Wójciak-Stothard B, Curtis A S, Monaghan W, McGrath M, Sommer I, Wilkinson C D
Department of Cell Biology, University of Glasgow, Scotland, United Kingdom.
Cell Motil Cytoskeleton. 1995;31(2):147-58. doi: 10.1002/cm.970310207.
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 microns width and 0.5, 1, 2, and 5 microns 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 intersection 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 microns. 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.
利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-槽基底形貌对齐过程中的作用。对正常细胞以及用细胞骨架修饰剂细胞松弛素D、秋水仙酰胺和紫杉醇处理的细胞进行了此项研究。用荧光鬼笔环肽定位肌动蛋白。通过间接免疫荧光观察微管蛋白、纽蛋白和细胞间黏附分子-1。在宽度为5、10和25微米以及深度为0.5、1、2和5微米的凹槽上测量了在这些条件下培养24小时后细胞的铺展、伸长和取向。我们还观察了细胞附着最初30分钟内的情况。细胞附着5分钟后，在靠近槽壁和脊顶的交点处，即地形不连续处，可见F-肌动蛋白凝聚物。这些凝聚物通常与槽边缘成直角，且显示出0.6微米的周期性。细胞铺展早期纽蛋白的排列与肌动蛋白相似。微管系统的组织随后出现，在细胞接种后约30分钟变得明显。这些结果支持了柯蒂斯和克拉克的理论（即细胞主要通过肌动蛋白成核在基底的不连续线处对形貌作出反应）。使用细胞骨架毒素并没有完全消除细胞对凹槽的反应。秋水仙酰胺增加了细胞铺展，降低了细胞取向和伸长。细胞松弛素D降低了细胞铺展、取向和伸长。紫杉醇降低了细胞伸长，但不影响细胞铺展和取向。我们得出结论，肌动蛋白沿槽/脊边界的聚集是决定成纤维细胞在微槽基底上取向的主要驱动事件。
