Snapshots of dynamin-mediated membrane fission by cryo-EM

Authors

• N Kundu
• S Nyenhuis
• JR Jimah
• H Breen
• JT Harrison
• B Canagarajah
• JE Hinshaw

Abstract

Dynamin drives the process of endocytosis by pinching off invaginating vesicles from the plasma membrane via its GTPase activity. Dynamin is a potential target for antiviral, anti-cancer, and anti-epilepsy treatment as dynamin-mediated endocytosis facilitates entry of viruses, including HIV and SARS-CoV-2, and dysregulation of dynamin is associated with many cancers and neurological disorders. Dynamin mediates endocytosis in three steps; 1) assembling as a helical polymer on the neck of invaginating vesicles, 2) constricting the membrane neck upon GTP binding and, 3) further constriction and fission upon GTP hydrolysis, which ultimately leads to release of vesicles. By a time-resolved cryoEM method, we showed that dynamin constricts the membranes within 150 milliseconds. Furthermore, by cryo-electron tomography, we provided evidence that dynamin may be involved in early stages of endocytosis by wrapping around large diameter vesicles (200-300nm).
Using GTPase-defective dynamin K44A mutant, we determined atomic structures of the dynamin helical polymer assembled on lipid tubules, akin to necks of budding endocytic vesicles, in GDP-bound, super-constricted state. In this state, dynamin is assembled as a 2-start helix on lipid tubules with an inner luminal diameter of 3.4 nm, primed for spontaneous fission. To better resolve the membrane binding of the PH domains of dynamin assembly, we screened different buffer and phospholipid conditions and obtained the first high-resolution structures of the PH domains inserting into the membrane using cryoEM. Our work illuminates how dynamin binds to the membrane and how membrane composition such as different phospholipids alters the membrane constriction mechanism mediated by dynamin.

Scientific Focus Area: Structural Biology

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