A Chemical Controller of SNARE-Driven Membrane Fusion That Primes Vesicles for Ca²⁺-Triggered Millisecond Exocytosis

Abstract

Membrane fusion is mediated by the SNARE complex which is formed through a zippering process. Here, we developed a chemical controller for the progress of membrane fusion. A hemifusion state was arrested by a polyphenol myricetin which binds to the SNARE complex. The arrest of membrane fusion was rescued by an enzyme laccase that removes myricetin from the SNARE complex. The rescued hemifusion state was metastable and long-lived with a decay constant of 39 min. This membrane fusion controller was applied to delineate how Ca2+ stimulates fusion-pore formation in a millisecond timescale. We found, using a single-vesicle fusion assay, that such myricetin-primed vesicles with synaptotagmin 1 respond synchronously to physiological concentrations of Ca2+. When 10 µM Ca2+ was added to the hemifused vesicles, the majority of vesicles rapidly advanced to fusion pores with a time constant of 16.2 ms. Thus, the results demonstrate that a minimal exocytotic membrane fusion machinery composed of SNAREs and synaptotagmin 1 is capable of driving membrane fusion in a millisecond time scale when a proper vesicle priming is established. The chemical controller of SNARE-driven membrane fusion should serve as a versatile tool for investigating the differential roles of various synaptic proteins in discrete fusion steps.

Energetics, kinetics, and pathway of SNARE folding and assembly revealed by optical tweezers

“…As a result, a large energy barrier separates the folded and the unfolded CTD states, leading to a large hysteresis of the CTD transitions in the FEC. Finally, the half‐zippered state is supported by many other experiments, especially imaging by electron microscopy . Combining with earlier evidence, these observations corroborate that a half‐zippered trans‐SNARE structure is required for the calcium‐triggered synaptic vesicle fusion.…”

Spatially encoded fast single-molecule fluorescence spectroscopy with full field-of-view

“…Single-molecule imaging was carried out on an objective-type total internal reflection fluorescence (TIRF) microscope built around Olympus IX73 (Supplementary Fig. 1a ) similar to previous work 3 . Combined via a polarizing beam splitter, a green laser (Cobolt 06-DPL,532 nm), for excitation of Cy3B) and a red laser (Cobolt 06-MLD, 638 nm) with a laser clean-up filter were coupled to a single mode fiber.…”

“…We used polyethylene glycol (PEG)-coated flow chambers as described elsewhere 1 , 3 except the experiments of Atto647N blinking dynamics.…”

“…electron multiplying charge-coupled device (EMCCD) or scientific complementary metal-oxide semiconductor (sCMOS) camera is still at 50–100 frames per second. Cropping the field-of-view (FOV) is the simplest way to improve temporal resolution, however, it decreases the number of molecules that can be observed simultaneously 3 . This is especially disadvantageous for studying weakly interacting biomolecules because the complex formation will occur very rarely 4 .…”

Simulations Reveal Multiple Intermediates in the Unzipping Mechanism of Neuronal SNARE Complex

“…In the last decade, biologists have discovered several details about the function of this protein complex, about its structure, and about the mechanism that enables the lipid bilayers fusion. Specifically, several experiments have shown that a partially assembled SNARE complex exists as an intermediate state during vesicle exocytosis (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). The presence of such an intermediate state is critical for the kinetic of exocytosis.…”