Dynamin, a 100 kDa GTPase, is believed to be involved in the constriction of clathrin coated pits and may cause fission of clathrin coated vesicles during receptor mediated endocytosis and during membrane retrieval in nerve terminals. We have shown that purified recombinant dynamin assembles into rings and spirals, and binds to lipid vesicles to form helical tubes which are similar to the dense material seen at the necks of clathrin-coated pits. When GTP is added to the dynamin tubes they constrict and form small vesicles. These results provide strong evidence that dynamin is the structural component necessary for the formation of the constricted necks of coated pits, and support the hypothesis that dynamin is the force- generating molecule responsible for membrane fission.To further clarify the role of dynamin in membrane fission we have recently calculated a three-dimensional map of a dynamin mutant using cryo-electron microscopy and helical reconstruction methods. We found that dialyzing dynamin into GMP-PCP resulted in tubular crystals which diffract to approximately ~20 angstroms. The tubular crystals are similar in size and helical repeat to the constricted tubes seen when dynamin is treated with GTP. By helical processing methods we have calculated a 3D map of the dynamin tubes and found that the dynamin molecules extend out from the lipid bilayer in a T-shape with a bilobed stalk. Our STEM analysis of dynamin spirals treated with GMP-PCP indicates there are approximately 32 dynamin molecules per helical turn. We are presently examining wild type and other dynamin mutants in different nucleotide states by this method. We are also overexpressing and purifying a recombinant dynamin fragment (N-terminal fragment) for X-ray crystallography studies. Once crystallized we will be able to dock the high-resolution X-ray data into our low-resolution 3D map and determine which regions of dynamin occupy which domains within the 3D map. This work will help elucidate how dynamin molecules interact and change conformation upon GTP addition and ultimately provide clues to how dynamin is regulated in the cell during endocytosis and its precise role in membrane fission. - dynamin, endocytosis, membrane recycling, helical reconstruction, shibire, cryo-electron microscopy, 3- dimensional reconstruction, GTPase, clathrin
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