Highly Charged Membrane Templates for Studying the Mechano-Chemistry of Dynamin 1

Abstract

Dynamin 1 (Dyn1) is a large GTPase mediating membrane fission during synaptic vesicle recycling. Dyn1 converts the energy obtained from GTP-hydrolysis into mechanical work required to constrict and cut the neck of endocytic vesicle. Mechano-chemical activity of Dyn1 is coupled to membrane curvature. Dyn1 preferentially binds to highly curved membrane surfaces, such as vesicle necks. The binding is followed by oligomerization of Dyn1 into helices augmenting membrane constriction. The helical arrangement dramatically boosts the GTP hydrolysis that furthers constriction to the critical curvatures triggering fission. Molecular mechanisms behind this coupling, constituting the basis of Dyn1 function, remain a subject of debate. Membrane binding of Dyn1 is regulated by membrane charge. Here we present highly charged membrane templates that allow uncoupling of membrane binding and oligomerization of Dyn1 from its curvature and fission activities. We reveal that even on flat templates Dyn1 self-assembles into clusters that undergo GTP-dependent transformations. We further quantified the stoichiometry and dynamics of such clusters using GFP-conjugated dynamin and compared the effect of GTP on flat and curved templates. Our data provide new mechanistic insights into curvature stimulation of mechano-chemical activity of Dyn1.