Regulated internalization of caveolae.
Regulated internalization of caveolae.
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DOI：
10.1083/jcb.127.5.1199
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发表时间：
1994-12
影响因子：
7.8
通讯作者：
Simons, K
中科院分区：
文献类型：
--
作者：
Parton, R G;Joggerst, B;Simons, K
小窝是细胞膜上的一种特殊的内陷结构，在细胞内吞和信号转导等多种细胞过程中发挥重要作用。我们已经开发了一种测定方法，以确定内部与质膜小窝的分数。GPI锚定的蛋白，碱性磷酸酶，聚集在小窝抗体诱导的交联后，在低温下，然后，经过各种处理，碱性磷酸酶的相对量的细胞表面上进行了测定。使用该测定，我们能够显示依赖于抗体诱导的聚集的细胞表面碱性磷酸酶活性的时间和温度依赖性降低。细胞表面碱性磷酸酶活性的下降大大加快了磷酸酶抑制剂，冈田酸，但不是由蛋白激酶C激活剂。细胞松弛素D和激酶抑制剂星形孢菌素阻断了存在或不存在冈田酸的成簇碱性磷酸酶的内化。电子显微镜证实冈田酸诱导细胞表面小窝的去除。在高渗介质的存在下，这是其次是由小窝组的重新分配到靠近微管组织中心的细胞中心。这一过程是可逆的，细胞松弛素D阻断，并集中的小窝集群被证明是依赖于一个完整的微管网络。虽然内化的确切机制仍然未知，但结果表明小窝是可以内化到细胞中的动态结构。这个过程可能受到激酶活性的调节，并且需要完整的肌动蛋白网络。
Caveolae are specialized invaginations of the plasma membrane which have been proposed to play a role in diverse cellular processes such as endocytosis and signal transduction. We have developed an assay to determine the fraction of internal versus plasma membrane caveolae. The GPI-anchored protein, alkaline phosphatase, was clustered in caveolae after antibody-induced crosslinking at low temperature and then, after various treatments, the relative amount of alkaline phosphatase on the cell surface was determined. Using this assay we were able to show a time- and temperature-dependent decrease in cell-surface alkaline phosphatase activity which was dependent on antibody-induced clustering. The decrease in cell surface alkaline phosphatase activity was greatly accelerated by the phosphatase inhibitor, okadaic acid, but not by a protein kinase C activator. Internalization of clustered alkaline phosphatase in the presence or absence of okadaic acid was blocked by cytochalasin D and by the kinase inhibitor staurosporine. Electron microscopy confirmed that okadaic acid induced removal of caveolae from the cell surface. In the presence of hypertonic medium this was followed by the redistribution of groups of caveolae to the center of the cell close to the microtubule-organizing center. This process was reversible, blocked by cytochalasin D, and the centralization of the caveolar clusters was shown to be dependent on an intact microtubule network. Although the exact mechanism of internalization remains unknown, the results show that caveolae are dynamic structures which can be internalized into the cell. This process may be regulated by kinase activity and require an intact actin network.