Molecular regulation of membrane resealing in 3T3 fibroblasts
Molecular regulation of membrane resealing in 3T3 fibroblasts
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DOI：
10.1074/jbc.m410136200
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发表时间：
2005-01-14
影响因子：
4.8
通讯作者：
Steinhardt, RA
中科院分区：
文献类型：
--
作者：
Shen, SS;Tucker, WC;Steinhardt, RA
哺乳动物细胞损伤后的膜重新密封依赖于细胞内小泡与质膜的钙离子依赖的融合。当细胞受到两次损伤时，随后的重新密封通常会更快。生理生化研究表明，有两条不同的修复信号通路被启动，它们被称为易化反应和增强反应。易化反应依赖于新小泡的产生和募集，而增强反应则不是。在这里，我们报告了这两种反应可以从分子上区别定义。利用突触素-2和突触素C2结构域的重组片段，我们能够分离囊泡胞吐对初始膜重新密封的分子需求以及促进和增强的反应。最初的再密封反应被Synaptobrevin-2的片段和Synaptopagmin VII的C2B结构域所阻断，促进的和增强的反应也被Synaptopagmin VII的C2B结构域阻断。尽管最初的再密封反应不被Synaptopagmin I的C2AB结构域或Synaptopagmin VII的C2a结构域阻止，但促进反应的新小泡的募集被抑制。我们还利用钙离子结合突变研究表明，突触素对膜重新密封的影响是钙依赖的。我们观察到的突触素C2片段的抑制模式不能用来指定膜修复中的囊泡间隔，如溶酶体。
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.