Projet ANR-10-JCJC-1210 – Growth and Division : Les mécanismes moléculaires qui coordonnent la croissance et la division cellulaire
Programme : JCJC : Sciences de la vie, de la santé et des écosystèmes :  Biologie cellulaire, développement (JCJC SVSE 2)
Année de financement : 2010
Coordinateur du projet : Monsieur Derek MCCUSKER (CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION  AQUITAINE LIMOUSIN)
Partenaires : Centre national de la recherche scientifique
Montant : 200000.0 euros
Durée : 36 mois
Résumé : Molecular Mechanisms Underlying the Coordination of Cell Growth and Cell DivisionGrowth and Division
1) Cdk1 dependent polarized growth. 2) Spatial organization of endocytosis and exocytosis during polarized growth.Diverse cell types, from yeast to mammalian cells, often exhibit a dynamic pattern of growth throughout the cell cycle, dramatically changing cell polarity as cell cycle transitions occur. Communication between the cell cycle machinery and cytoskeletal components that organize the secretory apparatus ensures that cell morphology, cell growth and the cell cycle are coordinated appropriately.  Despite the fact that these control mechanisms ensure the fidelity of cell division and often fail in cancer, surprisingly little is known about them.In the genetically tractable model budding yeast, a core component of the cell cycle machinery, cyclin dependent kinase 1 (Cdk1), triggers polarization of the actin cytoskeleton and growth of a new cell, or bud, early in the cell cycle.  This places Cdk1 activity at a nexus between the control of polarized cell growth and cell division.  Cdk1 associated with G1 cyclins promotes polarized growth, while Cdk1 associated with mitotic cyclins leads to a cessation of polarized growth later in the cell cycle.  However, the mechanisms used by Cdk1 to trigger these events are poorly understood. We have found that Cdk1 phosphorylates multiple components of the Cdc42 GTPase regulatory module, which in turn initiates polarized growth by activating the conserved GTPase Cdc42. The activation of Cdc42 triggers the reorganization of endo- and exocytic membrane trafficking pathways into a polarized state that establishes the polarity axis for the ensuing cell cycle. These results raise the following questions that we are addressing:1) The molecular mechanisms by which Cdk1 activates Cdc42 to initiate and maintain polarized growth.2) Mechanisms that contribute to endocytic and exocytic vesicle segregation to establish cell polarization.
High-resolution live cell imaging, mathematical modeling, genetics, biochemistry.The mechanisms that estblish a polarity axis during the cell cycle are highly dynamic, involving signaling proteins such as Cdc42 and its regulators that are present in very low amounts in the cell. The methods employed to monitor these proteins must therefore be sufficiently sensitive.We are using interdisciplinary approaches including high spatial and temporal resolution live cell imaging to monitor vesicle dynamics during the cell cycle. Moreover, we are developing new quantitative tools to understand how vesicle dynamics change during the cell cycle-dependent establishment of a polarity axis. These approaches are complimented by mathematical modelling. Modelling is important, since the behaviour of complex signaling systems is often not intuitive and modelling provides a means of predicting the behaviour of a system that would not have been evident from experiments alone. To test the predictions of such modelling, we use the genetically tractable model eukaryote budding yeast. Budding yeast are also amenable to biochemistry and we have been testing how phosphorylation of Cdc42 regulators by cyclin dependent kinase 1 (Cdk1) results in the establishment of a polarity axis.
Résumé de la soumission : Les mécanismes moléculaires qui coordonnent la croissance et la division cellulaireLes propriétés dynamiques du cytosquelette facilitent la croissance et la division cellulaire. La distribution et le taux de croissance cellulaire liés à la progression du cycle cellulaire permettent de générer le spectre de taille et forme cellulaire qui existe dans la nature. Malgré l’importance fondamentale de ces processus, les mécanismes qui coordonnent la croissance et la division cellulaire restent mal compris.Il a été découvert il y a plus de 30 ans, que l’activité de Cdk1, un constituant majeur de la machinerie du cycle cellulaire chez la levure était requis pour initier la polarisation du cytosquelette d’actine et la morphogenèse de la cellule fille en fin de phase G1 du cycle cellulaire. Cdk1 associé avec les cyclines de G1 (cycline G1-Cdk1) induit la croissance polarisée, cependant, les mecanismes par lesquels Cdk1 déclenche ces événements sont mal connus. La levure S. cerevisae est un excellent modèle pour étudier ces mecanismes car les protéines qui contrôlent le cycle et la croissance cellulaires sont conservées de la levure aux mammifères. Durant mon post-doctorat, j’ai identifié les substrats du complexe cycline G1-Cdk1 qui induisent la polarisation cellulaire chez la levure. J’ai fait la surprenante découverte que l’activité de Cdk1 est également nécessaire pour maintenir la croissance cellulaire. J’ai découvert spécifiquement que:(1)	Cdk1 est nécessaire à la croissance polarisée de la cellule pendant un cycle cellulaire normal.(2)	une des cibles majeures de l’activité cycline G1-Cdk1 est un nouveau complexe multi-protéiques que j’ai purifié et identifié par spectrométrie de masse. Il comprend la GEF, la GAP et les adapteurs du module de la GTPase Cdc42.(3)	l’identification des sites de phosphorylation par spectrométrie de masse et l’étude des mutants pour ces sites de phosphorylation a révélé que la régulation du module par cycline G1-Cdk1 est générale et essentielle pour établir la croissance cellulaire polarisée.L’exploration des mécanisme par lesquels Cdk1 active Cdc42 permettra de déterminer comment la croissance et la division cellulaire sont coordonnées, un problème clé de biologie cellulaire qui reste encore à résoudre. Les objectifs décrits dans mon projet de recherche vont permettre spécifiquement d’améliorer nos connaisances au niveau:(1)	des mécanismes moléculaires par lesquels Cdk1 active Cdc42 pour initier et maintenir la croissance cellulaire polarisée.(2)	de mécanismes qui contribuent à la ségrégation des vésicules d’endocytose et d’exocytose pour établir la polarisation de la cellule.
Résultats : The high resolution imaging system that we have built has enabled us to carry out a quantitative imaging-based screen to identify mutants that disrupt the cell-cycle specific organization of membrane trafficking domains. This work is ongoing and we will submit a manuscript on this project in late 2012/early 2013.
The imaging system has enabled us to identify a role for Cyclin Dependent Kinase 1 (Cdk1) in the control of polarized growth that extends beyond Cdk1's known role in regulating actin dynamics (See Publication 1 and 3 below).
We have also used the high spatial and temporal resolution of the system to track the movement of intracellular vesicles in live cells and identify the presence of a Rho GTPase Activating Protein (Rho GAP) on these vesicles. These results provide a mechanism that explains the spatial regulation of Rho GTPase activation, a feature of Rho GTPase biology that is presently poorly understood (See publication 2 below).
We have also used our imaging system with complimentary quantitative approaches and mathematical modelling to understand how a polarity axis is established in a cell cycle-specific fashion. Our exciting new results have uncovered a new mechanism involved in the cell cycle-dependent establishment of a polarity axis involving endocytosis. This work is submitted for publication and received encouraging reviews to which we are currently responding (See publication 4 below).
Perspectives : Our imaging system and quantitative image approaches are facilitating the analysis of dynamic processes including cell growth and cell cycle progression in live cells. The combined use of this system with powerful mathematical modelling and genetic approaches has enabled us to identify new mechanisms and players involved in specifying the axis of cell growth and division. A major question that emerges is whether these newly identified mechanisms are conserved in other eukaryotic cells. The fact that the proteins that we have identified are highly conserved, and that some have been implicated in human diseases including cancer suggest that our results may be relevant and that such proteins may represent good candidates for therapeutic intervention.
Publications et brevets : Publications:
1) Cdk1-dependent control of membrane trafficking dynamics.
Kellogg D*, Royou A, Velours C, McCusker D*. Mol Biol Cell. 2012 Jul 5. [Epub ahead of print]. * Equal contribution.
2) Secretory pathway-dependent localization of the Saccharomyces cerevisiae Rho GTPase-activating protein Rgd1p at growth sites.
Lefèbvre F, Prouzet-Mauléon V, Hugues M, Crouzet M, Vieillemard A, McCusker D, Thoraval D, Doignon F. Eukaryot Cell. 2012 May;11(5):590-600. Epub 2012 Mar 23.
3) Coordinating membrane addition and cell cycle progression.
McCusker D. and Kellogg D. Curr. Opinion Cell Biol. Invited submission. In preparation.
4) Robust polarity establishment via an endocytosis-based cortical corralling mechanism.
Jose M, Tollis S, Nair D, Sibarita, JB and McCusker D. Journal of Cell Biology. Submitted.