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Reconstitution of Myosin IIA-driven membrane fission in vitro

Final Report Summary - MYOII-DRIVEN FISSION (Reconstitution of Myosin IIA-driven membrane fission in vitro)

Eukaryotic cells are subdivided in distinct membrane enclosed compartments that are connected by intracellular transport processes facilitated by vesicles. The temporal and spatial regulation of these transport processes is essential for the survival of cells. The transport vesicles are fissioned from the donor membrane after their formation. This events seems to be coordinated with the elongation of tubular precursors pulled by myosins and/or kinesins. Recent results show an unexpected function of nonmusle Myosin II A (nmMyoIIA) in fission of Rab6A transport carriers at the trans-Golgi network.
The aim of this project is the development of an in vitro system to mimic the fission of vesicles from the trans-Golgi network. We have developed a system in which we can recruit Rab6A to Giant Unilamellar Vesicles (GUV) after prenylation of the protein in vitro. Several Rab6 effectors can be recruited to these GUVs in a Rab6:GTP dependent manner. Furthermore we have isolated Golgi membranes on which we were able to detect Rab6A, nmMyoIIA and actin and have pulled tubes from these membranes.