Mikhailov A, Gundersen G G
Department of Pathology, Columbia University, New York, New York 10032, USA.
Cell Motil Cytoskeleton. 1998;41(4):325-40. doi: 10.1002/(SICI)1097-0169(1998)41:4<325::AID-CM5>3.0.CO;2-D.
Microtubules (MTs) contribute to the directional locomotion of many cell types through an unknown mechanism. Previously, we showed that low concentrations (<200 nM) of nocodazole or taxol reduced the rate of locomotion of NRK fibroblasts over 60% without altering MT polymer level [Liao et al., 1995: J. Cell Sci. 108:3473-3483]. In this paper, we directly measured the dynamics of MTs in migrating NRK cells injected with rhodamine tubulin and treated with low concentrations of nocodazole or taxol. Both drug treatments caused statistically significant reductions (approx. twofold) in growth and shortening rates and less dramatic effects on rescue and catastrophe transition frequencies. The percent time MTs were inactive (i.e., paused) increased greater than twofold in nocodazole- and taxol-treated cells, while the percent time growing was substantially reduced. Three parameters of MT dynamics were linearly related to the rates of locomotion determined previously: rate of shortening, percent time pausing and percent time growing. The number of MTs that came within 1 microm of the leading edge was reduced in drug-treated cells, suggesting that reduced MT dynamics may affect actin arrays necessary for cell locomotion. We examined two such structures, lamellipodium and adhesion plaques, and found that lamellipodia area was coordinately reduced with MT dynamics. No effect was detected on adhesion plaque density or distribution. In time-lapse recordings, MTs did not penetrate into the lamellipodium of untreated cells, suggesting that MTs affect lamellipodia either through their interaction with factors at the base of the lamellipodium or by releasing factors that diffuse into the lamellipodia. In support of the latter hypothesis, when all MTs were rapidly depolymerized by 20 microM nocodazole, we detected the rapid formation of exaggerated protrusions from the leading edge of the cell. Our results show for the first time a linear relationship between MT dynamics and the formation of the lamellipodium and support the idea that MT dynamics may contribute to cell locomotion by regulating the size of the lamellipodium, perhaps through diffusable factors.
微管（MTs）通过一种未知机制参与多种细胞类型的定向运动。此前，我们发现低浓度（<200 nM）的诺考达唑或紫杉醇可使NRK成纤维细胞的运动速率降低60%以上，而不改变MT聚合物水平[廖等人，1995：《细胞科学杂志》108：3473 - 3483]。在本文中，我们直接测量了注射了罗丹明微管蛋白并经低浓度诺考达唑或紫杉醇处理的迁移NRK细胞中MTs的动力学。两种药物处理均导致生长和缩短速率出现统计学上的显著降低（约两倍），对挽救和灾难转换频率的影响较小。在诺考达唑和紫杉醇处理的细胞中，MTs不活跃（即暂停）的时间百分比增加了两倍多，而生长时间百分比大幅降低。MT动力学的三个参数与先前确定的运动速率呈线性相关：缩短速率、暂停时间百分比和生长时间百分比。在药物处理的细胞中，距离前沿1微米内的MT数量减少，这表明MT动力学降低可能会影响细胞运动所需的肌动蛋白阵列。我们检查了两种这样的结构，片状伪足和黏着斑，发现片状伪足面积与MT动力学协同降低。未检测到对黏着斑密度或分布的影响。在延时记录中，MTs未穿透未处理细胞的片状伪足，这表明MTs要么通过与片状伪足基部的因子相互作用，要么通过释放扩散到片状伪足中的因子来影响片状伪足。为支持后一种假设，当所有MTs被20 microM诺考达唑迅速解聚时，我们检测到细胞前沿迅速形成夸张的突起。我们的结果首次表明MT动力学与片状伪足的形成之间存在线性关系，并支持这样一种观点，即MT动力学可能通过调节片状伪足的大小，也许是通过可扩散因子，来促进细胞运动。
