Caveolae are small plasma membrane invaginations present in many different cell types, which have been linked to diverse cellular functions, including cell signalling, membrane rearrangements and lipid regulation. The caveolae markers, members of the caveolin family of proteins, are essential for caveola formation and function. Recently, however, a protein named PTRF (Polymerase I and Transcript Release Factor) or cavin, originally identified as a nuclear factor that regulates transcription in vitro, was shown to be associated with caveolae in adipocytes. In the first chapter of this thesis, I have used the zebrafish Danio rerio to investigate the relation of PTRF/cavin to caveolae as well as caveola function in vivo. During zebrafish development, PTRF/cavin was highly expressed in the notochord in 18 h, 24 h and 35 h post-fertilization embryos, as detected by in situ hybrydization. Analysis of later development stages showed that PTRF/cavin is also present in the otic vesicle, brachial arches, and periderm. Disruption of PTRF/cavin expression, via morpholino-mediated inhibition, caused severely defective development of the notochord as well as heart edema, in a dose-dependent manner. PTRF/cavin knockdown embryos had curved notochords and were shorter than the controls. Examination of the notochord by electron microscopy showed that the number of caveolae was greatly reduced in PTRF/cavin-morpholino-injected embryos. Similar effects were observed when caveolin-1, the major protein of caveolae in non-muscle cells, was down-regulated. Altogether, these results indicated a role for PTRF/cavin during formation and/or stabilization of caveolae as well as an essential role for caveolae during zebrafish embryo development. Combined with results obtained in mammalian cells, these findings identify PTRF/cavin as the first component of a caveolar coat, required for caveola formation and function (Hill et al., 2008). We subsequently identified a family of PTRF/cavin-related proteins, the cavins, that all associate with caveolae. Using biochemistry, light microscopy, and FRET-based approaches we characterised PTRF/cavin and the new members of this family of proteins SDR/cavin-2, SRBC/cavin-3 and MURC/cavin-4. We have shown that the four members of the cavin family form a multi-protein complex that associates with caveolae. This complex can constitutively assemble in the cytosol and then associate with caveolin at the plasma membrane caveolae; interestingly, caveolin is essential for the plasma membrane translocation of the cavin complex, and in caveolin-1 knockout cells the four cavin proteins are restricted to the cytosol. PTRF/cavin-1, but not other cavins, can induce caveola formation in a heterologous system and is required for the recruitment of the cavin complex to caveolae. The four cavin proteins present distinct patterns of tissue expression, which suggests that caveolae may perform tissue-specific functions regulated by the composition of the cavin complex. MURC/cavin-4 is expressed predominantly in muscle and its distribution is perturbed in human muscle disease associated with caveolin-3 dysfunction, identifying MURC/cavin-4 as a novel muscle disease candidate caveolar protein. To functionally investigate the relation of cavins and caveolae, we explored a caveolar function in mechanosensation. Through the use of hypo-osmotic media, we induced membrane-stretch and showed that the increased membrane tension leads to dissociation of the caveolin-cavin module and caveola disassembly as observed by immunofluorescence and FLIM/FRET techniques. Once released from caveolae, caveolin was seen internalized in late endosomes and lysosomes. Cavin-1, on the other hand, was found to be diffused in the cytosol and from there it was translocated to the nuclear compartment. The nuclear translocation was observed in several different cell types, which suggests a universal role for nuclear cavin-1, and was independent of caveolin expression. Analysis of live cells using real-time FLIM/FRET showed that cells quickly respond to variations in membrane tension by dissociation/re-association of caveolin and cavin-1. Altogether, in the course of this project, I was able to show that cavin-1 is an essential regulator of caveola biogenesis in cultured cells and in vivo. Cavin-1 and the other members of the PTRF/Cavin family form a multiprotein complex that is recruited to caveolae by caveolin and coats plasma membrane caveolae. The association between cavin-1 and caveolin is crucial for caveolae assembly and this interaction has a role in the cellular sensation of plasma membrane tension. Under high membrane tensions, caveolin and cavin-1 dissociate with the consequent flattening of caveolae. Under these circumstances, caveolin is internalized into enlarged endosomes and lysosomes while cavin-1 is translocated to the nucleus, identifying for the first time a caveola- to nucleus signalling pathway. The exact role of nuclear cavin-1 under plasma membrane stretch is now amenable to analysis.

L’évolution d’une cellule tumorale initiée à une tumeur solide nécessite, à chaque étape, un microenvironnement favorable à sa survie et à sa croissance. Le microenvironnement tumoral est comparé à un foyer d’inflammation chronique dont la composition cellulaire et moléculaire est complexe. Les cellules souches mésenchymateuses (CSM) représentent l’un des principaux acteurs cellulaires présents. Elles migrent vers les sites tumoraux où elles soutiennent l’inflammation, l’angiogenèse et le développement tumoral en activant plusieurs voies de signalisation. Une des voies majeures qui contribuent à l’inflammation est la voie de signalisation NF-B. L’initiation de cette voie provient de la membrane cellulaire entre autres des cavéoles. Nous soumettons l’hypothèse que l’une des cavines, protéines associées aux cavéoles, modulerait le phénotype inflammatoire etou migratoire dans les CSM traitées à la cytokine TNF- (facteur de nécrose tumorale ) en modulant la voie de signalisation NF-B. En effet, nous avons observé une régulation à la hausse de l’expression de la COX-2 (cyclooxygénase-2) et une diminution de l’expression d’IκB qui sont synonymes de l’activation de la voie NF-B dans les CSM que nous avons traitées au TNF-. Nous avons trouvé que le TNF- induit la migration des CSM, et que la répression génique de la Cavine-2 augmente significativement la migration des CSM traitées par le TNF-. La répression génique de la Cavine-2 vient aussi amplifier la tubulogenèse dans les CSM en réponse au TNF-. D’un point de vue moléculaire, la répression génique de la Cavine-2 a montré une très forte amplification de l'expression protéique de la COX-2 dans les CSM en réponse au TNF-. Dans ces mêmes cellules où la Cavine-2 a été réprimée, et suite à un traitement au TNF-, le pic de phosphorylation est plus intense et la courbe de phosphorylation est plus prolongée dans le temps. Ces observations nous permettent d’affirmer que la Cavine-2 a un rôle répresseur sur l’expression de COX-2. Collectivement, nos résultats montrent que la Cavine-2 peut être proposée comme un gène suppresseur de tumeur et est de ce fait, une bonne cible thérapeutique dans les CSM qui permettraient d’agir à des stades précoces du développement tumoral. / The evolution of an initiated tumor cell into a solid tumor requires at each stage a favorable microenvironment for its survival and growth. The tumor microenvironment is compared to a chronic inflammation site with a cellular and molecular complex composition. Mesenchymal stem cells (MSC) have important roles in tumor microenvironment. They migrate to tumor sites where they maintain the inflammation, angiogenesis and tumor development by activating multiple signaling pathways. One of the major pathways that contribute to inflammation is the NF-B signaling pathway. The initiation of this pathway comes from the cell membrane and caveolae. Our hypothesis is that one of cavins, proteins associated to caveolae, modulates the inflammatory phenotype and migration in MSC treated with TNF-. We suggest that this process is modulated by a NF-B signaling pathway. Indeed, we observed an up-regulation of the expression of the cyclooxygenase-2 (COX-2) and a decrease in the expression of IκB which suggest that activation of the NF-B pathway is involved in the MSC treated with TNF. We found that the TNF- induced migration in the MSC, and the knockout of Cavin-2 significantly increased migration of MSC treated with TNF-. The silencing of Cavin-2 considerably increased tubulogenesis of MSC treated with TNF-. At the molecular level, knockout of Cavin-2 showed a very strong amplification of protein expression of COX-2 in the MSC in response to TNF-. In these same cells where Cavin-2 was repressed and treated with TNF-, the peak of phosphorylation of pIB is more intense and the phosphorylation curve is sustained in time. These observations allow us to assert that Cavin-2 has a repressing role on the expression of COX-2. Collectively, our results show that the gene encoding Cavin-2 can be proposed as tumor suppressor gene. This study allowed us to identify new therapeutic targets: Cavins proteins.