Single-Molecule Optical Tweezers Study of Regulated SNARE Assembly

Abstract

Intracellular membrane fusion mediates material and information exchange among different cells or cellular compartments with high accuracy and spatiotemporal resolution. Fusion is driven by ordered folding and assembly of soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptors (SNAREs) and regulated by many other proteins. Understanding regulated SNARE assembly is key to dissecting mechanisms and physiologies of various fusion processes and their associated diseases. Yet, it remains challenging to study regulated SNARE assembly using traditional ensemble-based experimental approaches. Here, we describe our new method to measure the energy and kinetics of neuronal SNARE assembly in the presence of α-SNAP, using a single-molecule manipulation approach based on high-resolution optical tweezers. Detailed experimental protocols and methods of data analysis are shown. This approach can be widely applied to elucidate effects of regulatory proteins on SNARE assembly and membrane fusion.

Munc18-1 catalyzes neuronal SNARE assembly by templating SNARE association

“…All pulling experiments were performed using dual-trap high-resolution optical tweezers as previously described (Gao et al, 2012; Ma et al, 2019; Ma et al, 2015). Briefly, an aliquot of the crosslinked protein-DNA mixture containing 10–100 ng DNA was mixed with 10 μL 2.1 μm diameter anti-digoxigenin antibody coated polystyrene beads (Spherotech) and incubated at room temperature for 15 min.…”