contrôle de la forme des endosomes par la myosine 1b – EndoMyoShape

Recent progress in studding the role of myosins in membrane traffic has provided new insight on the link between these motors and other protein machineries involved in trafficking as well as their involvement in the morphology of the organelles The challenge now is to understand the molecular mechanisms by which these myosins contribute to membrane traffic and whether they act in a coordinated way with the other protein complexes to control the shape of organelles. The goal of our project is to unravel the molecular mechanism by which one of the single headed myosin involved in membrane traffic, myosin1b (Myo1b) (primary named myosin I alpha or Myr1) regulates the transport along the endocytic pathway. We have previously shown that Myo1b localizes to endosomes and regulates the transport of internalized molecules along the endocytic pathway (Raposo et al. 1999, Cordonnier et al. 2001, Salas Cortes et al. 2005). Preliminary observations suggest that Myo1b functions between endosomes and TGN. The mechanochemical properties of Myo1b suggest that it is a membrane associated motor able to develop tension on membrane domains (Laasko et al. 2008). In agreement with this hypothesis we observed that overexpression of Myo1b increases the formation of membrane extension at the surface of endosomes (Salas Cortes et al. 2005). In controlling the site of polymerisation of actin on sorting endosomes as suggested for xenopus myosin 1 (Sokac et al. 2006) Myo1b may localise and transduce the force generated by actin polymerization on the membrane of endosomes and thereby be one of the players involved in membrane budding or membrane scission. To understand the endosome-actin interplay mediated by Myo1b in endocytic trafficking, our aims are: 1) to precise the step(s) of the pathway between endosomes and TGN that require(s) Myo1b; 2) to determine the requirement of Myo1b motor activity for its function along the pathway between endosomes and TGN; 3) to determine the mechanism by which Myo1b interacts with the endosomal membranes; 4) to identify the molecular mechanism that links Myo1b and actin structures on endosomes; 5) to measure the force generated by Myo1b and determine whether this force contributes to membrane tension of endosomes and controls their shape. Three teams of the Institut Curie will combine multidisciplinary approaches including biochemistry, direct visualization of endosomes and of actin and myosin using live cell imaging, analysis at the ultrastructural level and novel biophysical biomimetic assays. Evelyne Coudrier's team (UMR 144) will carry out the analysis in vivo using life cell biology imaging, and will develop molecular biochemical tools. Graça Raposo's team (UMR 144) will carry out studies at the ultrastructural level. Finally, Patricia Basserau team's (UMR 168), including Daniel Lévy and Cécile Sykes will develop biophysical biomimetic assays and perform quantitative measurements. Biomimetics assays have been recently reported to mimic the role of actin polymerisation at the surface of organelles, the role of coat and dynamin or the role of a kinesin in presence of microtubules. But to our knowledge no biomimetic assay is available to study the molecular mechanism by which a myosin could regulate the transport of a cargo. This project at the interface of the cell biology and biophysics is therefore an important step to understand how myosins and actin filaments regulate the shape of organelles. The purification of Myo1b and its rigor mutant on a large scale will be an important tool to set up our bio-mimetic assay. It may also in the future allow to attempt to crystallise and to study Myo1b structure at high resolution. If we succeed it will be the first full length myosin to be crystallised. Our in vitro actin assembly assay using isolated magnetic endosomes could be used at longer term to understand the role of other proteins that have been implicated in actin dynamics at the surface of the endosomes. Finally, the biomimetic assay could at longer term be applied to other myosins that have been reported to contribute to membrane traffic.