Ca-dependent nonsecretory vesicle fusion in a secretory cell.
Ca-dependent nonsecretory vesicle fusion in a secretory cell.
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分泌细胞中 Ca 依赖性非分泌囊泡融合。
DOI：
10.1085/jgp.200709950
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发表时间：
2008
期刊：
影响因子：
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通讯作者：
Hilgemann,DonaldW
中科院分区：
文献类型：
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作者：
Wang,Tzu-Ming;Hilgemann,DonaldW
我们已经比较了钙依赖性胞吐在切除的巨膜补丁和全细胞膜片钳强调大鼠分泌细胞系，RBL。在用latrunculin A部分破坏肌动蛋白细胞骨架后，容易从RBL细胞中切下2-4 pF的稳定斑块。通过将贴片切换到含有100-200 μM游离Ca的细胞质溶液来触发膜融合。电容和安培记录显示，含有5-羟色胺的大分泌颗粒（SG）大多从斑块中丢失。保留的小囊泡（非SG）不释放血清素或电流分析法检测到的其他物质，尽管破伤风毒素轻链减少了它们的融合。非SG融合不受N-乙基马来酰亚胺、磷脂酰肌醇-4，5-二磷酸（PI（4，5）P2）配体（如新霉素，一种可从膜上除去PI的PI转移蛋白）、PI（3）-激酶抑制剂LY 294002和PI（4，5）P2、PI（3）P和PI（4）P抗体的影响。在斑片记录，但不是全细胞记录，融合可以强烈减少ATP去除和非特异性PI激酶抑制剂渥曼青霉素和腺苷。在全细胞记录中，非SG融合强烈地减少了细胞肿胀，随后收缩后的恢复被渥曼青霉素抑制。因此，膜拉伸过程中发生的补丁形成可能是切除补丁和全细胞融合反应之间的差异的主要原因。关于非SG融合的Ca传感器，在突触结合蛋白（Syt）VII−/−小鼠胚胎成纤维细胞（MEF）以及PLCδ1、PLC δ1/δ4和PLCγ1−/− MEF中，融合仍然稳健。因此，不需要Syt VII和几个PLC。此外，非SG融合的Ca依赖性反映了比这些含C2结构域的蛋白预期的更低的Ca亲和力（KD <71 μM）。总之，我们发现，非SG膜融合的行为和调节实质上不同于SG融合，我们已经确定了一个ATP依赖的过程，恢复非SG融合能力后，它是由膜拉伸或细胞扩张扰动。
We have compared Ca-dependent exocytosis in excised giant membrane patches and in whole-cell patch clamp with emphasis on the rat secretory cell line, RBL. Stable patches of 2–4 pF are easily excised from RBL cells after partially disrupting actin cytoskeleton with latrunculin A. Membrane fusion is triggered by switching the patch to a cytoplasmic solution containing 100–200 μM free Ca. Capacitance and amperometric recording show that large secretory granules (SGs) containing serotonin are mostly lost from patches. Small vesicles that are retained (non-SGs) do not release serotonin or other substances detected by amperometry, although their fusion is reduced by tetanus toxin light chain. Non-SG fusion is unaffected byN-ethylmaleimide, phosphatidylinositol-4,5-bis-phosphate (PI(4,5)P2) ligands, such as neomycin, a PI-transfer protein that can remove PI from membranes, the PI(3)-kinase inhibitor LY294002 and PI(4,5)P2, PI(3)P, and PI(4)P antibodies. In patch recordings, but not whole-cell recordings, fusion can be strongly reduced by ATP removal and by the nonspecific PI-kinase inhibitors wortmannin and adenosine. In whole-cell recording, non-SG fusion is strongly reduced by osmotically induced cell swelling, and subsequent recovery after shrinkage is then inhibited by wortmannin. Thus, membrane stretch that occurs during patch formation may be a major cause of differences between excised patch and whole-cell fusion responses. Regarding Ca sensors for non-SG fusion, fusion remains robust in synaptotagmin (Syt) VII−/− mouse embryonic fibroblasts (MEFs), as well as in PLCδ1, PLC δ1/δ4, and PLCγ1−/− MEFs. Thus, Syt VII and several PLCs are not required. Furthermore, the Ca dependence of non-SG fusion reflects a lower Ca affinity (KD∼71 μM) than expected for these C2 domain–containing proteins. In summary, we find that non-SG membrane fusion behaves and is regulated substantially differently from SG fusion, and we have identified an ATP-dependent process that restores non-SG fusion capability after it is perturbed by membrane stretch or cell dilation.