Projet ANR-07-BLAN-0045 – Tyr-TIPs : Cell regulations at microtubule +ends: shifting from a Darwinist to a deterministic view of microtubule organization and of cell morphogenesis
Programme : Blanc (BLANC) 2007
Année de financement : 2007
Coordinateur du projet : Didier JOB (COMMISSARIAT ENERGIE ATOMIQUE REGION PARISIENNE)
Partenaires : Commissariat à l'énergie atomique et aux énergies alternatives
Montant : 400000.0 euros
Date de début : 2007
Durée : 36 mois
Résumé : "The discovery of microtubule dynamic instability, a process in which individual microtubules go stochastically through successive phases of growth and of catastrophic shrinkage, has promoted a ""darwinist"" view of microtubule organization and of cell morphogenesis. In this view, microtubules explore the cell through random fluctuations and natural selection operates by stabilization of the lucky polymers whose + ends happen to hit a stabilizing complex at the cell cortex. However there is evidence that the behaviour of microtubule + end is often under tight cellular controls, which affect complexes of proteins (+TIPs) able to associate specifically with microtubule + ends and depolymerising motors. At least in yeast + TIP complexes can spatially localize transitions between microtubule growth and shrinkage, at defined membrane domains, which is central for cell morphogenesis. -  We have recently demonstrated that the tubulin tyrosination cycle, in which the C-terminal amino-acid of alpha tubulin is sequentially removed from tubulin by a still ill defined tubulin carboxypeptidase (TCP) and re-added to tubulin by a tubulin tyrosine ligase (TTL) has a major effect on the recruitment of a subclass of + TIPs (CAP-Gly +TIPs) at microtubule + ends. Such recruitment is strongly inhibited by TTL suppression and resulting tubulin detyrosination. TTL loss is a frequent occurrence in human cancer cells and tubulin detyrosination apparently favors tumor growth. TTL suppression in mice induces lethal anomalies in neuronal differentiation and organization. Many of the effects of TTL loss can be traced down to abnormal control of spindle positioning and of cell shape and polarity, which all involve microtubule interactions with the cell membrane. -  In the present application, we propose a program to extend our work on microtubule tyrosination and to test whether, in addition to being regulated by a special modification of tubulin, microtubule + end behaviour is also controlled by, or acts on, more general signalling cascades involving small GTPases, phosphoinositide signalling or calcium signalling. We then intend to localize such regulations in cells using micro-patterned matrices which constrain the cell organization. -  1) tubulin tyrosination: a) we have evidence that in addition to affecting + TIPs, tubulin de tyrosination inhibits the activity of microtubule depolymerising motors of the KIF2 family. We intend to provide a firm demonstration of depolymerising motor inhibition by tubulin detyrosination, to describe its major cellular consequences, and to gather enough biochemical or biophysical data to propose a plausible structural basis for depolymerising motor dependence on tubulin tyrosination. b) In mammals, tubulin detyrosination is extensive in terminally differentiated cells, and its significance is unknown. Understanding the function of tubulin detyrosination requires the identification of TCP, which seems now at hands, based on data base searches. We intend to establish the molecular identity of TCP with the prospect of subsequent TCP suppression in cells or in whole animals, for functional studies. -  2) Small GTPases: we have observed that both in yeast and in TTL deficient fibroblasts, small GTPase activation triggers the re-localization of CAP-Gly + TIPs at detyrosinated microtubule + ends. We intend to use yeast genetics to identify a mechanism for such strong + TIP regulation, which will guide subsequent work in mammalian cells. -  3) Phosphoinositides: phosphoinositide signalling couples signalling events with the generation of specialized membrane micro-domains within seconds. We have genetic evidence in yeast for a functional link between microtubule + end behaviour and phosphoinositide signalling and we intend to decipher the underlying molecular mechanisms. In mammalian cells, we will principally test whether microtubule + TIPs are involved in the effect of lithium and valproate, which are major mood stabilizers, w