Polarized cell movements are essential to the cell rearrangements that occur during morphogenesis. In Xenopus, cell polarity is reflected in the directional cell intercalations that drive the morphogenetic movements characterizing gastrulation. While these cell behaviours are well described, the molecular mechanism underlying this cell polarity is unknown. PAR-3 is a multi-domain scaffolding protein and a key regulator of cell polarity. I have isolated a cDNA encoding Xenopus PAR-3 and generated several mutant constructs, each lacking a conserved domain. Initial characterization of GFP-tagged PAR-3 in A6 cells demonstrates localization to points of cell-cell contact in epithelial sheets, as well as at the leading edge of migrating cells. PAR-3 constructs lacking the CR1 or PDZ1 domain fail to compartmentalize properly and are found in the cytoplasm. Eliminating the PDZ3 domain resulted in a loss of contact inhibition. Mutation of the aPKC phosphorylation site created a membrane hyper-accumulation phenotype. Together these data suggest that the CR1 and PDZ1 domains mediate membrane compartmentalization that is modulated through aPKC phosphorylation, while the PDZ3 domain is required for contact inhibition. In embryos, PAR-3 is expressed throughout gastrulation and over-expression of PAR-3 inhibits blastopore closure indicating a requirement during gastrulation. Inhibition is relieved when the construct lacking the CR1 domain is over-expressed. PAR-3 was localized to the cell periphery in axial mesoderm. Localization was abolished with deletion of the CR1 domain indicating that membrane targeting of PAR-3 is required for gastrulation and this targeting is dependent on oligomerization of PAR-3. This investigation also suggests PAR-3 functions independent of the PAR complex in Xenopus embryos indicating involvement of a different PAR-3 signaling pathway.

Polarized cell movements are essential to the cell rearrangements that occur during morphogenesis. In Xenopus, cell polarity is reflected in the directional cell intercalations that drive the morphogenetic movements characterizing gastrulation. While these cell behaviours are well described, the molecular mechanism underlying this cell polarity is unknown. PAR-3 is a multi-domain scaffolding protein and a key regulator of cell polarity. I have isolated a cDNA encoding Xenopus PAR-3 and generated several mutant constructs, each lacking a conserved domain. Initial characterization of GFP-tagged PAR-3 in A6 cells demonstrates localization to points of cell-cell contact in epithelial sheets, as well as at the leading edge of migrating cells. PAR-3 constructs lacking the CR1 or PDZ1 domain fail to compartmentalize properly and are found in the cytoplasm. Eliminating the PDZ3 domain resulted in a loss of contact inhibition. Mutation of the aPKC phosphorylation site created a membrane hyper-accumulation phenotype. Together these data suggest that the CR1 and PDZ1 domains mediate membrane compartmentalization that is modulated through aPKC phosphorylation, while the PDZ3 domain is required for contact inhibition. In embryos, PAR-3 is expressed throughout gastrulation and over-expression of PAR-3 inhibits blastopore closure indicating a requirement during gastrulation. Inhibition is relieved when the construct lacking the CR1 domain is over-expressed. PAR-3 was localized to the cell periphery in axial mesoderm. Localization was abolished with deletion of the CR1 domain indicating that membrane targeting of PAR-3 is required for gastrulation and this targeting is dependent on oligomerization of PAR-3. This investigation also suggests PAR-3 functions independent of the PAR complex in Xenopus embryos indicating involvement of a different PAR-3 signaling pathway.

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Les carcinomes rénaux représentent environ 3% des cancers chez l’adulte. Les plus fréquents parmi ces tumeurs sont les carcinomes rénaux à cellules claires (CRCC) (70% des cas). Dans une première partie, nous avons analysé le caryotype de 89 patients ayant subi une néphrectomie pour CRCC et avons corrélé les déséquilibres chromosomiques avec les principaux facteurs histo-pronostiques et cliniques de ces tumeurs. Cette étude nous a permis de confirmer l’impact diagnostique et/ou pronostique d’anomalies chromosomiques. Certaines étaient déja connues dans la littérature comme la perte du bras court d’un chromosome 3 à impact diagnostique ou la perte d’un chromosome 9 ou de son bras court associée à un pronostic défavorable. Nous avons ensuite, dans une seconde partie, sélectionné selon des critères cliniques et histologiques, deux lignées cellulaires R-180 et R-305 établies à partir de prélèvements chirurgicaux de CRCC de patients dont l’évolution clinique était défavorable pour le patient R-180 (survie de 1 an) et favorable pour le patient R-305 (survie de 7 ans). Nous avons analysé les profils cytogénétiques des deux lignées cellulaires et recherché des marqueurs d’intérêt. Nous avons mis en évidence une amplification du gène pard3 dans la lignée R-180 correspondant au patient qui est décédé 1 an après le diagnostic. Cette amplification a été associée à la surexpression de la protéine correspondante PAR-3 et à des modifications de l’organisation du cytosquelette. La diminution de l’expression de PAR-3 par transfection de siRNA dans les cellules R-180 a permis la restauration de l’organisation du cytosquelette et la réduction des capacités de migration cellulaire par rapport aux cellules non transfectées. Ce résultat suggère un rôle de PAR-3 dans la migration cellulaire des cellules R-180. Afin de valider la pertinence de ce nouveau biomarqueur dans le CRCC, nous avons étudié 101 tumeurs par immunohistochimie. Nous avons montré une corrélation significative entre la surexpression de PAR-3 dans la tumeur primitive des patients et une diminution de la survie globale et de la survie sans progression indépendamment d’autres facteurs pronostiques importants comme les métastases. De plus la surexpression de PAR-3 a été significativement associée aux facteurs histopathologiques et cliniques de mauvais pronostic : grades nucléaires de Fuhrman III ou IV, nécrose tumorale, composante sarcomatoide, atteinte surrénale, invasion de la graisse rénale ou hilaire, composante éosinophile, statut non-inactivé du gène VHL, grade tumoral plus élevé, envahissement ganglionnaire ou métastatique, et score ECOG (Eastern Cooperative Oncology Group) péjoratif. L’ensemble de nos résultats suggèrent que la surexpression de PAR-3 est associée à un risque significatif de progression et de mortalité dans le CRCC. Sa mise en évidence par immunohistochimie en routine hospitalière pourrait être utile pour identifier les patients à haut risque de progression, même en l’absence des paramètres pronostiques habituels. Des études complémentaires sont en cours pour intégrer ce biomarqueur dans les nomogrammes ainsi que pour évaluer l’impact de cette dérégulation dans la résistance des CRCC aux thérapies ciblées. / Kidney cancers represent about 3% of all adults’ malignancies. The most common form of kidney cancer is renal carcinoma of which 70 % of cases are defined as clear cell Renal Cell Carcinoma (ccRCC). We undertook a systematic review of all ccRCCs with a total of 89 patients who underwent nephrectomy surgery. We assessed the karyotype profile of all patients that we correlate with an immunohistochemical features and tumor symptoms. This study demonstrates a high impact of chromosomal abnormalities on patients’ diagnosis and prognosis. Some of these abnormalities have been submitted in other publications as the loss of the chromosome 3 p-arm which has a diagnosis impact, and the loss of the chromosome 9 or it s p-arm that have a poor prognosis impact. We selected two cell lines (R-180 and R-305) derived from ccRCC surgical specimens of a patient with unfavorable clinical course (R-180 cells) and a patient with favorable prognosis (R-305 cells) to identify genetic and molecular features that may explain the survival difference of the two patients. The cytogenetic analysis of these cell lines revealed that the pard3 gene was amplified only in the R-180 cell line that was derived from an aggressive ccRCC. The pard3 gene amplification was associated with overexpression of the encoded protein and altered cytoskeleton organization. PAR-3 knockdown in R-180 cell restored the cytoskeleton organisation and reduced cell migration in comparison to non-transfected cells. These results suggest PAR-3 role in R-180 migration cells line. With a view to corroborate the relevance of this new biomarker PAR-3 in ccRCC, we have studied 101 tumors using immunohistochemical methods. We proved a significant correlation between PAR-3 overexpression in the primitive tumor and, the decreasing of overall and free progression survival independently of other risk factors as metastasis. We also fund that the overexpression of PAR-3 is associated with an unfavorable clinical and immunohistochemical prognosis factors such as: stage III -IV in fuhrman system grading ,tumor necrosis, sarcomatoide component, supra renal metastasis, cancer spreading (surrounding fat and hilar), eosinophil component , none inactivate VHL gene, high tumor stage, lymph nodes spread, metastasis and ECOG scale. Our results reveal that the PAR-3 overexpression is associated with significant risk of ccRCCs mortality and spreading tumor. Immunohistochemical screening may be usefulness to identify patient’s high spreading risk whether the lack of the habitual prognosis parameters. Other studies are in progress to integrate this biomarker in nomograms and also to evaluate the impact on ccRCC’s resistance to targeted therapy.

Orderliness, speed, and rhythm in biochemistry are vital for cellular function. In order to achieve this, cells implement compartmentalization via several methods, one of which is the formation of membrane-less compartments. These compartments, often referred to as “biomolecular condensates”, are understood to be formed by separation of proteins and other biomolecules into dense and dilute phases. While the formation of the resulting protein-rich condensates is fundamental for spatiotemporal organization of biochemistry within the cell, a vast majority of proteins found to phase separate in vitro do so at a concentration an order of magnitude above their endogenous expression levels. Recently, a theoretical study has shown that membrane binding of phase separating proteins can result in phase separation spatially occurring at the membrane well below bulk saturation concentrations. However, much remains unknown about the formation mechanism and function of these condensates. To that end, for my doctoral project, I used a synthetic system composed of supported lipid bilayers decorated with lipid-bound NTA(Ni) to allow for coordination and thus membrane binding of the well-characterized protein FUS via a C-terminal His-tag. Through this model system I found that 2D phase separation of FUS could occur an order of magnitude below the experimentally determined bulk saturation concentration. FUS was able to form dense and dilute phases in 2D and the transition point to form these phases could be controlled by modulating buffer conditions. Additionally, membrane-bound FUS condensates were able to further recruit FUS from the bulk to form a multilayer of protein through a prewetting transition. With this characterization of 2D phase separation of FUS, I then explored a physiologically relevant protein in the form of PAR-3, a fundamental protein of the PAR polarity system, which is necessary for the establishment of polarity in the C. elegans zygote. I found that full-length PAR-3 was able to phase separate under physiological salt conditions with a Csat of 100nM. Further, I identified a C-terminal predicted prion-like domain to act as a driver for phase separation. Additionally, I determined PAR-3’s affinity and specificity for PI(4,5)P2 and found that it could form 2D condensates upon binding to the membrane at physiological concentrations. Furthermore, these condensates were able to recruit PAR-6 alone and PAR-6 in complex with PKC-3 to the membrane, ultimately resulting the reconstitution of the anterior PAR complex which is known to exist in a condensed clustered form in vivo. Taken together, this work provides insight into a mechanism where phase separation can be locally triggered by membrane binding under sub-saturation concentration, offering a robust and potentially universal mechanism by which cells can spatially control phase separation and pattern cellular membranes.