Hansen S H, Sandvig K, van Deurs B
Department of Anatomy, Panum Institute, University of Copenhagen, Denmark.
J Cell Biol. 1993 Apr;121(1):61-72. doi: 10.1083/jcb.121.1.61.
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.
通过免疫荧光和超微结构免疫金显微镜技术，以转铁蛋白的内化作为功能检测，研究了已知会扰乱网格蛋白包被小窝内吞作用的方法对HEp-2癌细胞中网格蛋白和HA2衔接蛋白定位的影响。低钾以及在高渗培养基中孵育会去除膜相关的网格蛋白晶格：质膜上的扁平网格蛋白晶格和包被小窝，以及细胞质中的网格蛋白包被囊泡，还有从反式高尔基体网络出芽的那些囊泡。相反，分别使用对α-和β-衔接蛋白特异的抗体进行免疫荧光显微镜观察，以及用抗α-衔接蛋白抗体对冷冻切片进行免疫金标记显示，在这些条件下HA2衔接蛋白在质膜上聚集的程度与对照细胞相同。用含等渗钾离子的培养基重构后，衔接蛋白聚集体和网格蛋白晶格在质膜上正常共定位，转铁蛋白的内化恢复。通过免疫荧光显微镜研究发现，胞质溶胶酸化对网格蛋白和HA2衔接蛋白均无影响。然而，定量超微结构观察表明，胞质溶胶酸化会导致质膜上形成大小不一且平均较小的网格蛋白包被小窝以及反式高尔基体网络上的芽。总体而言，我们的观察结果表明，扰乱网格蛋白包被囊泡形成的方法通过不同机制起作用：胞质溶胶酸化通过影响网格蛋白包被的膜结构域，以干扰网格蛋白包被囊泡从质膜以及从反式高尔基体网络出芽；低钾以及在高渗培养基中孵育则是通过阻止网格蛋白和衔接蛋白相互作用。此外，我们的观察结果表明，衔接蛋白聚集体可以独立于网格蛋白晶格存在于质膜上，并增加了衔接蛋白聚集体可以形成网格蛋白晶格成核位点的可能性。
