Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 110, Issue 35, Pages 14249-14254Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1306699110
Keywords
TIR-FRET imaging; electrochemical imaging; time superresolution microscopy; image analysis; transmitter release
Categories
Funding
- National Institutes of Health [GM085808, MH060600]
- European Research Council Advanced Grant [322699]
- National Science Foundation [ECS-0335765]
- European Research Council (ERC) [322699] Funding Source: European Research Council (ERC)
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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 similar to 90 ms. The FRET change correlated temporally with the opening of the fusion pore and not with its dilation.
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