Notch signaling is important for cell-cell signaling during development. Notch signaling is highly conserved across all metazoans and failure in Notch signaling is causative in many human diseases. In the Drosophila eye, activation of the Notch pathway requires Lqf (Drosophila Epsin)-dependent and Clathrin-dependent internalization of the Notch receptor ligands, Delta or Serrate, by the signal-sending cells. However, it is unclear why ligand must be internalized into the signal-sending cells to activate Notch signaling in the signal-receiving cells. Evidence suggests that in addition to Clathrin and Epsin, Auxilin is essential for signaling and is indirectly required for internalization of the Notch receptor ligand Delta. Auxilin functions in uncoating Clathrin-coated vesicles to maintain a pool of free Clathrin and Epsin in the cell. auxilin mutants were used as an entryway to identify previously unknown components of the Notch signaling pathway. An F1, FLP/FRT, EMS screen was performed and enhancers of an auxilin mutant rough eye defect were isolated. The enhancers ultimately formed one complementation group on the 2nd chromosome and fourteen complementation groups on the 3rd chromosome. Three of the 3rd chromosome complementation groups were each identified as Delta, lqf, or hsc70. A single allele was identified as faf. Delta and Epsin have known roles in signaling cells to activate Notch as described above. Hsc70 is an ATPase that functions with Auxilin to uncoat Clathrin-coated vesicles and Faf is a deubiquitinating enzyme that maintains levels of active Epsin in the cell. These results suggest I have isolated mutations in genes closely tied to Notch signaling or functioning directly with Auxilin. Mutations in two genes previously undescribed in Notch signaling in the developing Drosophila eye were also isolated from the screen and identified. The second chromosome complementation group was identified as α-adaptin. α-Adaptin is a subunit of the heterotetrameric Clathrin adaptor protein AP-2. One of the third chromosome complementation groups was identified as crumbs. Crumbs is an integral membrane protein that functions at adherens junctions and in establishing apical/basal polarity in cells. Characterizing roles for α-Adaptin and Crumbs during Notch signaling may elucidate the purpose for Delta internalization to activate Notch signaling. / text

Deregulation of platelet-derived growth factor receptor (PDGFR) signaling is a driving event in glioblastoma, promotes tumor progression epithelial to mesenchymal transition (EMT) in multiple cancers, modulates the tumor stroma to facilitate tumorigenesis and reduces tumor uptake of chemotherapeutics. Previous studies identified the 105 kDa isoform of ankyrin3 (Ank105) as a binding partner of the PDGFR signaling machinery and demonstrated that expression of Ank105 promoted PDGFR degradation (Ignatiuk et al., 2006)(Ignatiuk et al., 2006)(Ignatiuk et al., 2006). Receptor tyrosine kinases are targeted for degradation via endocytosis and ubiquitin-dependent trafficking to the lysosome. It was hypothesized that Ank105 promoted the constitutive degradation of the PDGFR and attenuation of PDGFR signaling by facilitating endocytosis of the PDGFR and targeting the PDGFR for lysosomal degradation via an ubiquitin-dependent mechanism. The studies in this thesis characterized the effects of Ank105 expression on PDGFR signaling and protein expression levels, determined the endocytic pathways involved in Ank105-mediated PDGFR degradation and studied the role of ubiquitin binding in Ank105 function. The most robust effect of Ank105 expression on the PDGFR was constitutive degradation as PDGFR protein expression levels in Ank105-expressing cells were significantly reduced compared to NIH 3T3 cells in the absence of PDGF ligand. Low constitutive PDGFR levels resulted in attenuated pro-proliferative AKT and mitogen-activated protein kinase (MAPK) signaling in response to ligand stimulation. To determine the endocytic requirements for Ank105-mediated constitutive PDGFR degradation, a constitutive PDGFR degradation assay was developed and the effects of several small molecule endocytosis inhibitors were evaluated. Additionally, the small molecule endocytosis inhibitors were validated by determining the effects of these inhibitors on low density lipoprotein (LDL) uptake and ligand-induced PDGFR degradation in Ank105-expressing cells. Both LDL uptake and ligand induced PDGFR degradation are known to proceed by a clathrin and dynamin dependent mechanism of endocytosis. In Ank105-expressing cells, both LDL uptake and ligand incuded PDGFR degradation were dependent upon clathrin and dynamin function. Interestingly, constitutive PDGFR degradation in Ank105-expressing cells was not dependent upon CME, but required dynamin activity. Expression of Ank105 may promote clathrin-independent, dynamin-dependent, constitutive endocytosis of the PDGFR. Additionally, acute inhibition of either lysosomal or proteasomal degradation strongly impaired constitutive PDGFR degradation, whereas ligand-induced PDGFR degradation was less sensitive to protein degradation inhibitors, while LDL uptake was unaffected. It was unclear if PDGFR was degraded in the proteasome or if the proteasome was involved in sorting of PDGFR to the lysosome for degradation. Ubiquitination of receptors is required to target them for degradation. Ank105 was assayed for the ability to interact with ubiquitin and ubiquitinated proteins. Interestingly, Ank105 bound ubiquitin in vitro via the spectrin binding domain and co-immunoprecipitated with several ubiquitinated proteins, suggesting a role for Ank105 in the sorting of ubiquitinated proteins for degradation. Furthermore, Ank105 co-immunoprecipitated with a number of high and low molecular weight proteins in the absence of PDGF stimulation. Identification of Ank105 binding partners would provide further insight in the mechanism of Ank105-mediated constitutive PDGFR degradation. In summary, Ank105 promoted the attenuation of PDGFR signaling via alteration of constitutive PDGFR endocytosis and targeting of constitutive PDGFR for degradation, potentially through interaction with ubiquitin and ubiquitinated proteins. Reduction of constitutive PDGFR levels in cancers with PDGFR driver mutations, acquired PDGF responsiveness and stromal expression of PDGFR, could significantly reduce tumor proliferation, tumorigenesis and increase effectiveness of chemotherapeutics.

Endocytosis effectuates a critical interface between the eukaryotic cell and its apposing environment. It is, subsequently, paramount for many physiologically important processes and encompasses a diverse array of mechanisms and pathways. The classical endocytic routes mediated by clathrin and caveolin are the best understood and the molecular roles of their major regulators, such as dynamin, adaptor proteins and various lipid species, are the most comprehensively described. Recent identification of an assortment of constitutive, noncaveolar, clathrin-independent endocytic (CIE) pathways has expanded the endocytic system. Unlike the classical endocytic pathways, little is known about the guiding parameters of CIE routes. Consequently, it is not possible to understand the important cellular roles these pathways may be fulfilling. This study has begun to characterise the very basic parameters governing the morphologically striking Clathrin-Independent Carrier (CLIC) pathway. Development of a diverse molecular toolkit has now allowed the quantitation of endocytic capacity provided by CLICs, the visualisation of subtle sorting components of the CLIC pathway, the isolation of novel CLIC cargo and regulators, and has linked this mechanism to the critical cellular processes of cellular migration and membrane repair. Calculation of the individual capacity of endocytic routes provides important information about the contribution of each pathway to total plasma membrane (PM) uptake and turnover. Quantitation of the volume, surface area and number of structures forming per minute in this study shows that CLICs provide the vast majority of constitutive endocytosis, up to four times the capacity of the clathrin mediated endocytic (CME) pathway. As the equivalent of the entire PM area could pass through the CLIC pathway within 12 minutes it is evident that CLICs are fundamental housekeepers of bulk membrane internalisation. Thus, they are likely to be central regulators of PM homeostasis and turnover. High-resolution tomography, in conjunction with analysis of CLIC cargo trafficking, identifies these carriers as complex, pleiomorphic structures that sort the bulk of membrane to early endosomes and recycle cargo back to the cell surface. Such vast internalisation combined with an ability to rapidly recycle components quickly attributes the CLIC pathway as a complex sorting station. Isolation of novel cargo and regulators has identified a striking array of proteins now associated with the CLIC pathway for the first time. A significant proportion of identified targets localise to lipid-rafts and recycle from the PM, facets consistent with association to the CLIC pathway. Numerous targets have also been directly implicated in clathrin-independent endocytosis by independent groups. Verification of selected cargo, such as CD44, Thy-1 and myoferlin, showing specific internalisation through the CLIC pathway, has provided insight into the sorting ability of the CLIC pathway and links to adhesion turnover and membrane recycling. Consistent with a role in cellular adhesion turnover, it was found that CLICs become polarised within migrating cells. This has shown the first instance of spatial separation between three major endocytic routes, CLICs, caveolae and CME and highlights the important and coordinated roles of multiple endocytic pathways during physiologically significant processes. The specific internalisation of paxillin, Thy-1 and CD44 through CLICs at the leading edge of migrating cells suggests that CLICs rapidly turnover adhesion components for dynamic extracellular sensation during directional cell migration. Indeed, specific ablation of the CLIC pathway significantly impedes cellular migration, implying coordination with CME at the leading edge. This study has defined numerous parameters of the CLIC pathway, developing the current understanding of this poorly defined route and places the CLIC pathway as a unique player during critical cellular processes.

Endocytosis effectuates a critical interface between the eukaryotic cell and its apposing environment. It is, subsequently, paramount for many physiologically important processes and encompasses a diverse array of mechanisms and pathways. The classical endocytic routes mediated by clathrin and caveolin are the best understood and the molecular roles of their major regulators, such as dynamin, adaptor proteins and various lipid species, are the most comprehensively described. Recent identification of an assortment of constitutive, noncaveolar, clathrin-independent endocytic (CIE) pathways has expanded the endocytic system. Unlike the classical endocytic pathways, little is known about the guiding parameters of CIE routes. Consequently, it is not possible to understand the important cellular roles these pathways may be fulfilling. This study has begun to characterise the very basic parameters governing the morphologically striking Clathrin-Independent Carrier (CLIC) pathway. Development of a diverse molecular toolkit has now allowed the quantitation of endocytic capacity provided by CLICs, the visualisation of subtle sorting components of the CLIC pathway, the isolation of novel CLIC cargo and regulators, and has linked this mechanism to the critical cellular processes of cellular migration and membrane repair. Calculation of the individual capacity of endocytic routes provides important information about the contribution of each pathway to total plasma membrane (PM) uptake and turnover. Quantitation of the volume, surface area and number of structures forming per minute in this study shows that CLICs provide the vast majority of constitutive endocytosis, up to four times the capacity of the clathrin mediated endocytic (CME) pathway. As the equivalent of the entire PM area could pass through the CLIC pathway within 12 minutes it is evident that CLICs are fundamental housekeepers of bulk membrane internalisation. Thus, they are likely to be central regulators of PM homeostasis and turnover. High-resolution tomography, in conjunction with analysis of CLIC cargo trafficking, identifies these carriers as complex, pleiomorphic structures that sort the bulk of membrane to early endosomes and recycle cargo back to the cell surface. Such vast internalisation combined with an ability to rapidly recycle components quickly attributes the CLIC pathway as a complex sorting station. Isolation of novel cargo and regulators has identified a striking array of proteins now associated with the CLIC pathway for the first time. A significant proportion of identified targets localise to lipid-rafts and recycle from the PM, facets consistent with association to the CLIC pathway. Numerous targets have also been directly implicated in clathrin-independent endocytosis by independent groups. Verification of selected cargo, such as CD44, Thy-1 and myoferlin, showing specific internalisation through the CLIC pathway, has provided insight into the sorting ability of the CLIC pathway and links to adhesion turnover and membrane recycling. Consistent with a role in cellular adhesion turnover, it was found that CLICs become polarised within migrating cells. This has shown the first instance of spatial separation between three major endocytic routes, CLICs, caveolae and CME and highlights the important and coordinated roles of multiple endocytic pathways during physiologically significant processes. The specific internalisation of paxillin, Thy-1 and CD44 through CLICs at the leading edge of migrating cells suggests that CLICs rapidly turnover adhesion components for dynamic extracellular sensation during directional cell migration. Indeed, specific ablation of the CLIC pathway significantly impedes cellular migration, implying coordination with CME at the leading edge. This study has defined numerous parameters of the CLIC pathway, developing the current understanding of this poorly defined route and places the CLIC pathway as a unique player during critical cellular processes.

Les étapes précoces de l’infection virale sont des évènements critiques dans le déroulement du cycle viral. Notamment, l’entrée virale est la première étape d’interaction entre un virus et une cellule permettant d’initier, de maintenir et de propager l’infection. De ce fait, cette étape constitue une cible majeure de la réponse immunitaire adaptative de l’hôte. C’est ainsi que cette étude bipartite s’est intéressée aux étapes précoces de l’infection du virus de l’hépatite C (HCV). Dans un premier temps, l’obtention de clones de cellules Huh-7.5 n’exprimant plus le récepteur néonatal des immunoglobulines, le FcRn, a permis d’analyser l’implication de ce récepteur dans la neutralisation du HCV par des anticorps neutralisants. Les résultats obtenus nous informent que le récepteur FcRn n’intervient vraisemblablement pas dans la modulation de la neutralisation du HCV. Dans un second temps, nous avons réalisé une étude préliminaire afin d’approfondir les mécanismes régissant l’endocytose du HCV : à savoir, quelles sont les protéines adaptatrices responsables du déclenchement de l’endocytose dépendante de la clathrine et s’il existe une potentielle voie d’entrée alternative pour ce virus. A ces fins, nous avons opté pour une stratégie basée sur la transfection de siRNA, couplée à l’utilisation des pseudoparticules HCVpp qui constituent une approche encore pertinente appliquée à l’étude de l’entrée du HCV. / The early steps during a viral infection are critical events in the course of the viral cycle. Particularly, the viral entry is the first step allowing the interaction between a virus and a cell to initiate, maintain and propagate an infection. Therefore, this very step is a major target for the host adaptive immunity. This bipartite study is focused on the early steps of the infection by the hepatitis C virus (HCV). First of all, generation of Huh-7.5 clones whose expression of the neonatal Fc receptor, FcRn, has been deleted gave us the opportunity to analyze the involvement of this receptor during the neutralization of HCV by neutralizing antibodies. Regarding the results, it appears unlikely that the FcRn modulates the neutralization of HCV. Then we conducted a preliminary study to further explore the mechanisms underlying the endocytosis of HCV: that is, which are the adaptor proteins that trigger the clathrine-mediated endocytosis and if there is another putative entry pathway for the virus. To these ends we opted for a RNAi based strategy coupled to the use of HCV-derived pseudo-particles (HCVpp) that are still useful and relevant tools dedicated to HCV-entry studies.

Endocytosis is important for the regulation of signal transduction and for the movement of essential cellular components from outside the cell to their appropriate intracellular compartment(s). Two established mechanisms of endocytosis are clathrinmediated (CME) and clathrin-independent endocytosis, and they are responsible for internalization of different ligands. In this study, the newly established technique of total internal reflection fluorescent microscopy (TIRF-M) was used, along with standard biochemical and molecular biological tools, to systematically study the sorting and early trafficking of two established ligands of endocytosis, transferrin (Tf) and epidermal growth factor (EGF). TIRF-M studies revealed that Tf binds its receptor that is located in large clathrin arrays positioned just below the surface of the cell and that these large clathrin platforms serves as the major site of CME at the plasma membrane. EGF endocytosis is very different and occurs as follows 1) the liganded EGFR recruits Rab5 to the plasma membrane, 2) Rab5 concentrates around vesicles containing liganded EGFR and 3) these vesicles co-localize with EEA1 enriched endosomes. EEA1 was shown to play a pivotal role in EGF endocytosis, establishing a new role for EEA1 in vesicle trafficking in addition to its role in tethering and fusion. Finally, WDFY2, a new FYVE domain protein was shown to decorate a specific subset of vesicles, upstream of the EEA1 vesicle pool that appear to participate in Tf endocytosis. These studies establish new functions and components of endocytosis that enhances our understanding of this complex process.

Les carcinomes hépatocellulaires (CHC) sont la deuxième cause de décès par cancer dans le monde. La signalisation du récepteur du facteur de croissance épidermique (EGFR) joue un rôle au cours du développement des CHC. Il a été montré que le trafic intracellulaire régulait la signalisation des récepteurs de la famille ErbB dans les CHC. De façon intéressante, l'expression de la clathrine, un acteur majeur de l'endocytose, est anormalement élevée dans les CHC. Ainsi, les objectifs de nos travaux étaient de définir si l'endocytose module la signalisation de la famille des récepteurs ErbB en réponse à divers ligands. Les expériences ont été effectuées dans trois lignées cellulaires issues de CHC, qui expriment des niveaux variables des différents récepteurs ErbB. Nos résultats montrent que l'inhibition de l'expression de la clathrine par ARN interférence était associée à une diminution significative de la phosphorylation des récepteurs EGFR, ErbB2 et ErbB3. La phosphorylation de STAT3 était significativement augmentée dans toutes les lignées. Les conséquences de l'inhibition pharmacologique de la dynamine et de la régulation négative de SMAP1 par ARN interférence ont été étudiées dans la lignée Hep3B. L'inhibition de la dynamine entraînait une augmentation significative des niveaux de phosphorylation d'EGFR, d'ErbB3 et d'AKT, alors que SMAP1 ne jouait aucun rôle dans la signalisation. Au total, nos observations soulignent que la signalisation des récepteurs ErbB dans les CHC est un processus complexe qui dépend de l'expression des différents membres de la famille ErbB et de la disponibilité de leurs ligands dans l'environnement tumoral. / Hepatocellular carcinoma (HCC) is the second leading cause of death by cancer in the world. The epidermal growth factor receptor (EGFR) signalling axis plays a key role in HCC. Intracellular trafficking has been shown to regulate receptor signalling, and to be altered in HCC. Our aim was to investigate whether endocytosis may modulate signalling of the ErbB receptor family in response to various ligands. The experiments have been performed in three HCC cell lines, which express variable levels of ErbB receptors. We investigated the role of clathrin, dynamin, and SMAP1 (Small ArfGAP1). Our results show that the effects of down-regulating clathrin by siRNA varied among HCC cell lines, depending on the ligand. Upon clathrin down-regulation by RNA interference, EGFR phosphorylation decreased in Hep3B and in PLC/PRF/5 cells stimulated with AR, EGF or HB-EGF, as well as in HRG-stimulated PLC/PRF/5 cells. Clathrin inhibition decreased ErbB2 phosphorylation in HepG2 cells stimulated with EGF, HB-EGF or HRG, and in HRG-stimulated PLC/PRF/5 cells. Phosphorylation of ErbB3 significantly decreased in all cell lines upon stimulation with EGF, HB-EGF or HRG. STAT3 phosphorylation significantly increased in all cell lines. Dynamin inhibition by dynasore led to a significant increase in the phosphorylation levels of EGFR, ErbB3 and AKT, in the Hep3B cell line. SMAP1 played no role in the early signalling of ErbB receptors upon stimulation with whatever ligand. Altogether, our observations underline that ErbB signalling in HCC is a complex process that may depend on the expression of the various ErbB family members and on the availability of their ligands in the tumour environment.

Clathrin coated vesicles were the first type of coated vesicle to be characterised. The coat consists of two components, clathrin and adaptor (or AP) complexes, the AP-1 complex is associated with the clathrin coated vesicles that bud from the TGN and the AP-2 complex is associated with the clathrin coated vesicles that bud from the plasma membrane. A new type of adaptor-like complex was discovered in our laboratory and was published in 1996. The complex has been shown to consist of two known proteins, beta3B and mu3B, and two unknown proteins of 160kD and 22kD. Unlike the conventional adaptor complexes this complex is not associated with clathrin. The aim of this thesis was to complete the characterisation of the adaptor-like complex and to establish its function. My studies have shown that, the adaptor-like complex consist of an alpha/gamma like subunit, delta, a beta subunit (beta3A/B), a mu subunit (mu3A/B) and a sigma subunit (sigma3A/B). We named the adaptor-like complexAP-3, by analogy with the AP-1 and AP-2 complexes. The AP-3 complex is localised to perinuclear and more peripheral membranes in non-neuronal cells, with little overlap with endocytic markers. The beta subunit of the AP-3 complex is the major target for phosphorylation. Analysis of mice with mutations in the beta3A subunit, and in the delta subunit of the AP-3 complex, have revealed that the beta subunit is required for the stability of the mu subunit and that the delta subunit is essential for the stability of the whole complex. Further analysis of the mutant mice indicated that the mice lack significant levels of functional AP-3 complex. Studies on fibroblasts generated from these mice revealed that the AP-3 complex plays a role in the trafficking of LAMPI to lysosomes.

L’endocytose dépendante de la clathrine (EDC) est la principale voie d’internalisation des récepteurs de surface et de leurs ligands. L’internalisation se fait suite à l’invagination de la membrane plasmique vers l’intérieur de la cellule suite à la formation, dans un premier temps, de puits recouverts de clathrine (PRCs) qui bourgeonnent ensuite en vésicules recouvertes de clathrine dans le cytosol. L’EDC est un processus très dynamique qui a lieu en l’espace de 30 sec-1mn. Elle est impliquée dans de multiples fonctions et permet ainsi à la cellule de réguler l’expression de ses protéines en surface, de répondre aux signaux de prolifération ou migration envoyés par l’environnement immédiat via l’activation de voies de signalisation spécifiques ou encore de réguler le renouvellement des composants de la membrane plasmique. De par son importance, des dérégulations de l’endocytose dépendante de la clathrine ont déjà été observées dans les cancers. Ces modifications peuvent impliquer directement l’EDC en modifiant ses composants ou indirectement lors d’altérations de récepteurs régulés par celle-ci. La progression tumorale est elle-même régulée par de multiples facteurs, notamment l’accumulation de mutations qui ont des conséquences sur les cellules cancéreuses elle-même ou bien sur l’environnement immédiat, formant ainsi la « niche tumorale ». Ces changements agissent réciproquement sur la progression tumorale afin de l’amplifier. Lors de la croissance tumorale, les cellules cancéreuses recrutent des fibroblastes qui vont participer au remodelage et à l’augmentation de la rigidité autour de la tumeur. La rigidité de la matrice extracellulaire est détectée par les cellules ce qui envoie des signaux déclencheurs de prolifération et de migration en conséquence. Cette détection passe essentiellement par les intégrines à la surface membranaire qui vont s’agréger et induire des cascades de signalisation impliquées dans ces réponses. Ces intégrines peuvent se regrouper dans deux types de structures, les adhésions focales et les structures recouvertes de clathrine. En ce qui concerne ces dernières, il a été démontré précédemment que la rigidité du substrat augmente sa force d’interaction avec les intégrines, et empêche ainsi l’internalisation des vésicules recouvertes de clathrine, on parle alors d’ « endocytose frustrée ». Cette rétention des structures recouvertes de clathrine à la surface provoque une signalisation soutenue à la surface au lieu de l’arrêter par dégradation ultérieure des récepteurs dans les lysosomes. Le laboratoire a démontré que les structures de clathrine frustrées capturent ainsi différent récepteurs conduisant à une signalisation accrue dans la voie de la MAP Kinase Erk. Mon projet de thèse repose sur ces observations en s’intéressant plus particulièrement au rôle d’une autre modification induite par la croissance tumorale, le confinement. En effet, en se multipliant de manière incontrôlée dans un environnement spatialement restreint, les cellules tumorales se retrouvent soumises à des forces de compression. Les résultats mis en évidence au cours de ma thèse ont montré que le confinement provoque, comme la rigidité, une frustration des structures de clathrine qui ne sont donc plus capables de soutenir l’endocytose des récepteurs. De plus, la compression cellulaire induit le clivage d’un pro-ligand de l’EGFR, le HB-EGF, ce qui conduit à l’activation paracrine de l’EGFR et à l’activation de la voie Erk. En effet, l’absence de facteurs de croissance dans le milieu ainsi que l’inhibition de ce clivage démontrent la nécessité de la mise en place de ce mécanisme. En résumé, le confinement induit le clivage du pro-ligand HB-EGF, qui à son tour va activer le récepteur à l’EGF. En parallèle, l’endocytose est ralentie et provoque une signalisation accrue à la membrane. Ces deux évènements coopèrent pour mener à une très forte activation de la voie Erk. / Clathrin-mediated endocytosis (CME) is the major route of endocytosis for many cargos in eukaryotic cells. Endocytosis takes place at clathrin-coated pits (CCPs), small assemblies of clathrin and clathrin adaptors randomly distributed at the plasma membrane. Clathrin polymerization induces the progressive bending of the plasma membrane resulting in the formation of a vesicle budding off into the cytosol. CME is a highly dynamic process with an average lifetime of CCPs in the order of 30 seconds. In this manner, CME fulfills a range of different functions and enables cells to regulate the surface expression of proteins, to sample the cell’s environment for growth and guidance cues, to control the activation of signaling pathways and to turn over membrane components by sending these components for degradation in the endo-lysosomal pathway. A deregulation of the endocytic pathways was previously shown to be involve in cancer. These modifications can affect CME directly by modifying its actors, or indirectly with mutations on receptors or cargoes undertaken by CME. Tumor progression is dependent of several factors, the first one involving the accumulation of mutations which results in modifications in the cells themselves or on their surrounding environment by changing its biochemical and physical properties, leading to the formation of the tumor niche. These changes reciprocally foster cancer progression. During tumor growth, fibroblasts will be recruited around tumor cells, leading to the remodeling of the microenvironment and to an increase of rigidity nearby the tumor. This stiffness is sensed by the cells and send signals for proliferation and migration as a result. Stiffness sensing engages mainly integrins at the cell surface which will aggregate and initiate signaling cascades accountable for these responses. Integrins are capable of clustering into two types of structures: focal adhesions and clathrin-coated structures (CCSs). Regarding CCSs, it was shown previously that high stiffness strengthen the interaction between integrins and the substrate, hence preventing the budding off of the vesicle, and this is referred to as “frustrated endocytosis”. This holding of CCSs at the cell surface promotes a sustained signaling at the plasma membrane instead of a signal termination after internalization and further degradation in lysosomes. My PhD project relied on these previous findings, with a particular focus on another mechanical alteration observed in tumors, the confinement. Indeed, during the uncontrolled proliferation of cancer cells in a spatially restricted area, cells become subjected to compressive forces. The results I obtained indicate that confinement leads to frustrated endocytosis and hence to sustained signaling from the plasma membrane. In addition, compression also leads to HB-EGF shedding at the cell surface, and the resulting EGF product activate the EGFR in a paracrine manner, thus leading to the activation of the MAP kinase Erk signaling pathway. Indeed, both the absence of EGFR ligands in the medium and the inhibition of the shedding demonstrate the necessity of this mechanism in EGFR activation. To sum up, confinement induces the shedding of the EGFR pro-ligand HB-EGF necessary to EGFR activation in these conditions. Simultaneously, endocytosis is delayed and frustrated endocytosis leads to sustained signaling at the cell surface. Together, these events cooperate to strongly activate the Erk pathway. These findings highlight the interplay between the physical feature of the tumor environment and signaling pathways known to govern tumor growth.

La migration cellulaire est un processus fondamental au maintien des fonctions physiologiques de l’organisme. Elle est également centrale dans de nombreuses pathologies et entre notamment en jeu lors de la dissémination métastatique. Lorsqu’elles migrent, les cellules utilisent des structures d’adhésion afin de s’appuyer sur leur environnement. Nous avons récemment montré que les puits recouverts de clathrine, plus connus pour leur rôle dans l’endocytose, peuvent également servir de structures d’adhésion. Dans ce manuscrit, je démontre que certains ligands internalisés par la voie d’endocytose clathrine peuvent également se lier à la matrice et orienter la migration cellulaire en régulant les structures adhésives de clathrine.J’ai commencé par montrer que le collagène est associé à plus de structures de clathrine et a plus de protrusions lorsqu’il est recouvert par des ligands. J’ai ensuite montré que les cellules appliquaient plus de forces sur des fibres de collagènes décorées par des ligands et que ce surplus de force nécessite la présence de structures de clathrine. Enfin j’ai montré que les cellules suivent les ligands liés à des réseaux de collagène en 3D et que cette migration dirigée nécessite également la présence de structures de clathrine. Ce mécanisme de migration pourrait notamment permettre aux cellules de suivre des gradients de ligands liés à la matrice in vivo et ainsi de s’orienter dans l’organisme. / Cell migration is a fundamental process in the development and homeostasis of multicellular organisms. It is also central to many pathologies and it is especially important for metastatic dissemination. When migrating, cells use adhesion structures to push on their substrate in order to move forward. We recently showed that clathrin coated structures, primarily known as endocytic structures, can also serve as adhesion structures. In this manuscript, I show that some ligands internalized through clathrin mediated endocytosis can also bind to the extracellular matrix and orient cell migration using adhesive clathrin structures.I first showed that ligand-decorated collagen fibers are associated with more clathrin structures and more protrusions. I then showed that cells applied more forces to the ligand-decorated collagen fibers and this extra amount of forces requires the presence of clathrin structures. Finally, I showed that cells can migrate following collagen-bound ligands in 3D, this directed migration also requiring the presence of clathrin structures. Such migration mechanism could be used by cells to follow in vivo gradient of matrix-bound ligands and thus find their way when migrating inside the body.