Monitoring clathrin-mediated endocytosis during synaptic activity.
To visualize clathrin redistribution during endocytosis in hippocampal boutons, we used a fusion protein of clathrin light chain with enhanced green fluorescent protein. Both high potassium and electric field stimulation lead after a stimulus-dependent delay to a transient increase of fluorescence in synapses, but a slight and transient decrease in adjacent axonal segments. We conclude that the rise and fall of the signal in boutons, with decay kinetics remarkably similar to previous estimates of the endocytic time course, reflects coat assembly and disassembly. Thus, we could selectively measure clathrin-mediated endocytosis and separate its kinetics from other modes of membrane retrieval in CNS synapses. A long-lasting delay preceding the fluorescent transients shows that endocytosis during the first few seconds of continuing stimulation cannot be mediated by newly formed clathrin-coated pits. Therefore, a fast mode of endocytosis is either clathrin-independent or involves preassembled (easily retrievable) clathrin lattices at sites of endocytosis.
Department of Membrane Biophysics, Max-Planck Institute for Biophysical Chemistry, D-37077 Goettingen, Germany.
Veronika J
VJ
Mueller
Martin
M
Wienisch
Ralf B
RB
Nehring
Jurgen
J
Klingauf
D000200:Action Potentials / Q000502:physiology; D000818:Animals; D002478:Cells, Cultured; D002966:Clathrin / Q000235:genetics / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism / Q000648:ultrastructure; D004058:Diffusion; D004558:Electric Stimulation; D004705:Endocytosis / Q000502:physiology*; D049452:Green Fluorescent Proteins; D006624:Hippocampus / Q000648:ultrastructure; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics; D008959:Models, Neurological; D009474:Neurons / Q000378:metabolism* / Q000648:ultrastructure; D017729:Presynaptic Terminals / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013268:Stimulation, Chemical; D009435:Synaptic Transmission / Q000502:physiology*
D002966:Clathrin; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins