Auswahl der wissenschaftlichen Literatur zum Thema „Regulation of Cdc42“

Les mutations sont responsables de diverses pathologies du développement, en particulier chez les patients atteints de maladies rares ou pour lesquels il n’y a pas de diagnostic clinique clair. Cdc42 est une protéine clé pour la polarité cellulaire, une étape cruciale de nombreux processus cellulaires, comme la migration cellulaire, la division cellulaire ou la réponse immunitaire. Les mutations de Cdc42 entrainent une variété de pathologies, par exemple des dérégulations de la croissance ou de la morphologie faciale ainsi que des anomalies immunologiques, hématologiques et du développement neuronal. Les fonctions de Cdc42 reposent en grande partie sur la localisation de cette protéine dans la cellule. La comparaison des différentes formes de Cdc42 et de certaines formes mutantes montrent que les derniers acides aminés de la protéine jouent un rôle clé dans sa localisation et donc dans sa fonction. Nous avons centré notre étude sur l’identification : 1) des acides aminés essentiels à la localisation de la protéine ; et 2) de nouveaux mécanismes de régulation de Cdc42 responsables de sa localisation intracellulaire. Nous avons aussi montré que les deux isoformes jouent des rôles différents au cours de la migration cellulaire. Ce travail devrait nous permettre de mieux comprendre les pathologies liées aux mutations de Cdc42<br>Mutations in proteins cause diverse developmental disorders, particularly for individuals with rare diseases or for whom a unifying clinical diagnosis is unknown. Cdc42 is one such protein; vital for establishing cell polarity, a crucial step in many biological processes such as cell migration, division and immune responses. Not surprisingly, mutations in Cdc42 cause a range of diseases such as growth dysregulation, facial dysmorphism and neurodevelopmental, immunological, and hematological abnormalities. In vertebrates there are two isoforms of Cdc42. The first being the ubiquitous isoform, has almost exclusively been studied and the role of the second isoform, being the brain isoform, is largely unknown. We have shown that the two isoforms are localized differently in cells. The ubiquitous isoform is mostly found in the cell cytoplasm and at the plasma membrane, while the Brain isoform localizes at the Golgi apparatus and on intracellular vesicles. We have also shown that the two isoforms carry out different functions during cell migration, suggesting that the differences between these two isoforms which only differs by the last 10 amino acids are responsible for their distinct localisation and function. Interestingly, a mutation in the C-ter sequence of Cdc42 ubiquitous isoform alters Cdc42 localisation and causes a generalized pustular psoriasis disease. Two main objectives have been studied in this project 1) the impact of the last amino acids of the protein in Cdc42 localization; and 2) new regulatory mechanisms of Cdc42 responsible for its intracellular localization. These findings will bring a better understanding of pathologies related to Cdc42 mutations

The generation of cell polarity is essential for the development of multi-cellular organisms as well as for the function of epithelial organs in the mature animal. Small GTPases regulate the establishment and maintenance of polarity through effects on cytoskeleton, membrane trafficking, and signaling. Using short-term 3-dimensional culture of MDCK cells, we find that the small GTPase Rab14 is required for apical membrane specification. Rab14 knockdown results in disruption of polarized lipid domains and failure of the Par/aPKC/Cdc42 polarity complex to localize to the apical membrane. These effects are mediated through tight control of lipid localization, as overexpression of the phosphatidylinositol 4-phosphate 5-kinase a [PtdIns(4) P5K] activator Arf6 or PtdIns(4) P5K alone, or treatment with the phosphatidylinositol 3-kinase (PtdInsI3K) inhibitor wortmannin, rescued the multiple-apical domain phenotype observed after Rab14 knockdown. Rab14 also co-immunoprecipitates and colocalizes with the small GTPase Cdc42, and Rab14 knockdown results in increased Cdc42 activity. Furthermore, Rab14 regulates trafficking of vesicles to the apical domain, mitotic spindle orientation, and midbody position, consistent with Rab14' s reported localization to the midbody as well as its effects upon Cdc42. These results position Rab14 at the top of a molecular cascade that regulates the establishment of cell polarity.

All eukaryotic cells rely on endocytic events to satisfy a constant need for nutrient and fluid uptake from their surroundings. Endocytosis-dependent turnover of cell surface constituents also serves to control signal transduction and establish morphological changes in response to extracellular stimuli. During endocytosis, distinct protein machineries re-sculpt the plasma membrane into vesicular carriers that enclose molecules that are to be taken up into the cell. Besides those produced from the canonical clathrin-mediated endocytic machinery, it is becoming increasingly clear that other membrane carriers exist. The indisputable connection between the function of these uptake systems and various disease states, highlights why it is so important to increase our knowledge about the underlying molecular machineries. The aim of this thesis was therefore to characterise the function of GRAF1, a protein suggested to be a tumour suppressor due to that the gene has been found to be mutated in certain cancer patients. My work focused on understanding how this protein operates during formation of clathrin-independent carriers, with possible implications for disease development. Previous in vitro studies showed that GRAF1 harbours a GTPase activating domain to inactivate Rho GTPase Cdc42, a major actin cytoskeleton regulator. Herein, microscopy based approaches used to analyse HeLa cells demonstrated the importance of a transient interaction between GRAF1 and Cdc42 for proper processing of GRAF1-decorated carriers. Although GRAF1-mediated inactivation of Cdc42 was not vital for the budding of carriers from the plasma membrane, it was important for carrier maturation. In addition, studies of purified GRAF1 and its association with lipid bilayers identified a membrane scaffolding-dependent oligomerisation mechanism, with the ability to sculpt membranes. This was consistent with the assumption that GRAF1 possesses an inherent banana shaped membrane binding domain. Remarkably, this function was autoinhibited and in direct competition with the Cdc42 interaction domain. Finally, other novel GRAF1 interaction partners were identified in this study. Interestingly, many of these partners are known to be associated with protein complexes involved in cell adherence, spreading and migration. Although never actually seen localising to mature focal adhesions that anchor cells to their growth surface, dynamic GRAF1 carriers were captured travelling to and from such locations. Moreover, GRAF1 was recruited specifically to smaller podosome-like structures. Consistent with this, the tracking of GRAF1 in live cells uncovered a clear pattern of dynamic carrier formation at sites of active membrane turnover – notably protrusions at the cell periphery. Furthermore, the silencing of GRAF1 gave rise to cells defective in spreading and migration, indicating a targeting of GRAF1-mediated endocytosis to aid in rapid plasma membrane turnover needed for morphological changes that are a prerequisite for cell movement. Since these cells exhibited an increase in active Rab8, a GTPase responsible for polarised vesicle transport, the phenotype could also be explained by a defect in Rab8 trafficking that results in hyperpolarisation. Taken together, the spatial and temporal regulation of GRAF1 membrane sculpting function is likely to be accomplished via its membrane binding propensity, in concert with various protein interactions. The importance of GRAF1 in aiding membrane turnover during cell movement spans different functional levels – from its local coordination of membrane and actin dynamics by interacting with Cdc42, to its global role in membrane lipid trafficking.

Cell polarisation is a key biological process crucial for the functioning of essentially all cells. Regulation of cell polarity is achieved through various processes determined by both internal and external factors. An example of the latter is that cell polarity can be disrupted or lost as a consequence of a variety of external stresses. When facing such stresses, cells adapt to unfavourable conditions by activating a range of molecular signalling pathways, collectively termed ‘stress response’. Despite the connections between external stress and cell polarity, whether stress-response signalling regulates cell polarisation and what the molecular basis for such regulation remains an open question. The fission yeast Schizosaccharomyces pombe presents an excellent biological platform to study the complexity of cell polarity regulation on a systematic level. This study is aimed at understanding the functional relationship between stress-response signalling and maintenance of cell polarity in this model organism. The findings presented in this thesis set the basis for establishing a functional link between the activation of the S.pombe stress-response pathway and the activity of the master regulator of cell polarity- the Rho GTPase Cdc42. Here, I describe experiments that identify an active involvement of the stress-response mitogen-activated kinase (MAPK) Sty1 in the dispersal of active Cdc42 from the sites of growth. This new role for Sty1 occurs independently from its involvement in transcription regulation and other previously identified signalling pathways involving Sty1. Furthermore, I also find that Sty1’s involvement in Cdc42 regulation has direct implications for fission yeast physiology as it is essential for the maintenance of cellular quiescence upon nitrogen starvation. This thesis also focuses on identifying the targets of Sty1 orchestrating the active Cdc42 disruption. Here, I describe a candidate-based approach, where I investigate the role of proteins from the Cdc42 regulatory network during Sty1 activation. Additionally, I present a global phospho-proteomics approach to identify novel targets of Sty1 and offer preliminary findings which might explain Sty1’s involvement in Cdc42 regulation.

The Rho GTPases form a family of enzymes that control numerous cellular processes including cell migration and proliferation through effects on the cytoskeleton, membrane trafficking and cell adhesion. The activity of these molecular switches is modulated by GTPase-activating proteins (GAPs), a group of negative-regulators which includes Cdc42-GTPase-Activating Protein (CdGAP). This protein specifically negatively-regulates the Rho GTPases Cdc42 and Rac1. In this study, we show that CdGAP is regulated by lipid-, protein- and intramolecular-interactions. First, we demonstrate that a polybasic region (PBR) of CdGAP preceding the GAP domain and found in numerous Rho family GAPs is required for CdGAP specific association with phosphatidilinositol-3,4,5-trisphosphate (PI(3,4,5)P3). We show that the binding of PI(3,4,5)P3 is required for CdGAP-mediated GAP activity in vitro, and that an intact PBR is required for its CdGAP-mediated GAP activity in vivo. Second, we characterize the binding site for the negative-regulator of CdGAP Intersectin-1 located in the Basic-Rich (BR) domain of CdGAP. We present evidence that this interaction mediated by the SH3D domain of Intersectin requires one to three lysine residues located in the Basic-Rich (BR) domain of CdGAP. Thirdly, we show that CdGAP is negatively-regulated by its C-terminal domain. This observation is part of a study that links two human CdGAP gene mutations to a syndrome which presents a combination of aplasia cutis congenita (ACC) and terminal transverse limb defects (TTLD). In this syndrome, the deletion-mutant gene products which lack the residual amino-acid of CdGAP at its C-terminus have an increased activity compared to wild-type proteins. We show that this C-terminus can bind to the GAP domain of CdGAP, providing a model to explain how the absence of the C-terminus induces this syndrome. In summary, this work provides novel insight into understanding the mechanisms of regulation of CdGAP, a protein involved in cell migration and adhesion with unexpected roles related to human diseases.<br>Les Rho GTPases forment une famille d'enzymes qui contrôlent de nombreux processus cellulaires, tels que la migration cellulaire et la prolifération, grâce à leurs effets sur le cytosquelette, le trafic membranaire et l'adhésion cellulaire. L'activité de ces interrupteurs moléculaires est modulée par les protéines activatrices de GTPases (GAPs), un groupe de régulateurs négatifs qui inclu CdGAP (Cdc42-GTPase activating protein). Cette protéine régule négativement les Rho GTPases Cdc42 et Rac1 de façon spécifique. Dans la présente étude, nous montrons que CdGAP est régulée par des interactions lipidiques, protéiques et intramoléculaire. Premièrement, nous démontrons qu'une région polybasique (PBR), précédant le domaine GAP et retrouvée dans plusieurs GAP de la famille Rho, est requise pour l'association spécifique de CdGAP avec le phosphatidilinositol-3,4,5-trisphosphate (PI(3,4,5)P3). Nos résultats suggèrent que l'activation des GAP requiert la liaison du PI(3,4,5)P3 à CdGAP dans un contexte in vitro et un PBR intact pour que CdGAP provoque ses effets GAP-dépendants dans un contexte in vivo. Deuxièmement, nous caractérisons le site de liaison du régulateur négatif de CdGAP Intersectin-1. Ce site est localisé dans le domaine riche en résidus basiques (BR) de CdGAP. Nous suggérons que cette interaction, médiée par le domaine SH3D d'Intersectin, requiert de un à trois résidus lysine dans le domaine BR de CdGAP. Troisièmement, nous montrons que CdGAP est régulé de manière négative par son propre domaine C-terminal. Cette observation fait partie d'une étude qui associe deux mutations humaines du gène CdGAP à un syndrôme présentant une combinaison d'aplasie cutis congenita (ACC) et de malformation des doigts et des orteils (TTLD). Les gènes mutants produisent des protéines tronquées qui ont une activité GAP supérieure à la protéine de type sauvage. Nous montrons que ce C-terminal peut lier le domaine GAP de CdGAP, supportant un modèle expliquant comment l'absence du C-terminal induit ce syndrome. En bref, ce travail présente un nouvel aperçu des mécanismes de régulation de CdGAP, une protéine impliquée dans la migration cellulaire et dans l'adhésion des cellules en plus d'être directement impliquée dans une maladie humaine.

The Epidermal Growth Factor Receptor (EGFR) is overexpressed in several solid tumours including squamous cell carcinoma of the head &amp; neck (SCCHN). Drugs that directly block the action of EGFR are currently available. However, a major unanswered question is; how best to select patients most likely to respond to these new treatments, as the response rate to EGFR-targeted mono-therapy in SCCHN, as well as other solid tumours, such as lung and colorectal cancer, is very low. Resistance to EGFR therapy may stem from aberrant receptor trafficking. Cdc42 is known to affect EGFR downregulation by sequestering c-Cbl, preventing it from catalyzing receptor ubiquitination. The aim of this project is to determine the role of Cdc42 in mediating cancer cell response to EGFR and ErbB-family targeted therapy. Using optical imaging techniques such as, Fluorescence Resonance Energy Transfer (FRET) and Fluorescence Lifetime Imaging Microscopy (FUM) I have analysed Cdc42 activity in Squamous Carcinoma Cell Lines after EGFR tyrosine kinase inhibitor (TKI) treatment, using the FRET biosensor Raichu-Cdc42. I have demonstrated that conversely, Raichu-Cdc42, and consequently endogenous Cdc42 activity increases significantly following EGFR TKI treatment. Further investigation revealed that the serine/threonine kinase, c-Jun NH2 Terminal Kinase 1 (JNK1) may modulate Cdc42 activity via a negative feedback mechanism, and JNK1 in turn regulates EGFR ubiquitination and downregulation. I have also demonstrated for the first time protein-protein interactions in pathological SCCHN tissue between members of the ErbB receptor family (EGFR and HER2) and between PKCa and Ezrin, using FRET and FUM. The novel results from this thesis provides further knowledge on factors influencing EGFR downregulation in SCCHN, that could account for the resistance to EGFR targeted therapies observed in a clinical setting. In addition the optical proteomic assays could be translated into new diagnostic/predictive tests, potentially allowing us to improve the outcome for head and neck cancer patients.

L'exocytose nécessite la formation d'un pore de fusion. Le pore initial est étroit ; seules de petites molécules sont libérées. Quand le pore s'élargit les macromolécules sont libérées. J'ai étudié le rôle de deux protéines sur la dilatation du pore: la protéine SNARE synaptobrévine 2 (Syb2), et la Rhô GTPase Cdc42. L'assemblage des SNAREs fournirait l'énergie nécessaire à la fusion. L'insertion d'un espacer dans le domaine juxtamembranaire de Syb2 ne modifie pas la fréquence des événements d'exocytose détectés par ampérométrie à 1|jM [Ca2+], mais empêche l'apparition d'une composante de sécrétion à de plus fortes [Ca2+]. Les événements peuvent être classés en deux groupes, liés à la vitesse et au degré de dilatation des pores; l'allongement de Syb2 réduit la population de pics rapides, mais n'affecte pas celle des pics lents. Les événements lents seraient dus à un assemblage partiel des SNAREs, alors que ceux dits rapides résulteraient d'un assemblage plus serré, assurant ainsi une dilatation rapide du pore. Cdc42 contrôle la dynamique de l'actine. Diminuer son expression dans les cellules BON réduit le nombre de granules fusionnant complètement avec la membrane, mais n'affecte pas leur recrutement et leur liaison à la membrane. Réduire l'expression de Cdc42 diminue le nombre de hauts pics dus à une dilatation rapide et complète des pores, et augmente le nombre de pieds seuls, dus à des pores ne s'élargissant pas. L'augmentation de tension de la membrane corrige les effets dus à l'absence de Cdc42 ; sa diminution par dépolymérisation de l'actine imite les effets obtenus en son absence. Cdc42 contrôlerait la dilatation du pore en modulant la tension de membrane<br>Exocytosis ends with the formation of a fusion pore. The initial pore is narrow, only small molecules flow through it. The pore then enlarges, releasing larger secretory products. I studied the role of two proteins on the dilation of the pore: the SNARE protein synaptobrevin 2 (Syb2), and the Rho GTPase Cdc42. Zippering of SNAREs in opposed membranes might give energy to catalyze fusion. Inserting a linker between the SNARE core and the transmembrane domain of Syb2 did not modify the frequency of exocytotic events detected by amperometry at 1|jM free [Ca2+] but prevented the occurrence of an extra component of release at higher [Ca2+]. Analysis of these events led to their classification into two groups, due to the rate and extent of dilation of the pore; lengthening Syb2 reduced the population of fast spikes, leaving the slow one unchanged. Slow fusion events might be due to a partial zippering of the SNAREpin while fast fusion events require a tight one, i. E. A short intermembrane distance to assure rapid dilation of the pore. Cdc42 controls actin dynamics. TIRFM experiments showed that its silencing in BON cells reduced the number of granules undergoing full fusion, with little effect on their recruitment and docking at the membrane. Using amperometry, we showed that this silencing reduced the number of high spikes due to fast and complete dilation of the pore, and increased stand-alone foot signals reflecting pores failing to enlarge. Increasing membrane tension rescued the effects of silencing while decreasing it through actin depolymerization mimicked Cdc42 silencing. Cdc42 might control fusion pore dilation by modulating membrane tension