Capturing dynamin-mediated fission intermediates in Drosophila with Cryo-ET

Authors

• P Kunamaneni
• KA Sochacki
• CJ Fagerstrom
• MR Hannaford
• C Khan
• NM Rusan
• JW Taraska
• JE Hinshaw

Abstract

Dynamin, a large GTPase, mediates membrane fission through GTP hydrolysis, facilitating the endocytosis of molecules at the plasma membrane in many physiological systems. Despite extensive studies, capturing fission intermediates in cells remains difficult due to the dynamic and rapid involvement of many regulatory factors. Dynamin was first shown to play a role in endocytosis from studies of a temperature-sensitive (ts) dynamin mutant in Drosophila (shibire ts) that results in synaptic vesicle defects and paralytic phenotypes at restrictive temperatures (30°C). Under these conditions, dynamin fails to pinch off vesicles from the plasma membrane and remains assembled around the necks of invaginated pits. Our objective is to capture intermediates in dynamin-mediated fission by characterizing the structures of shibire ts at restrictive temperatures. We expressed shibire ts in Drosophila flies with the UAS-Gal4 system and are currently optimizing cryo-electron tomography (ET) methods to obtain high-resolution images of the samples at the plasma membrane. These methodologies will be applied to capture shibire ts under restrictive temperatures and ultimately gain deeper insights into its structural aspects. By targeting the restrictive state in shibire, we aim to uncover mechanistic insights and expand understanding of the complex events involved in dynamin-mediated fission.

Scientific Focus Area: Structural Biology

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