Grapes(Chk1) prevents nuclear CDK1 activation by delaying cyclin B nuclear accumulation

Entry into mitosis is characterized by a dramatic remodeling of nuclear and cytoplasmic compartments. These changes are driven by cyclin-dependent kinase 1 (CDK1) activity, yet how cytoplasmic and nuclear CDK1 activities are coordinated is unclear. We injected cyclin B (CycB) into Drosophila melanogaster embryos during interphase of syncytial cycles and monitored effects on cytoplasmic and nuclear mitotic events. In untreated embryos or embryos arrested in interphase with a protein synthesis inhibitor, injection of CycB accelerates nuclear envelope breakdown and mitotic remodeling of the cytoskeleton. Upon activation of the Grapes(checkpoint kinase 1) (Grp(Chk1))-dependent S-phase checkpoint, increased levels of CycB drives cytoplasmic but not nuclear mitotic events. Grp(Chk1) prevents nuclear CDK1 activation by delaying CycB nuclear accumulation through Wee1-dependent and independent mechanisms

The Septins Function in G1 Pathways that Influence the Pattern of Cell Growth in Budding Yeast

The septins are a conserved family of proteins that have been proposed to carry out diverse functions. In budding yeast, the septins become localized to the site of bud emergence in G1 but have not been thought to carry out important functions at this stage of the cell cycle. We show here that the septins function in redundant mechanisms that are required for formation of the bud neck and for the normal pattern of cell growth early in the cell cycle. The Shs1 septin shows strong genetic interactions with G1 cyclins and is directly phosphorylated by G1 cyclin-dependent kinases, consistent with a role in early cell cycle events. However, Shs1 phosphorylation site mutants do not show genetic interactions with the G1 cyclins or obvious defects early in the cell cycle. Rather, they cause an increased cell size and aberrant cell morphology that are dependent upon inhibitory phosphorylation of Cdk1 at the G2/M transition. Shs1 phosphorylation mutants also show defects in interaction with the Gin4 kinase, which associates with the septins during G2/M and plays a role in regulating inhibitory phosphorylation of Cdk1. Phosphorylation of Shs1 by G1 cyclin-dependent kinases plays a role in events that influence Cdk1 inhibitory phosphorylation

WSES guidelines for management of Clostridium difficile infection in surgical patients

In the last two decades there have been dramatic changes in the epidemiology of Clostridium difficile infection (CDI), with increases in incidence and severity of disease in many countries worldwide. The incidence of CDI has also increased in surgical patients. Optimization of management of C difficile, has therefore become increasingly urgent. An international multidisciplinary panel of experts prepared evidenced-based World Society of Emergency Surgery (WSES) guidelines for management of CDI in surgical patients.Peer reviewe

Régulation par la phosphorylation d'un module Rho GTPase dans la levure Saccharomyces cerevisiae

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.BORDEAUX2-Bib. électronique (335229905) / SudocBORDEAUX1-Bib.electronique (335229901) / SudocSudocFranceF

Cdk1-dependent control of membrane-trafficking dynamics

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Robust polarity establishment occurs via an endocytosis-based cortical corralling mechanism

Formation of a stable polarity axis underlies numerous biological processes. Here, using high-resolution imaging and complementary mathematical modeling we find that cell polarity can be established via the spatial coordination of opposing membrane trafficking activities: endocytosis and exocytosis. During polarity establishment in budding yeast, these antagonistic processes become apposed. Endocytic vesicles corral a central exocytic zone, tightening it to a vertex that establishes the polarity axis for the ensuing cell cycle. Concomitantly, the endocytic system reaches an equilibrium where internalization events occur at a constant frequency. Endocytic mutants that failed to initiate periodic internalization events within the corral displayed wide, unstable polarity axes. These results, predicted by in silico modeling and verified by high resolution in vivo studies, identify a requirement for endocytic corralling during robust polarity establishment

Robust polarity establishment occurs via an endocytosis-based cortical corralling mechanism

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