Low concentrations of nocodazole interfere with fibroblast locomotion without significantly affecting microtubule level: implications for the role of dynamic microtubules in cell locomotion.
Low concentrations of nocodazole interfere with fibroblast locomotion without significantly affecting microtubule level: implications for the role of dynamic microtubules in cell locomotion.
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发表时间：
1995-11
影响因子：
4
通讯作者：
G. Liao;Takayuki Nagasaki;G. Gundersen
中科院分区：
文献类型：
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作者：
G. Liao;Takayuki Nagasaki;G. Gundersen
微管（MT）在细胞运动中的作用是不确定的：虽然MT不是某些细胞的运动所必需的，但MT是大细胞如成纤维细胞、内皮细胞和神经生长锥定向移位所必需的。基于先前的研究，我们假设细胞运动可能以两种可能的方式涉及MT：（1）细胞运动的速率与MT水平成正比;或（2）细胞运动与MT水平不成正比，但需要临界水平的MT才能进行。为了检验这些假设，我们测量了迁移到体外伤口的NRK成纤维细胞的运动速率，在用不同浓度的诺考达唑处理以产生具有不同MT水平的细胞之前和之后。使用时间推移记录直接监测细胞的运动，并用Image-1图像分析程序进行分析。加入诺考达唑（>或= 50 nM）导致运动迅速降低至新的速率，并维持> 2小时。我们发现，添加低至100 nM的诺考达唑使运动速率降低60%以上;并且300 nM的诺考达唑完全停止细胞运动。虽然100 nM诺考达唑减少运动超过60%，我们检测到MT分布免疫荧光没有质的变化。免疫荧光染色制剂中MT荧光的定量分析表明，100 nM诺考达唑对MT水平没有可检测的影响，300 nM诺考达唑仅将MT水平降低至对照组的约40%。通过细胞提取和蛋白质印迹法对微管蛋白聚合物水平进行定量分析，得到的结果与通过MT荧光定量获得的结果相似。运动速率测量值与MT水平测量值的比较表明，超过一半的细胞运动速率可以被诺考达唑阻断，而不会显著影响细胞中的MT水平;剩余的运动速率与MT水平成比例降低。这些结果不支持细胞运动需要临界水平的MT的观点，并且表明细胞的速度的仅一部分（< 50%）与MT水平成比例。相反，通过与MT拮抗剂对有丝分裂纺锤体的研究类比，他们提出了第三种可能性：低浓度的诺考达唑干扰MT动力学，因此，MT动力学对细胞运动的最大速度至关重要。紫杉醇和长春碱对细胞运动的类似作用进一步支持了这一观点：在据报道引起MT水平变化不大的浓度下，紫杉醇和长春碱也显著降低了NRK细胞的运动速率。总之，我们的研究结果建立了微管水平和运动速率之间的关系，并表明动态MT是成纤维细胞运动的限速。
The role of microtubules (MTs) in cell locomotion is uncertain: while MTs are not essential for motility of certain cells, MTs are necessary for the directed translocation of large cells such as fibroblasts, endothelial cells and neuronal growth cones. Based on previous studies, we hypothesize that cell locomotion may involve MTs in two possible ways: (1) the rate of cell locomotion is proportional to MT level; or (2) cell locomotion is not proportional to MT level but requires a critical level of MTs to proceed. To test these hypotheses, we measured the rate of locomotion of NRK fibroblasts migrating into an in vitro wound, before and after treatment with different concentrations of nocodazole to generate cells with different levels of MTs. Locomotion of cells was monitored directly using timelapse recording and analyzed with an Image-1 image analysis program. Addition of nocodazole (> or = 50 nM) resulted in a rapid reduction in locomotion to a new rate that was maintained for > 2 hours. We found that addition of as little as 100 nM nocodazole decreased the rate of locomotion by more than 60%; and that 300 nM nocodazole completely stopped cell locomotion. Although 100 nM nocodazole decreased locomotion over 60%, we detected no qualitative change in MT distribution by immunofluorescence. Quantitative analysis of MT fluorescence in immunofluorescently stained preparations showed that 100 nM nocodazole had no detectable effect on MT levels and that 300 nM nocodazole only decreased MT levels to approximately 40% of controls. Quantitative analysis of tubulin polymer levels by cell extraction and western blotting yielded results similar to those obtained by quantification of MT fluorescence. A comparison of the locomotion rate measurements with the MT level measurements indicated that over half of the cell locomotion rate could be blocked by nocodazole without significantly affecting MT levels in the cell; the remaining locomotion rate was reduced proportionally to MT levels. These results do not support the notion that a critical level of MTs is required for cell locomotion and suggest that only a portion (< 50%) of the speed of the cells is proportional to MT levels. Rather, by analogy with studies of MT antagonists on the mitotic spindle, they suggest a third possibility: that low concentrations of nocodazole interfere with MT dynamics and thus, MT dynamics are critical for the maximal speed of cell locomotion. This notion was further supported by analogous effects of taxol and vinblastine on cell locomotion: at concentrations that reportedly cause little change in the level of MTs, taxol and vinblastine also dramatically decreased the rate of locomotion of NRK cells. In summary, our results establish the relationship between microtubule levels and locomotion rate and suggest that dynamic MTs are rate-limiting for fibroblast locomotion.