Synaptotagmin I is necessary for compensatory synaptic vesicle endocytosis in vivo.
Neurotransmission requires a balance of synaptic vesicle exocytosis and endocytosis. Synaptotagmin I (Syt I) is widely regarded as the primary calcium sensor for synaptic vesicle exocytosis. Previous biochemical data suggest that Syt I may also function during synaptic vesicle endocytosis; however, ultrastructural analyses at synapses with impaired Syt I function have provided an indirect and conflicting view of the role of Syt I during synaptic vesicle endocytosis. Until now it has not been possible experimentally to separate the exocytic and endocytic functions of Syt I in vivo. Here, we test directly the role of Syt I during endocytosis in vivo. We use quantitative live imaging of a pH-sensitive green fluorescent protein fused to a synaptic vesicle protein (synapto-pHluorin) to measure the kinetics of endocytosis in sytI-null Drosophila. We then combine live imaging of the synapto-pHluorins with photoinactivation of Syt I, through fluorescein-assisted light inactivation, after normal Syt I-mediated vesicle exocytosis. By inactivating Syt I only during endocytosis, we demonstrate that Syt I is necessary for the endocytosis of synaptic vesicles that have undergone exocytosis using a functional Syt I protein.
Poskanzer
Kira E
KE
Department of Biochemistry and Biophysics, Program in Neuroscience, University of California, San Francisco, 513 Parnassus Avenue, San Francisco, California 94143, USA.
Marek
Kurt W
KW
Sweeney
Sean T
ST
Davis
Graeme W
GW
D000818:Animals; D002135:Calcium-Binding Proteins; D004331:Drosophila melanogaster / Q000166:cytology* / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis; D017353:Gene Deletion; D049452:Green Fluorescent Proteins; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008561:Membrane Fusion; D008562:Membrane Glycoproteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D018377:Neurotransmitter Agents / Q000378:metabolism; D013572:Synaptic Vesicles / Q000378:metabolism*; D050863:Synaptotagmin I; D050857:Synaptotagmins
D002135:Calcium-Binding Proteins; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D018377:Neurotransmitter Agents; D050863:Synaptotagmin I; D050857:Synaptotagmins; D049452:Green Fluorescent Proteins