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J. Biol. Chem., Vol. 280, Issue 2, 1652-1660, January 14, 2005

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Molecular Regulation of Membrane Resealing in 3T3 Fibroblasts^*

Sheldon S. Shen, Ward C. Tucker¶||, Edwin R. Chapman¶**, and Richard A. Steinhardt

From the Department of Genetics, Development, and Cell Biology, Iowa State University, Ames, Iowa 50011, the ^¶Department of Physiology, University of Wisconsin School of Medicine, Madison, Wisconsin 53706, and the Department of Molecular and Cell Biology, University of California, Berkeley, California 94720

Membrane resealing in mammalian cells after injury depends on Ca²⁺-dependent fusion of intracellular vesicles with the plasma membrane. When cells are wounded twice, the subsequent resealing is generally faster. Physiological and biochemical studies have shown the initiation of two different repair signaling pathways, which are termed facilitated and potentiated responses. The facilitated response is dependent on the generation and recruitment of new vesicles, whereas the potentiated response is not. Here, we report that the two responses can be differentially defined molecularly. Using recombinant fragments of synaptobrevin-2 and synaptotagmin C2 domains we were able to dissociate the molecular requirements of vesicle exocytosis for initial membrane resealing and the facilitated and potentiated responses. The initial resealing response was blocked by fragments of synaptobrevin-2 and the C2B domain of synaptotagmin VII. Both the facilitated and potentiated responses were also blocked by the C2B domain of synaptotagmin VII. Although the initial resealing response was not blocked by the C2AB domain of synaptotagmin I or the C2A domain of synaptotagmin VII, recruitment of new vesicles for the facilitated response was inhibited. We also used Ca²⁺ binding mutant studies to show that the effects of synaptotagmins on membrane resealing are Ca²⁺-dependent. The pattern of inhibition by synaptotagmin C2 fragments that we observed cannot be used to specify a vesicle compartment, such as lysosomes, in membrane repair.

Received for publication, September 3, 2004 , and in revised form, November 8, 2004.

^* This study was supported in part by National Institutes of Health (NIH) Grant EY 13436 (to R. A. S.) and NIGMS, NIH Grant GM 56827 and NIMH NIH Grant MH 61876 (to E. R. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| Supported by a Postdoctoral Individual National Research Service Award from the National Institutes of Health.

^** A Pew Scholar in the Biomedical Sciences.

To whom correspondence should be addressed. Tel.: 510-520-1073; Fax: 510-643-6791; E-mail: rsteinha{at}socrates.berkeley.edu.

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