Adams J C
MRC Laboratory for Molecular Cell Biology, Department of Biology, University College London, UK.
J Cell Sci. 1995 May;108 ( Pt 5):1977-90. doi: 10.1242/jcs.108.5.1977.
The organisation of the actin cytoskeleton was examined in H9c2 and human intestinal smooth muscle cells adherent on fibronectin or thrombospondin-1. Whereas cells adherent on fibronectin adopted a polygonal shape and rapidly assembled prominent stress fibres and focal contacts, cells adherent on thrombospondin-1 assumed a more irregular morphology with large lamellae containing radial actin microspikes. Focal contacts were not detected in cells adherent on thrombospondin-1, as determined by indirect immunofluorescence staining for vinculin and other focal contact components. Instead, the radial microspikes stained positively for the actin-bundling protein, 55 kDa/fascin, and myosins. In cells adherent on fibronectin, 55 kDa/fascin immunoreactivity was diffuse and tended to be concentrated in the perinuclear region. In long-term adherent cells cultured in serum-containing medium, 55 kDa/fascin was detected in membrane ruffles, in stress fibres and in the perinuclear region. The microspikes formed within 40 minutes of plating cells on thrombospondin-1 and remained present when cells were treated with sodium orthovandate and hydrogen peroxide to increase intracellular phosphotyrosine levels. Indeed, although vanadate-treated cells tended to retract, the microspikes became more prominent and showed an increased intensity of staining for fascin. Under these conditions, a proportion of the microspikes did not appear to be in contact with the substratum: these spikes stained weakly for focal adhesion kinase, talin and vinculin. Cells treated with genistein also spread and formed fascin-containing microspikes which tended to be more slender than those of control cells. In contrast, cells adherent on fibronectin displayed a complex rearrangement of the actin cytoskeleton and a transient enrichment of 55 kDa/fascin-containing structures at the cell surface when treated with sodium orthovanadate and hydrogen peroxide. These observations indicate that cell interactions with fibronectin or thrombospondin-1 send distinct organisational signals to the actin cytoskeleton and may offer a mechanistic framework for further investigations of the anti-adhesive properties of thrombospondin-1.
在纤连蛋白或血小板反应蛋白-1上贴壁的H9c2细胞和人肠道平滑肌细胞中，对肌动蛋白细胞骨架的组织情况进行了检测。贴壁于纤连蛋白的细胞呈多边形，能迅速组装出明显的应力纤维和黏着斑，而贴壁于血小板反应蛋白-1的细胞形态更不规则，有大的片状伪足，其中含有放射状肌动蛋白微刺。通过对纽蛋白和其他黏着斑成分进行间接免疫荧光染色确定，在贴壁于血小板反应蛋白-1的细胞中未检测到黏着斑。相反，放射状微刺对肌动蛋白成束蛋白55 kDa/成束蛋白和肌球蛋白呈阳性染色。在贴壁于纤连蛋白的细胞中，55 kDa/成束蛋白的免疫反应性呈弥漫性，且倾向于集中在核周区域。在含血清培养基中培养的长期贴壁细胞中，55 kDa/成束蛋白在膜皱褶、应力纤维和核周区域中被检测到。将细胞接种于血小板反应蛋白-1上40分钟内即可形成微刺，当用原钒酸钠和过氧化氢处理细胞以提高细胞内磷酸酪氨酸水平时，微刺仍然存在。实际上，尽管经钒酸盐处理的细胞倾向于回缩，但微刺变得更加突出，且成束蛋白染色强度增加。在这些条件下，一部分微刺似乎未与基质接触：这些微刺对黏着斑激酶、踝蛋白和纽蛋白的染色较弱。用染料木黄酮处理的细胞也会铺展并形成含成束蛋白的微刺，这些微刺往往比对照细胞的微刺更细长。相比之下，当用原钒酸钠和过氧化氢处理时，贴壁于纤连蛋白的细胞会表现出肌动蛋白细胞骨架的复杂重排以及细胞表面含55 kDa/成束蛋白结构的短暂富集。这些观察结果表明，细胞与纤连蛋白或血小板反应蛋白-1的相互作用会向肌动蛋白细胞骨架发送不同的组织信号，并且可能为进一步研究血小板反应蛋白-1的抗黏附特性提供一个机制框架。
