ROLE OF THE CYTOSKELETON IN THE REACTION OF FIBROBLASTS TO MULTIPLE GROOVED SUBSTRATA
ROLE OF THE CYTOSKELETON IN THE REACTION OF FIBROBLASTS TO MULTIPLE GROOVED SUBSTRATA
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DOI：
10.1002/cm.970310207
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发表时间：
1995-01-01
影响因子：
--
通讯作者：
WILKINSON, CDW
中科院分区：
文献类型：
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作者：
WOJCIAKSTOTHARD, B;CURTIS, ASG;WILKINSON, CDW
使用共聚焦扫描显微镜研究了细胞骨架和细胞附着在幼仓鼠肾成纤维细胞与脊和沟基质地形排列中的作用。这是用正常细胞和用细胞骨架修饰剂细胞松弛素 D、秋水酰胺和紫杉醇处理的细胞进行的。肌动蛋白定位于荧光鬼笔环肽。通过间接免疫荧光观察微管蛋白、纽蛋白和细胞内粘附分子-1。在这些条件下培养24小时后，在宽度为5、10和25μm、深度为0.5、1、2和5μm的凹槽上测量细胞的铺展、伸长和方向。我们还观察了细胞附着前 30 分钟的事件。细胞附着五分钟后，在靠近凹槽壁和脊顶部的相交处（即在地形不连续处）观察到F-肌动蛋白凝结。凝结通常与凹槽边缘成直角，并显示出0.6μm的周期性。细胞扩散早期阶段的纽蛋白排列与肌动蛋白的排列相似。微管系统的组织随后出现，在细胞铺板后约 30 分钟变得明显。这些结果支持了柯蒂斯和克拉克理论（细胞主要通过肌动蛋白成核作用在基质的不连续线上对地形做出反应）。细胞骨架毒物的使用并没有完全消除细胞对凹槽的反应。秋水仙胺增加细胞铺展并减少细胞定向和伸长。细胞松弛素 D 减少细胞扩散、定向和伸长。紫杉醇减少细胞伸长，但不影响细胞铺展和方向。我们得出的结论是，肌动蛋白沿着凹槽/脊边界的聚集是确定微凹槽基质上成纤维细胞方向的主要驱动事件。 (C) 1995 Wiley-Liss, Inc.
The role of the cytoskeleton and cell attachments in the alignment of baby hamster kidney fibroblasts to ridge and groove substratum topography was investigated using confocal scanning microscopy. This was carried out with normal cells and cells treated with the cytoskeleton modifiers cytochalasin D, colcemid, and taxol. Actin was localised with fluorescent phalloidin. Tubulin, vinculin, and intracellular adhesion molecule-1 were visualised by indirect immunofluorescence. The spreading, elongation, and orientation of the cells after 24 h of culture in these conditions were measured on grooves of 5, 10, and 25 mu m width and 0.5, 1, 2, and 5 mu m depth. We have also observed events over the first 30 min of cell attachment. Five minutes after cell attachment, F-actin condensations were seen close to the intesection of groove wall and ridge top, that is, at a topographic discontinuity. The condensations were often at right angles to the groove edge and showed a periodicity of 0.6 mu m. Vinculin arrangement at the early stages of cell spreading was similar to that of actin. Organisation of the microtubule system followed later, becoming obvious at about 30 min after cell plating. The Curtis and Clark theory (that cells react to topography primarily at lines of discontinuity in the substratum by actin nucleation) is supported by these results. The use of cytoskeletal poisons did not entirely abolish cell reaction to grooves. Colcemid increased cell spreading and reduced cell orientation and elongation. Cytochalasin D reduced cell spreading, orientation, and elongation. Taxol reduced cell elongation but did not affect cell spreading and orientation. We conclude that the aggregation of actin along groove/ridge boundaries is a primary driving event in determining fibroblast orientation on microgrooved substrata. (C) 1995 Wiley-Liss, Inc.