The Synaptotagmin-7 C2AB Domain Alters Membrane Morphology in a Calcium-Dependent Manner

Abstract

The proper execution of Ca2+-triggered exocytosis requires extreme changes in bilayer shape to be precisely regulated. Because membranes alone are unlikely to undergo the required changes, proteins are necessary to mediate events. However, the identity of these proteins and the conditions under which they act are not well understood. Within this context, our studies focus on membrane-targeting C2 domains from synaptotagmin-7 (Syt-7) - an isoform of the Syt protein family that is important for secretion in neuroendocrine cells. To define how Syt-7 drives changes in membrane morphology, we have used recombinant C2AB protein fragments, supported lipid bilayers (SLBs), and total internal reflection fluorescence microscopy (TIRFM). We have developed conditions for forming SLBs under which the membrane spontaneously forms long tubule-like structures extending away from the support surface. Upon addition of purified Syt-7 C2AB in the presence of Ca2+, these long tubules disappear and are replaced by shorter tubules or vesicles. The effect is reversible upon removal of Ca2+. These studies demonstrate that Syt-7 can alter membrane morphology, ostensibly by driving changes in membrane curvature. They also demonstrate the utility of a novel experimental system in which protein-mediated changes in membrane topology can be studied in aqueous media and in real-time.