Abstract
Synaptotagmin I (syt1) is required for normal rates of synaptic vesicle endo- and exocytosis. However, whether the kinetic defects observed during endocytosis in Syt1 knockout neurons are secondary to defective exocytosis or whether syt1 directly regulates the rate of vesicle retrieval remains unknown. To address this question, we sought to dissociate these two activities. We uncoupled the function of syt1 in exo- and endocytosis in mouse neurons either by re-targeting the protein or via mutagenesis of its tandem C2 domains. The effect of these manipulations on exo- and endocytosis were analyzed using electrophysiology, in conjunction with optical imaging of the vesicle cycle. Our results indicate that syt1 is directly involved in endocytosis. Notably, either of the C2 domains of syt1, C2A or C2B, was able to function as a Ca2+ sensor for endocytosis. Thus, syt1 functions as a dual Ca2+ sensor for both endo- and exocytosis, potentially coupling these two components of the vesicle cycle.
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Acknowledgements
We thank E. Hui for providing constructs used for the electrophysiology experiments shown in Figure 3. We also thank M.B. Jackson, X. Lou and members of the Chapman laboratory for their comments on this manuscript. This study was supported by a grant from the US National Institutes of Health (MH 61876). J.Y. is supported by an American Heart Association postdoctoral fellowship (11POST5720016). S.E.K. was supported by an Epilepsy Foundation predoctoral fellowship (09PRE2060819). E.R.C. is an Investigator of the Howard Hughes Medical Institute.
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J.Y., S.E.K. and E.R.C. designed the experiments. J.Y. conducted the electrophysiological and immunostaining experiments. S.E.K. and J.Y. conducted the pHluorin imaging experiments. F.M.D., S.E.K. and J.Y. conducted the molecular biology experiments. J.D.G. conducted the cell fractionation experiments. J.D.G. and J.Y. conducted the immunoblot experiments. J.Y., S.E.K. and E.R.C. analyzed the data and wrote the manuscript.
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Yao, J., Kwon, S., Gaffaney, J. et al. Uncoupling the roles of synaptotagmin I during endo- and exocytosis of synaptic vesicles. Nat Neurosci 15, 243–249 (2012). https://doi.org/10.1038/nn.3013
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DOI: https://doi.org/10.1038/nn.3013
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