Abstract
Munc13-1 is a large banana-shaped soluble protein that is involved in the regulation of synaptic vesicle docking and fusion. Recent studies suggest that multiple copies of Munc13-1 form nanoassemblies in active zones of neurons. However, it is not known if such clustering of Munc13-1 is correlated with multivalent binding to synaptic vesicles or specific plasma membrane domains at docking sites in the active zone. The functional significance of putative Munc13-1 clustering is also unknown. Here we report that nano-clustering is an inherent property of Munc13-1, and is indeed required for vesicle binding to bilayers containing Munc13-1. Purified Munc13-1 protein reconstituted onto supported lipid bilayers assembled into clusters containing from 2 to ~20 copies as revealed by a combination of quantitative TIRF microscopy and step-wise photobleaching. Surprisingly, only clusters containing a minimum of 6 copies of Munc13-1 were capable of efficiently capturing and retaining small unilamellar vesicles. The C-terminal C 2 C domain of Munc13-1 is not required for Munc13-1 clustering, but is required for efficient vesicle capture. This capture is largely due to a combination of electrostatic and hydrophobic interactions between the C 2 C domain and the vesicle membrane.