Rapid constriction of lipid bilayers by the mechanochemical enzyme dynamin

Dynamin, a large GTPase, is located at the necks of clathrin-coated pits where it facilitates the release of coated vesicles from the plasma membrane upon GTP binding, and hydrolysis. Previously, we have shown by negative stain electron microscopy that wild-type dynamin and a dynamin mutant lacking the C-terminal proline-rich domain, DeltaPRD, form protein-lipid tubes that constrict and vesiculate upon addition of GTP. Here, we show by time-resolved cryo-electron microscopy (cryo-EM) that DeltaPRD dynamin in the presence of GTP rapidly constricts the underlying lipid bilayer, and then gradually disassembles from the lipid. In agreement with the negative stain results, the dynamin tubes constrict from 50 to 40 nm, and their helical pitch decreases from approximately 13 to 9.4 nm. However, in contrast to the previous results, examination by cryo-EM shows that the lipid bilayer remains intact and small vesicles or fragments do not form upon GTP binding and hydrolysis. Therefore, the vesicle formation seen by negative stain may be due to the lack of mobility of the dynamin tubes on the grid during the GTP-induced conformational changes. Our results confirm that dynamin is a mechanochemical enzyme and suggest that during endocytosis dynamin is directly responsible for membrane constriction. In the cell, other proteins may enhance the activity of dynamin or the constraints induced by the surrounding coated pit and plasma membrane during constriction may cause the final membrane fission event.