Cdc42 is a member of Rho family of Ras GTPase superfamily and has been shown to regulate actin cytoskeleton re-organization and filopodia formation. Calmodulin (CaM) is a calcium modulating protein and regulates calcium dependent signal transduction pathways in the cell. According to CaM target database analysis, amino acid region 151-163 of Cdc42 has a potential CaM binding domain that interacts with CaM. In the present work, we have investigated putative CaM binding region in Cdc42. In addition the role of basic amino acids K153 and K163 within this region in Cdc42 interaction with CaM and effect on Cdc42 activity was elucidated. GST-Cdc42 M (Δ151-163), GST-Cdc42K153A and GST-Cdc42K163A mutants were generated. Binding assay experiments showed that amino acid region 151-163 in Cdc42 is an important regulatory domain for CaM binding. Results also demonstrated that K163A mutant showed significantly reduced binding to CaM, whereas Cdc42 K153A showed reduced but non-significant decrease in its interaction with CaM. A previous study in our laboratory has shown that CaM plays critical role in maintaining basal activity of Cdc42 suggesting that K153A and K163A mutants may play a role in regulating this basal activity. In CHRF 288-11 cells expressing mutant forms of Cdc42 (K153A & K163A), basal activation was significantly decreased as compared to wild type Cdc42. The decrease in basal activity in Cdc42 mutants was not due to an inability to bind GTP. In summary, the results demonstrated that K163 in Cdc42 is a critical amino acid for CaM interaction and in the regulation of basal activity of Cdc42. / October 2015

Epithelial sheets line the surfaces of the body, forming a barrier between the external environment and internal tissues. During development, regulation of epithelial architechture can drive morphogenesis and build the three-dimensional structures of the body. Epithelial form and function derive from the polarized morphology of epithelial cells, which have apical surfaces that face the external environment, lateral surfaces containing cell-cell junctions and basal surfaces that connect to the underlying tissue. A network of polarity proteins establishes the apico-basolateral axis, while a system of polarized membrane traffic ensures delivery of specialized cargo to distinct membrane surfaces. How these systems of polarity and trafficking are integrated is still poorly understood. The focus of my study was to investigate how the apical polarity proteins Cdc42, Par6, Bazooka and aPKC (the “Par complex”) regulate polarity and adherens junction (AJ) integrity during Drosophila development. Upon perturbation of Cdc42/Par activity during embryogenesis, apical membrane proteins accumulate in sorting endosomes. This trafficking defect occurs throughout the ectoderm, but in the ventral neuroectoderm (VNE) is accompanied by a concomitant depletion of the apical proteins from the plasma membrane (PM) and a loss of AJ integrity. I have demonstrated that the VNE phenotype is a consequence of the relatively high morphogenetic activity of this tissue. Furthermore, I have shown that the AJ defects are likely a downstream consequence of the depletion of important apical polarity factors, such as Crumbs, from the PM. To further characterize the mechanism of apical trafficking, I searched for interactors of Cdc42/Par in the membrane trafficking machinery. I describe interactions between several trafficking genes and Cdc42/Par and provide evidence that Vps26, a component of the retromer complex that retrieves proteins from endosomal membrane and delivers them to the Golgi for re-secretion, is phosphorylated by aPKC and acts as an aPKC effector in the recycling of apical membrane proteins. I propose that Cdc42/Par regulate the retromer to promote the PM localization of apical proteins, which is important to maintain AJ integrity in morphogenetically active tissues.

Epithelial sheets line the surfaces of the body, forming a barrier between the external environment and internal tissues. During development, regulation of epithelial architechture can drive morphogenesis and build the three-dimensional structures of the body. Epithelial form and function derive from the polarized morphology of epithelial cells, which have apical surfaces that face the external environment, lateral surfaces containing cell-cell junctions and basal surfaces that connect to the underlying tissue. A network of polarity proteins establishes the apico-basolateral axis, while a system of polarized membrane traffic ensures delivery of specialized cargo to distinct membrane surfaces. How these systems of polarity and trafficking are integrated is still poorly understood. The focus of my study was to investigate how the apical polarity proteins Cdc42, Par6, Bazooka and aPKC (the “Par complex”) regulate polarity and adherens junction (AJ) integrity during Drosophila development. Upon perturbation of Cdc42/Par activity during embryogenesis, apical membrane proteins accumulate in sorting endosomes. This trafficking defect occurs throughout the ectoderm, but in the ventral neuroectoderm (VNE) is accompanied by a concomitant depletion of the apical proteins from the plasma membrane (PM) and a loss of AJ integrity. I have demonstrated that the VNE phenotype is a consequence of the relatively high morphogenetic activity of this tissue. Furthermore, I have shown that the AJ defects are likely a downstream consequence of the depletion of important apical polarity factors, such as Crumbs, from the PM. To further characterize the mechanism of apical trafficking, I searched for interactors of Cdc42/Par in the membrane trafficking machinery. I describe interactions between several trafficking genes and Cdc42/Par and provide evidence that Vps26, a component of the retromer complex that retrieves proteins from endosomal membrane and delivers them to the Golgi for re-secretion, is phosphorylated by aPKC and acts as an aPKC effector in the recycling of apical membrane proteins. I propose that Cdc42/Par regulate the retromer to promote the PM localization of apical proteins, which is important to maintain AJ integrity in morphogenetically active tissues.

La polarité cellulaire, organisation asymétrique du matériel cellulaire dans l'espace et le temps, est fréquemment observée en biologie. Elle est nécessaire pour de nombreux mécanismes cellulaires essentiels allant de la division cellulaire et la migration au développement et la croissance polarisée. Comprendre comment la cellule génère et maintient cette polarité est crucial, les défauts de polarité étant liés à des maladies graves comme le cancer ou les maladies neurodégénératives. Chez la levure Saccharomyces cerevisiae, la polarité cellulaire est établie lorsque le module de la Rho GTPase Cdc42, qui comprend le facteur d'échange de nucléotide guanine (GEF) Cdc24 et la protéine scaffold Bem1, localise à un unique site à la membrane plasmique pour activer Cdc42 et ainsi, établir un axe de polarité utilisé pour la croissance et la division cellulaire. Les mécanismes responsables de l'activation de Cdc42 à un site unique au cortex pendant l'établissement de la polarité sont essentiels mais largement inconnus. En utilisant des expériences complémentaires d'imagerie in vivo et des expériences in vitro, je mis en évidence que le ciblage avide du module de Cdc42 à la membrane plasmique implique des interactions multivalentes entre des lipides anioniques et le module de Cdc42. En détail, j'ai démontré que la combinaison de plusieurs phospholipides anioniques, comprenant PS, PI4P et PI(4,5)P2, est nécessaire à la localisation de Bem1 et Cdc24 in vivo. J'ai identifié des groupements cationiques interagissant avec des lipides (CLICs) dans l'extrémité N-terminale de Bem1 qui étaient nécessaires et suffisants pour interagir avec des phospholipides anioniques. Réduire l’interaction de Bem1 avec les lipides en mutant la séquence CLICs a fortement diminué la localisation de Bem1 au niveau du cortex ainsi que la signalisation de Cdc42. En plus des CLICs de Bem1, le domaine PX de Bem1 et le domaine PH de Cdc24 augmentent davantage l'avidité du module GTPase pour les lipides anioniques et la combinaison des trois domaines est essentielle pour l'établissement de la polarité cellulaire. Ces résultats définissent pour la première fois le mécanisme de ciblage avide des activateurs de Cdc42 à la membrane plasmique pendant l'établissement de l'axe de polarité. / Cell polarity, the asymmetric organization of cell material in space and time, is frequently observed in biology. It is required for numerous essential cellular processes ranging from cell division and migration to development and polarized growth. Addressing how cells generate and maintain polarity is crucial, since defects in polarity are linked to severe diseases including cancer and neurodegeneration. In the budding yeast Saccharomyces cerevisiae, cell polarity is established when the Cdc42 Rho GTPase module, which includes the Guanine nucleotide Exchange Factor (GEF) Cdc24 and the scaffold protein Bem1, accumulate at a unique site on the plasma membrane to activate Cdc42 and establish the polarity axis used for cell growth and division. The mechanisms responsible for the site-specific activation of Cdc42 at the cortex during polarity establishment are essential but are largely unknown. Using complementary in vivo imaging and in vitro experiments, I found that the avid targeting of the Cdc42 GTPase module to the plasma membrane involves multivalent anionic lipid-Cdc42 module interactions. I found that a combination of anionic phospholipids, including PS, PI4P and PI(4,5)P2, are necessary for Bem1 and Cdc24 localization in vivo. I identified Cationic-enriched Lipid Interacting Clusters (CLICs) in the N-terminus of Bem1 that were necessary and sufficient for anionic phospholipid interactions. Reducing Bem1 lipid binding by mutating the CLICs strongly diminished the localization of Bem1 at the cortex and Cdc42 signaling. In addition to the Bem1 CLICs, the Bem1 PX domain and the Cdc24 PH domain increased the avidity of the GTPase module for anionic lipids, and a combination of all three domains was essential for the establishment of cell polarity. The results of my thesis define a mechanism of avid targeting of Cdc42 activators to the cortex during polarity axis establishment.

In vitro, la dégradation de la matrice extracellulaire (MEC) par ces cellules invasives est effectuée par les invadopodes, des protrusions cellulaires dotées d’une activité de protéolyse émanant de la membrane ventrale de cellules cultivées sur des substrats bidimensionnels. Le facteur d'échange (GEF) Fgd1, un GEF spécifique de Cdc42, a été montré comme étant un des composants des invadopodes. Nonobstant, la façon dont Fgd1 est régulé n’est pas encore connue. Mon projet visait à élucider la régulation de Fgd1 dans les cellules cancéreuses invasives. J'ai montré que la distribution subcellulaire de la protéine endogène varie au cours des différentes étapes de la formation des invadopodes et que Fgd1 s'accumule aux sites de formation des invadopodes et ce de façon concomittante à l'assemblage du noyau F-actine/cortactine. La partie N –terminale de la molécule est essentielle pour la localisation aux invadopodes. En outre, la Filamine A (FLNa) a été identifiée comme un nouveau partenaire de liaison in vitro et in vivo. J’ai de plus montré que FLNa colocalise avec Fgd1 aux invadopodes et est nécessaire pour leur formation et leur fonction. J'ai émis l'hypothèse que FLNa puisse être impliquée dans la dégradation de Fgd1 par le protéasome, un processus étroitement lié au niveau de l'activation de Cdc42. Dans mon modèle, FLNa agirait comme une protéine d’échafaudage pour connecter Fgd1 et Cdc42 et ainsi réguler localement l'activation de la GTPase, mais également pour stabiliser Fgd1 en empêchant sa dégradation par le protéasome. Dans l'ensemble, mes résultats apportent de nouveaux éléments concernant le rôle de Fgd1 dans la biogénèse des invadopodes / In vitro, degradation of the extracellular matrix (ECM) by invasive tumor cells is carried out by invadopodia, proteolytically active protrusions emanating from the ventral membrane of cells cultured on two dimensional ECM substrates. Identification and characterization of novel molecular components of the invadopodia machinery is now witnessing a relevant interest. The guanine nucleotide exchange factors (GEFs) Fgd1, a specific GEF for Cdc42, has been recently brought into the picture of invadopodia components and regulators. Notwithstanding, how Fgd1 is regulated in invasive cancer cells remains poorly understood. My project aimed to further elucidate Fgd1 regulation in cancer cells. I showed that the cellular distribution of endogenous Fgd1 quantitatively changes along the different stages of invadopodia formation, and that Fgd1 accumulates at invadopodia sites concomitantly with the initial assembling of the actin/cortactin core. The N-terminal proline rich domain (PRD) of Fgd1 is essential for its localization and function at invadopodia. Furthermore, using a yeast two hybrid approach, Filamin A (FLNa) was identified as a novel Fgd1 binding partner and this interaction was validated in vivo. I report that FLNa co-localizes with Fgd1 at invadopodia and is required for their formation and function. I hypothesized that FLNa may be involved in the SCF FWD1/β-TrCP –mediated proteasome degradation of Fgd1, a process that is strictly connected to the Cdc42 activation rate. In my model, FLNa may act as a scaffold to connect Fgd1 and Cdc42 for local activation of the small GTPase, and to increase Fgd1 stability by preventing its proteasomal degradation. Taken together, my findings provide novel insights on the role of Fgd1 in invadopodia biogenesis

The vegetal cortex of the Xenopus oocyte is enriched for several mRNAs critical for early embryonic developmental processes, including germ layer specification and dorsoventral axis formation. A recent microarray screen for other vegetally localized RNAs identified several hundred novel cortex-enriched transcripts, which may have undiscovered roles in early development. In order to better elucidate the functions of localized mRNAs in early development, I characterized the spatiotemporal expression patterns and developmental functions of two novel transcripts, TRIO and F-actin binding protein (triobp) and Cdc42 effector protein 4-like (cep4l). Overexpression and loss-of-function experiments failed to identify a critical role for TrioBP in early Xenopus development. For Cep4l, I found that overexpression of Cep4l induced primary neuron formation throughout the epidermis, preferentially inducing primary sensory neurons. This increase came at the expense of neighboring non-neuronal ciliated and ion-secreting cells, suggesting a role for Cep4l in neural boundary formation. Additionally, I have shown that Cep4l binds specifically to Cdc42 through its known Cdc42/Rac-interactive binding (CRIB) domain, and that this activation was necessary for Cep4l function. Morpholino (MO) oligonucleotide based inhibition of Cep4l protein synthesis resulted in decreased primary sensory neurogenesis. Additionally, I have shown that Cdc42 itself is required for sensory neurogenesis. Furthermore, I find that Fgf8a, an isoform of Fgf8 previously known to regulate neuronal development, but not the Fgf8b isoform, regulates the association of Cep4l and Cdc42. Importantly, I further show that Cep4l and Cdc42 are required for the ability of Fgf8a to induce sensory neurons. Overall, this work suggests a novel role for Cep4l and Cdc42 in the regulation of primary sensory neuronal fate downstream of a unique Fgf8 signaling pathway. I propose that binding of Fgf8a to its receptors activates Cdc42 and recruits Cep4l, which could serve as a scaffold for integrating additional signaling pathways involved in controlling sensory neuron fate.

How proliferating cells maintain the copy number and overall size of their organelles is not clear. In the budding yeast Saccharomyces cerevisiae the G1 cyclins Cln1,2,3p control initiation of cell division by regulating the activity of the cyclin-dependent kinase (Cdk) Cdc28p. We show that Cln3p controls vacuolar (lysosomal) biogenesis and segregation. First, loss of Cln3p, but not Cln1p or Cln2p, resulted in vacuolar fragmentation. Although the vacuoles of cln3Δ cells were fragmented, together they occupied a large space, which accounted for a significant fraction of the overall cell size increase in cln3Δ cells. Second, cytosol prepared from cells lacking Cln3p had reduced vacuolar homotypic fusion activity in cell-free assays. Third, vacuolar segregation was perturbed in cln3Δ cells. Our findings reveal a novel role for a eukaryotic G1 cyclin in cytoplasmic organelle biogenesis and segregation. Furthermore we show that the scaffold protein Bem1p, a critical regulator of Cdc42p activity, is a downstream effector of Cln3p/Cdc28p complex. The Cdc42p GTPase is known to be required for vacuole fusion. Our results suggest that Ser72 on Bem1p is phosphorylated by Cdc28p in a Cln3p-dependent manner to promote vacuole fusion. Replacing Ser72 with Asp, to mimic phosphorylation at an optimal Cdkconsensus site located in the first SH3 domain of Bem1p, suppressed vacuolar fragmentation in cells lacking Cln3p. Using in vivo and in vitro assays, we found that Cln3p was unable to promote vacuole fusion in the absence of Bem1p or in the presence of a non-phosphorylatable Bem1p-Ser72Ala mutant. Furthermore, activation of Cdc42p also suppressed vacuolar fragmentation in the absence of Cln3p. Our results provide a mechanism that links cyclin-dependent kinase activity with vacuole fusion through Bem1p and the Cdc42p GTPase cycle.

Rho GTPases are highly conserved regulators of cell polarity and the actin cytoskeleton. The role of the Rho GTPase Cdc42 and its regulation during cell division is not well understood. Using biochemical and imaging approaches in budding yeast, I demonstrate that Cdc42 activation peaks during the G1/S transition and during anaphase, but drops during mitotic exit and cytokinesis. Cdc5/Polo kinase is an important upstream cell cycle regulator that suppresses Cdc42 activity. Failure to downregulate Cdc42 during mitotic exit prevents the normal localization of key cytokinesis regulators - Iqg1 and Inn1- at the division site, and results in an abnormal septum. The effects of Cdc42 hyperactivation are largely mediated by the Cdc42 effector p21-activated kinase (PAK) kinase, Ste20. Inhibition of Cdc42 and related Rho GTPases may be a general feature of cytokinesis in eukaryotes.

Prior work has indicated that preventing Cdc42 interacting with its downstream effector proteins can reverse Ras-driven oncogenesis. The experiments demonstrating this used the G protein binding region (GBD) of the Cdc42-specific effector ACK, which is 42 amino acids long and binds with 40 nM affinity. The aim of this work was to find a peptide which exhibits similar effects in reversing oncogenic characteristics in cells but with more promising therapeutic properties: for example, a smaller size, improved binding and improved protease resistance. Firstly, the Cdc42-ACK binding interface was characterised thermodynamically, producing a comprehensive dataset of the contribution of individual ACK residues to complex affinity. This information revealed regions of the ACK GBD with higher concentrations of vital interactions but also residues that were unimportant for maintaining affinity. An \emph{in vitro} display technology (CIS-display) was employed to select for shorter peptides with improved binding to Cdc42. By selecting the best binders from libraries based on the ACK GBD, peptides have been generated that are half the length of the full binding domain but bind with affinities approaching the wild-type ($\approx$100 nM). More interestingly, a serendipitous discovery in a na\"{\i}ve cyclic library revealed a 16-residue sequence that binds with 350 nM affinity, subsequently called C1. The biophysical properties of selected peptides were characterised and demonstrate that C1 is reliant on an intramolecular disulphide bond for tight binding. An N-terminal nona-arginine sequence was added to C1 to facilitate entry into cells: internalisation was confirmed by confocal microscopy. Subsequently it was found that C1 reduced signalling through the ACK and PAK effector pathways, negatively impinging on MAPK signalling. Levels of signalling returned to baseline within 24 hours, however, and the transiency of the effect was thought to be linked to peptide degradation: an effect likely in a construct dependent on a disulphide bond for cyclisation and binding affinity. Subsequent maturation of C1 using a focussed CIS-display library has selected second generation peptides. These have been characterized and display binding affinities to Cdc42 of $\approx$20 nM: a 17-fold increase in binding over C1. This has enhanced the affinity to a level even higher than wild-type ACK and should improve the cellular potency of the peptides identified in this selection. %any more?! Having successfully selected peptides both shorter in length and with higher affinity than the wild-type sequence, efforts were channelled into developing a method to cyclize the peptide by a chemical linkage that would be stable in a cellular environment. The most successful approach involved desulphurisation of the disulphide bond to a thioether, making the cyclic linkage non-labile in the reducing environment of the cytosol, while retaining original binding affinity. The peptides developed in this work bind to Cdc42 with nanomolar affinity \emph{in vitro} and can enter cells and impact upon signalling processes connected with oncogenesis. As such, they provide a potential therapeutic lead for future development.

L’activation d’un lymphocyte T (LT) CD4+ par une cellule présentatrice d’antigène (CPA) estune étape cruciale pour la mise en place d’une réponse immune adaptatrice efficace contre unpathogène ou une cellule tumorale. Elle nécessite un contact prolongé entre les deux types cellulaires,initié par la reconnaissance, par le récepteur des LT (TCR), d’un complexe CMH-peptide spécifiqueprésenté à la surface de la CPA. Cette interaction LT-CPA induit la formation d’une zone de contactorganisée dans le temps et l’espace, appelée la synapse immunologique. La mise en place de cettestructure entraîne le remodelage des cytosquelettes d’actine et de microtubules dans les LT. Quelquesminutes après la reconnaissance de l’antigène par le TCR, l’actine se polymérise à la zone synaptiqueet le centre organisateur des microtubules (MTOC) se polarise en face de la CPA.Le but du travail de thèse présenté ici a été d’étudier le rôle de la réorganisation ducytosquelette des LT lors de la mise en place de la synapse, dans la sécrétion des cytokines et lemaintien de l’interaction permettant l’activation des LT. Nous nous sommes particulièrementintéressés au rôle de deux protéines qui régulent le remodelage du cytosquelette : la petite RhoGTPaseCdc42 et un de ses partenaires IQGAP1. Cette étude a été réalisée essentiellement dans des LT CD4+primaires humains, grâce au développement d’une approche permettant d’inhiber l’expression de cesdeux protéines par introduction de shRNAs à l’aide de vecteurs lentiviraux.Nous avons ainsi mis en évidence que le remaniement du cytosquelette d’actine à la synapseétait dépendant de Cdc42, et contrôlait : 1/ la formation d’un anneau d’actine polymérisée enpériphérie de la synapse, 2/ le recrutement et la concentration des vésicules contenant l’IFN-g aucentre de la synapse, 3/ la sécrétion de l’IFN-g dans la zone synaptique. Nous avons également montréque la protéine IQGAP1 contrôlait le remaniement de l’actine des LT à la synapse, la signalisation enaval du TCR, et la dynamique de contact entre LT et CPA.Cette étude participe à une meilleure compréhension du rôle du remodelage du cytosqueletted’actine et de sa régulation dans la mise en place de l’interaction entre LT et CPA, l’activation des LTet une de leur fonction clé : la sécrétion de lymphokines. / CD4+ T lymphocyte activation by an antigen presenting cell (APC) is a crucial step in theestablishment of an adaptive immune response against pathogens or tumor cells. It requires contactbetween the two cell types, initiated by the recognition, by the T cell receptor (TCR), of a specificpeptide-MHC complex presented by the APC. This T cell-APC interaction induces the formation of aparticular zone organized in time and space, called the immunological synapse. The establishment ofthis structure induces the remodeling of the actin and microtubule cytoskeleton in T cells. Few minutesafter antigen recognition by the TCR, actin polymerizes at the synaptic zone, and the microtubuleorganizing center (MTOC) polarizes toward the APC.The goal of the work presented here was to study the role of T cell cytoskeleton remodelingduring the establishment of the synapse, in cytokine secretion and in the maintenance of the interactionallowing T cell activation. We mainly studied the role of two proteins regulating the cytoskeleton: thesmall RhoGTPase Cdc42 and one of its partners IQGAP1. This study was performed mostly in humanprimary CD4+ T cells, thanks to the development of an approach allowing the inhibition of theexpression of these two proteins by introducing shRNAs through lentiviral vectors.Altogether, our results show that remodeling of the actin cytoskeleton at the synapse isdependent on Cdc42 and controls : 1/ the formation of a polymerized actin ring at the periphery of thesynapse, 2/ the recruitment and concentration of vesicles containing IFN-g at the center of the synapse,3/ the secretion of IFN-g in the synaptic cleft. They also show that IQGAP1 controls T cell actinremodeling at the synapse, signaling downstream the TCR, and the dynamic of interactions between Tcells and APC.This study allows a better understanding of the role of cytoskeleton remodeling and of itsregulation in the establishment of Tcell-APC interaction, the activation of T cells and one of their keyfunctions: lymphokine secretion.