TY - JOUR
T1 - Ultrafast endocytosis at mouse hippocampal synapses
AU - Watanabe, Shigeki
AU - Rost, Benjamin R.
AU - Camacho-Pérez, Marcial
AU - Davis, M. Wayne
AU - Söhl-Kielczynski, Berit
AU - Rosenmund, Christian
AU - Jorgensen, Erik M.
N1 - Funding Information:
Acknowledgements We would like to thank D. Lorenz and A. Muenster-Wandowski for providing access to electron microscopes. We would like to thank P. Hegemann and F.Schneiderforprovidingthe ChetaTC construct,A.Feliesfor cellcultures,B.Brokowski for generating lentivirus, E. Hujber for image processing and freezing calculations, C.Ebelingfor calculatingthetimeconstant for synaptic vesicle collapse, andJ. Iwasafor drawingthe modelfigure.Wewouldlike tothank C.TomovaandLeicaMicrosystemsfor providing us with technical details of the controller of the high-pressure freezer for precise temporal control of light stimulation. We thank EMBO for providing the travel funds (S.W.). The research was funded by the US National Institutes of Health (NS034307; E.M.J.), European Research Council grant (249939 SYNVGLUT; C.R.), and German Research Council grants (EXC 257, SFB 665, SFB958; C.R.). E.M.J. is an Investigator of the Howard Hughes Medical Institute and is an Alexander von Humboldt Scholar.
PY - 2013
Y1 - 2013
N2 - To sustain neurotransmission, synaptic vesicles and their associated proteins must be recycled locally at synapses. Synaptic vesicles are thought to be regenerated approximately 20 s after fusion by the assembly of clathrin scaffolds or in approximately 1 s by the reversal of fusion pores via 'kiss-and-run' endocytosis. Here we use optogenetics to stimulate cultured hippocampal neurons with a single stimulus, rapidly freeze them after fixed intervals and examine the ultrastructure using electron microscopy - 'flash-and-freeze' electron microscopy. Docked vesicles fuse and collapse into the membrane within 30 ms of the stimulus. Compensatory endocytosis occurs within 50 to 100 ms at sites flanking the active zone. Invagination is blocked by inhibition of actin polymerization, and scission is blocked by inhibiting dynamin. Because intact synaptic vesicles are not recovered, this form of recycling is not compatible with kiss-and-run endocytosis; moreover, it is 200-fold faster than clathrin-mediated endocytosis. It is likely that 'ultrafast endocytosis' is specialized to restore the surface area of the membrane rapidly.
AB - To sustain neurotransmission, synaptic vesicles and their associated proteins must be recycled locally at synapses. Synaptic vesicles are thought to be regenerated approximately 20 s after fusion by the assembly of clathrin scaffolds or in approximately 1 s by the reversal of fusion pores via 'kiss-and-run' endocytosis. Here we use optogenetics to stimulate cultured hippocampal neurons with a single stimulus, rapidly freeze them after fixed intervals and examine the ultrastructure using electron microscopy - 'flash-and-freeze' electron microscopy. Docked vesicles fuse and collapse into the membrane within 30 ms of the stimulus. Compensatory endocytosis occurs within 50 to 100 ms at sites flanking the active zone. Invagination is blocked by inhibition of actin polymerization, and scission is blocked by inhibiting dynamin. Because intact synaptic vesicles are not recovered, this form of recycling is not compatible with kiss-and-run endocytosis; moreover, it is 200-fold faster than clathrin-mediated endocytosis. It is likely that 'ultrafast endocytosis' is specialized to restore the surface area of the membrane rapidly.
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U2 - 10.1038/nature12809
DO - 10.1038/nature12809
M3 - Article
C2 - 24305055
AN - SCOPUS:84890442090
SN - 0028-0836
VL - 504
SP - 242
EP - 247
JO - Nature
JF - Nature
IS - 7479
ER -