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Les épithéliums jouent le rôle fondamental de barrière physique et chimique chez les Métazoaires. Les jonctions adhérentes, par le biais de la protéine transmembranaire E-cadhérine (E-cad), assurent une grande partie de l’adhésion intercellulaire. Malgré cette robustesse, les épithéliums peuvent subir des remodelages considérables pendant l’embryogenèse ou la cicatrisation. Lors de la gastrulation de l’embryon de Drosophile, l’épithélium ventro-latéral (la bandelette germinale) subit une élongation le long de l’axe antéropostérieur induite par l’intercalation cellulaire. Le remodelage polarisé des jonctions cellulaires est à la base de ce phénomène: les jonctions parallèles à l’axe dorsoventral (DV) rétrécissent et forment de manière irréversible de nouvelles jonctions parallèles à l’axe antéropostérieur (AP). Ce remodelage dépend de l’enrichissement du moteur moléculaire Myosine II (MyoII) dans les jonctions DV, qui induit une anisotropie de tension. Les protéines des jonctions adhérentes (E-cad, β-catenin) sont, elles aussi, polarisées : elles sont enrichies dans les jonctions AP. Néanmoins, nous ne savions pas si cette polarité de l’adhésion avait un rôle dans le remodelage des jonctions, et nous ne connaissions pas les mécanismes contrôlant cette localisation asymétrique. L’un des mécanismes les mieux connus de la modulation de l’adhésion cellulaire est l’endocytose des protéines d’adhésion. A ce titre, je me suis intéressé au rôle de l’endocytose Clathrine dépendante (ECD) pendant l’intercalation cellulaire. J’ai ainsi pu montrer que l’ECD de E-cad est régulée à la hausse dans la bandelette germinale au niveau jonctionnelle, plus particulièrement au niveau des jonctions DV (qui rétrécissent). L’ECD d’E-cad est nécessaire à l’intercalation et à la distribution polarisée d’E-cad. Elle est régulée par l’organisation de l’actine: la formine Diaphanous ainsi que le moteur moléculaire Myosine II accélèrent le recrutement de la machinerie d’endocytose (AP2 et Clathrine) et régulent la polarité de l’ECD dans l’embryon. Elles sont contrôlées par RhoGEF2, qui est enrichie dans les jonctions DV, et induisent l’endocytose par un mécanisme de clustering latéral d’E-cad. Dans la seconde partie de ma thèse, je me suis intéressé au couplage entre E-cad et la dynamique de MyoII. En effet, l’intercalation dépend aussi de flux contractiles de MyoII qui ont lieu préférentiellement en direction des jonctions DV. J’ai ainsi pu montrer que la direction des flux est induite par les anisotropies de forces d’ancrage de MyoII. Les faibles niveaux d’E-cad et le fort taux d’endocytose dans les jonctions DV augmentent la probabilité de générer une anisotropie d’ancrage et induisent davantage de flux de MyoII vers les jonctions DV. Ce projet met en lumière le rôle fondamental du couplage entre E-cad et MyoII dans la régulation de la morphogenèse. / Epithelia build up strong mechanical and chemichal barriers in Metazoans. Adherens junctions, through the adhesion provided by the transmembrane protein E-cadherin (E-cad), are essential for the mechanical integrity of the tissue. Yet, epithelia can be dramatically remodeled during embryogenesis or wound healing. During gastrulation of Drosophila embryo, the ventrolateral epithelium (the germ band) undergoes a massive elongation along the anteroposterior (AP) axis, driven by cell-cell intercalation. This is based on the polarized remodeling of intercellular junctions whereby junctions parallel to the dorsoventral axis (DV) shrink and form new junctions along AP axis. This remodeling is mediated by the planar polarized enrichment of Myosin II (MyoII) in DV junctions, which generates high tension. Adhesion proteins are also planar polarized, E-cad is enriched in AP junctions, but we did not know if this polarity contributed to cell-cell intercalation and the mechanism driving this polarity. As such, I have studied the role of Clathrin mediated endocytosis (CME) during germ band extension. I have shown that E-cad CME is specifically upregulated at the junction plane in the germ band, and planar polarized (enriched in DV shrinking junctions). It is required for cell-cell intercalation and the planar polarized distribution of E-cad. E-cad CME is regulated by the concerted action of the Formin Diaphanous and Myosin-II, which accelerates CME through the lateral clustering of E-cad. They are controlled by RhoGEF2, which is also enriched in DV junctions. In the second part of my PhD, I have studied the coupling between E-cad and MyoII dynamics. Indeed, planar polarized contractile flows of MyoII are required for DV junction shrinkage, but we did not know the mechanism driving the polarity of these flows. I have shown that the transient anisotropy of anchoring forces between two facing junctions triggers flow. As such, the low steady state amount of E-cad and the high rate of CME in DV junctions trigger more anisotropy and polarize the flow. These results outline the strong crossregulation between E-cad and MyoII and their concerted action in morphogenesis.

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Vesicular traffic along the biosynthetic and endocytic pathways is essential for homeostasis of eukaryotic cells. However, it raised the question of how the proteins characteristic for each compartment are transported to their destination (Bonifacino and Glick, 2004). This study is especially focusing on the connection between the Golgi apparatus and the endosomal compartment, mediated by two parallel trafficking pathways regulated by the clathrin adaptors AP-1A and AP-3 (Owen et al., 2004). Typical cargo molecules sorted along the AP-1A regulated pathway are mannose 6-phosphate receptors (MPRs) (Ghosh et al., 2003) or the gpI envelop glycoprotein of the Vesicular Zoster virus (Alconada et al., 1996), while sorting of lysosomal membrane proteins like Lamp-1 and LimpII is AP-3 regulated (Eskelinen et al., 2003). To study how AP-1A and AP-3 coats are stabilized on membranes and to identify the protein networks involved, a liposome based in vitro assay that recapitulates the fidelity of protein sorting in vivo was developed and combined with proteomic screens. Therefore, liposomes carrying cytoplasmic domains of gpI or Lamp-1/LimpII were used as affinity matrix to recruit selectively AP-1A or AP-3 and associated protein machineries. The coated liposomes were then analyzed by mass spectrometry. Using the in vitro recruitment assay, it was possible to demonstrate that efficient and selective recruitment of AP-1A and AP-3 coats depends on the presence of several low affinity binding sites on membranes. Thus, AP-1A and AP-3 recognize their target membranes by activated Arf1 GTPases, organelle specific phosphoinositides, PI-4P and PI-3P respectively, and distinct cargo molecules carrying intact signals in their cytoplasmic domains. The implication of PI-3P in AP-3 recruitment was further supported by in vivo experiments. During the biochemical characterization of the assay, several lines of evidence indicated that cargo tails containing intact sorting signals stabilize not only AP-1A and AP-3 coats on membranes but also influence the membrane recruitment of Arf1. It is possible that cargo molecules indirectly drive an Arf1 amplification loop, thereby ensuring efficient AP coat assembly. The proteomic screens identified protein networks of ≈40 proteins selectively recruited on AP-1A coated structures. The most appealing result of the analysis was the presence of two additional protein machineries, one involved in actin nucleation the other involved membrane fusion. More precisely, the AP-1A analysis identified the selective recruitment of the AP-1A subunits and interacting molecules (clathrin, g-synergin), Arf1 and Arf1 effectors (Big2, Git1), Rac1 including Rac1 effectors (b-PIX, RhoGEF7) and a Rac1 dependent actin nucleation machinery (Wave/Scar complex, Arp2/3 complex, associated effectors) as well as members of a Rab machinery (Rab11, Rab14). This finding was further supported by in vivo colocalization studies of the AP-1A cargo CI-MPR with CYFIP2, a protein of the Wave/Scar complex, and the localization of Big2 and Git1 on Rab11 positive membranes (Matafora et al., 2001; Shin et al., 2004). The biochemical characterization revealed that the stabilization of AP-1A coats, most probably driven by cargo molecules that stabilize AP-1A and Arf1 on membranes, leads as well to the stabilization of the two other machineries. Thus, the results support the notion that cargo sorting, vesicular movement and membrane fusion are coordinated during early steps of vesicular traffic. In analogy, the proteomic screens on AP-3 coated structures identified as well ≈40 selectively recruited proteins, which constituted a similar supramolecular network of protein machineries involved in coat formation, action nucleation and membrane fusion via Rab proteins. Thus, beside the AP-3 coat including the AP-3 subunits, Arf1 and Arf effectors (Big1, ARAP1, AGAP1), members of the septin family involved in actin rearrangements and most of the already described effectors of Rab5 microdomains (EEA1, Rabaptin-5, Rabex-5, Vps45) involved in early endosomal dynamics were selectively recruited together with Rab5 and Rab7. Thus, the proteomic analysis of AP-1A and AP-3 coated structures suggest that both AP coats use similar principles - coats, actin nucleation devices and Rab fusion machineries - to assemble supramolecular structures needed for membrane traffic. Although we do not have the ultimate proves yet, it seems as AP-1A and AP-3 use different members of subcomplexes, hence different GTPase effectors, different actin nucleation machineries and different Rab GTPases, to regulate their specific transport pathways and to link the different protein machineries. The proteomic analysis revealed for example that they probably use different Arf and Rho GTPase effectors to link the coat with actin nucleation. However, this has to be proven experimentally. In order to understand the networks of protein interactions, bioinformatic tools were used as a first approach. Even though some clues about the overall organization of the supramolecular protein complexes were provided, the direct links to the Rab machinery are still elusive. Maybe the proteins with thus far unknown functions could be involved. The biochemical analysis, especially the role of PIPs, and the Rab GTPases identified in the context of AP-1A and AP-3, provide indications about AP-1A and AP-3 function in vivo. The results could be interpreted in a way that AP-1A functions either in traffic from PI-4P positive membranes towards Rab11/Rab14 positive membranes or AP-1A coats assemble on PI-4P and Rab11 or Rab14 positive membranes, hence, TGN to endosomes traffic. The same holds true for AP-3, the results either suggest AP-3 mediates traffic from PI-3P positive towards Rab5/Rab7 positive membranes or they could be interpreted in a way that AP-3 assembles on PI-3P and Rab5 positive membranes for subsequent transport to Rab7 positive membranes, thus traffic from early to late endosomes. Overall, the results of this thesis research provided important insight into the formation of AP-1A and AP-3 coated structures and the potential interconnection between AP coats, actin nucleation and membrane fusion machineries. Alconada, A., U. Bauer, and B. Hoflack. 1996. A tyrosine-based motif and a casein kinase II phosphorylation site regulate the intracellular trafficking of the varicella-zoster virus glycoprotein I, a protein localized in the trans-Golgi network. Embo J. 15:6096-110. Bonifacino, J.S., and B.S. Glick. 2004. The mechanisms of vesicle budding and fusion. Cell. 116:153-66. Eskelinen, E.L., Y. Tanaka, and P. Saftig. 2003. At the acidic edge: emerging functions for lysosomal membrane proteins. Trends Cell Biol. 13:137-45. Ghosh, P., N.M. Dahms, and S. Kornfeld. 2003. Mannose 6-phosphate receptors: new twists in the tale. Nat Rev Mol Cell Biol. 4:202-12. Matafora, V., S. Paris, S. Dariozzi, and I. de Curtis. 2001. Molecular mechanisms regulating the subcellular localization of p95-APP1 between the endosomal recycling compartment and sites of actin organization at the cell surface. J Cell Sci. 114:4509-20. Owen, D.J., B.M. Collins, and P.R. Evans. 2004. Adaptors for clathrin coats: structure and function. Annu Rev Cell Dev Biol. 20:153-91. Shin, H.W., N. Morinaga, M. Noda, and K. Nakayama. 2004. BIG2, a guanine nucleotide exchange factor for ADP-ribosylation factors: its localization to recycling endosomes and implication in the endosome integrity. Mol Biol Cell. 15:5283-94.

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