Clathrin and HA2 adaptors: effects of potassium depletion, hypertonic medium, and cytosol acidification.
Clathrin and HA2 adaptors: effects of potassium depletion, hypertonic medium, and cytosol acidification.
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DOI：
10.1083/jcb.121.1.61
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发表时间：
1993-04
期刊：
影响因子：
--
通讯作者：
van Deurs B
中科院分区：
文献类型：
--
作者：
Hansen SH;Sandvig K;van Deurs B
通过免疫荧光和超微结构免疫金显微镜，使用转铁蛋白的内化作为功能测定，研究了已知干扰网格蛋白包被的凹点的内吞作用的方法对HEp-2癌细胞中网格蛋白和HA 2衔接子定位的影响。钾消耗以及在高渗培养基中孵育，去除膜相关网格蛋白晶格：来自质膜的平坦网格蛋白晶格和包被的小凹，和来自细胞质的网格蛋白包被的囊泡，以及从TGN出芽的那些。相比之下，免疫荧光显微镜检查使用特异性抗体的α-和β-适应素，分别和免疫金标记的冷冻切片与抗α-适应素抗体显示，在这些条件下，HA 2衔接子聚集在质膜上的程度与对照细胞相同。在用含等渗K（+）的培养基重构后，衔接子聚集体和网格蛋白晶格像正常情况一样共定位在质膜上，并且转铁蛋白的内化恢复。酸化的细胞质既不影响网格蛋白，也不HA 2衔接研究免疫荧光显微镜。然而，定量的超微结构观察表明，酸化的胞质溶胶的结果在形成的异质性大小和平均较小的网格蛋白包被的坑在质膜和芽上的TGN。总的来说，我们的观察结果表明，干扰网格蛋白包被的囊泡形成的方法通过不同的机制起作用：酸化的胞质溶胶通过影响网格蛋白包被的膜结构域的方式，干扰出芽的网格蛋白包被的囊泡从质膜以及从TGN;钾耗竭和高渗介质中的孵育，通过防止网格蛋白和衔接子相互作用。此外，我们的观察表明，适配器聚集体可以独立于网格蛋白晶格存在于质膜上，并提高了适配器聚集体可以形成网格蛋白晶格成核位点的可能性。
The effects of methods known to perturb endocytosis from clathrin- coated pits on the localization of clathrin and HA2 adaptors in HEp-2 carcinoma cells have been studied by immunofluorescence and ultrastructural immunogold microscopy, using internalization of transferrin as a functional assay. Potassium depletion, as well as incubation in hypertonic medium, remove membrane-associated clathrin lattices: flat clathrin lattices and coated pits from the plasma membrane, and clathrin-coated vesicles from the cytoplasm, as well as those budding from the TGN. In contrast, immunofluorescence microscopy using antibodies specific for the alpha- and beta-adaptins, respectively, and immunogold labeling of cryosections with anti-alpha- adaptin antibodies shows that under these conditions HA2 adaptors are aggregated at the plasma membrane to the same extent as in control cells. After reconstitution with isotonic K(+)-containing medium, adaptor aggregates and clathrin lattices colocalize at the plasma membrane as normally and internalization of transferrin resumes. Acidification of the cytosol affects neither clathrin nor HA2 adaptors as studied by immunofluorescence microscopy. However, quantitative ultrastructural observations reveal that acidification of the cytosol results in formation of heterogeneously sized and in average smaller clathrin-coated pits at the plasma membrane and buds on the TGN. Collectively, our observations indicate that the methods to perturb formation of clathrin-coated vesicles act by different mechanisms: acidification of the cytosol by affecting clathrin-coated membrane domains in a way that interferes with budding of clathrin-coated vesicles from the plasma membrane as well as from the TGN; potassium depletion and incubation in hypertonic medium by preventing clathrin and adaptors from interacting. Furthermore our observations show that adaptor aggregates can exist at the plasma membrane independent of clathrin lattices and raise the possibility that adaptor aggregates can form nucleation sites for clathrin lattices.