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Published online
doi:10.1083/jcb.200804162
The Journal of Cell Biology, Vol. 182, No. 5, 1007-1016
The Rockefeller University Press, 0021-9525 $30.00
© Kasprowicz et al.
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Article

Inactivation of clathrin heavy chain inhibits synaptic recycling but allows bulk membrane uptake



Jaroslaw Kasprowicz1,2, Sabine Kuenen1,2, Katarzyna Miskiewicz1,2, Ron L.P. Habets1,2, Liesbet Smitz1,2, and Patrik Verstreken1,2

1 Department of Molecular and Developmental Genetics, VIB (Flemish Institute for Biotechnology), 3000 Leuven, Belgium
2 Laboratory of Neuronal Communication, Program in Cognitive and Molecular Neuroscience, Program in Molecular and Developmental Genetics, Center for Human Genetics, Katholieke Universiteit Leuven, 3000 Leuven, Belgium

Correspondence to Patrik Verstreken: patrik.verstreken{at}med.kuleuven.be

Synaptic vesicle reformation depends on clathrin, an abundant protein that polymerizes around newly forming vesicles. However, how clathrin is involved in synaptic recycling in vivo remains unresolved. We test clathrin function during synaptic endocytosis using clathrin heavy chain (chc) mutants combined with chc photoinactivation to circumvent early embryonic lethality associated with chc mutations in multicellular organisms. Acute inactivation of chc at stimulated synapses leads to substantial membrane internalization visualized by live dye uptake and electron microscopy. However, chc-inactivated membrane cannot recycle and participate in vesicle release, resulting in a dramatic defect in neurotransmission maintenance during intense synaptic activity. Furthermore, inactivation of chc in the context of other endocytic mutations results in membrane uptake. Our data not only indicate that chc is critical for synaptic vesicle recycling but they also show that in the absence of the protein, bulk retrieval mediates massive synaptic membrane internalization.

J. Kasprowicz and S. Kuenen contributed equally to this paper.

Abbreviations used in this paper: ANOVA, analysis of variance; Chc, clathrin heavy chain; Clc, clathrin light chain; EJP, excitatory junctional potential; Endo, endophilin; FALI, fluorescein-assisted light inactivation; FlAsH, 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein; Kan, kanamycin; NMJ, neuromuscular junction; Synj, synaptojanin; sytI, synaptotagmin I.

© 2008 Kasprowicz et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).


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