Rapid structural change in synaptosomal-associated protein 25 (SNAP25) precedes the fusion of single vesicles with the plasma membrane in live chromaffin cells

Abstract

The SNARE complex consists of the three proteins synaptobrevin-2, syntaxin, and synaptosomal-associated protein 25 (SNAP25) and is thought to execute a large conformational change as it drives membrane fusion and exocytosis. The relation between changes in the SNARE complex and fusion pore opening is, however, still unknown. We report here a direct measurement relating a change in the SNARE complex to vesicle fusion on the millisecond time scale. In individual chromaffin cells, we tracked conformational changes in SNAP25 by total internal reflection fluorescence resonance energy transfer (FRET) microscopy while exocytotic catecholamine release from single vesicles was simultaneously recorded using a microfabricated electrochemical detector array. A local rapid and transient FRET change occurred precisely where individual vesicles released catecholamine. To overcome the low time resolution of the imaging frames needed to collect sufficient signal intensity, a method named event correlation microscopy was developed, which revealed that the FRET change was abrupt and preceded the opening of an exocytotic fusion pore by ∼90 ms. The FRET change correlated temporally with the opening of the fusion pore and not with its dilation.TIR-FRET imaging | electrochemical imaging | time superresolution microscopy | image analysis | transmitter release N eurotransmitters, hormones, and many other mediators are stored in secretory vesicles, and their release occurs by the mechanism of exocytosis that begins with formation of a narrow fusion pore (1). Fusion-pore formation in neurosecretory vesicles is stimulated by an increase of intracellular [Ca 2+ ] and is thought to be induced by a large conformational change in the SNARE complex (2-4). Such changes may be involved in various steps from preparing vesicles for fusion (5) to fusion-pore dilation (6). To determine whether a conformational change in SNAREs is linked to fusion, the synaptosomal-associated protein 25 (SNAP25) mutant SCORE (SNARE COmplex REporter) has been developed (7), which contains two fluorescent proteins, CFP as a fluorescence resonance energy transfer (FRET) donor and Venus as a FRET acceptor. SCORE and constructs like it (5, 7-9) have the advantage that donor and acceptor exist at fixed stoichiometry, facilitating the analysis and interpretation of the measurements. Like SNAP25, SCORE forms SNARE complexes with syntaxin and vesicle-associated membrane protein (VAMP)/synaptobrevin (7,8), and when endogenous SNAP25 in chromaffin cells is cleaved by botulinum toxin E, a toxin-resistant SCORE rescues exocytosis (8). In PC12 cells expressing SCORE, a FRET change was evoked by high [K + ] stimulation, but with this method the time scale was tens of seconds (7). This FRET change was abolished in the absence of extracellular Ca 2+ , indicating that it is dependent on Ca 2+ entry. In contrast, the FRET change was not affected by tetanus neurotoxin treatment (7), which causes the blockade of exocytosis (10). In beta cells, a SCORE-like construct indicated a FRET chan...

How Could SNARE Proteins Open a Fusion Pore?

“…These experiments revealed a rapid conformational change in SNAP-25 that occurred specifically at the sites of fusion events and preceded fusion pore formation by ϳ90 ms (70). This FRET change may result from the formation of trans SNARE complexes that form after cross-linking the vesicle and plasma membranes via the synaptotagmin C2B domain, as recently proposed (22,63).…”

Membrane shaping by actin and myosin during regulated exocytosis

“…Especially, the temporal interplay between myosin II, dynamin, PtdIns(4,5)P 2 and Arp2/3 seems a logical target for an in-depth analysis. High sensitivity FRET and correlation measurements (Zhao et al, 2013) exocytosis by controlling fusion profiles through plasma membrane tension (Wen et al, 2016).…”

Imaging Analysis of Insulin Secretion with Two-Photon Microscopy

“…0.5 µm 2 . 43) In chromaffin cells, the conformational changes in SNAP25 during exocytosis were examined with the combined use of a FRET probe of SNAP25 (SNARE complex reporter), 42,44) TIRF microscopy, and amperometry. The average FRET signal from 900 fusion events was reported to precede the opening of the fusion pore by 90 ms, and ca.…”

“…7% of the FRET probe molecules were estimated to be involved in the transient FRET increase within the area of the exocytic site (0.1 µm 2 ). 44) Because two-photon microscopy utilizes an ultra-short pulse laser, it is also suited for fluorescence lifetime imaging, 45) which enables the quantification of intermolecular as well as intramolecular FRET. Combined with simultaneous multicolor imaging, the molecular mechanisms of exocytosis are expected to be elucidated in living tissues.…”