Rapid structural change in synaptosomal-associated protein 25 (SNAP25) precedes the fusion of single vesicles with the plasma membrane in live chromaffin cells.
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.
School of Applied and Engineering Physics, Cornell University, Ithaca, NY 14850, USA.
Ying
Y
Zhao
Qinghua
Q
Fang
Adam Drew
AD
Herbst
Khajak N
KN
Berberian
Wolfhard
W
Almers
Manfred
M
Lindau
D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D019439:Chromaffin Cells / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D008561:Membrane Fusion; D011487:Protein Conformation; D050825:Synaptosomal-Associated Protein 25 / Q000737:chemistry / Q000378:metabolism*
D050825:Synaptosomal-Associated Protein 25
TIR-FRET imaging; electrochemical imaging; image analysis; time superresolution microscopy; transmitter release