Depletion of intracellular potassium disrupts coated pits and reversibly inhibits cell polarization during fibroblast spreading.
Depletion of intracellular potassium disrupts coated pits and reversibly inhibits cell polarization during fibroblast spreading.
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DOI：
10.1083/jcb.120.6.1449
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发表时间：
1993-03
期刊：
影响因子：
--
通讯作者：
Grinnell F
中科院分区：
文献类型：
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作者：
Altankov G;Grinnell F
为了进一步了解包被窝内吞途径在细胞粘附中的可能作用，我们研究了包被窝因细胞间钾的耗尽而被破坏的成纤维细胞的附着和扩散。在无钾培养基中悬浮培养的成纤维细胞在10分钟内失去了80%的细胞内钾，根据网格蛋白的荧光染色显示出被破坏的包被坑。缺钾细胞在与对照细胞相同的时间过程（15分钟-2小时）附着和扩散在纤维连接蛋白包被的基质上。然而，与对照组不同的是，缺钾成纤维细胞获得了具有周向组织的肌动蛋白细丝束的径向形态，并且无法过渡到具有应力纤维的极化形态。在呈放射状扩散的成纤维细胞中，纤维连接蛋白受体和血管蛋白共定位于局灶黏附位点，似乎是周向排列的肌动蛋白细丝束的膜插入点，但这些位置远小于极化细胞中的局灶黏附斑块。缺钾对细胞粘附的影响是可逆的。在将K（+）缺失的成纤维细胞切换到含有KCl的培养基后1小时内，细胞形成极化形态，肌动蛋白应激纤维插入局灶性粘附斑块。在此期间，细胞表面也重新形成了包覆的凹坑。由于局灶性粘附斑块的形成先于细胞边缘网格蛋白包被的凹坑的重新出现，因此包被凹坑似乎不太可能在粘附斑块的组装中起直接作用。加入KCl后，成纤维细胞的极化被瓦巴因抑制，这表明细胞内钾是活性所必需的。当贫钾细胞在高渗或酸化条件下切换到含钾介质时，极化也被抑制，这两种情况都被证明可以抑制受体介导的内吞作用。我们的研究结果表明，包被凹坑内吞途径不是成纤维细胞初始附着、扩散和局灶粘附形成所必需的，但可能在细胞极化中发挥作用。
To learn more about the possible role of the coated pits endocytic pathway in cell adhesion, we studied attachment and spreading of fibroblasts whose coated pits were disrupted by depletion of intercellular potassium. Fibroblasts incubated in suspension in potassium-free medium lost 80% of their intracellular potassium within 10 min and showed disrupted coated pits based on fluorescence staining of clathrin. Potassium-depleted cells attached and spread on fibronectin-coated substrata over the same time course (15 min-2 h) as control cells. Unlike controls, however, potassium-depleted fibroblasts attained a radial morphology with circumferentially organized actin filament bundles and were unable to make the transition to a polarized morphology with stress fibers. In the radially spread fibroblasts, fibronectin receptors and vinculin colocalized in focal adhesion sites and appeared to be membrane insertion points for circumferentially arranged actin filament bundles, but these sites were much smaller than the focal adhesion plaques in polarized cells. The effects of potassium depletion on cell adhesion were reversible. Within 1 h after switching K(+)-depleted fibroblasts to medium containing KCl, cells developed a polarized morphology with actin stress fibers inserting into focal adhesion plaques. Coated pits also reformed on the cell surface during this time. Because formation of focal adhesion plaques preceded reappearance of clathrin-coated pits at the cell margins, it seems unlikely that coated pits play a direct role in adhesion plaque assembly. Polarization of fibroblasts upon addition of KCl was inhibited by ouabain showing that intracellular potassium was required for activity. Polarization also was inhibited when potassium-depleted cells were switched to potassium-containing medium under hypertonic or acidified conditions, both of which have been shown to inhibit receptor- mediated endocytosis. Our results suggest that the coated pit endocytic pathway is not required for initial attachment, spreading, and formation of focal adhesions by fibroblasts, but may play a role in cell polarization.