One of the hallmarks of postprandial glucose homeostasis is the ability of insulin to promote glucose uptake into skeletal muscles. Insulin achieves this feat by enhancing the recruitment of glucose transporter 4 (GLUT4) from an intracellular compartment to the plasma membrane of muscles in order to create a net increase in surface GLUT4, which results in elevated glucose uptake. From a molecular perspective, this insulin-regulated GLUT4 traffic action requires the independent activation of Akt and Rac-1 in muscle cells because perturbation of either molecule results in an impaired response. Although Rac-1 has been validated as key component of insulin response, its downstream signalling capacity contributing to GLUT4 translocation remains unexplored. Studies on Rac-1 have shown that it is responsible for the formation of cortical remodelled actin that facilitates GLUT4 translocation following insulin stimulation. However, the downstream Rac-dependent molecules governing this actin remodelling are undetermined. Here we identified Arp2/3 and cofilin as the Rac-dependent regulators of insulin-induced actin remodelling in muscle cells. While Arp2/3 acts to initiate a burst of actin polymerization, cofilin balances out the actin dynamics through its severing/depolymerizing activity. Inhibition of either molecule’s function leads to defective GLUT4 translocation mediated by insulin in muscle cells, suggesting the requirement of actin dynamics to facilitate GLUT4 traffic to the plasma membrane. Furthermore, given the importance of Rac-1 in insulin-mediate GLUT4 traffic, its application potential to reverse insulin resistance has never been explored. We discovered that providing muscle cells with additional Rac-1 activity produces an insulin-independent gain in surface GLUT4 with magnitude comparable to that normally elicited by insulin. This phenotype is accomplished because of the concomitant cross-activation of Akt pathway when supplying the cells with active Rac-1. Interestingly, this response can bypass signalling defects imposed by cellular insulin resistance conditions, leading to restoration of GLUT4 translocation in muscle cells. Overall, these results not only reinforce the functional impact of Rac-1 on GLUT4 traffic but also identify additional molecules governed by Rac-1 contributing to the integrity of this insulin-mediated response in muscle cells.

GTPases are found in all domains of life and are highly conserved. In eukaryotes, they serve as signalling molecules for many cellular processes. However, the prokaryotic GTPases play a very different role and are found to be associated with ribosome function. Among the 11 conserved GTPases, Der is the most interesting in prokaryotes. It possesses a unique structure with two GTPase domains (G-Domains) tethered by a variable length acidic linker and a carboxyl terminal KH-like domain. The exact function of Der is still under investigation and most of the data suggest that it is important for 50S ribosomal assembly or stability. In order to investigate the function of Escherichia coli Der (Ec-Der), expression plasmids for wild-type and mutated proteins were created and the proteins were successfully expressed. The expression of the mutant protein that lacked G-Domain 1 was toxic to the cells and it was found that some large ribosomal proteins were missing from the ribosomes of these cells. In addition, other macromolecular complexes such as the GroEL/GroES chaperonin appeared not to be assembled under these conditions. The activities of both wild-type and mutated proteins were also tested and found to be dependent on potassium ions (K+), which enhanced nucleotide binding. Additionally, intra-molecular control over nucleotide binding and release was also observed for Ec-Der. The in vitro selection of RNA aptamers with nanomolar affinity for Ec-Der produced aptamers that contained short variable sequences. These aptamers affected the growth of the E. coli cells and caused a change in cellular morphology that had been noted previously during Ec-Der over-expression. Ec-Der showed high affinity (nM) to both selected RNA and the unselected RNA library. The activity of Ec-Der and Era was inhibited in the presence of any sequence of RNA that has the length of greater than 16 nucleotides. RNA was also cross-linked to Ec-Der in the presence of GTP, but not GDP, suggesting that RNA was a regulator of the Ec-Der GTPase cycle. Based on these results, it is speculated that Ec-Der might be involved in more than one function. It may be acting at the level of the membrane (based on cellular morphology reported here and by Hwang and Inouye 2001) and may also take part in processes related to ribosome function. Regulation of protein activity by RNA length has not been predicted or described and this may represent a novel mean of regulation of the Era subfamily of GTPases.

Thesis (Ph. D.)--University of Texas at Austin, 2004. / Supervisor: John J.G. Tesmer. Vita. Includes bibliographical references. Also available from UMI.

Thèse (M.Sc.)--Université Laval, 2001. / Bibliogr.: f. 67-74. Publié aussi en version électronique.

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The Ras superfamily of GTPases are important regulators of morphogenesis involved in control of cytoskeletal dynamics, intracellular trafficking, apical-basal polarity and cell migration. Mis-regulation of GTPase signaling interferes with development and is linked to pathogenesis. Traditionally, GTPase signaling has been depicted as a series of independent linear pathways. However, recently it has become apparent that multiple GTPases can interact to regulate a single cellular process, functioning in poorly understood networks of cross talk between pathways during development. Jim Fristrom (unpublished data) identified a mutation (18-5) that interacts with components of the GTPases Rho1, Rala, and Cdc42 signaling in multiple developmental contexts. Genetic analysis, physical mapping studies, and sequencing of the mutant allele have indicated that the gene was an allele of GEFmeso (CG30115), which encodes guanine nucleotide exchange factor. To show that 18-5 is an allele of GEFmeso, I demonstrated that a GEFmeso transgene could functionally rescue developmental defects associated with the 18-5 mutation. I also investigated cross talk and network variation in signaling interactions between GEFmeso and other GTPase signaling components in the Drosophila wing. My data provide evidence for microenvironment-dependent variation in GTPase signaling networks in specific domains of the wing, and reveal intercellular variation in GTPase signaling within an otherwise uniform epithelium.

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第23334号 / 生博第452号 / 新制||生||60(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 永尾 雅哉, 教授 渡邊 直樹, 教授 安達 泰治 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

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No description available.

Le cycle cellulaire eucaryote est caractérisé par des changements abrupts et dynamiques de la polarité cellulaire lorsque les chromosomes sont dupliqués et ségrégés. Ces évènements nécessitent une coordination entre la machinerie du cycle cellulaire et les régulateurs de la polarité. Les mécanismes qui contrôlent cette coordination ne sont pas totalement compris. Dans la levure S. cerevisiae, comme dans d’autres organismes eucaryotes, la GTPase Cdc42 joue un rôle important dans la régulation de la polarité cellulaire. En effet ses régulateurs constituent un module GTPase qui subit une phosphorylation dynamique, au cours du cycle cellulaire, par des kinases évolutivement conservées dont la Cycline-Dependent Kinase 1 (Cdk1) et la p21-Activated Kinase (PAK). Ces kinases et substrats pourraient relier la polarité et la progression dans le cycle cellulaire. En utilisant une approche in vitro, nous avons reconstitué la phospho-régulation du Guanine nucléotide Exchange Factor (GEF) de Cdc42, la protéine Cdc24. Nous avons identifié un possible mécanisme de régulation de la phosphorylation impliquant une protéine d’échafaudage qui augmente la phosphorylation de Cdc24 par la PAK et Cdk1. Cette phosphorylation accroit modérément l’affinité de Cdc24 pour cette même protéine d’échafaudage, Bem1. De plus, en testant les effets d’autres composants du module GTPase sur la phosphorylation de Cdc24, nous avons identifié un effet antagoniste pour une GTPase Activating Protein (GAP), Rga2. Cette protéine est présente dans le même complexe que Cdc24 et Bem1, les membres de ce complexe sont tous phosphorylés par Cdk1. Des mutants rga2 suggèrent que la phosphorylation que subie Rga2 inhibe son activité GAP. Nous proposons un modèle provisoire pour expliquer la présence de Rga2 dans ce complexe et l’inhibition qu’elle oppose à la phosphorylation de Cdc24. La présence de la protéine GAP dans le complexe pourrait être un mécanisme de contrôle de la phosphorylation de Cdc24 dans le but de déstabiliser son intéraction avec la protéine Bem1 en cas de mauvaise localisation du complexe. Par ailleurs, la PAK est activée par l’activité de Cdc42, nos résultats sont consistants avec un modèle dans lequel des signaux du cycle cellulaire engendreraient une auto-amplification de l’activation du module GTPase. Chez S. pombe, la croissance polarisée nécessite un gradient d’activation de Cdc42 dû à une ségrégation de GEF et de GAP. Dans ces travaux nous montrons que toutes les protéines GAPs de Cdc42 localisent aux sites de croissance au cours du cycle cellulaire. Ces localisations sont consistantes avec le besoin de cyclage de Cdc42 pour maintenir sa polarisation. Ces résultats suggèrent que la localisation des protéines GAP régulant Cdc42 chez S. cerevisiae semble différente de ce qui est connu chez S. pombe. / The eukaryotic cell cycle is characterized by abrupt and dynamic changes in cellular polarity as chromosomes are duplicated and segregated. Those dramatic cellular events require coordination between the cell cycle machinery and polarity regulators. The mechanisms underlying this coordination are not well understood. In the yeast S. cerevisiae, as in other eukaryotes, the GTPase Cdc42 plays an important role in the regulation of cell polarity. Cdc42 regulators constitute a GTPase module that undergoes dynamic phosphorylation during the cell cycle by conserved kinases including Cyclin-Dependent Kinase 1 (Cdk1) and p21-activated kinase (PAK). These kinases and substrates may link cell polarity to the cell cycle progression. Using in vitro approaches, we have reconstituted the phospho-regulation of the Cdc42 Guanine Nucleotide Exchange Factor (GEF), Cdc24. We have identified a possible mechanism of Cdc24 regulation involving a scaffold-dependent increase in Cdc24 phosphorylation by Pak and Cdk1. This phosphorylation moderately increases the affinity of Cdc24 for another GTPase module component, the scaffold Bem1. Moreover, by testing the effect of other GTPase module components on the phosphorylation of Cdc24, and thus on its interaction with the scaffold, we identified an antagonistic function for the GTPase Activating Protein (GAP) Rga2. Our in vivo data of rga2 mutants suggest that Rga2 phosphorylation by Cdk1 inhibits its GAP activity. We propose a tentative model to explain the inhibition of Cla4 by Rga2 and its presence in a complex containing Cdc24 and Bem1. The presence of the GAP protein in the complex may be a mechanism that reduces Cdc24 phosphorylation in case of a mistargetting of the complex in order to downregulate the GEF/Scaffold dimer. Since the PAK component of the GTPase module is itself activated by Cdc42 activity, our results are consistent with a model in which inputs from the cell cycle lead to auto-amplification of the Cdc42 GTPase module. In S. pombe, polarised growth requires a gradient of activation of Cdc42 due to GEF and GAP segregation. Here we show that all Cdc42 GAPs localise to the polarised site during the cell cycle. Those localisations are consistent with a requirement of Cdc42 cycling to maintain a polarity cap. Our results may suggest that Cdc42 GAPs localisations in S. cerevisiae are different from current knowledge in S. pombe.