Fluorescence resonance energy transfer detection of synaptophysin I and vesicle-associated membrane protein 2 interactions during exocytosis from single live synapses.
To investigate the molecular interactions of synaptophysin I and vesicle-associated membrane protein 2 (VAMP2)/synaptobrevin II during exocytosis, we have used time-lapse videomicroscopy to measure fluorescence resonance energy transfer in live neurons. For this purpose, fluorescent protein variants fused to synaptophysin I or VAMP2 were expressed in rat hippocampal neurons. We show that synaptophysin I and VAMP2 form both homo- and hetero-oligomers on the synaptic vesicle membrane. When exocytosis is stimulated with alpha-latrotoxin, VAMP2 dissociates from synaptophysin I even in the absence of appreciable exocytosis, whereas synaptophysin I oligomers disassemble only upon incorporation of the vesicle with the plasma membrane. We propose that synaptophysin I has multiple roles in neurotransmitter release, regulating VAMP2 availability for the soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex and possibly participating in the late steps of exocytosis.
Department of Neuroscience, S. Raffaele Scientific Institute and Vita-Salute University, Milan, Italy.
Maria
M
Pennuto
David
D
Dunlap
Andrea
A
Contestabile
Fabio
F
Benfenati
Flavia
F
Valtorta
D000818:Animals; D002478:Cells, Cultured; D005089:Exocytosis / Q000502:physiology*; D031541:Fluorescence Resonance Energy Transfer; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D018715:Microscopy, Video; D008954:Models, Biological; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D050683:R-SNARE Proteins; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D013111:Spider Venoms / Q000494:pharmacology; D013569:Synapses / Q000378:metabolism*; D013572:Synaptic Vesicles / Q000378:metabolism; D016708:Synaptophysin / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors
D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins; D013111:Spider Venoms; D016708:Synaptophysin; C020763:alpha-latrotoxin