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Supplementary Figures. Fig.S1: Additional confocal images showing that expression of EFA6Bvsvg in MCF7 cells restores normal apico-basal polarity and promotes lumen formation Fig.S2: Analysis of the specificity of EFA6B repression using siRNAs Fig.S3: Additional confocal images showing that repression of EFA6B in MCF7 cells abrogates the assembly of TJ and leads to unorganized aggregates Fig.S4: Exogenous expression of EFA6Bvsvg abrogates the TGFβ-induced EMT in MDCK cells Fig.S5: Analysis of EFA6B/PSD4 mutations and copy number alterations from the cBioPortal website

Golgi lipid environment regulates sorting and cargo secretion. However, the mechanisms that spatiotemporally control the lipid composition of the secretory membranes to drive cargo trafficking are poorly understood. Lipid transfer proteins regulate the concentration of specific lipids at membrane contact sites. We hypothesised that by catalysing cholesterol/PI(4)P exchange at ER-trans-Golgi membrane contact sites the lipid transfer protein oxysterol binding protein (OSBP) affects the secretion of a subset of cargoes. Here, we report that OSBP is a major epithelial protein as its inhibition leads to complete loss of apico-basal polarity. By mapping the OSBP proximity proteome with the biotin ligase TurboID, we found that OSBP controls the secretion of multiple membrane associated proteins, including key polarity determinants such as E-cadherin. Mechanistically, we established that OSBP contributes to E-cadherin secretion by supplying cholesterol to post-Golgi membranes. Importantly, when cells downregulate cell-cell junctions upon epithelial-to-mesenchymal transition, they re-wire their lipid homeostasis and downregulate OSBP as well, thus altering the trafficking of the OSBP-dependent secretory cargoes.

Wnt signalling is a core pathway involved in a wide range of developmental processes throughout the metazoa. In vitro studies have suggested that the small GTP binding protein Arf6 regulates upstream steps of Wnt transduction, by promoting the phosphorylation of the Wnt co-receptor, LRP6, and the release of β-catenin from the adherens junctions. To assess the relevance of these previous findings in vivo, we analysed the consequence of the absence of Arf6 activity on Drosophila wing patterning, a developmental model of Wnt/Wingless signalling. We observed a dominant loss of wing margin bristles and Senseless expression in Arf6 mutant flies, phenotypes characteristic of a defect in high level Wingless signalling. In contrast to previous findings, we show that Arf6 is required downstream of Armadillo/β-catenin stabilisation in Wingless signal transduction. Our data suggest that Arf6 modulates the activity of a downstream nuclear regulator of Pangolin activity in order to control the induction of high level Wingless signalling. Our findings represent a novel regulatory role for Arf6 in Wingless signalling., Summary: Arf6 is necessary for the patterning of the wing margin by Wingless in Drosophila melanogaster, acting downstream of stabilized Armadillo but upstream or at the level of Pangolin activity.

Wnt signalling is a core pathway involved in a wide range of developmental processes throughout the metazoa.In vitrostudies have suggested that the small GTP binding protein Arf6 regulates upstream steps of Wnt transduction, by promoting the phosphorylation of the Wnt co-receptor, LRP6, and the release of β-catenin from the adherens junctions. To assess the relevance of these previous findingsin vivo, we analyse the consequence of the absence of Arf6 activity onDrosophilawing patterning, a developmental model of Wnt/Wingless signalling. We observed a dominant loss of wing margin bristles and Senseless expression in Arf6 mutant flies, phenotypes characteristic of a defect in high level Wingless signalling. In contrast to previous findings, we show that Arf6 is required downstream of Armadillo/β-catenin stabilisation in Wingless signal transduction. Our data suggest that Arf6 modulates the activity of a downstream nuclear regulator of Pangolin activity in order to control the induction of high level Wingless signalling. Our findings represent a novel regulatory role for Arf6 in Wingless signalling.

Cancer is initiated by somatic mutations in oncogenes or tumor suppressor genes. However, additional alterations provide selective advantages to the tumor cells to resist treatment and develop metastases. Their identification is of paramount importance. Reduced expression of EFA6B (Exchange Factor for ARF6, B) is associated with breast cancer of poor prognosis. Here, we report that loss of EFA6B triggers a transcriptional reprogramming of the cell-to-ECM interaction machinery and unleashes CDC42-dependent collective invasion in collagen. In xenograft experiments, MCF10 DCIS.com cells, a DCIS-to-IDC transition model, invades faster when knocked-out for EFA6B. In addition, invasive and metastatic tumors isolated from patients have lower expression of EFA6B and display gene ontology signatures identical to those of EFA6B knock-out cells. Thus, we reveal an EFA6B-regulated molecular mechanism that controls the invasive potential of mammary cells; this finding opens up avenues for the treatment of invasive breast cancer., Reduced expression of EFA6B is found in highly metastatic breast cancer subtypes. Here, the authors report that loss of EFA6B promotes collective invasion through activation of the epithelial-to-mesenchymal transition program and CDC42-dependent signalling pathways in breast cancer.

International audience; Breast cancer is a major disease for women worldwide, where mortality is associated with tumour cell dissemination to distant organs. While the number of efficient anticancer therapies increased in the past 20 years, treatments targeting the invasive properties of metastatic tumour cells are still awaited. Various studies analysing invasive breast cancer cell lines have demonstrated that Arf6 is an important player of the migratory and invasive processes. These observations make Arf6 and its regulators potential therapeutic targets. As of today, no drug effective against Arf6 has been identified, with one explanation being that the activation of Arf6 is dependent on the presence of lipid membranes that are rarely included in drug screening. To overcome this issue we have set up a fluorescence-based high throughput screening that follows overtime the activation of Arf6 at the surface of lipid membranes. Using this unique screening assay, we isolated several compounds that affect Arf6 activation, among which the antibiotic chlortetracycline (CTC) appeared to be the most promising. In this report, we describe CTC in vitro biochemical characterization and show that it blocks both the Arf6-stimulated collective migration and cell invasion in a 3D collagen I gel of the invasive breast cancer cell line MDA-MB-231. Thus, CTC appears as a promising hit to target deadly metastatic dissemination and a powerful tool to unravel the molecular mechanisms of Arf6-mediated invasive processes.

Ciliogenesis is a coordinated process initiated by the recruitment and fusion of pre-ciliary vesicles at the distal appendages of the mother centriole through mechanisms that remain unclear. Here, we report that EFA6A (also known as PSD), an exchange factor for the small G protein Arf6, is involved in early stage of ciliogenesis by promoting the fusion of distal appendage vesicles forming the ciliary vesicle. EFA6A is present in the vicinity of the mother centriole before primary cilium assembly and prior to the arrival of Arl13B-containing vesicles. During ciliogenesis, EFA6A initially accumulates at the mother centriole and later colocalizes with Arl13B along the ciliary membrane. EFA6A depletion leads to the inhibition of ciliogenesis, the absence of centrosomal Rab8-positive structures and the accumulation of Arl13B-positive vesicles around the distal appendages. Our results uncover a novel fusion machinery, comprising EFA6A, Arf6 and Arl13B, that controls the coordinated fusion of ciliary vesicles docked at the distal appendages of the mother centriole.

The Arf6-specific exchange factor EFA6 is involved in the endocytic/recycling pathway for different cargos. In addition EFA6 acts as a powerful actin cytoskeleton organizer, a function required for its role in the establishment of the epithelial cell polarity and in neuronal morphogenesis. We previously showed that the C-terminus of EFA6 (EFA6-Ct) is the main domain which contributes to actin reorganization. Here, by in vitro and in vivo experiments, we sought to decipher, at the molecular level, how EFA6 controls the dynamic and structuring of actin filaments. We showed that EFA6-Ct interferes with actin polymerization by interacting with and capping actin filament barbed ends. Further, in the presence of actin mono-filaments, the addition of EFA6-Ct triggered the formation of actin bundles. In cells, when the EFA6-Ct was directed to the plasma membrane, as is the case for the full-length protein, its expression induced the formation of membrane protrusions enriched in actin cables. Collectively our data explain, at least in part, how EFA6 plays an essential role in actin organization by interacting with and bundling F-actin.

A key step of epithelial morphogenesis is the creation of the lumen. Luminogenesis by hollowing proceeds through the fusion of apical vesicles at cell-cell contacts. The small nascent lumens grow through extension, coalescence and enlargement, coordinated with cell division, to give rise to a single central lumen. Here, by using MDCK cells grown in 3D-culture, we show that EFA6A (also known as PSD) participates in luminogenesis. EFA6A recruits α-actinin 1 (ACTN1) through direct binding. In polarized cells, ACTN1 was found to be enriched at the tight junction where it acts as a primary effector of EFA6A for normal luminogenesis. Both proteins are essential for the lumen extension and enlargement, where they mediate their effect by regulating the cortical acto-myosin contractility. Finally, ACTN1 was also found to act as an effector for the isoform EFA6B (also known as PSD4) in the human mammary tumoral MCF7 cell line. EFA6B restored the glandular morphology of this tumoral cell line in an ACTN1-dependent manner. Thus, we identified new regulators of cyst luminogenesis essential for the proper maturation of a newly-formed lumen into a single central lumen.

A key step of epithelial morphogenesis is the creation of the lumen. Luminogenesis by hollowing proceeds through the fusion of apical vesicles at cell-cell contacts. The small nascent lumens grow through extension, coalescence and enlargement, coordinated with cell division, to give rise to a single central lumen. Here, by using MDCK cells grown in 3D-culture, we show that EFA6A (also known as PSD) participates in luminogenesis. EFA6A recruits α-actinin 1 (ACTN1) through direct binding. In polarized cells, ACTN1 was found to be enriched at the tight junction where it acts as a primary effector of EFA6A for normal luminogenesis. Both proteins are essential for the lumen extension and enlargement, where they mediate their effect by regulating the cortical acto-myosin contractility. Finally, ACTN1 was also found to act as an effector for the isoform EFA6B (also known as PSD4) in the human mammary tumoral MCF7 cell line. EFA6B restored the glandular morphology of this tumoral cell line in an ACTN1-dependent manner. Thus, we identified new regulators of cyst luminogenesis essential for the proper maturation of a newly-formed lumen into a single central lumen.

International audience; Polymeric immunoglobulin A (pIgA) transcytosis, mediated by the polymeric immunoglobulin receptor (pIgR), is a central component of mucosal immunity and a model for regulation of polarized epithelial membrane traffic. Binding of pIgA to pIgR stimulates transcytosis in a process requiring Yes, a Src family tyrosine kinase (SFK). We show that Yes directly phosphorylates EGF receptor (EGFR) on liver endosomes. Injection of pIgA into rats induced EGFR phosphorylation. Similarly, in MDCK cells, pIgA treatment significantly increased phosphorylation of EGFR on various sites, subsequently activating extracellular signal-regulated protein kinase (ERK). Furthermore, we find that the Rab11 effector Rab11-FIP5 is a substrate of ERK. Knocking down Yes or Rab11-FIP5, or inhibition of the Yes-EGFR-ERK cascade, decreased pIgA-pIgR transcytosis. Finally, we demonstrate that Rab11-FIP5 phosphorylation by ERK controls Rab11a endosome distribution and pIgA-pIgR transcytosis. Our results reveal a novel Yes-EGFR-ERK-FIP5 signalling network for regulation of pIgA-pIgR transcytosis.

We have previously reported that EFA6, exchange factor for Arf6, is implicated upon E-cadherin engagement in the process of epithelial cell polarization. We had found that EFA6 acts through stabilization of the apical actin ring onto which the tight junction is anchored. Mutagenesis experiments showed that both the catalytic domain of EFA6 and its C-terminal domain were required for full EFA6 function. Here we address the contribution of the specific substrate of EFA6, the small G protein Arf6. Unexpectedly, depletion of Arf6 by RNA interference or expression of the constitutively active fast-cycling mutant (Arf6T157N) revealed that Arf6 plays an opposing role to EFA6 by destabilizing the apical actin cytoskeleton and the associated tight junction. However, in complementation experiments, when the C-terminal domain of EFA6 is co-expressed with Arf6T157N, it reverts the effects of Arf6T157N expressed alone to faithfully mimic the phenotypes induced by EFA6. In addition, we find that the two signaling pathways downstream of EFA6, i.e. the one originating from the activated Arf6GTP and the other one from the EFA6 C-terminal domain, need to be tightly balanced to promote the proper reorganization of the actin cytoskeleton. Altogether, our results indicate that to regulate the tight junction, EFA6 activates Arf6 through its Sec7 catalytic domain as it modulates this activity through its C-terminal domain.

Summary The spread of diseases transmitted by Anopheles and Culex mosquitoes, such as malaria and West Nile fever, is a growing concern for human health. Bacillus sphaericus binary toxin (Bin) is one of the few avail- able bioinsecticides able to control populations of these mosquitoes efficiently. We previously showed that Bin binds to Cpm1, an a -glucosidase located on the apical side of Culex larval midgut epithelium. We analysed the effects of Bin by expressing a construct encoding Cpm1 in the mammalian epithelial MDCK cell line. Cpm1 is targeted to the apical side of polar- ized MDCK, where it is anchored by glycosylphos- phatidylinositol (GPI) and displays a a a -glucosidase activity. Bin bound to transfected cells and induced a non-specific current presumably related to the open- ing of pores. The formation of these pores may be related to the location of the toxin/receptor complex in lipid raft microdomains. Finally, Bin promoted the time-dependent appearance of intracytoplasmic vac- uoles but did not drive cell lysis. Thus, the dual func- tionality (enzyme/toxin receptor) of Cpm1 is fully conserved in MDCK cells and Cpm1 is an essential target protein for Bin cytotoxicity in Culex mosquitoes.

One of the earliest events in epithelial carcinogenesis is the dissolution of tight junctions and cell polarity signals that are essential for normal epithelial barrier function. Here, we report that EFA6B, a guanine nucleotide exchange factor for the Ras superfamily protein Arf6 that helps assemble and stabilize tight junction, is required to maintain apico-basal cell polarity and mesenchymal phenotypes in mammary epithelial cells. In organotypic three-dimensional cell cultures, endogenous levels of EFA6B were critical to determine epithelial–mesenchymal status. EFA6B downregulation correlated with a mesenchymal phenotype and ectopic expression of EFA6B hampered TGFβ-induced epithelial-to-mesenchymal transition (EMT). Transcriptomic and immunohistochemical analyses of human breast tumors revealed that the reduced expression of EFA6B was associated with loss of tight junction components and with increased signatures of EMT, cancer stemness, and poor prognosis. Accordingly, tumors with low levels of EFA6B were enriched in the aggressive triple-negative and claudin-low breast cancer subtypes. Our results identify EFA6B as a novel antagonist in breast cancer and they point to its regulatory and signaling pathways as rational therapeutic targets in aggressive forms of this disease. Cancer Res; 74(19); 5493–506. ©2014 AACR.

International audience; Members of the Arf family of small G proteins are involved in membrane traffic and organelle structure. They control the recruitment of coat proteins, and modulate the structure of actin filaments and the lipid composition of membranes. The ADP-ribosylation factor 6 (Arf6) isoform and the exchange factor for Arf6 (EFA6) are known to regulate the endocytic pathway of many different receptors. To determine the molecular mechanism of the EFA6/Arf6 function in vesicular transport, we searched for new EFA6 partners. In a two-hybrid screening using the catalytic Sec7 domain as a bait, we identified endophilin as a new partner of EFA6. Endophilin contains a Bin/Amphiphysin/Rvs (BAR) domain responsible for membrane bending, and an SH3 domain responsible for the recruitment of dynamin and synaptojanin, two proteins involved, respectively, in the fission and uncoating of clathrin-coated vesicles. By using purified proteins, we confirmed the direct interaction, and identified the N-BAR domain as the binding motif to EFA6A. We showed that endophilin stimulates the catalytic activity of EFA6A on Arf6. In addition, we observed that the Sec7 domain competes with flat but not with highly curved lipid membranes to bind the N-BAR. In cells, expression of EFA6A recruits endophilin to EFA6A-positive plasma membrane ruffles, whereas expression of endophilin rescues the EFA6A-mediated inhibition of transferrin internalization. Overall, our results support a model whereby EFA6 recruits endophilin on flat areas of the plasma membrane to control Arf6 activation and clathrin-mediated endocytosis.

Transcytosis of polymeric immunoglobulin A (pIgA) across epithelial cells is mediated by the polymeric immunoglobulin receptor (pIgR). Binding of pIgA to pIgR stimulates transcytosis of the pIgA-pIgR complex via a signal transduction pathway that is dependent on a protein tyrosine kinase (PTK) of the SRC family. Here we identify the PTK as p62(yes). We demonstrate the specific physical and functional association of the pIgR with p62(yes) in rodent liver. Analysis of p62(yes) knockout mice revealed a dramatic reduction in the association of tyrosine kinase activity with the pIgR and in transcytosis of pIgA. We conclude that p62(yes) controls pIgA transcytosis in vivo.

The polymeric Ig receptor (pIgR) transcytoses its ligand, dimeric IgA (dIgA), from the basolateral to the apical surface of epithelial cells. Although the pIgR is constitutively transcytosed in the absence of ligand, binding of dIgA stimulates transcytosis of the pIgR. We recently reported that dIgA binding to the pIgR induces translocation of protein kinase C, production of inositol triphosphate, and elevation of intracellular free calcium. We now report that dIgA binding causes rapid, transient tyrosine phosphorylation of several proteins, including phosphatidyl inositol-specific phospholipase C-γl. Protein tyrosine kinase inhibitors or deletion of the last 30 amino acids of pIgR cytoplasmic tail prevents IgA-stimulated protein tyrosine kinase activation, tyrosine phosphorylation of phospholipase C-γl, production of inositol triphosphate, and the stimulation of transcytosis by dIgA. Analysis of pIgR deletion mutants reveals that the same discrete portion of the cytoplasmic domain, residues 727–736 (but not the Tyr734), controls both the ability of pIgR to cause dIgA-induced tyrosine phosphorylation of the phospholipase C-γl and to undergo dIgA-stimulated transcytosis. In addition, dIgA transcytosis can be strongly stimulated by mimicking phospholipase C-γl activation. In combination with our previous results, we conclude that the protein tyrosine kinase(s) and phospholipase C-γl that are activated upon dIgA binding to the pIgR control dIgA-stimulated pIgR transcytosis.

β2-adrenergic receptor (β2AR), a member of the GPCR (G-Proteins Coupled Receptor) family, is internalized in a ligand- and β-arrestin-dependent manner into early endosomes, and subsequently recycled back to the plasma membrane. Here we report that β-arrestin promotes the activation of the small G protein Arf6, which regulates the recycling and degradation of β2AR. We demonstrate in vitro that the C-terminal region of β-arrestin1 interacted directly and simultaneously with Arf6GDP and its specific exchange factor EFA6, to promote Arf6 activation. Similarly, the ligand-mediated activation of β2AR leads to the formation of Arf6GTP in vivo in a β-arrestin-dependent manner. Expression of either EFA6 or an activated Arf6 mutant caused accumulation of β2AR in the degradation pathway. And this phenotype could be rescued by the expression of an activated mutant of Rab4, suggesting that Arf6 acts upstream of Rab4. We propose a model in which Arf6 plays an essential role for the β2AR desensitization. The ligand-mediated stimulation of β2AR relocates β-arrestin to the plasma membrane, and triggers the activation of Arf6 by EFA6. The activated Arf6 leads to accumulation of β2AR to the degradation pathway, and negatively controls the Rab4-dependent fast recycling to prevent the re-sensitization of β2AR.

International audience; In epithelial cells, the tight junction (TJ) functions as a permeability barrier and is involved in cellular differentiation and proliferation. Although many TJ proteins have been characterized, little is known about the sequence of events and temporal regulation of TJ assembly in response to adhesion cues. We report here that the deubiquitinating enzyme USP9x has a critical function in TJ biogenesis by controlling the levels of the exchange factor for Arf6 (EFA6), a protein shown to facilitate TJ formation, during a narrow temporal window preceding the establishment of cell polarity. At steady state, EFA6 is constitutively ubiquitinated and turned over by the proteasome. However, at newly forming contacts, USP9x-mediated deubiquitination protects EFA6 from proteasomal degradation, leading to a transient increase in EFA6 levels. Consistent with this model, USP9x and EFA6 transiently co-localize at primordial epithelial junctions. Furthermore, knockdown of either EFA6 or USP9x impairs TJ biogenesis and EFA6 overexpression rescues TJ biogenesis in USP9x-knockdown cells. As the loss of cell polarity is a critical event in the metastatic spread of cancer, these findings may help to understand the pathology of human carcinomas.

International audience; Polarized epithelial cells contain apical and basolateral surfaces with distinct protein compositions. To establish and maintain this asymmetry, newly made plasma membrane proteins are sorted in the trans Golgi network for delivery to apical or basolateral surfaces. Signals for basolateral sorting are generally located in the cytoplasmic domain of the protein, whereas signals for apical sorting can be in any part of the protein and can depend on N-linked glycosylation of the protein. Signals for constitutive transcytosis to the apical surface have not been reported. In this study, we used the polymeric immunoglobulin receptor (pIgR), which is biosynthetically delivered to the basolateral surface. There the pIgR can bind a ligand and, with or without bound ligand, the pIgR can then be transcytosed to the apical surface. We found that the glycosylation of the pIgR did not affect the biosynthetic transport of the pIgR. However, glycosylation had an effect on pIgR apical transcytosis. Importantly, analysis of the cytoplasmic tail of the pIgR suggested that a short peptide segment was sufficient to transcytose the pIgR or a neutral reporter from the basolateral to the apical surface. This apical transcytosis sorting signal was not involved in polarized biosynthetic traffic of the pIgR.

The Arf6-specific exchange factor EFA6 coordinates membrane trafficking with actin cytoskeleton remodeling. It localizes to the plasma membrane where it catalyzes Arf6 activation and induces the formation of actin-based membrane ruffles. We have shown previously that the pleckstrin homology (PH) domain of EFA6 was responsible for its membrane localization. In this study we looked for the partners of the PH domain at the plasma membrane. Mutations of the conserved basic residues suspected to be involved in the binding to phosphoinositides redistribute EFA6-PH to the cytosol. In addition, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) breakdown also leads to the solubilization of EFA6-PH. Direct binding measured by surface plasmon resonance gives an apparent affinity of approximately 0.5 microm EFA6-PH for PI(4,5)P2. Moreover, we observed in vitro that the catalytic activity of EFA6 is strongly increased by PI(4,5)P2. These results indicate that the plasma membrane localization of EFA6-PH is based on its interaction with PI(4,5)P2, and this interaction is necessary for an optimal catalytic activity of EFA6. Furthermore, we demonstrated by fluorescence recovery after photobleaching and Triton X-100 detergent solubility experiments that in addition to the phophoinositides, EFA6-PH is linked to the actin cytoskeleton. We observed both in vivo and in vitro that EFA6-PH interacts directly with F-actin. Finally, we demonstrated that EFA6 could bind simultaneously filamentous actin and phospholipids vesicles. Our results explain how the exchange factor EFA6 via its PH domain could coordinate at the plasma membrane actin cytoskeleton organization with membrane trafficking.

Aluminum fluoride (AlFx) is known to activate directly the α subunit of G-proteins but not the homologous small GTP-binding proteins. However, AlFx can stabilize complexes formed between Ras, RhoA or Cdc42 and their corresponding GTPase-activating proteins (GAPs). Here, we demonstrate that Arf1GDP can be converted into an active conformation by AlFx to form a complex with the Arf-GAP ASAP1 in vitro and in vivo. Within this complex ASAP1, which GAP activity is inoperative, can still alter the recruitment of paxillin to the focal complexes, thus indicating that ASAP1 interferes with focal complexes independently of its GAP activity.

Integrin cytoplasmic domain-associated protein 1 (ICAP-1) has been shown to interact specifically with the β1 integrin cytoplasmic domain and to control cell spreading on fibronectin. Interestingly, ICAP-1 also is observed in the nucleus, by immunocytochemical staining, and after biochemical cell fractionation, suggesting that it has additional roles that have yet to be determined. We show that the nucleocytoplasmic shuttling capability of ICAP-1 is dependent on a functional nuclear localization signal. In addition, overexpression of β1 integrin strongly reduced this nuclear localization, suggesting that integrin activity could modulate ICAP-1 shuttling by sequestering it in the cytoplasm. Indeed, the nuclear localization of ICAP-1 is dependent on the stage of cell spreading on fibronectin, and we also show that ICAP-1 expression stimulates cellular proliferation in a fibronectin-dependent manner. This function is dependent on its nuclear localization. Moreover, ICAP-1 is able to activate the c-myc promoter in vitro. Together, these results demonstrate that ICAP-1 shuttles between the nucleus and cytoplasm in a β1 integrin-dependent manner. It could act as a messenger that relays information from sites of integrin-dependent cell adhesion to the nucleus for controlling gene expression and cell proliferation.

In polarized epithelial cells, the tight junction has been ascribed several functions including the regulation of the paracellular permeability, an impediment to the diffusion of molecules between the apical and basolateral domains, a site of delivery of transport vesicles for basolateral proteins, and a scaffold for structural and signaling molecules. The tight junction is anchored physically into the apical actin cytoskeleton circumscribing the cell, which is known as the perijunctional actomyosin ring. This connection was first suggested by experiments using the actin depolymerizing drug cytochalasin, which was also found to disrupt the transepithelial permeability. Since then a large number of studies have reported the effects of drugs, molecular tools, or physiological and pathological conditions that alter coordinately actin organization and the tight junction. In support of this model, proteins of the tight junction, such as the members of the ZO family and occludin, have been shown to bind to actin. However, very little is known regarding the molecular mechanisms by which the actin cytoskeleton modulates tight junction functions. We have studied the role of the Exchange Factor for Arf6, EFA6, in tight junction assembly. By combining a large panel of methods, including morphological, physiological, and biochemical, described in detail hereafter we demonstrated that EFA6 plays a role in the physical association of the tight junction to the perijunctional actomyosin ring.

The small GTP-binding protein ADP-ribosylation factor 6 (Arf6) is involved in plasma membrane/endosomes trafficking. However, precisely how the activation of Arf6 regulates vesicular transport is still unclear. Here, we show that, in vitro, recombinant Arf6GTP recruits purified clathrin-adaptor complex AP-2 (but not AP-1) onto phospholipid liposomes in the absence of phosphoinositides. We also show that phosphoinositides and Arf6 tightly cooperate to translocate AP-2 to the membrane. In vivo, Arf6GTP (but not Arf6GDP) was found associated to AP-2. The expression of the GTP-locked mutant of Arf6 leads to the plasma membrane redistribution of AP-2 in Arf6GTP-enriched areas. Finally, we demonstrated that the expression of the GTP-locked mutant of Arf6 inhibits transferrin receptor internalization without affecting its recycling. Altogether, our results demonstrated that Arf6GTP interacts specifically with AP-2 and promotes its membrane recruitment. These findings strongly suggest that Arf6 plays a major role in clathrin-mediated endocytosis by directly controlling the assembly of the AP-2/clathrin coat.

The function of Arf6 has been investigated largely by using the T27N and the Q67L mutants, which are thought to be blocked in GDP- and GTP-bound states, respectively. However, these mutants have been poorly characterized biochemically. Here, we found that Arf6(T27N) is not an appropriate marker of the inactive GDP-bound form because it has a high tendency to lose its nucleotide in vitro and to denature. As a consequence, most of the protein is aggregated in vivo and localizes to detergent-insoluble structures. However, a small proportion of Arf6(T27N) is able to form a stable complex with its exchange factor EFA6 at the plasma membrane, accounting for its dominant-negative phenotype. To define the cellular localization of Arf6-GDP, we designed a new mutant, Arf6(T44N). In vitro, this mutant has a 30-fold decreased affinity for GTP. In vivo, it is mostly GDP bound and, in contrast to the wild type, does not switch to the active conformation when expressed with EFA6. This GDP-locked mutant is found at the plasma membrane, where it localizes with EFA6 and Ezrin in actin- and phosphatidylinositol (4,5)-bisphosphate-enriched domains. From these results, we conclude that the Arf6 GDP-GTP cycle takes place at the plasma membrane.

Epithelial cells have separate apical and basolateral plasma membrane domains with distinct compositions. After delivery to one surface, proteins can be endocytosed and then recycled, degraded or transcytosed to the opposite surface. Proper sorting into the transcytotic pathway is essential for maintaining polarity, as most proteins are endocytosed many times during their lifespan. The polymeric immunoglobulin receptor (pIgR) transcytoses polymeric IgA (pIgA) from the basolateral to the apical surface of epithelial cells and hepatocytes. However, the molecular machinery that controls polarized sorting of pIgR-pIgA and other receptors is only partially understood. The retromer is a multimeric protein complex, originally described in yeast, which mediates intracellular sorting of Vps10p, a receptor that transports vacuolar enzymes. The yeast retromer contains two sub-complexes. One includes the Vps5p and Vps17p subunits, which provide mechanical force for vesicle budding. The other is the Vps35p-Vps29p-Vps26p subcomplex, which provides cargo specificity. The mammalian retromer binds to the mannose 6-phosphate receptor, which sorts lysosomal enzymes from the trans-Golgi network to the lysosomal pathway. Here, we show a function for the mammalian Vps35-Vps29-Vps26 retromer subcomplex in promoting pIgR-pIgA transcytosis.

We addressed the role of EFA6, exchange factor for ARF6, during the development of epithelial cell polarity in Madin-Darby canine kidney cells. EFA6 is located primarily at the apical pole of polarized cells, including the plasma membrane. After calcium-triggered E-cadherin–mediated cell adhesion, EFA6 is recruited to a Triton X-100–insoluble fraction and its protein level is increased concomitantly to the accelerated formation of a functional tight junction (TJ). The expression of EFA6 results in the selective retention at the cell surface of the TJ protein occludin. This effect is due to EFA6 capacities to promote selectively the stability of the apical actin ring onto which the TJ is anchored, resulting in the exclusion of TJ proteins from endocytosis. Finally, our data suggest that EFA6 effects are achieved by the coordinate action of both its exchange activity and its actin remodeling C-terminal domain. We conclude that EFA6 is a signaling molecule that responds to E-cadherin engagement and is involved in TJ formation and stability.

Large clostridial toxins glucosylate some small G proteins on a threonine residue, thereby preventing their interactions with effector molecules and regulators. We show that the glucosyltransferase domain of lethal toxin from Clostridium sordellii (LT(cyt); amino acids 1-546), which is released into the cytosol during cell infection, binds preferentially to liposomes containing phosphatidylserine as compared with other anionic lipids. The binding of LT(cyt) to phosphatidylserine increases by two orders of magnitude the rate of glucosylation of liposome-bound geranyl-geranylated Rac-GDP. Limited proteolysis and deletion studies show that the binding site for phosphatidylserine lies within the first 18 N-terminal residues of LT(cyt). Deletion of these residues abolishes the effect of phosphatidylserine on the activity of LT(cyt) on liposome-bound geranyl-geranylated Rac-GDP and prevents the morphological effects induced by LT(cyt) microinjection into various cells, but it does not affect the intrinsic activity of LT(cyt) on non-geranyl-geranylated Rac-GDP in solution. We conclude that the avidity of LT(cyt) for phosphatidylserine facilitates its targeting to the cytosolic leaflet of cell membranes and, notably, the plasma membrane, where this anionic lipid is abundant and where several targets of lethal toxin reside.

Transcytosis of the polymeric immunoglobulin receptor (pIgR) is stimulated by binding of its ligand, dimeric IgA (dIgA). During this process, dIgA binding at the basolateral surface of the epithelial cell transmits a signal to the apical region of the cell, which in turn stimulates the transport of dIgA–pIgR complex from a postmicrotubule compartment to the apical surface. We have previously reported that the signal of stimulation was controlled by a protein-tyrosine kinase (PTK) activated upon dIgA binding. We now show that this signal of stimulation moves across the cell independently of pIgR movement or microtubules and acts through the tyrosine kinase activity by releasing Ca++from inositol trisphosphate–sensitive intracellular stores. Surprisingly we have found that a second independent signal is required to achieve dIgA-stimulated transcytosis of pIgR. This second signal depends on dIgA binding to the pIgR solely at the basolateral surface and the ability of pIgR to dimerize. This enables pIgR molecules that have bound dIgA at the basolateral surface to respond to the signal of stimulation once they reach the postmicrotubule compartment. We propose that the use of two signals may be a general mechanism by which signaling receptors maintain specificity along their signaling and trafficking pathways.

Primary biliary cirrhosis (PBC) is a chronic autoimmune liver disease of unknown etiology characterized by high-titer anti-mitochondrial antibodies. The major autoantigen has been identified as the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2). The fact that PDC-E2 is present in all nucleated cells, but autoimmune damage is confined to biliary epithelial cells, prompted us to investigate the possibility that mucosally-derived IgA may be pathogenic for biliary epithelial cells. Serum IgA was purified from six patients with PBC and its localization and ability to penetrate cells was studied using Madine–Darby canine kidney (MDCK) cells transfected with the human IgA receptor (MDCK-pIgR). The potential of IgA to be transported through the cells was studied by a combination of immunohistochemistry and dual color fluorescent microscopy. Interestingly, IgA from all PBC patients co-localized with PDC-E2 (the major autoantigen of PBC) inside the cells; this was demonstrated by dual staining with anti-human IgA and a mouse monoclonal antibody directed to PDC-E2. In contrast, no co-localization was observed for IgA controls. Furthermore, dual staining of liver sections from PBC patients demonstrated co-localization of IgA and PDC-E2, both cytoplasmically and at the apical surface. We postulate that there may be a direct effect of these autoantibodies on the mitochondrial function of biliary epithelial cells.

My PhD research project aims to demonstrate a novel negative regulatory pathway of breast cancer tumor invasion, regulated by EFA6B, an exchange factor for Arf6. Breast cancer (BC) is the second leading cause of cancer death in women worldwide. Primary tumor metastasis is responsible for most of the mortality. Despite the benefits of breast cancer targeted therapies there is still no effective treatments against metastases. Cancer initiation is caused by somatic mutations in oncogenes or tumor suppressor genes, but additional mutations can provide selective advantages to the tumor cells to spread into nearby tissue or distant organs and develop metastatic cancers. The identification of pro-invasive factors and the comprehension of their mechanism of action is therefore of paramount importance.My laboratory has recently proposed that EFA6B acts as an antagonist to BC development. They have demonstrated that EFA6 is a tight junction regulator and that its level of expression determines the epithelial status of mammary cells grown in 3D culture. Additionally, they observed that the loss of expression of EFA6B is correlated with a poor prognosis and the metastatic Claudin-low breast cancer subtype that is characterized by the loss of expression of the tight junction components, and transcriptomic signatures of epithelial-to-mesenchymal transition (EMT) and cancer stem cells. In order to identify the molecular mechanisms by which EFA6B acts as an antagonist to the development of BC we invalidated the gene PSD4/EFA6B in normal human mammary cells using the CRISPR/Cas9 technology. We found that EFA6B depletion is sufficient to induce the formation of cellular branched structures in collagen I by collective invasion. My PhD project aimed at describing the cellular and molecular mechanisms that promote invasion of the EFA6B knock-out (EFA6B KO) cells. We demonstrated that the loss of EFA6B induces an up-regulation of EMT transcription factors, leads to the modification of the matrisome and integrin repertoire, and increases contractility. All these results are accompanied by the formation of integrin 1-based invadopodia enriched with the metalloprotease MMP-14 responsible for the degradation of collagen I matrix, hence contributing to collective invasion of the EFA6B KO cells. Furthermore, we demonstrated that the invasion of EFA6B KO cells depends on the activation of the small G protein Cdc42, which in turn elicits two signaling pathways: Cdc42-MRCK-pMLC and Cdc42-N-WASP-Arp2/3 that control cell contractility and invadopodia formation respectively. In addition, invasive and metastatic tumors isolated from patients have lower expression of EFA6B and display gene ontology signatures identical to those of EFA6B knock out-cells. Collectively, our results reveal a new EFA6B-regulated molecular mechanism that controls the invasive capacity of mammary cells and opens up new avenues for the treatment of invasive breast cancer.; Mon projet de recherche a pour but d’identifier et de décrire une nouvelle voie de régulation négative de l’invasion tumorale contrôlée par EFA6B, un facteur d’échange d’Arf6, dans un modèle de cancer du sein. Ce dernier est la seconde cause de décès par cancer chez les femmes dans le monde. Les métastases issues de la tumeur primaires sont responsables de la quasi-totalité des décès. Pourtant, il n’existe toujours pas de traitement qui cible spécifiquement le développement métastatique.L’initiation tumorale est le fait de mutations somatiques dans les oncogènes ou les gènes suppresseurs de tumeur, mais des mutations supplémentaires dans d’autres gènes peuvent procurer des avantages sélectifs aux cellules tumorales pour se disséminer dans les tissus voisins ou dans des organes distants pour y développer des métastases. L’identification d’altérations génétiques et de facteurs pro-invasifs associée à la compréhension de leurs mécanismes d’action est donc cruciale.Mon laboratoire d’accueil a récemment proposé que la protéine EFA6B agit comme un antagoniste au développement de cancers du sein. Ils ont démontré qu’EFA6B est un régulateur des jonctions serrées, et que son niveau d’expression détermine le statut épithélial ou mésenchymal de cellules mammaires cultivées en matrice 3D de collagène de type I. En plus, ils ont observé dans les tumeurs de patientes que la perte d’expression d’EFA6B est corrélée avec un mauvais pronostic, et enrichie dans le sous-type métastatique Claudin-Low qui est caractérisé par la perte d’expression des composants des jonctions serrées, et les signatures transcriptomiques de la transition épithélio-mésenchimateuse (EMT) et des cellules souches cancéreuses.Pour identifier par quels mécanismes EFA6B agit comme un antagoniste au développement du cancer du sein, nous avons invalidé le gène PSD4 codant pour EFA6B dans des cellules mammaires humaines normales en utilisant la technologie CRISPR/Cas9. Nous avons observé que la perte d’EFA6B suffit à induire la formation de structures cellulaires branchées en collagène I par invasion collective. Mon projet de thèse a consisté à décrire les mécanismes moléculaires et cellulaires qui supportent l’invasion des cellules « knock-out » EFA6B (EFA6B KO).Nous avons démontré que la perte d’EFA6B induit une augmentation des facteurs de transcription de l’EMT, conduit à la modification du matrisome et du répertoire des intégrines, et augmente la contractilité. Tous ces changements sont accompagnés par la formation d’invadopodes enrichis en intégrine β1, en métalloprotéase MMP14 responsable de la dégradation de la matrice de collagène I, pour contribuer à l’invasion collective des cellules EFA6B KO. En outre, nous avons démontré que l’invasion des cellules EFA6B KO dépend de l’activation de la petite protéine G Cdc42, qui elle-même recrute deux voies de signalisation : Cdc42-MRCK-pMLC et Cdc42-NWASP-Arp2/3 qui contrôlent respectivement la contractilité cellulaire et la formation des invadopodes. De plus, les tumeurs invasives et les métastases isolées de patientes qui ont une expression réduite d’EFA6B présentent des signatures géniques similaires à celles des lignées cellulaires EFA6B KO. L’ensemble de nos résultats révèle un nouveau mécanisme moléculaire de l’invasion régulé par EFA6B ouvrant de nouvelles perspectives pour chercher des traitements contre les cancers du sein invasif.

Epithelial tissue homeostasis is fundamental for the survival and maintenance of a healthy organism. During mammary gland morphogenesis, epithelial cells communicate with their surrounding stroma in order to orchestrate the formation of a functional organ by undergoing multiple cycles of controlled proliferation, epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition, migration and invasion. In cancer cells, these processes are dysregulated. Thus, understanding how normal mammary cells preserve their homeostasis during development is crucial to identify the molecular events inducing breast cancer. Homeostasis of epithelia relies on key specific features: apico-basal polarity, cell cohesion and lumen formation. Here stands our protein of interest, EFA6B, exchange factor for Arf6, hence my interest in studying its role in mammary epithelial cells. During my PhD work, I investigated the role of EFA6B on epithelial integrity.Using CRISPR/Cas9 EFA6B knock-out cells, I showed that the loss of EFA6B deregulates the homeostasis of normal mammary epithelial cells at different levels. First, deleting EFA6B allows the formation of invadopodia rich in integrin ITGBI and metalloprotease MMPI 4. My results indicate that EFA6B KO cells invasion is Cdc42-dependent, supported by increased cell contractility and the formation of protrusive branched structures through the Cdc42/MRCK/pMLC and Cdc42/N-WASP/Arp2/3 signaling pathways. Second, we showed that the loss of EFA6B is associated with the engagement of cells into EMT, revealed by a cadherin switch and an upregulation of EMT transcription factors. Third, coherently with EFA6B roles on junction assembly, its depletion prevented cells from polarizing and forming normal acini with central lumens. Collectively, our data are in agreement with previous results showing a correlation between the loss of EFA6B and the breast cancer claudin-low subtype defined by EMT properties, invasion capacities and a decrease in TJ proteins expression. I also contributed to the characterization of the role of a-actininl as an effector of EFA6A in normal epithelial cells (MDCK), and of EFA6B in a tumorigenic mammary cell line (MCF7). I show that EFA6B and a-actininl by regulating together the apical cellular contractility coordinate the establishment of apico-basal polarity and luminogenesis, two essential processes for functional epithelia. Altogether, these results demonstrate that the loss of EFA6B triggers invasive potentials in normal epithelial cells, and modifies their microenvironment (contractility, degradative invadopodia, alteration of the matrisome). We propose the gene PSD4 encoding for EFA6B as an invasion-suppressor gene that will preserve cells from losing their epithelial features in order to maintain tissue integrity.; L'homéostasie des tissus épithéliaux est fondamentale à la survie et au maintien d'un organisme sain. Au cours de la morphogenèse des glandes mammaires, les cellules épithéliales communiquent avec leur stroma environnant afin d'orchestrer la formation d'un organe fonctionnel en passant par de multiples cycles régulés de prolifération, de transitions épithéliomésenchymateuse (TME) et mésenchymateuse-épithéliale, de migration et d'invasion. Dans les cellules cancéreuses, ces processus sont dérégulés. Il est donc important d'étudier comment les cellules mammaires normales conservent leur homéostasie au cours du développement pour appréhender les événements moléculaires induisant le cancer du sein. L'homéostasie de l'épithélium repose sur le contrôle de propriétés spécifiques : polarité apico-basale, cohésion cellulaire et formation de lumen. Notre protéine d'intérêt EFA6B, facteur d'échange d'Arf6, se trouve au cœur de ces caractéristiques structurelles, d'où mon intérêt à étudier son rôle dans les cellules épithéliales mammaires. Au cours de mes travaux de thèse, j'ai étudié l'impact de la perte d'EFA6B sur l'intégrité épithéliale. En utilisant des cellules épithéliales normales mammaires invalidées pour EFA6B par la technique de CRISPR/Cas9, j'ai démontré que la perte d'EFA6B dérégule fortement l'homéostasie épithéliale à différents niveaux. Premièrement, sa déplétion entraîne la formation d'invadopodes riches en intégrine ITGBI et en métalloprotéase MMP 14. Mes résultats indiquent que l'invasion des cellules EFA6B knock-out dépend de l'activation de Cdc42. Elle est soutenue par une contractilité accrue des cellules et la formation de structures branchées protrusives contrôlées par les voies de signalisation Cdc42/MRCK/P-MLC et Cdc42/NWASP/Arp2/3. Deuxièmement, nous avons montré que la perte d'EFA6B est associée à l'engagement des cellules EFA6B knock-out dans une TME, révélée par un échange de cadhérine et une augmentation de l'expression de facteurs de transcription mésenchymateux. Troisièmement, conformément au rôle d'EFA6B dans l'assemblage des jonctions serrées, sa déplétion empêche les cellules de se polariser et de former des acini avec un lumen central. Collectivement, nos données sont en accord avec des résultats précédemment publiés montrant une corrélation entre la perte d'EFA6B et le sous-type de cancer du sein invasif Claudin-low défini par une signature TME et une perte d'expression des protéines des jonctions serrées. J'ai aussi contribué à la caractérisation du rôle de l'a-actinine 1 comme effecteur d'EFA6A dans des cellules épithéliales normales (MDCK) et d'EFA6B dans une lignée cellulaire mammaire tumorigène (MCF 7). Je montre qu'EFA6B et l'a-actinine 1, en régulant la contractilité cellulaire apicale, coordonnent l'établissement de la polarité apico-basale, la formation des jonctions serrées et du lumen. L'ensemble de ces résultats démontre que la perte d'EFA6B stimule les propriétés invasives de cellules épithéliales normales et impacte leur microenvironnement (contractilité, dégradation de la matrice, altération du matrisome). Nous proposons que le gène PSD4 codant pour EFA6B est un gène suppresseur d'invasion qui contribue à préserver l'intégrité des tissus épithéliaux.

Epithelial tissue homeostasis is fundamental for the survival and maintenance of a healthy organism. During mammary gland morphogenesis, epithelial cells communicate with their surrounding stroma in order to orchestrate the formation of a functional organ by undergoing multiple cycles of controlled proliferation, epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition, migration and invasion. In cancer cells, these processes are dysregulated. Thus, understanding how normal mammary cells preserve their homeostasis during development is crucial to identify the molecular events inducing breast cancer. Homeostasis of epithelia relies on key specific features: apico-basal polarity, cell cohesion and lumen formation. Here stands our protein of interest, EFA6B, exchange factor for Arf6, hence my interest in studying its role in mammary epithelial cells. During my PhD work, I investigated the role of EFA6B on epithelial integrity.Using CRISPR/Cas9 EFA6B knock-out cells, I showed that the loss of EFA6B deregulates the homeostasis of normal mammary epithelial cells at different levels. First, deleting EFA6B allows the formation of invadopodia rich in integrin ITGBI and metalloprotease MMPI 4. My results indicate that EFA6B KO cells invasion is Cdc42-dependent, supported by increased cell contractility and the formation of protrusive branched structures through the Cdc42/MRCK/pMLC and Cdc42/N-WASP/Arp2/3 signaling pathways. Second, we showed that the loss of EFA6B is associated with the engagement of cells into EMT, revealed by a cadherin switch and an upregulation of EMT transcription factors. Third, coherently with EFA6B roles on junction assembly, its depletion prevented cells from polarizing and forming normal acini with central lumens. Collectively, our data are in agreement with previous results showing a correlation between the loss of EFA6B and the breast cancer claudin-low subtype defined by EMT properties, invasion capacities and a decrease in TJ proteins expression. I also contributed to the characterization of the role of a-actininl as an effector of EFA6A in normal epithelial cells (MDCK), and of EFA6B in a tumorigenic mammary cell line (MCF7). I show that EFA6B and a-actininl by regulating together the apical cellular contractility coordinate the establishment of apico-basal polarity and luminogenesis, two essential processes for functional epithelia. Altogether, these results demonstrate that the loss of EFA6B triggers invasive potentials in normal epithelial cells, and modifies their microenvironment (contractility, degradative invadopodia, alteration of the matrisome). We propose the gene PSD4 encoding for EFA6B as an invasion-suppressor gene that will preserve cells from losing their epithelial features in order to maintain tissue integrity.; L'homéostasie des tissus épithéliaux est fondamentale à la survie et au maintien d'un organisme sain. Au cours de la morphogenèse des glandes mammaires, les cellules épithéliales communiquent avec leur stroma environnant afin d'orchestrer la formation d'un organe fonctionnel en passant par de multiples cycles régulés de prolifération, de transitions épithéliomésenchymateuse (TME) et mésenchymateuse-épithéliale, de migration et d'invasion. Dans les cellules cancéreuses, ces processus sont dérégulés. Il est donc important d'étudier comment les cellules mammaires normales conservent leur homéostasie au cours du développement pour appréhender les événements moléculaires induisant le cancer du sein. L'homéostasie de l'épithélium repose sur le contrôle de propriétés spécifiques : polarité apico-basale, cohésion cellulaire et formation de lumen. Notre protéine d'intérêt EFA6B, facteur d'échange d'Arf6, se trouve au cœur de ces caractéristiques structurelles, d'où mon intérêt à étudier son rôle dans les cellules épithéliales mammaires. Au cours de mes travaux de thèse, j'ai étudié l'impact de la perte d'EFA6B sur l'intégrité épithéliale. En utilisant des cellules épithéliales normales mammaires invalidées pour EFA6B par la technique de CRISPR/Cas9, j'ai démontré que la perte d'EFA6B dérégule fortement l'homéostasie épithéliale à différents niveaux. Premièrement, sa déplétion entraîne la formation d'invadopodes riches en intégrine ITGBI et en métalloprotéase MMP 14. Mes résultats indiquent que l'invasion des cellules EFA6B knock-out dépend de l'activation de Cdc42. Elle est soutenue par une contractilité accrue des cellules et la formation de structures branchées protrusives contrôlées par les voies de signalisation Cdc42/MRCK/P-MLC et Cdc42/NWASP/Arp2/3. Deuxièmement, nous avons montré que la perte d'EFA6B est associée à l'engagement des cellules EFA6B knock-out dans une TME, révélée par un échange de cadhérine et une augmentation de l'expression de facteurs de transcription mésenchymateux. Troisièmement, conformément au rôle d'EFA6B dans l'assemblage des jonctions serrées, sa déplétion empêche les cellules de se polariser et de former des acini avec un lumen central. Collectivement, nos données sont en accord avec des résultats précédemment publiés montrant une corrélation entre la perte d'EFA6B et le sous-type de cancer du sein invasif Claudin-low défini par une signature TME et une perte d'expression des protéines des jonctions serrées. J'ai aussi contribué à la caractérisation du rôle de l'a-actinine 1 comme effecteur d'EFA6A dans des cellules épithéliales normales (MDCK) et d'EFA6B dans une lignée cellulaire mammaire tumorigène (MCF 7). Je montre qu'EFA6B et l'a-actinine 1, en régulant la contractilité cellulaire apicale, coordonnent l'établissement de la polarité apico-basale, la formation des jonctions serrées et du lumen. L'ensemble de ces résultats démontre que la perte d'EFA6B stimule les propriétés invasives de cellules épithéliales normales et impacte leur microenvironnement (contractilité, dégradation de la matrice, altération du matrisome). Nous proposons que le gène PSD4 codant pour EFA6B est un gène suppresseur d'invasion qui contribue à préserver l'intégrité des tissus épithéliaux.

A major challenge in biology is to understand how complex gene expression patterns in organismal development are encoded in the genome. While transcriptional enhancers have been studied extensively, few transcriptional silencers have been identified and they remain poorly understood. Here we used a novel strategy to screen hundreds of sequences for tissue-specific silencer activity in whole Drosophila embryos. Strikingly, 100% of the tested elements that we found to act as transcriptional silencers were also active enhancers in other cellular contexts. These elements were enriched in highly occupied target (HOT) region overlap (Roy et al., 2010) and specific transcription factor (TF) motif combinations. CRM bifunctionality complicates the understanding of how gene regulation is specified in the genome and how it is read out differently in different cell types. Our results challenge the common practice of treating elements with enhancer activity identified in one cell type as serving exclusively activating roles in the organism and suggest that thousands or more bifunctional CRMs remain to be discovered in Drosophila and perhaps 104-105 in human (Heintzman et al., 2009). Characterization of bifunctional elements should aid in investigations of how precise gene expression patterns are encoded in the genome.; Un des enjeux majeurs de la biologie moderne est de comprendre les mécanismes complexes régissant l’expression de gènes d’un organisme en développement. Alors que les activateurs (enhancers) ont été abondamment étudiés et analysés, seul un relatif petit nombre de répresseurs (silencers) a été identifié à ce jour et restent jusqu’à présent assez mal compris. Un nombre non négligeable de CRMs jouent par ailleurs un double rôle à la fois d’amplificateurs et d’inactivateurs de transcription en fonction de l’état ou du type cellulaire dans lequel ils se trouvent, rajoutant un niveau supplémentaire de à la régulation génique dans différents types cellulaires et tissus. De façon surprenante, nous avons découvert que tous les éléments ayant une activité de répression transcriptionnelle que nous avons identifiés, s’avèrent aussi avoir une activité d’activation transcriptionnelle dans d’autres contextes cellulaires. Nos résultats remettent donc en question le paradigme de deux catégories distinctes de CRMs et suggèrent que des milliers, ou plus, d’éléments bifonctionnels restent à être découverts chez la Drosophile et potentiellement 104-105 chez l’humain. Le référencement et la caractérisation de ces éléments devraient s’avérer utiles, si ce n’est cruciaux, afin de comprendre la façon par laquelle ces motifs d’expression sont encodés au sein des génomes d'organismes métazoaires et donc éventuellement chez l’Homme.