Single-Molecule Diffusion Measurements Indicate Independent Membrane Insertion by the Tandem C2 Domains of Synaptotagmin 7

Abstract

The synaptotagmin (Syt) family of proteins is characterized by the presence of tandem C2 domains, C2A and C2B, which sense Ca2+ to trigger vesicle fusion during exocytosis. The widely studied Syt1 is central to rapid neurotransmitter release, while Syt7 is involved in slower secretion of large dense-core vesicles and has C2 domains that dock much more tightly to target membranes. Despite recent progress in understanding mechanisms of vesicle fusion, the molecular mechanisms of synaptotagmin C2AB membrane docking remain incompletely characterized. For example, the two C2 domains of Syt1 are reported to cooperatively insert into target membranes, but specific interdomain contacts have not been identified. To test whether the two C2 domains from Syt7 interact on a planar lipid bilayer, lateral diffusion constants of fluorescent-tagged C2A, C2B, and C2AB domains from human Syt7 were measured on PC:PS (3:1) bilayers using total internal reflection fluorescence microscopy with single-particle tracking. The Syt7 C2AB tandem exhibits a lateral diffusion constant half the value of the isolated single domains, and does not change when additional residues are engineered into the C2A-C2B linker. This is the expected result if C2A and C2B are separated when membrane-bound; theory predicts that C2AB diffusion would be faster if the two domains interact. Furthermore, ensemble stopped-flow measurements of membrane dissociation kinetics also support an absence of interdomain interactions, as EDTA-induced dissociation kinetics of the C2AB tandem are similar to the isolated C2A domain and remain unchanged when rigid or flexible linker extensions are included. Together, the results suggest that the two C2 domains of Syt7 bind independently to membranes. Ongoing efforts seek to perform analogous measurements with Syt1, whose C2 domains have much shorter membrane-bound lifetimes.