Calcium-membrane resealing

Calcium-membrane resealing - Published December 15, 1995...

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Calcium-regulated Exocytosis Is Required for Cell Membrane Resealing Guo-Qiang Bi,* Janet M. Alderton,* and Richard A. Steinhardt** *Group in Biophysics; and ~Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3200 Abstract. Using confocal microscopy, we visualized exocytosis during membrane resealing in sea urchin eggs and embryos. Upon wounding by a laser beam, both eggs and embryos showed a rapid burst of local- ized Ca 2+-regulated exocytosis. The rate of exocytosis was correlated quantitatively with successfully reseal- ing. In embryos, whose activated surfaces must first dock vesicles before fusion, exocytosis and membrane resealing were inhibited by neurotoxins that selectively cleave the SNARE complex proteins, synaptobrevin, SNAP-25, and syntaxin. In eggs, whose cortical vesicles are already docked, vesicles could be reversibly un- docked with externally applied stachyose. If cortical vesicles were undocked both exocytosis and plasma membrane resealing were completely inhibited. When cortical vesicles were transiently undocked, exposure to tetanus toxin and botulinum neurotoxin type C1 ren- dered them no longer competent for resealing, al- though botulinum neurotoxin type A was still ineffec- tive. Cortical vesicles transiently undocked in the presence of tetanus toxin were subsequently fusion in- competent although to a large extent they retained their ability to redock when stachyose was diluted. We conclude that addition of internal membranes by exo- cytosis is required and that a SNARE-like complex plays differential roles in vesicle docking and fusion for the repair of disrupted plasma membrane. C ELt membranes are able to reseal after mechanical injury or disruption by microinjection, chemical per- meabilization, or electroporation, but the mecha- nism of resealing has not been well-characterized (Cajal, 1928; Celis, 1984; Yawo and Kuno, 1985; McNeil and Ito, 1989; Xie and Barrett, 1991; McNeil, 1991; Tsong, 1991; McNeil and Khakee, 1992; Yu and McNeil, 1992; Clarke et al., 1993: Spira et al., 1993; Weaver, 1993; Krause et al., 1994). Our previous study of plasma membrane repair led to the hypothesis that a calcium-regulated fusion of vesi- cles is necessary for resealing (Steinhardt et al., 1994). We found that membrane resealing in sea urchin eggs, early embryos, and Swiss 3T3 fibroblasts required threshold levels of extracellular Ca 2÷ that could be antagonized by Mg 2÷. In addition, we found that both botulinum neurotoxin types B (BNT-B) 1 and A (BNT-A) inhibited membrane resealing in Swiss 3T3 fibroblasts and in sea urchin embryos. BNT-B and BNT-A are known to be proteases that block neu- rotransmission by specific cleavages of the SNARE synaptic vesicle docking/fusion proteins, synaptobrevin, and SNAP- Address all correspondence to Dr. Richard Steinhardt, 391 LSA, Depart- ment of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3200. Tel.: (510) 642-3517. Fax: (510) 643-6791.
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This note was uploaded on 10/12/2010 for the course BC BC367 taught by Professor Millard during the Spring '10 term at Colby.

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Calcium-membrane resealing - Published December 15, 1995...

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