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. It has been shown that purified dynamin incubated under low salt conditions forms rings and spirals which, in dimension and appearance, resemble the dense material occasionally observed at the necks of coated pits. We have show that purified dynamin can also be induced to form spirals under physiological salt conditions by incubating it with GDP and g-phosphate analogues (BeF or AlF) or by dialyzing it into GTPgS. This demonstrates that the polymerized state of dynamin is markedly stabilized by sustaining its GTP or GDP/Pi-bound conformation. The formation of dynamin spirals was quantified by a sedimentation assay and imaged by electron microscopy using either negative staining techniques. Currently we are overexpressing and purifying recombinant dynamin fragments to determine which regions of dynamin are responsible for assembly. Our new ability to stabilize dynamin spirals in physiological salt solution strengthens the idea that in vivo, even in the absence of other protein cofactors, dynamin can assemble into spirals around the necks of coated pits and thereby promote vesicle fission. We have also shown by negative stain electron microscopy that purified recombinant dynamin is able to bind to lipid vesicles to form helical tubes which are similar in dimensions to dense material seen around the necks of clathrin-coated pits. By using both light and electron microscopy, we have evidence that GTP hydrolysis causes a major structural change in the dynamin/lipid tubes which may represent the process which occurs upon vesicle scission. When GTP is added to dynamin tubes they appear to 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 understand dynamin?s role in membrane fission we are currently calculating a three-dimensional map of dynamin using cryo-electron microscopy and helical reconstruction methods. We have been able to obtain tubes that diffract to approximately ~23 angstroms which have been used to compute a preliminary 3D map. These results clearly show a lipid bilayer with dynamin molecules extending out in the shape of a ?T?. We are presently examining wild-type and mutant dynamin by this method as well as dynamin in different nucleotide states. 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.
| Mears, Jason A; Hinshaw, Jenny E (2008) Visualization of dynamins. Methods Cell Biol 88:237-56 |
| Mears, Jason A; Ray, Pampa; Hinshaw, Jenny E (2007) A corkscrew model for dynamin constriction. Structure 15:1190-202 |
| Naylor, Kari; Ingerman, Elena; Okreglak, Voytek et al. (2006) Mdv1 interacts with assembled dnm1 to promote mitochondrial division. J Biol Chem 281:2177-83 |
| Hinshaw, Jenny E (2006) Filling the GAP for dynamin. Nat Cell Biol 8:432-3 |
| Nie, Zhongzhen; Hirsch, Dianne S; Luo, Ruibai et al. (2006) A BAR domain in the N terminus of the Arf GAP ASAP1 affects membrane structure and trafficking of epidermal growth factor receptor. Curr Biol 16:130-9 |
| Hickman, Alison B; Perez, Zhanita N; Zhou, Liqin et al. (2005) Molecular architecture of a eukaryotic DNA transposase. Nat Struct Mol Biol 12:715-21 |
| Ingerman, Elena; Perkins, Edward M; Marino, Michael et al. (2005) Dnm1 forms spirals that are structurally tailored to fit mitochondria. J Cell Biol 170:1021-7 |
| Kochs, Georg; Reichelt, Mike; Danino, Dganit et al. (2005) Assay and functional analysis of dynamin-like Mx proteins. Methods Enzymol 404:632-43 |
| Zhang, P; Hinshaw, J E (2001) Three-dimensional reconstruction of dynamin in the constricted state. Nat Cell Biol 3:922-6 |
| Danino, D; Hinshaw, J E (2001) Dynamin family of mechanoenzymes. Curr Opin Cell Biol 13:454-60 |
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