The dynamin family of proteins consists of unique GTPases involved in membrane fission and fusion events throughout the cell. The founding member, dynamin, is crucial for endocytosis, synaptic membrane recycling, membrane trafficking within the cell and, more recently, has been associated with filamentous actin. Dynamin was first implicated in endocytosis when it was discovered to be the mammalian homologue of the shibire gene product in Drosophila. A temperature sensitive shibire allele causes a defect in clathrin-mediated endocytosis. Since then, overexpressing human dynamin mutants in mammalian cells was found to block clathrin-mediated endocytosis. Over the years, our structural work has played a leading role in dissecting the function of dynamin in membrane fission. We have shown that purified dynamin readily assembles into rings and spirals and it forms similar structures on liposomes, generating dynamin-lipid tubes that constrict upon GTP hydrolysis. A potential mechanism for dynamin constriction was revealed when we solved the first three-dimensional structure of dynamin. All evidence supports the hypothesis that dynamin assembles around the necks of clathrin-coated pits where it assists in membrane fission. The tension created by dynamin constricting the neck of coated pits may be sufficient for membrane fission in the cell. The ability of dynamin to constrict and generate a force on the underlying lipid bilayer makes it unique among GTPases as a mechanochemical enzyme. Previously, we solved the structure of a dynamin mutant (lacking its C-terminus) in the constricted and non-constricted states using helical reconstruction and the IHRSR methods. The 3D volumes reveal three distinct radial densities, outer, middle and inner layers. During constriction the most obvious change is a decrease in the axial repeat and radius. However, the volume interiors shows a large conformational change within the middle layer, which provides a clue to the mechanism of constriction. Dynamin contains five identifiable domains: GTPase, middle, pleckstrin homology (PH), GTPase effector (GED) and proline/arginine-rich (PRD). Using molecular modeling tools, we docked the crystal structures of the GTPase and PH domains of dynamin into the 3D maps using a rigid-body Monte Carlo algorithm. The GTPase domain docked into the outer radial density while the PH domain docks into the inner radial density. The GED would then reside in the middle layer, which fits with previous findings that GED directly interacts in trans with a GTPase domain to stimulate the GTPase activity of dynamin. The results show how adjacent GTPase structures associate with one another in an arrangement consistent with the cryo-EM structure and suggest a mechanism for self-assembly and corkscrew motion during constriction. The positioning of the PH domain within the inner radial density places the variable loops facing toward the membrane. This positioning is consistent with the Charcot-Marie-Tooth mutation in the PH domain having an effect on lipid binding. During synaptic membrane retrieval, dynamin and endophilin are recruited to the necks of clathrin-coated pits and play a crucial role in vesiculation. Recently we have shown that endophilin and dynamin co-localize at the necks of clathrin-coated pits, proximal to the coat, after microinjection of GTPγS. The interaction between dynamin and endophilin was further shown to be important for endocytosis by microinjection studies using the SH3-domain of endophilin and the synaptojanin derived peptide (PP19). Both perturbed the assembly of the proteins and inhibited endocytosis. In support of the role of endophilin enhancing the assembly of dynamin, we found that addition of endophilin increased the recruitment of dynamin to liposomes and the formation of tubules. These results suggest that endophilin and dynamin form a """"""""pre-fission complex"""""""" at the necks of the coated pits proximal to the clathrin-coat, which coordinates dynamin-mediated budding of newly formed vesicles in vertebrate synapses.

Project Start
Project End
Budget Start
Budget End
Support Year
16
Fiscal Year
2011
Total Cost
$627,481
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Antonny, Bruno; Burd, Christopher; De Camilli, Pietro et al. (2016) Membrane fission by dynamin: what we know and what we need to know. EMBO J 35:2270-2284
Sambuughin, Nyamkhishig; Goldfarb, Lev G; Sivtseva, Tatiana M et al. (2015) Adult-onset autosomal dominant spastic paraplegia linked to a GTPase-effector domain mutation of dynamin 2. BMC Neurol 15:223
Mattila, Juha-Pekka; Shnyrova, Anna V; Sundborger, Anna C et al. (2015) A hemi-fission intermediate links two mechanistically distinct stages of membrane fission. Nature 524:109-113
Sundborger, Anna C; Hinshaw, Jenny E (2015) Dynamins and BAR Proteins-Safeguards against Cancer. Crit Rev Oncog 20:475-84
Sundborger, Anna C; Fang, Shunming; Heymann, J├╝rgen A et al. (2014) A dynamin mutant defines a superconstricted prefission state. Cell Rep 8:734-42
Sundborger, Anna C; Hinshaw, Jenny E (2014) Regulating dynamin dynamics during endocytosis. F1000Prime Rep 6:85
Hinshaw, J (2012) Structural analysis of dynamin reveals a mechanism of membrane fission. Microsc Microanal 18 Suppl 2:58-9
Hurley, James H; Hinshaw, Jenny E (2012) Dynamin: membrane scission meets physics. Curr Biol 22:R1047-8
Shiba, Yoko; Luo, Ruibai; Hinshaw, Jenny E et al. (2011) ArfGAP1 promotes COPI vesicle formation by facilitating coatomer polymerization. Cell Logist 1:139-154
Sundborger, Anna; Soderblom, Cynthia; Vorontsova, Olga et al. (2011) An endophilin-dynamin complex promotes budding of clathrin-coated vesicles during synaptic vesicle recycling. J Cell Sci 124:133-43

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