Parton R G, Joggerst B, Simons K
European Molecular Biology Laboratory, Heidelberg, Germany.
J Cell Biol. 1994 Dec;127(5):1199-215. doi: 10.1083/jcb.127.5.1199.
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.
小窝是质膜的特化内陷结构，有人提出其在多种细胞过程中发挥作用，如内吞作用和信号转导。我们开发了一种测定方法来确定内部小窝与质膜小窝的比例。糖基磷脂酰肌醇（GPI）锚定蛋白碱性磷酸酶在低温下经抗体诱导交联后聚集在小窝中，然后，经过各种处理后，测定细胞表面碱性磷酸酶的相对含量。使用该测定方法，我们能够显示细胞表面碱性磷酸酶活性呈现时间和温度依赖性下降，这取决于抗体诱导的聚集。磷酸酶抑制剂冈田酸可大大加速细胞表面碱性磷酸酶活性的下降，但蛋白激酶C激活剂则无此作用。无论有无冈田酸，细胞松弛素D和激酶抑制剂星形孢菌素均可阻断聚集的碱性磷酸酶的内化。电子显微镜证实冈田酸可诱导小窝从细胞表面去除。在高渗培养基存在的情况下，随后小窝群体会重新分布到靠近微管组织中心的细胞中央。这个过程是可逆的，被细胞松弛素D阻断，并且小窝簇的集中化显示依赖于完整的微管网络。虽然内化的确切机制仍然未知，但结果表明小窝是动态结构，可以被内化到细胞中。这个过程可能受激酶活性调节，并且需要完整的肌动蛋白网络。
