Secretory granules are recaptured largely intact after stimulated exocytosis in cultured endocrine cells
Secretory granules are recaptured largely intact after stimulated exocytosis in cultured endocrine cells
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DOI：
10.1073/pnas.0337526100
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发表时间：
2003-02-18
影响因子：
11.1
通讯作者：
Almers, W
中科院分区：
文献类型：
--
作者：
Taraska, JW;Perrais, D;Almers, W
经典细胞生物学教导，胞吐作用导致胞吐囊泡的膜分散到细胞表面，并且细胞随后必须通过分子分选来检索它希望保留在内部的任何膜成分。我们已经测试了这种观点是否适用于完整 PC-12 细胞中的分泌颗粒。三种颗粒蛋白用不同颜色的荧光蛋白标记，并使用双色渐逝场显微镜观察胞吐期间和之后的单个颗粒。神经肽 Y 在几秒钟内就从颗粒中丢失，而组织纤溶酶原激活剂 (tPA) 和膜蛋白噬蛋白在颗粒位点保留超过 1 分钟，从而为胞吐后颗粒提供了标记。当 tPA 与作为胞质标记的青色荧光蛋白 (CFP) 同时成像时，颗粒所占的体积显示为黑点，其中排除了 CFP。即使在 tPA 报告胞吐作用后，该斑点仍然存在，表明颗粒未能压平到细胞表面。 Phogrin 在其管腔末端用 GFP 标记，用于感测颗粒中的 pH 值。当胞吐作用导致酸性颗粒内部中和时，GFR-噬菌蛋白首先变亮，然后随着内部与细胞外空间分离并重新酸化而再次变暗。再酸化和变暗可以通过使用 NH4Cl 来逆转。我们的结论是，大多数颗粒重新密封在
Classical cell biology teaches that exocytosis causes the membrane of exocytic vesicles to disperse into the cell surface and that a cell must later retrieve by molecular sorting whatever membrane components it wishes to keep inside. We have tested whether this view applies to secretory granules in intact PC-12 cells. Three granule proteins were labeled with fluorescent proteins in different colors, and two-color evanescent-field microscopy was used to view single granules during and after exocytosis. Whereas neuropeptide Y was lost from granules in seconds, tissue plasminogen activator (tPA) and the membrane protein phogrin remained at the granule site for over 1 min, thus providing markers for postexocytic granules. When tPA was imaged simultaneously with cyan fluorescent protein (CFP) as a cytosolic marker, the volume occupied by the granule appeared as a dark spot where it excluded CFP. The spot remained even after tPA reported exocytosis, indicating that granules failed to flatten into the cell surface. Phogrin was labeled with GFP at its luminal end and used to sense the pH in granules. When exocytosis caused the acidic granule interior to neutralize, GFR-phogrin at first brightened and later dimmed again as the interior separated from the extracellular space and reacidified. Reacidification and dimming could be reversed by application of NH4Cl We conclude that most granules reseal in