Real-time measurements of vesicle-SNARE recycling in synapses of the central nervous system.
Following the fusion of synaptic vesicles with the presynaptic plasma membrane of nerve terminals by the process of exocytosis, synaptic-vesicle components are recycled to replenish the vesicle pool. Here we use a pH-sensitive green fluorescent protein to measure the residence time of VAMP, a vesicle-associated SNARE protein important for membrane fusion, on the surfaces of synaptic terminals of hippocampal neurons following exocytosis. The time course of VAMP retrieval depends linearly on the amount of VAMP that is added to the plasma membrane, with retrieval occurring between about 4 seconds and 90 seconds after exocytosis, and newly internalized vesicles are rapidly acidified. These data are well described by a model in which endocytosis appears to be saturable, but proceeds with an initial maximum velocity of about one vesicle per second. We also find that, following exocytosis, a portion of the newly inserted VAMP appears on the surface of the axon.
Sankaranarayanan
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Department of Biochemistry, Weill Medical College of Cornell University, 1300 York Avenue, New York, New York 10021, USA.
Ryan
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D000200:Action Potentials / Q000502:physiology; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004705:Endocytosis / Q000502:physiology; D005456:Fluorescent Dyes; D006624:Hippocampus / Q000166:cytology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008561:Membrane Fusion / Q000502:physiology; D008565:Membrane Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D017729:Presynaptic Terminals / Q000378:metabolism; D050683:R-SNARE Proteins; D051381:Rats; D017207:Rats, Sprague-Dawley; D050600:SNARE Proteins; D013572:Synaptic Vesicles / Q000378:metabolism*; D033921:Vesicular Transport Proteins
D005456:Fluorescent Dyes; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050683:R-SNARE Proteins; D050600:SNARE Proteins; D033921:Vesicular Transport Proteins