La maladie de Charcot-Marie-Tooth de type 4H est une neuropathie héréditaire sensitivo-motrice démyélinisante à transmission récessive. Elle est causée par à des mutations dans le gène FGD4 codant la protéine FRABIN, RhoGEF comportant cinq domaines fonctionnels : un domaine FAB de liaison à l’actine, un domaine DH à activité d’échange GDP/GTP sur les petites RhoGTPases, deux domaines PH et un domaine FYVE impliqués tous trois dans la liaison aux polyphosphoinositides. D’une part, nous avons pu identifier trois nouvelles mutations, portant à dix le nombre de mutations dans FGD4. D’autre part, des études transcriptionnelles ont permis de caractériser huit transcrits alternatifs pouvant coder pour différentes isoformes de FRABIN, dépourvues de différents domaines. Ces résultats suggèrent que FRABIN pourrait être une protéine modulaire. J’ai pu montrer que, dans les fibroblastes des patients, la protéine FRABIN était absente. Dans les lymphoblastes, nous supposons que l’isoforme FRABIN présente est dépourvue du domaine de liaison à l’actine, mais nous n’avons pas pu analyser l’effet des mutations sur la production de la protéine. Dans les fibroblastes et les lignées lymphoblastoïdes des patients, j’ai pu mettre en évidence une diminution drastique de l’activation des RhoGTPases CDC42 et RAC1. Cependant, cette diminution n’a pas pu être corrélée avec des anomalies du cytosquelette ou de la migration des fibroblastes des patients. Toutefois, ces RhoGTPases sont primordiales pour la myélinisation, il est donc fort possible que dans les cellules du système nerveux périphérique, la perte de FRABIN résultant en une diminution de plus de 50% de l’activation des RhoGTPases entraîne des défauts majeurs dans le processus de « radial sorting ». L’arrivée des souris KO conditionnelles pour fgd4 dans les cellules de Schwann ou dans les motoneurones, devrait nous permettre de valider ou d’infirmer plusieurs hypothèses qui ont été émises durant ce travail et ainsi de mieux comprendre la physiopathologie de la maladie. / CMT4H is an autosomal recessive demyelinating CMT due to mutations in FGD4, which encodes FRABIN. FRABIN has five functional domains: a F-actin binding domain, a RhoGEF domain with GDP/GTP exchange activity, two PH and one FYVE domains which interact with polyphosphoinositides. In this study, we identified three novel FGD4 mutations, bringing to ten the number of mutations in this gene. Moreover, I characterized eight alternative transcripts, and all of them could lead to a functional FRABIN isoform, deprived of one or more functional domains. This led us to consider FRABIN as a modular protein. In patient’s fibroblasts, I have been able to show that FRABIN is degraded. Unlike in patient’s lymphoblastoïd cells line where we were unable to characterize the mutation effect on the protein. In these cells, we proposed that FRABIN is present without the F actin binding domain. Nevertheless, in patient’s fibroblasts and lymphoblastoïde cells line, I showed a major diminution of the CDC42 and RAC1 active forms, which is not in correlation with the absence of abnormalities in cytoskeleton and migration of patient‘s fibroblasts. We suggested that, in peripheral nerve system cells, the diminution of these RhoGTPases activation is damaging for the myelination. We are waiting for two fgd4 conditional KO mice model (one in Schwann cells and one in neuron). Exploration of these models will allow us to explain the physiopathological mechanism of CMT4H.

Biological outputs of insulin/IGF signaling are regulated through essential mediators, such as IRSs, PI3-kinase, and PKB/Akt. These mediators serve critical roles in signal propagation, feedback, and as junctions for crosstalk with other pathways. Abnormal insulin/IGF signaling results in disease, such as obesity, diabetes, and cancer. Given the vital role of this signaling pathway to human health, unraveling its regulatory mechanisms is crucial. Components of this pathway are highly conserved throughout evolution. PTEN, one of the well-defined regulators of this pathway, functions as a lipid phosphatase that negatively regulates insulin/IGF-1 signaling at the PIP3 level, a phosphoinositol that is upregulated by activated PI3-kinase in both Drosophila and mammals. To discover genetic modulators of PTEN in Drosophila, we performed a loss-of-function genetic screen to identify molecules that modify the phenotype elicited by PTEN overexpression in the Drosophila eye. From this screen, we identified a member of the Dbl-family, the guanine nucleotide exchange factor DRhoGEF2, which suppresses the PTEN-overexpression eye phenotype via its effects on dPKB/dAkt activation. By conducting a genetic rescue, we established that PDZ-RhoGEF, a member of the regulator of G-protein signal (RGS)-like domain containing Rho GEFs (RGS-RhoGEFs) subfamily of Dbl-family GEFs, is the mammalian counterpart of DRhoGEF2. PDZ-RhoGEF is essential for cell proliferation and survival through ROCK-dependent activation of IRS/PI3-kinase signaling cascade, which has a major impact on adipose tissue homeostasis. Through an integrative approach, we have demonstrated that DRhoGEF2/PDZ-RhoGEF-dependent signaling has tissue-specific effects on insulin/IGF-signaling throughput in both Drosophila and mammals. Particularly, we have demonstrated the role played by PDZ-RhoGEF in diet related pathology, provides an alternative therapeutic opportunity in disease intervention.

The gene pebble (pbl) encodes a Rho GEF required for the migration of mesoderm cells during Drosophila gastrulation. The spreading of mesoderm cells is controlled by the FGF signalling pathway acting through the FGF receptor Heartless (Htl). Pbl represents an important downstream component of this FGF pathway and activates the Rho GTPase Rac, but the regulation of Pbl by FGF signalling is unclear. Furthermore Pbl is required for the formation of the actin-myosin contractile ring during cytokinesis by activation of RhoA. The purpose of this work is to find molecular links between Pbl and the Htl signalling pathway and get insight into the localization and regulation of Pbl during mesoderm cell migration A genetic screen is carried out to find genes that interact with Pbl and are involved in mesoderm development. A gain-of-function variant of Pbl that causes defects in eye morphology was used to find genetic interactors. Results of a screen using chromosomal deletions and an EMS-based screen revealed candidates, which genetically interact with Pbl and are required for mesoderm cell migration. In addition, a structure-function analysis of the Pbl protein was performed. The data revealed an important role of the PH domain for the localization of Pbl at the cell cortex. Moreover the PH domain is indispensable for the function of Pbl in mesoderm migration. Furthermore an important role for the C-terminal tail of Pbl for the regulation of the protein was shown, which might be regulated by FGF signalling. The C-terminal tail is required for the stability of the protein outside the nucleus and it regulates the substrate preference of Pbl for Rac and Rho. Furthermore indication was found that the function of the C-terminal tail possibly is regulated by phosphorylation of Ser825 in the C-terminal tail. Mutation of this site affects the function of Pbl during mesoderm migration but not in cytokinesis. Therefore phosphorylation of the C-terminal tail might regulate or enhance the exchange activity of Pbl for Rac. The localization and function of Pbl depends on the PH domain and the C-terminal tail of Pbl. Both domains have distinct roles during Pbl function in mesoderm cell migration.

Biological outputs of insulin/IGF signaling are regulated through essential mediators, such as IRSs, PI3-kinase, and PKB/Akt. These mediators serve critical roles in signal propagation, feedback, and as junctions for crosstalk with other pathways. Abnormal insulin/IGF signaling results in disease, such as obesity, diabetes, and cancer. Given the vital role of this signaling pathway to human health, unraveling its regulatory mechanisms is crucial. Components of this pathway are highly conserved throughout evolution. PTEN, one of the well-defined regulators of this pathway, functions as a lipid phosphatase that negatively regulates insulin/IGF-1 signaling at the PIP3 level, a phosphoinositol that is upregulated by activated PI3-kinase in both Drosophila and mammals. To discover genetic modulators of PTEN in Drosophila, we performed a loss-of-function genetic screen to identify molecules that modify the phenotype elicited by PTEN overexpression in the Drosophila eye. From this screen, we identified a member of the Dbl-family, the guanine nucleotide exchange factor DRhoGEF2, which suppresses the PTEN-overexpression eye phenotype via its effects on dPKB/dAkt activation. By conducting a genetic rescue, we established that PDZ-RhoGEF, a member of the regulator of G-protein signal (RGS)-like domain containing Rho GEFs (RGS-RhoGEFs) subfamily of Dbl-family GEFs, is the mammalian counterpart of DRhoGEF2. PDZ-RhoGEF is essential for cell proliferation and survival through ROCK-dependent activation of IRS/PI3-kinase signaling cascade, which has a major impact on adipose tissue homeostasis. Through an integrative approach, we have demonstrated that DRhoGEF2/PDZ-RhoGEF-dependent signaling has tissue-specific effects on insulin/IGF-signaling throughput in both Drosophila and mammals. Particularly, we have demonstrated the role played by PDZ-RhoGEF in diet related pathology, provides an alternative therapeutic opportunity in disease intervention.

La constriction apicale des cellules du mésoderme et l'intercalation des cellules de l'ectoderme sont contrôlées par des réseaux contractiles d'acto-myosine dans l'embryon de Drosophile. Le niveau d'activation et la polarisation du cytosquelette d'acto-myosine détermine la nature des déformations cellulaires observées. Nous montrons que le GPCR Smog et les protéines G (Gα,Gβγ) en aval, activent la signalisation Rho1 et donc la Myosine-II dans les deux tissus. Dans l'ectoderme, Gα12/13 active Rho1 à la membrane apicale (aussi appelé compartiment médio-apical) tandis que les sous-unités Gβ13F-Gγ1 activent Rho1 en médio-apical et aux jonctions cellulaires. Les mécanismes contrôlant l’activation polarisée de Rho1 dans ce tissu demeurent incompris. Nous montrons ici que deux RhoGEFs, RhoGEF2 et une nouvelle RhoGEF Wireless/p114RhoGEF, activent Rho1 sous le contrôle des protéines G dans l’ectoderme. RhoGEF2 stimule Rho1 en médio-apical sous la dépendance de Gα12/13 alors que Wireless/p114RhoGEF contrôle l’activité de Rho1 aux jonctions avec Gβ13F-Gγ1. RhoGEF2 est présente aux jonctions et en médio-apical tandis que Wireless/p114RhoGEF est uniquement jonctionnelle où elle est recrutée par Gβ13F-Gγ1. Pour finir, Wireless/p114RhoGEF est absente des jonctions dans les cellules du mésoderme. En résumé, des GPCRs contrôlent l’activité spatio-temporelle de Rho1 au moyen de deux modules régulatoires dans l’ectoderme. Les protéines G transduisent le signal en recrutant et en activant deux RhoGEFs complémentaires en médio-apical et aux jonctions. Une variation dans la nature des GPCRs, protéines G ou des RhoGEFs détermine le contrôle tissu-spécifique de Rho1 au cours de la morphogenèse. / Cell apical constriction in the mesoderm and cell intercalation in the ectoderm are controlled by contractile actomyosin networks in the developing Drosophila embryo. The extent of both actomyosin activation and polarization determines the nature of these cell deformations. We find that the GPCR Smog and the downstream G proteins (Gα,Gβγ) activate Rho1 signaling and thereby myosin-II in both tissues. In the ectoderm, Gα12/13 activates Rho1 at the apical membrane (also called medial-apical compartment) while Gβ13F-Gγ1 subunits promote Rho1 activity at the apical membrane and at cell junctions. How such a polarized activation of Rho1 is achieved remains unclear. Here, we show that two RhoGEFs, RhoGEF2 and a previously uncharacterized RhoGEF Wireless/p114RhoGEF, control Rho1 activity downstream of G proteins in the ectoderm. RhoGEF2 activates medial-apical Rho1 under control of Gα12/13 and Wireless/p114RhoGEF is required to mediate Gβ13F-Gγ1-dependent activation of Rho1 at junctions. RhoGEF2 is present both at junctions and at the apical membrane. In contrast, Wireless/p114RhoGEF only localizes at junctions together with Gβ13F-Gγ1 which recruit the GEF. Finally, we show that Wireless/p114RhoGEF is absent from junctions in the mesoderm. Collectively, GPCRs shape Rho1 activity through distinct biochemical modules in the ectoderm. Heterotrimeric G proteins transduce the signal by recruiting and activating two complementary RhoGEFs apically and at junctions. Variation in type of GPCRs, G proteins or RhoGEFs underlie the tissue-specific control of Rho1 signaling during morphogenesis.