Oakley C, Jaeger N A, Brunette D M
Department of Oral Biology, University of British Columbia, Vancouver, Canada.
Exp Cell Res. 1997 Aug 1;234(2):413-24. doi: 10.1006/excr.1997.3625.
Fibroblasts alter their shape, orientation, and direction of movement to align with the direction of micromachined grooves, exhibiting a phenomenon termed topographic guidance. In this study we examined the ability of the microtubule and actin microfilament bundle systems, either in combination with or independently from each other, to affect alignment of human gingival fibroblasts on sets of micromachined grooves of different dimensions. To assess specifically the role of microtubules and actin microfilament bundles, we examined cell alignment, over time, in the presence or absence of specific inhibitors of microtubules (colcemid) and actin microfilament bundles (cytochalasin B). Using time-lapse videomicroscopy, computer-assisted morphometry and confocal microscopy of the cytoskeleton we found that the dimensions of the grooves influenced the kinetics of cell alignment irrespective of whether cytoskeletons were intact or disturbed. Either an intact microtubule or an intact actin microfilament-bundle system could produce cell alignment with an appropriate substratum. Cells with intact microtubules aligned to smaller topographic features than cells deficient in microtubules. Moreover, cells deficient in microtubules required significantly more time to become aligned. An unexpected finding was that very narrow 0.5-microm-wide and 0.5-microm-deep grooves aligned cells deficient in actin microfilament bundles (cytochalasin B-treated) better than untreated control cells but failed to align cells deficient in microtubules yet containing microfilament bundles (colcemid treated). Thus, the microtubule system appeared to be the principal but not sole cytoskeletal substratum-response mechanism affecting topographic guidance of human gingival fibroblasts. This study also demonstrated that micromachined substrata can be useful in dissecting the role of microtubules and actin microfilament bundles in cell behaviors such as contact guidance and cell migration without the use of drugs such as cytochalasin and colcemid.
成纤维细胞会改变其形状、方向和移动方向，以与微加工凹槽的方向对齐，表现出一种称为地形引导的现象。在本研究中，我们研究了微管和肌动蛋白微丝束系统相互结合或独立作用时，对人牙龈成纤维细胞在不同尺寸微加工凹槽上排列的影响。为了具体评估微管和肌动蛋白微丝束的作用，我们在存在或不存在微管特异性抑制剂（秋水仙酰胺）和肌动蛋白微丝束特异性抑制剂（细胞松弛素B）的情况下，随时间观察细胞排列情况。通过延时视频显微镜、计算机辅助形态测量和细胞骨架共聚焦显微镜，我们发现凹槽的尺寸影响细胞排列的动力学，无论细胞骨架是完整还是受到干扰。完整的微管或完整的肌动蛋白微丝束系统都能使细胞与合适的基质对齐。与微管缺陷的细胞相比，微管完整的细胞能与更小的地形特征对齐。此外，微管缺陷的细胞需要显著更多的时间才能对齐。一个意外的发现是，非常窄的0.5微米宽、0.5微米深的凹槽能使肌动蛋白微丝束缺陷（经细胞松弛素B处理）的细胞比未处理的对照细胞排列得更好，但不能使微管缺陷但含有微丝束（经秋水仙酰胺处理）的细胞对齐。因此，微管系统似乎是影响人牙龈成纤维细胞地形引导的主要但不是唯一的细胞骨架与基质反应机制。本研究还表明，微加工基质可用于剖析微管和肌动蛋白微丝束在接触引导和细胞迁移等细胞行为中的作用，而无需使用细胞松弛素和秋水仙酰胺等药物。
