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Uncoupling the roles of synaptotagmin I during endo- and exocytosis of synaptic vesicles

Nature Neuroscience volume 15, pages 243–249 (2012)Cite this article

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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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Figure 1: The plasma membrane–targeted cytoplasmic domain of syt1 rescues rapid synaptic vesicle exocytosis in syt1 knockout neurons.
Figure 2: GAP43-C2AB cannot rescue defective synaptic vesicle endocytosis in syt1 knockout neurons.
Figure 3: Ca2+-binding activity is required for syt1 to accelerate synaptic vesicle endocytosis.
Figure 4: Ca2+-independent synaptic vesicle endocytosis does not rely on syt1.
Figure 5: C2A and C2B are redundant Ca2+ sensors for endocytosis.
Figure 6: The C2A domain is capable of internalizing syt1 into newly retrieved synaptic vesicles.

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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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  1. F Mark Dunning

    Present address: Present address: BioSentinel Pharmaceuticals, Madison, Wisconsin, USA.,

  2. Jun Yao and Sung E Kwon: These authors contributed equally to this work.

Authors and Affiliations

  1. Howard Hughes Medical Institute and Department of Neuroscience, University of Wisconsin, Madison, Wisconsin, USA

    Jun Yao, Sung E Kwon, Jon D Gaffaney, F Mark Dunning & Edwin R Chapman

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Contributions

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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Correspondence to Edwin R Chapman.

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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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