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Reconstitution of Myosin IIA-driven membrane fission in vitro (MYOII-DRIVEN FISSION)
Start date: Mar 1, 2014, End date: Feb 29, 2016 PROJECT  FINISHED 

Eukaryotic cells contain a variety of membrane-enclosed compartments that are connected by dynamic transport processes facilitated by vesicles. The temporal and spatial regulation of these transport processes is essential for cellular function. After initial formation transport vesicles are separated from their donor membrane and this fission event seems to be coordinated with the elongation of tubular precursors pulled by motor proteins. Recent results from the host laboratory demonstrated a direct interaction of Rab6 (a Golgi-associated RabGTPase) and nonmuscle Myosin II A (MyoIIA) and showed an unexpected function of MyoIIA in the fission of Rab6 transport carriers at the trans-Golgi network.The aim of this project is the development of an in vitro system to mimic the MyoIIA-driven fission process. In this system, we will pull membrane nanotubes from Giant Unilamellar Vesicles (GUVs) using optical tweezers and we will monitor tube stability in the presence of Rab6, MyoIIA, actin and ATP. Two hypotheses will be tested: either MyoIIA induces phase separation in membrane tubes by the formation of actomyosin patches, which then leads to fission; or MyoIIA contracts short actin filaments around the membrane tubes in a manner similar to its role during the formation of the cleavage furrow in cytokinesis. If we do not observe fission in this minimal system, we will add additional factors that have been implicated in fission at the trans-Golgi network.In the past years the cell biology host laboratory of Bruno Goud and the experimental physicists from Patricia Bassereau’s group have collaborated extensively to develop new interdisciplinary methods to investigate intracellular transport processes. As I have so far focused on biophysical measurements with Rab proteins in the absence of membranes, the development of the in vitro system in these laboratories will give me the possibility to get exposure to a variety of new techniques that will expand my current expertise.
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