REF] Taylor | Structure of the T4 baseplate and its function in triggering sheath contraction[END_REF][START_REF] Büttner | Baseplate assembly of phage Mu: Defining the conserved core components of contractile-tailed phages and related bacterial systems[END_REF][START_REF] Kostyuchenko | Three-dimensional structure of bacteriophage T4 baseplate[END_REF]. TssK has no homologue in Myoviridae, but shares architectural homologies with receptor-binding proteins (RBP) of Siphoviridae phages [START_REF] Nguyen | Type VI secretion TssK baseplate protein exhibits structural similarity with phage receptor-binding proteins and evolved to bind the membrane complex[END_REF].

The structure of the N-terminal domain of TssK is superimposable with that of Siphoviridae RBP shoulder domains that are anchored into the baseplate [START_REF] Nguyen | Type VI secretion TssK baseplate protein exhibits structural similarity with phage receptor-binding proteins and evolved to bind the membrane complex[END_REF]. Indeed, the TssK N-terminal domain establishes extensive contacts with the TssF 2 G complex [START_REF] Nguyen | Type VI secretion TssK baseplate protein exhibits structural similarity with phage receptor-binding proteins and evolved to bind the membrane complex[END_REF]. The TssK C-terminal domain has evolved to bind to the MC, and specifically to the TssL and TssM cytoplasmic domains (57, 64, 66, 67). Similar to the MC, the BP can be accessorized by additional subunits, such as TssA1 in P. aeruginosa [START_REF] Planamente | TssA forms a gp6-like ring attached to the type VI secretion sheath[END_REF], that may stabilize the complex or provide additional functions.

The tail tube/sheath complex

The tail tube/sheath complex comprises the needle and the contractile sheath (Fig. 1). It forms a ~ 1 µm-long tubular structure in the cytoplasm, that is assembled in 30-50 sec (52, 83).

The needle is composed of the inner tube topped by the spike complex. The inner tube is made of hexamers of the Hcp protein [START_REF] Ballister | In vitro self-assembly of tailorable nanotubes from a simple protein building block[END_REF][START_REF] Leiman | Type VI secretion apparatus and phage tail-associated protein complexes share a common evolutionary origin[END_REF][START_REF] Brunet | Type VI secretion and bacteriophage tail tubes share a common assembly pathway[END_REF]. These donut-shaped hexameric Hcp rings [START_REF] Mougous | A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus[END_REF][START_REF] Douzi | Crystal structure and self-interaction of the type VI secretion tailtube protein from enteroaggregative Escherichia coli[END_REF] stack on each other in a head-to-tail orientation to form a hollow tube [START_REF] Brunet | Type VI secretion and bacteriophage tail tubes share a common assembly pathway[END_REF].

Interestingly, despite very low sequence similarities between T6SS Hcps and tube proteins from other CISs, their structure is strictly conserved [START_REF] Brackmann | Using force to punch holes: mechanics of contractile nanomachines[END_REF]. Hcp tube polymerization starts at the baseplate, through direct recruitment of the first ring to the base of the VgrG hub/spike [START_REF] Renault | The gp27-like hub of VgrG serves as adaptor to promote Hcp tube assembly[END_REF].

The spike complex is composed of a trimer of the VgrG protein and, in most instances, of the PAAR-repeat protein [START_REF] Leiman | Type VI secretion apparatus and phage tail-associated protein complexes share a common evolutionary origin[END_REF][START_REF] Shneider | PAAR-repeat proteins sharpen and diversify the type VI secretion system spike[END_REF]. VgrG contains several conserved domains [START_REF] Pukatzki | Type VI secretion system translocates a phage tail spike-like protein into target cells where it cross-links actin[END_REF][START_REF] Leiman | Type VI secretion apparatus and phage tail-associated protein complexes share a common evolutionary origin[END_REF]. The Nterminal domain resembles the phage T4 gp27 protein, and acts as a symmetry adaptor between the six-fold symmetry of the inner tube and the three-fold symmetry of the VgrG central and C-terminal domains, which share homologies with the phage T4 gp5 N-terminal and β-prism domains [START_REF] Renault | The gp27-like hub of VgrG serves as adaptor to promote Hcp tube assembly[END_REF][START_REF] Leiman | Contractile tail machines of bacteriophages[END_REF][START_REF] Spínola-Amilibia | The structure of VgrG1 from Pseudomonas aeruginosa, the needle tip of the bacterial type VI secretion system[END_REF]. The VgrG β-prism domain is a triangular β-helix that forms, together with the conical PAAR protein, the penetration device of the T6SS needle [START_REF] Shneider | PAAR-repeat proteins sharpen and diversify the type VI secretion system spike[END_REF][START_REF] Uchida | Structure and properties of the C-terminal β-helical domain of VgrG protein from Escherichia coli O157[END_REF]. The VgrG trimer and the PAAR protein can be extended by additional domains that may act as effectors, or as adaptors for effectors [START_REF] Pukatzki | Type VI secretion system translocates a phage tail spike-like protein into target cells where it cross-links actin[END_REF][START_REF] Shneider | PAAR-repeat proteins sharpen and diversify the type VI secretion system spike[END_REF].

The sheath polymerizes from the baseplate. It is proposed that, similarly to its gp25like homologues in Myoviridae, the TssE BP subunit constitutes the sheath polymerization initiator [START_REF] Taylor | Structure of the T4 baseplate and its function in triggering sheath contraction[END_REF][START_REF] Leiman | Contractile tail machines of bacteriophages[END_REF]. By contrast to other CISs, the T6SS sheath is composed of two proteins, TssB and TssC [START_REF] Bönemann | Tubules and donuts: a type VI secretion story[END_REF]52,[START_REF] Leiman | Type VI secretion apparatus and phage tail-associated protein complexes share a common evolutionary origin[END_REF][START_REF] Bönemann | Remodelling of VipA/VipB tubules by ClpV-mediated threading is crucial for type VI protein secretion[END_REF][START_REF] Lossi | Structure-function analysis of HsiF, a gp25-like component of the type VI secretion system, in Pseudomonas aeruginosa[END_REF], forming a stable dimer that is the repeat unit for sheath polymerization [START_REF] Bröms | A functional VipA-VipB interaction is required for the type VI secretion system activity of Vibrio cholerae O1 strain A1552[END_REF][START_REF] Zhang | Dissection of the TssB-TssC interface during type VI secretion sheath complex formation[END_REF][START_REF] Kube | Structure of the VipA/B type VI secretion complex suggests a contraction-state-specific recycling mechanism[END_REF]. Six TssBC dimers form a strand that wraps a Hcp hexameric ring.

The TssBC dimer can be divided in three regions: Domains 1 and 2 that resemble CIS sheath proteins, and an additional Domain 3 inserted into Domain 2 [START_REF] Kudryashev | Structure of the type VI secretion system contractile sheath[END_REF]100). Extensive contacts between TssBC dimers from the same strand and from the neighboring -1 and +1 strands stabilize the extended conformation of the sheath polymer (100, 101).

In the T6SS, assembly of the inner tube and that of the extended sheath are interdependent [START_REF] Brunet | Type VI secretion and bacteriophage tail tubes share a common assembly pathway[END_REF]102). The TssA protein coordinates the polymerization of the tail tube/sheath complex (103) (Fig. 2). TssA localizes at the distal extremity of the growing tail tube/sheath (103), at the location in which hexameric tube rings and TssBC strands are incorporated (104). TssA presents a 6-arm starfish-like structure with a central core (103).

Protein-protein interaction studies have suggested that the central core of TssA may undergo large conformational changes to insert new Hcp hexamers, whereas the arms may facilitate sheath polymerization (103, 105). Tail tube/sheath polymerization proceeds in the cytoplasm, and is stopped when the distal end hits the membrane on the opposite membrane of the bacterial cell (104, 106). A recent study has identified TagA, a protein that interacts with TssA to stop the assembly of the tail and to maintain the sheath under the extended conformation (106) (Fig. 2). However, the TssA cap protein and the TagA stopper are not conserved in T6SS gene clusters, suggesting that different mechanisms control tail tube/sheath assembly and termination in different T6SS + species (105-107).

Contraction of the T6SS sheath, which occurs in less than 2-5 msec, is believed to start at the BP. The cryo-electron microscopy structure of the Vibrio cholerae T6SS sheath has been solved in the two states: extended and contracted, allowing a reconstitution of the molecular events leading to contraction [START_REF] Kudryashev | Structure of the type VI secretion system contractile sheath[END_REF]100). Contraction consists to a reorganization of the TssBC strands, and notably an outward rotation of the sheath subunits (100). By doing so, the sheath compacts on the BP, and contacts with the inner tube are abolished, thus, promoting its expulsion (5, 100, 101). The free energy released during contraction is estimated to > 44,000 kcal.mol -1 for a 1-µm-long sheath (100).

After contraction, the sheath is disassembled by a dedicated AAA + ATPase, ClpV (94, 102) (Fig. 2). ClpV binds to an N-terminal helix of TssC that belongs to sheath Domain 3 (108, 109), which is only accessible in the contracted conformation [START_REF] Kube | Structure of the VipA/B type VI secretion complex suggests a contraction-state-specific recycling mechanism[END_REF]100). Although this is not clearly established, it is proposed that contracted sheath subunits are recycled rather than conveyed to degradation (102).

LOADING AND TRANSPORT OF EFFECTORS

As summarized above, a broad repertoire of anti-bacterial and anti-host activities have been already described for T6SS effectors. In addition, the mode of loading and transfer of these effectors into target cells is also variable. The common theme is that these effectors are associated with needle components, as the needle is the only portion of the T6SS to be propelled into the target cell (12, 13) (Fig. 3). Effectors can be additional domains fused to needle components such as Hcp, VgrG, or PAAR, or independent proteins that directly or indirectly bind to Hcp, VgrG, or PAAR [START_REF] Durand | Tle, and beyond: the versatile arsenal of type VI secretion effectors[END_REF][START_REF] Diniz | Molecular weaponry: diverse effectors delivered by the Type VI secretion system[END_REF]. Recruitment of these independent cargo effectors to Hcp, VgrG or PAAR can be mediated by adaptors, which are themselves domains of the needle components, or independent proteins (110) (Fig. 3).

Specialized Hcp, VgrG and PAAR

When the effector module is on the same polypeptide as the needle component, the T6SS subunit is called "specialized" or "evolved". Although effectors fused to Hcp or PAAR have been described (36, 90, 111), the best-characterized examples are C-terminal extensions of specialized VgrGs such as V. cholerae VgrG1 that cross-links actin and VgrG3 that has peptidoglycan glycoside hydrolase activity, A. hydrophila VgrG1 that ADP-ribosylates actin, P. aeruginosa VgrG2b that interacts with tubule cap complex, and Burkholderia pseudomallei VgrG5 that induces host cell membrane fusion [START_REF] Pukatzki | Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system[END_REF][START_REF] Sana | Internalization of Pseudomonas aeruginosa strain PAO1 into epithelial cells is promoted by interaction of a T6SS effector with the microtubule network[END_REF][START_REF] Schwarz | VgrG-5 is a Burkholderia type VI secretion system-exported protein required for multinucleated giant cell formation and virulence[END_REF][START_REF] Pukatzki | Type VI secretion system translocates a phage tail spike-like protein into target cells where it cross-links actin[END_REF][START_REF] Suarez | A type VI secretion system effector protein, VgrG1, from Aeromonas hydrophila that induces host cell toxicity by ADP ribosylation of actin[END_REF][START_REF] Durand | Crystal structure of the VgrG1 actin cross-linking domain of the Vibrio cholerae type VI secretion system[END_REF](112)(113).

Cargo Effectors

Cargo effectors are independent proteins that need to recognize their Hcp, VgrG or PAAR carrier for transfer. This recognition could be direct, such as the case of effectors that bind Hcp, or may require an additional adaptor module that bind VgrG or PAAR [START_REF] Durand | Tle, and beyond: the versatile arsenal of type VI secretion effectors[END_REF][START_REF] Diniz | Molecular weaponry: diverse effectors delivered by the Type VI secretion system[END_REF]110) (Fig. 3). Usually the effector genes are genetically linked to genes encoding their vehicle, their adaptors (if any) and in case of antibacterial toxins, their immunity proteins. These genetic elements could be found within T6SS gene clusters, or as Hcp-VgrG islands scattered on the genome [START_REF] Cascales | The type VI secretion toolkit[END_REF][START_REF] Boyer | Dissecting the bacterial type VI secretion system by a genome wide in silico analysis: what can be learned from available microbial genomic resources?[END_REF].

When associated with Hcp, the effector is embedded in the lumen of the hexameric ring, and is thus likely found inside the channel of the inner tube during T6SS assembly [START_REF] Sana | Salmonella Typhimurium utilizes a T6SS-mediated antibacterial weapon to establish in the host gut[END_REF]114). As such, it is protected and stabilized (114, 115). However the available space in the Hcp ring lumen limits the size of the effector to be transported, which is estimated to be < 25 kDa (114).

Adaptors can be isolated proteins, or domains fused to the cargo or the vehicle (110).

Adaptors from distinct families, such as DUF1795 (EagT6, EagR), DUF2169, DUF2345, DUF4123 (Tap-1 or Tec), transthyretin (TTR) or Recombination hot-spot (Rhs) have been described and studied [START_REF] Whitney | An[END_REF][START_REF] Shneider | PAAR-repeat proteins sharpen and diversify the type VI secretion system spike[END_REF](116)(117)(118)(119)(120)(121)(122)(123)(124)(125). When several copies of VgrG or PAAR proteins are encoded within the genome, these adaptor modules specify the carriers on which the effector should be mounted (112,118,122,123,126). In addition to loading the effector on the vehicle, some of these adaptors have been shown to act as chaperones to stabilize the effector, or to wrap hydrophobic transmembrane segments to prevent effector aggregation (112,124,125).

CONCLUDING REMARKS

Although the Type VI secretion system is one of the most recently identified secretion apparati, we now have a detailed view on how the system is assembled, how it is structurally arranged, and how effectors are loaded and transported. The broad repertoire of effectors has only recently started to emerge, and it is likely that many effectors with interesting activities will be identified and characterized in the next years. The discovery of the T6SS 13 years ago and its role as an antibacterial weapon have altered our view of bacterial communities. It is now broadly admitted that bacteria do not only cohabitate peacefully but rather that complex interactions are established to maintain stable ecosystems, such as the human gut microbiota.

Further fundamental and translational works are required to better understand how T6SS activation or inhibition may impact microbial communities and may perturb complex ecosystems.

interbacterial NAD(P)(+) glycohydrolase toxin requires elongation factor Tu for delivery to target cells. Cell.

FIGURE 2

Assembly and mechanism of firing of the Type VI secretion system. T6SS biogenesis starts with the positioning and assembly of the membrane complex, and the assembly of the baseplate (1). The recruitment and docking of the baseplate on the membrane complex (2) initiates the TssA-mediated polymerization of the tail tube/sheath tubular structure [START_REF] Cascales | Microbiology: and Amoebophilus invented the machine gun![END_REF][START_REF] Taylor | Contractile injection systems of bacteriophages and related systems[END_REF][START_REF] Brackmann | Using force to punch holes: mechanics of contractile nanomachines[END_REF], which is stopped when hitting the opposite membrane by the TagA stopper [START_REF] Brackmann | Using force to punch holes: mechanics of contractile nanomachines[END_REF]. Sheath contraction propels the tube/spike needle into the target [START_REF] Cianfanelli | Aim, load, fire: the type VI secretion system, a bacterial nanoweapon[END_REF]. The ClpV ATPase is recruited to the contracted sheath to recycle sheath subunits [START_REF] Cianfanelli | Aim, load, fire: the type VI secretion system, a bacterial nanoweapon[END_REF]. Needle components, and effectors associated to them, are delivered inside the target [START_REF] Coyne | Type VI secretion systems and the gut microbiota[END_REF].

FIGURE 3

 3 FIGURE 3 Schematic representation of the mechanisms of effector loading. Effectors are

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  Schematic representation of the Type VI secretion system. The different subunits are labeled, as well as the different subcomplexes. IM, inner membrane; OM, outer membrane.

	LEGEND TO FIGURES
	FIGURE 1

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ACKNOWLEDGMENTS

We thank the laboratory members for helpful discussions and support. Our work on T6SS is supported by the Centre National de la Recherche Scientifique (CNRS), the Aix-Marseille Université (AMU), and grants from the Agence Nationale de la Recherche (ANR-14-CE14-0006 and ANR-17-CE11-0039 to EC, ANR-18-CE15-0013 to LJ, ANR-18-CE11-0023 to ED), the Fondation pour la Recherche Médicale (DEQ20180339165), and the Fondation Bettencourt-Schueller. YC is supported by the FRM (FRM-ECO20160736014).


Yoann G Santin
email: ysantin@imm.cnrs.fr

METHODS, MODELS & TECHNIQUES -BioEssays Uncovering the in vivo proxisome using proximity-tagging methods

Keywords: Protein-protein interaction, in vivo proximity labeling, APEX, BioID, PUP-IT, proxisome, bacteria

The development of new approaches is critical to gain further insights into biological processes that cannot be obtained by existing methods or technologies. The detection of protein-protein interaction is often challenging, especially for weak and transient interactions or for membrane proteins. Over the last decade, several proximity-tagging methodologies have been developed to explore protein interactions in living cells. Among those, the most efficient are based on protein partner modification, such as biotinylation or pupylation. Such technologies are based on engineered variants of enzymes like peroxidases or ligases that release reactive molecules, in the presence of specific substrates, that bind surrounding proteins. Fusing a protein of interest to these enzymes allows the definition of an unbiased "proxisome", that is all of the proteins in interaction or in close vicinity of the protein of interest. Here, I describe the different proximitylabeling tools available and provide a comprehensive comparison to discuss advantages and limitations.

Introduction

By analogy with a theater play, biological processes require different actors assigned to perform specific tasks in space and time. In living cells, such actors are mostly proteins that "communicate" between each other. Communication is achieved through different types of interactions such as weak, transient, stable or long interactions, resulting in specific biological effects. Protein-protein interaction (PPI) can lead to conformational effects which enable the formation of a structural complex, activate or inactivate a protein, create a new biding site for the interaction with other binding partners or a substrate, serve as regulatory pathway, or allow subcellular relocalization.

Detection of PPIs, in space and time, is therefore critical for deciphering each step of a biological process. A number of methods for assaying PPIs in vivo have been developed and are routinely used in laboratories. Co-immunoprecipitation (Co-IP) represents one of the most standard methods of identifying interacting partners in vivo [1,2] . Briefly, a protein of interest (POI) is immunoprecipitated from a cellular protein extract by using specific antibodies immobilized on beads (Fig. 1a). While unbound proteins are washed out, binding partners are co-precipitated and can be visualized and identified by Western Blot or mass spectrometry analysis. It is worthy to note that Co-IP is a variant of the pull-down assay, which used a tagged bait protein to capture protein complexes instead of antibodies. Tandem affinity purification (TAP) is also a systematic approach to detect PPIs at near proteome-scale under in vivo conditions. TAP consists in two consecutive purifications by using two different tags fused to a bait protein, then considerably reducing the amount of nonspecific contaminants [3,4] .

Additional methods such as two-hybrid systems, including bacterial (BACTH) and yeast (Y2H) two-hybrid assays (see [5] for comparative review), are powerful genetic approaches to characterize PPIs in native or near-native context [6][7][START_REF] Karimova | Proc. Natl. Acad. Sci. U. S. A[END_REF][START_REF] Karimova | [END_REF][10][11][12][13] . POIs are fused to the two isolated fragments from the Bordetella pertussis adenylate cyclase [6,[START_REF] Karimova | Proc. Natl. Acad. Sci. U. S. A[END_REF][START_REF] Karimova | [END_REF][10]13] or from the Gal4 transcription factor [7,11,12] for BACTH and Y2H respectively (Fig. 1b). Physical association between tested proteins pairs restores the activity of the adenylate cyclase or Gal4 that can be visualized by a transcriptional-activated reporter. Other interaction-mediated reconstitutionbased methods exist, such as the TOXCAT or GALLEX systems for studying transmembrane helix-helix oligomerization in a natural membrane environment [14][15][16] . With a similar conceptual approach, Förster resonance energy transfer (FRET) is a well-suited biophysical method for the investigation of PPIs that occur between two proteins positioned within 10 nanometers of each other, allowing study of molecular interactions in real time [17,18] . In brief, FRET relies on the energy transfer from a donor fluorescent molecule (i.e., diminution of fluorescence intensity) to an acceptor when these two probes are separated by 10 nanometers or less [19][20][21][22] (Fig. 1c).

Then, PPIs or conformational changes within a protein (if the two probes are fused to the same protein) can be visualized in real time, by using a fluorometer or a fluorescence microscope.

Furthermore, this method can be extend to several practical applications in biology such as the detection of protein cleavage, changes in micro-environment or quantitative analysis of protein interactions (see [23,24] for comprehensive reviews).

Although these methods have improved our knowledge about biological processes by the characterization of number of PPIs, some important limitations are still present. Due to the nature of stringent purifications, co-precipitation based-methods (i.e., Co-IP, pull down and TAP) are not reliable to detect weak or transient PPIs, leading to a lot of false negatives [25] . In two-hybrid systems, proteins are produced from multi-copy plasmids and not at the physiological concentration in the cell, which may cause unbalanced stoichiometry and effects on PPIs, or toxicity within the cell [26] . Proper re-assembly of the reporter is dependent of the spatial association between the two tested proteins, hence steric effects may occur and prevent detection of PPI. Screening only soluble domains of membrane proteins, which need the cell membrane for proper folding, can lead to false negative in PPIs. Further, bait and prey proteins are artificially put into the same subcellular compartment (i.e., the bacterial cytosol or the yeast nucleus for BACTH and Y2H respectively), which may be different from the native compartment of the POIs. Hence, protein pair could interact in a compartment where they normally may not encounter each other or not interact if the physicochemical conditions are too different from the native compartment(s). Finally, fluorescence based-methods suffer from inherent physical and technical limitations. In FRET, since the distance between the donor and the acceptor must be within the Förster radius (i.e., ~10 nm), poor positioning of fluorescent probes in protein pair might lead to defect in detection of PPI. In addition, spectral leakage, that correspond to the direct excitation of the acceptor and causing false positive detections, donor fluorescence background, photobleaching and trouble for measurements make real PPI detection quite difficult [27] .

Deciphering subtle PPIs in living cells is a challenge that cannot be achieved by those conventional methods. Detection of weak and transient interactions, which constitute the major part of PPIs in dynamic processes, interactions with membrane proteins and more importantly, the detection in true in vivo context (i.e., both in native cell and at the chromosomal expression level) must be improved to understand the complexity of biological processes. Over the last decade, proximity-tagging approaches have been developed to explore these questions. Based on protein partner modifications, proximity-tagging methodologies allow the definition of an unbiased "proxisome", that is all of the proteins in direct interaction or in close vicinity of the POI, in native conditions and without loss of elusive PPIs. Currently, three major proximitytagging approaches are used: the proximity-dependent biotin identification (BioID), the APEX proximity-dependent biotin labeling and the recent pupylation-based interaction tagging (PUP-IT). Here, I detail the principles of these proximity-tagging methodologies and their different applications in biology. Finally, I provide a comprehensive comparison to allow the definition of the most appropriate approach for your studies.

Proximity-dependent biotin identification (BioID)

BioID is a useful proximity-dependent labeling method to screen both interacting and neighboring proteins in their in vivo context. BioID assay is based on the promiscuous biotinylation generated by a modified variant of BirA [28,29] . BirA is a 35-kDa bacterial biotin ligase that catalyzes the biotinylation of BCCP, a subunit of the acetyl-CoA carboxylase in Escherichia coli [30,31] . By using endogenous biotin and ATP, BirA produces biotinoyl-5'-AMP molecules and biotinylates BCCP with an exquisite specificity. To perform proximitydependent labeling, the R118G mutant BirA protein (BirA R118G or BioID protein) is used. This variant, which normally binds ATP, displays a low affinity for biotinoyl-5'-AMP compared to the WT BirA [32] . Hence, biotinoyl-5'-AMP molecules are released from BioID protein and covalently react with proximate proteins, and specifically on lysine residues, in a range of approximately 10 nm [33,34] . For BioID assay, promiscuous biotinylation is induced in cells producing BioID fusion protein by adding a supraphysiological concentration of biotin in the medium during an optimal labeling period of 16-18h [34][35][36] (Fig. 2). Biotinylated proteins, which are candidate interactors for the POI, are then enriched on streptavidin beads and identified by mass spectrometry.

Recently, significant efforts have been made to improve BioID assay in living cells. Screen for substitutions in the biotin binding site that modulate BirA activity revealed that R118K substitution reduced biotin affinity and increased the biotinoyl-5'-AMP release [37] . In 2016, Kim and colleagues tested the potential of BirA protein from the thermophilic bacterium Aquifex aeolicus [38] . This variant, called BioID2, is 8-kDa smaller than BioID (i.e., 27 kDa) and requires less biotin supplementation for proximity labeling [38] . Interestingly, assaying for dimerization-dependent protein interactions was investigated by split-BioID, where inactive BioID-fragment complementation pair was generated as split-reporter and forms a functional BioID protein that biotinylates substrates and other proximate proteins upon heterodimerization [39] . Finally, by using yeast display-based directed evolution, the Ting lab recently engineered two enhanced BioID variants: the 35-kDa TurboID and the 28-kDa miniTurbo [40] . These variants display a remarkable labeling power, dropping from 16-18h to only 10 min labeling period with similar size and specificity than standard BioID [40] . Furthermore, proximity labeling activity of TurboID and miniTurbo is conserved in different ranges of pH and at low temperature [40] by contrast to BioID and BioID2 proteins [38] . Taken together, such improvements greatly extended the potentiality of the BioID system for assaying proxisome detection in plethora of contexts.

BioID was initially developed for the study of the in vivo nuclear lamin-A proxisome in mammalian cells [34] . Up to now, BioID has been successfully used in diverse contexts. In mammalian cells, BioID was applied to probe architecture of different subcellular locations such as the nuclear pore complex [33,41,42] , focal adhesion complexes [43] or the centrosome-cilium interface [44] , and also to get insights about molecular mechanisms such as mitochondrial proteostasis [45,46] , autophagy [47] , mitosis [48][49][50] or viral infection [51] . BioID was also elegantly adapted to investigate RNA-protein interactions that play important roles in cellular functions and diseases. Named RaPID (for RNA-protein interaction detection), this method enables the detection of proteins which interact with a specific RNA [52] . Briefly, the RNA of interest is flanked by bacteriophage BoxB stem loops which bind by the lN peptide with high affinity [53] .

The lN peptide is fused to the N-terminus of BioID protein. Then, BoxB stem loops recruit the lN-BioID fusion protein and thereby biotinylating proteins bound to the RNA of interest in living cells [52] . In addition, authors engineered BASU, a modified biotin ligase from Bacillus subtilis that displays faster kinetics (>1000-fold) and increased signal-to-noise ratio (>30-fold) compared to the standard Escherichia coli BioID protein. Although less active than miniTurbo and TurboID [40] , BASU enabled sufficient labeling of proteins for RaPID study in living cells [52] . Finally, BioID was used for proximity labeling in planta [54,55] and directly on animals such as in mouse [56] and recently in fruit flies (Drosophila melanogaster) and worms (Caenorhabditis elegans) [40] .

APEX proximity-dependent biotin labeling

APEX is a spatially resolved proteomic mapping assay to determine in vivo proxisome in dynamic systems in living cells. By contrast to BioID, APEX-dependent labeling is based on the activity of the engineered enhanced hemic ascorbate peroxidase APEX, a 27-kDa monomeric derivative of protein from soybean [57,58] . Briefly, APEX catalyzes the H2O2dependent oxidation of phenol derivatives (the biotin-phenol, BP, being the most specific substrate for APEX) into short lived phenoxyl radicals (<1 ms) that covalently react in a small labeling radius (<20 nm) with electron-rich amino acids such as Tyr, Trp, His and Cys [58] . It has been recently established that tyrosine residues are the principal site of biotinylation by APEX with more than 98 % labeling observed [59] . Similar to BioID workflow, APEX labeling starts with the construction of a functional fusion between APEX and the POI. After incubation with BP, cells producing APEX fusion are treated with a 1-min pulse of hydrogen peroxide enabling APEX-dependent biotinylation of proximate proteins in a short labeling radius (Fig. 3) [58,60] . Subsequently, cells are lysed and biotinylated proteins are purified and identified by mass spectrometry analysis.

Due to its low activity when expressed at physiological level, APEX protein was further improved to enhance its sensibility in living cells. By using directed evolution, a single point substitution (A134P), located in a loop at the vicinity of both heme and the aromatic substratebinding site was found to confer great improvements in stability, kinetics, heme binding and resistance to high H2O2 concentrations [61] . Named APEX2, this variant is much more active than APEX in the cell and is consequently better for proteomic mapping [61] . Very recently, a split-APEX (sAPEX) system has been developed [62] . Two fragments called "AP" (a 200-amino-acid N-terminal fragment) and "EX" (a 50-amino-acid C-terminal fragment) are each inactive but give peroxidase activity upon reconstitution. Authors proposed that the sAPEX system could be useful to address question about interaction-dependent proximity labeling at higher spatiotemporal resolution. Hence, sAPEX reconstitution can be applied for mapping specifically proxisome of target nucleic acids (e.g., mRNA, non-coding RNA or genomic locus) by considerably reducing background caused by the activity of APEX2 that is not bound to the target of interest [63,64] , or to capture molecular composition at the interface of two organellar structures such as at mitochondria-ER contact [62] .

Over the past 6 years, many applications of APEX labeling have emerged to map proxisome within specific locations and/or in dynamic molecular processes. Extensively used in eukaryotic cells, APEX has been applied for investigating complex proteomes such as in human or in Drosophila melanogaster mitochondria [58,60,61,[65][66][67] , in mammalian cilia [68] or in yeast Golgi [69] .

APEX proximity biotinylation was also used for exploring dynamic macromolecular complexes within specific genomic loci or for RNA subcellular location. In order to define the human mitochondrial nucleoid proteome, fusion between APEX2 and the mitochondrial DNA helicase Twinkle was constructed and revealed new nucleoid-associated proteins [70] . Last year, two independent groups reported the development of GLoPro (for genomic locus proteomics) and C-BERST (for dCas9 biotinylation at genomic elements by restricted spatial tagging), a combination of CRISPR-based genome targeting with APEX2-dependent proximity labeling for defining proteome at specific genomic locus [63,64] . While receptor signaling constitutes a critical process involved in numerous physiological regulations, tracking those processes requires high spatio-temporal resolution and high throughput, missing in current methods.

Back-to-back papers from Kruse and Krogan labs elegantly demonstrated the application of APEX assay to G-protein-coupled receptors signaling by tracking interactions at several different time points upon addition of activating ligand and within a native cellular environment [71,72] . They finally concluded that APEX is a powerful method for spatially resolved quantitative analysis of signaling, applicable to any other signal transduction monitoring [71,72] . Designed at the beginning for proteomic mapping, APEX has been recently used for transcriptomic mapping assay. Spatial organization of RNA within the cell has been at first determined by an indirect approach combining APEX proximity labeling with RNAprotein chemical crosslinking [73] . As this approach was inadequate for spatial specificity in unenclosed cellular locations, authors introduced APEX-seq, a direct APEX-catalyzed labeling of RNA combined with RNA-seq. This direct approach was successfully used in nine distinct subcellular compartments allowing determination of a the RNA atlas in human cell [74] and recently the organization of translation initiation complexes [75] . Finally, Zhou and colleagues recently developed promising biotin-aniline and biotin-naphthylamine probes (Btn-An and Btn-Nap, respectively), a new class of phenolic compounds with significantly higher reactivity towards nucleic acids [76] . Such developments open the field of new broad applications for in situ transcriptomic assay to shed light on RNA functions in cell physiology.

Over the last year, APEX biotinylation assay was also performed in different microbial pathogens such as for the mapping of the inclusion membrane proteins in Chlamydia trachomatis [77][78][79][80] , in Mycobacterium smegmatis periplam [81] , in the diplomonad fish parasite Spironucleus salmonicida [82] , and in enteroaggregative Escherichia coli where stage-blocking mutations were used to define in vivo temporal contacts of the TssA protein during type VI secretion system biogenesis [83] . With its broad range of applications in diverse organisms and for labeling of different types of macromolecules, APEX proximity-dependent biotin labeling constitutes a powerful tool for cellular processes that require high spatial specificity and short labeling windows.

Pupylation-based interaction tagging (PUP-IT)

As a new proximity tagging method, the PUP-IT system was developed from the ubiquitin-like process present in actinobacteria [84][85][86] . The pupylation-based modification was extensively studied in Mycobacterium tuberculosis for its implication in bacterial proteasome, a cellular system required for causing disease [85,87,88] . In this system, the Pup ligase PafA uses ATP to transfer an activated Pup(E) substrate to a lysine of a target protein [85,89,90] . Thereafter, the Pup(E)-protein complex is targeted to the mycobacterial proteasome for unfolding and degradation [85,90,91] . Taking advantage of this system, Liu and colleagues adapted the Pup system from Corynebacterium glutamicum as a PUP-IT tool to define membrane and intercellular PPI [92] . They first determined the potential of PafA to be a suitable proximitytagging enzyme and revealed that compared to BioID and APEX substrate, activated Pup(E) does not freely diffuse from PafA, suggesting that very close proximity between the bait protein and their partners is required for proper pupylation. They then tested in vivo PUP-IT by fusing PafA to the membrane receptor CD28 to identify its cytosolic tail interactome (Fig. 4a). By inducing the production of a biotinylated carboxylase-fused Pup(E) protein (bio-Pup(E)), which was under the control of the locked TET-ON system [START_REF] Urlinger | Proc. Natl. Acad. Sci. U. S. A[END_REF] , the authors initiated the pupylationbased labeling and successfully enriched known CD28-interacting proteins such as ITK, p85 or LCK [92] . They finally explored the possibility of extracellular PUP-IT labeling. To avoid long exposure of PafA at the cell surface, authors induced the external heterodimerization of CD28 and PafA by using the FKBP-rapamycin-FRB system [START_REF] Banaszynski | [END_REF] (Fig. 4b). Upon reconstitution, the addition of ATP and truncated biotin-fused Pup(E) DE28 peptides (bio-DE28) enabled cell surface labeling suggesting that PUP-IT can be used for extracellular pupylation [92] . While the PUP-IT has been only used in this study, it represents a promising method for assaying in vivo PPIs especially involved in cell-to-cell communication, cell-to-cell adhesion, or host-pathogen interactions.

What is the best approach (for you)?

Proximity-tagging methods can be used to circumvent inherent technical limitations that conventional approaches for assaying in vivo PPIs suffer from. However, each of them display differences which are both important or not depending on the nature of the study. In this section, I will compare the specifications of each method to help defining the most suitable approach for your studies.

Advantages compared to conventional methods

As mentioned in the introduction, proximity-tagging methods present many benefits compared to conventional methods. First, interactome/proxisome of a POI can be defined under native expression and stoichiometry by fusing tagging enzyme-coding gene at chromosomal locus of the bait POI. Because proximate proteins are covalently labeled before purification, transient, weak or hydrophobic interactions, which are lacking from some conventional methods, can be easily detected. Further, promiscuous labeling of non-interacting-proximate proteins might be useful to understand the environment of the POI and hence, to get new insights about its role and/or its subcellular location in the cell. Interestingly, it has been shown that proxisome can be expanded or reduced by modifying the length of the linker between the bait and the tagging protein [38] . As previously described, labeling molecules (i.e., biotinoyl-5'-AMP for BioID, biotin-phenoxyls for APEX and Pup(E) for PUP-IT) have been shown not to cross plasma membrane [34,58,92] , therefore proteome definition into cellular organelle or near to the membrane might be useful to determine topology of transmembrane proteins [58,67,95] . Finally, the use of proximity-tagging methods in several biological contexts, ranging from the bacterial cell to the living animals, makes them powerful versatile tools for investigating endogenous PPIs.

Common features to keep in mind before starting

As biological applications, proximity-tagging methods can be limited or be not suitable in specific context. First, characterization of the bait protein is a basic, but important prerequisite to confirm that its location and its function in the cell are conserved in fusion with the tagging enzyme. Hence, the use of proximity labeling method without preliminary results about the POI is strongly discouraged.

While biotin is commonly used for detection and enrichment, it is necessary to examine endogenous biotinylation occurring in your model organism. Overrepresentation of endogenous biotinylated proteins might be detrimental by preventing enrichment of proximity labeledproteins, especially if the POI is thought to have only few partners. For example, in Mycobacterium smegmatis, APEX2-dependent biotinylated proteins were confounded by endogenous biotinylated proteins [81] . To address this problem, authors successfully designed two alternative labeling molecules: tyramide alkyne and tyramide azide, which can be used both for detection and enrichment via a copper-catalyzed alkyne/azide cycloaddition "click" reaction [81] . Thus, the design of new probes could be a good option to circumvent this type of issue in specific organisms.

Proximity labeling is associated with the presence and the accessibility of reactive residues such as lysine in BioID [28,34] and PUP-IT [92] assay, and mostly tyrosine in APEX proximitylabeling [58,59] . Consequently, the efficiency of labeling is dictated by the composition and the folding of proximate proteins. The absence of labeling does not therefore means a lack of interacting or proximity partners, especially for proteins that lack of exposed Tyr/Lys. By contrast, abundance of biotinylated proteins cannot be used to quantitatively assess the presence or the strength of a putative interaction.

Except for PUP-IT system that requires contact for labeling [92] , labeling radius in BioID and APEX assay is not a fixed value but instead a "cloud" or a "labeling gradient" where contours depend on the physicochemical micro-conditions such as temperature or pH [28,60] . By defining a "proxisome", it could be necessary to determine whether identified biotinylated proteins are in direct interaction or in close proximity of the POI, especially if the POI is included within a multiprotein complex.

Although weak, labeling of endogenous proteins present at many nanometers away from tagging enzyme might be detected by MS analysis, especially in non-membrane-enclosed cellular regions. Hence, efficient discrimination from proximate biotinylated proteins to the cellular background is essential. To address this question, most studies combined proximitytagging methods with quantitative proteomics such as stable isotope labeling by amino acids in cell culture [43,60,63,66,[95][96][97] (SILAC), tandem mass tagging [64,70] (TMT), or label-free quantification [92] (LFQ). Schematic pipelines of these approaches applied to proximity-tagging methods are represented in Fig. 5. Although there is no any report for preferentially using one approach compared to another in the proximity-dependent labeling context, technical specifications should be considered before starting your study. For example, SILAC and TMT are much more expensive than LFQ but are more accurate for proteomic quantification.

Because LFQ is a non-ratiometric approach, there is no limit in multiplexing (i.e., the number of conditions that can be tested simultaneously) by contrast to SILAC and TMT, limited to 5plex and 10-plex respectively. SILAC requires metabolically active cells to incorporate labels whereas TMT can be directly performed on extracted peptides. Still, while LFQ presents a better proteome coverage, it requires significantly longer data acquisition time compared to SILAC or TMT. For the choice of the most suitable quantitative approach, see [98] for recent comprehensive comparison.

Comparative of the three approaches

To discriminate among those proximity-tagging methods, comparison of the main features could be important for specific biological studies. In this context, I decided to distinguish TurboID/miniTurbo assay (hereafter, (mini)TurboID assay) from BioID assay due to striking differences in labeling process. All of the data are summarized in Table 1.

APEX2, BioID2 and miniTurbo are relatively small proteins with a molecular mass of about 27 kDa [38,40,58,61] while Turbo-ID, BioID and PafA proteins are larger, with a molecular mass of about 35 kDa [40] , 35 kDa [34] and 54 kDa [92] respectively. Because the tagging-enzyme is fused to the bait protein, its molecular mass can affect the localization and/or the function of the bait protein in the cell. Thus, preliminary results about your POI can be useful to wisely determine where you have to fuse the tagging-enzyme (i.e., in N or C-terminus of the POI), the nature of the linker, in terms of residues and length, and the most suitable enzyme.

In BioID and (mini)TurboID assays, tagging-enzyme uses native ATP and exogenous input of biotin as substrates for proximity labeling [34,40] . Since biotin is essential for life, its incorporation in cells is ensured by specialized transporters [99] . By contrast, APEX2 uses H2O2 and non-natural phenol derivatives as substrates [58,76,81] . Although in mammalian cells no specific treatment is used for biotin-phenol incorporation, it has been reported that cell permeability can limit biotin-phenol incorporation in yeast, preventing efficient labeling [69] . In E. coli, biotin-phenol uptake is in part mediated by the major g-proteobacterium biotin transporter YigM (Santin et al., unpublished data). Therefore, biotin-phenol incorporation requires optimization for each organism. Finally, as non-natural compounds, whether phenol derivatives affect cell growth needs to be examined for each case. During labeling, H2O2 treatment could affect the general cell oxidative state and cause cellular stress. Hence, APEX assay is perhaps not suited for studies applied to oxidative stress or protein repair. In PUP-IT assay, as Pup(E) derivative-peptides are not commercially available [92] , purification of these substrates for extracellular labeling could be a limitation. However, Pup(E) substrates can be genetically expressed in cells, and hence PUP-IT assay might be useful in organisms in which substrate uptake is limiting.

To perform efficient in vivo labeling, the temperature in which the tagging-enzyme is active has to fit with the growth temperature of the studied organism. In contrast to the BioID protein that has an optimal temperature of 37°C [34,38] , BioID2 protein has optimal activity of 50°C, yielding this enzyme well suitable for labeling in thermophilic conditions [38] . Whereas BioID2 retains highly efficient biotinylation at 37°C, both BioID and BioID2 proteins exhibit reduced activity below 37ºC [38] . APEX2, miniTurbo and TurboID proteins have an optimal activity at 37°C. Because these proteins were evolved from directed evolution in yeast (growing at 30°C), they retain high activity at 30°C [40,61] . In addition, miniTurbo and TurboID have been successfully used in worms and flies which grow at 20°C and 25°C, respectively. While PafA has only been tested in mammalian cells [92] (37°C), the range of temperatures in which it retains its activity has to be determined.

An important aspect of the proximity labeling is the temporal resolution of the reaction, both in the control of the labeling initiation and in the labeling time. For example, definition of signaling networks upon ligand-induced activation requires high temporal resolution [71,72] in contrast to studies about proteome definition in specific subcellular locations [33,41,44,58,60,65,67] .

In BioID and (mini)TurboID assays, the enzyme is active just upon translation and can use endogenous biotin and ATP for proximity labeling, even before correct localization of the fusion protein [34,40] . In the PUP-IT assay, even though Pup(E) expression might be monitored, invariable delay exists between induction and PafA mediated-pupylation [92] . Hence, the initiation of the labeling process may not be precisely controlled in these approaches. Finally, as APEX-proximity labeling is nearly instant [58,60,61] , temporal control of the labeling can be achieved by addition of H2O2 in the culture at specific time points. APEX assay is therefore perfectly suited for taking snapshots of PPIs, for example in cellular response upon environmental changes [71,72] or during assembly of macromolecular complexes [83] . With slow tagging kinetics, the BioID and PUP-IT assays require 16-18 hours or 24-36 hours of labeling time for efficient biotinylation/pupylation, respectively [34,92] . These approaches are hence not suited for capturing transient protein interactions but rather provide a history of protein associations over long periods of time, as used in cell cycle [48][49][50] or in viral infection [51] .

TurboID and miniTurbo were designed to improve BioID assay by reducing the labeling time from 16-18 hours to 10 minutes [40] . However, recent studies showed that this time might be adapted depending on the organism studied. While 10 min are sufficient in mammalian cells [40] , TurboID-mediated biotinylation requires about 30 minutes to 3 hours in yeast [100] , 4 hours in flies [40] , 4 hours to several days in worms [40] and 12 hours in plants [101] . Optimization of the TurboID labeling time should therefore be done for each organism, especially to prevent toxicity via chronic endogenous biotinylation or endogenous biotin consumption due to the high activity of these enzymes [40] .

Finally, BioID and (mini)TurboID assays have never been tested for extracellular labeling by contrast to APEX [102] and PUP-IT [92] . Nevertheless, as PUP-IT and BioID-derivative assays use a similar combination of ATP and substrates (Pup(E) and biotin, respectively), there is no reason why BioID and (mini)TurboID should not work for extracellular labeling.

Conclusions and prospects

In molecular biology, the major question is: who acts in that process? To address this question, many PPI-assaying methodologies have been designed for decades. However, technical limitations in detection of PPIs in living cells, such as weak, transient or hydrophobic interactions, prevent global comprehension. By providing both in vivo spatiotemporal information and high-throughput analysis, proximity-tagging methods represent the next generation of in vivo PPIs assay. In addition to the major proximity-tagging methods presented in this review, other technologies exist including for example horseradish peroxidasedependent labeling approaches (EMARS [103,104] and SPPLAT [105][106][107] ), photoactivated in vivo proximity labeling [108] , NEDDylator system [109,110] and methylation-based chromatin profiling approach DamID [111,112] . While proximity-tagging methods have been used in a variety of biological contexts, further challenges remain to be overcome. For example, in thermophilic organisms, such as archaea, where tools available for PPIs assay are limited [113] , or in proteinlipid interactions, involved in many diseases including cancer, obesity, neurodegenerative disorders or cardiovascular pathologies [114] . However, over the last decade, the rapid development of these proximity-tagging methods, in specificity and spatio-temporal control, has revealed their potential to answer deeply questions in molecular biology. One may assume that other applications will be made in the near future to further unravel the molecular complexity in living cells.

Legend to Figures

Figure 1 .

 1 Figure 1. Schematic representation of conventional methods used for detection of PPIs. a) Co-

Figure 2 .

 2 Figure 2. BioID workflow. Fusion between BioID protein (blue) and a bait protein (black) is

Figure 3 .

 3 Figure 3. APEX proximity-dependent biotin labeling workflow. Fusion between APEX2

Figure 4 .

 4 Figure 4. PUP-IT workflow for intra and extracellular labeling. a) For intracellular labeling,

Figure 5 .

 5 Figure 5. Schematic pipelines for major quantitative approaches used for proximity-tagging

  Figure 1.

Figure 3 .

 3 Figure 3.

  Figure 5.

Table 1 .

 1 Comparative table of the main features of the different proximity-tagging methods

		BioID	(mini)TurboID	APEX	PUP-IT
	Origin	Bacteria	Bacteria	Plantae (APX from	Bacteria
		(BirA from	(BirA from	Glycine max)	(PafA from
		Escherichia	Escherichia coli)		Corynebacterium
		coli)			glutamicum)
	Molecular mass 35 kDa (for	28 kDa (for	27 kDa	54 kDa
		BioID)	miniTurbo)
		27 kDa (for	35 kDa (for
		BioID2)	TurboID)
	Substrates	Biotin and ATP Biotin and ATP	H2O2 and phenol	Pup(E)
				derivative-	derivative-
				molecules	peptides and ATP
	Range of	37°C for BioID	20°C to 37°C	30°C to 37°C	37°C
	temperature a	and 37°C to
		50°C for BioID2
	Enzyme	+	+++	++++	++
	activity b (at
	37°C)
	Time of labeling 16-18 hours	10 min to several	1 min	24-36 hours
			hours (depending
			on the organism)
	Labeling radius 10-15 nm	10-15 nm	20 nm	Very close
					contact
	Labeled	Proteins	Proteins	Proteins and	Proteins
	macromolecules			nucleic acids
	Labeled residues Lys	Lys	Tyr (98%) and	Lys
				Trp, His, Cys (2%)
				for proteins;
				mostly guanosine
				for nucleic acids
	Temporal	No	No	Yes	No
	control (labeling
	initiation)
	Information	Long history	Intermediate	Snapshot	Long history
	about the POI

a Enzyme works at these temperatures but temperatures between this range have not necessary been tested b ++++, strong activity; +++, very good activity; ++, good activity; +, weak activity

Acknowledgments

I would like to thank Eric Cascales for support, Dukas Jurėnas for language editing, Etienne Vanlioglu for discussions on mass spectrometry analysis, Camille Laberthonnière for helpful advices, the members of the Cascales research group for useful discussions, and Yamamoto Kadèrapé for encouragements. Research in Cascales laboratory is supported by the Centre National de la Recherche Scientifique, the Aix-Marseille Université, and grants from the Agence Nationale de la Recherche (ANR-14-CE14-0006-02, ANR-17-CE11-0039-01), the Fondation pour la Recherche Médicale (DEQ20180339165), and the Fondation Bettencourt-Schueller. My PhD work is supported by a doctoral fellowship from the French ministry of higher education and research. All the figures were created with BioRender.

Abbreviations

PPI, protein-protein interaction; POI, protein of interest; Co-IP, Co-Immunoprecipitation; TAP, Tandem Affinity Purification; BACTH, bacterial adenylate cyclase-based two-hybrid; Y2H, yeast two-hybrid assay; FRET, Förster resonance energy transfer; APEX, APEX proximitydependent biotin labeling BioID, proximity-dependent biotin identification; PUP-IT, pupylation-based interaction tagging; SILAC, stable isotope labeling by amino acids in cell culture; TMT, tandem mass tagging; LFQ, label-free quantitation.