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 i...

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Bibliographic Details
Published in:The Journal of neuroscience 2004-02, Vol.24 (8), p.2004-2012
Main Authors: Mueller, Veronika J, Wienisch, Martin, Nehring, Ralf B, Klingauf, Jurgen
Format: Article
Language:English
Subjects:
Action Potentials - physiology
Animals
Cells, Cultured
Cellular/Molecular
Clathrin - genetics
Clathrin - metabolism
Coated Pits, Cell-Membrane - metabolism
Coated Pits, Cell-Membrane - ultrastructure
Diffusion
Electric Stimulation
Endocytosis - physiology
Green Fluorescent Proteins
Hippocampus - ultrastructure
Humans
Kinetics
Luminescent Proteins - genetics
Models, Neurological
Neurons - metabolism
Neurons - ultrastructure
Presynaptic Terminals - metabolism
Rats
Rats, Wistar
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - metabolism
Stimulation, Chemical
Synaptic Transmission - physiology
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Summary: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.
ISSN:0270-6474
1529-2401
DOI:10.1523/JNEUROSCI.4080-03.2004