Peckham M, Wells C, Taylor-Harris P, Coles D, Zicha D, Dunn G A
Muscle and Cell Motility Research Centre, Randall Institute, King's College London, U.K.
Biochem Soc Symp. 1999;65:281-99.
We have used digitally recorded interference microscopy with automatic phase shifting (DRIMAPS) to investigate the crawling locomotion of normal and mutant mouse myoblasts. Contraction forces that give rise to cell body movement, tail retraction and cell adhesion to the substrate in myoblasts and other locomoting tissue cells arise from the interactions of actin and non-muscle myosin II. The activity of non-muscle myosin II is regulated differently from that of skeletal myosin. Using DRIMAPS, we found that crawling locomotion was altered in myoblasts that heterologously expressed human beta-cardiac myosin heavy chain (MHC); the cells moved more slowly and had reduced rates of protrusion and retraction. Immunolocalization demonstrated that MHC and non-muscle myosin II were not co-localized, suggesting that MHC does not compete directly with myosin II, but interferes with cell locomotion by binding inappropriately to actin filaments and possibly cross-linking them. Myosin I may be involved in protrusion of the lamellipodia. However, using DRIMAPS, we found that crawling locomotion was unaltered in myoblasts that heterologously expressed a truncated myosin I which lacked the membrane-binding tail domain. This suggests that, if endogenous myosin I is important for cell locomotion, this mutant was unable to interfere with its action. We conclude that the effects on locomotion of expressing foreign or mutant proteins of the cytoskeleton in vertebrate cells can be subtle and can be swamped by the intrinsic variability of the cells. Their characterization requires automated methods of acquiring data, such as DRIMAPS, and careful statistical analysis in order to take account of other sources of variation.
我们使用具有自动相移功能的数字记录干涉显微镜（DRIMAPS）来研究正常和突变小鼠成肌细胞的爬行运动。在成肌细胞和其他运动组织细胞中，导致细胞体移动、尾部收缩以及细胞与底物粘附的收缩力源于肌动蛋白和非肌肉肌球蛋白II的相互作用。非肌肉肌球蛋白II的活性调节方式与骨骼肌肌球蛋白不同。使用DRIMAPS，我们发现，在异源表达人β - 心脏肌球蛋白重链（MHC）的成肌细胞中，爬行运动发生了改变；细胞移动得更慢，突出和回缩速率降低。免疫定位表明，MHC和非肌肉肌球蛋白II没有共定位，这表明MHC不会直接与肌球蛋白II竞争，而是通过不恰当地结合肌动蛋白丝并可能使其交联来干扰细胞运动。肌球蛋白I可能参与片状伪足的突出。然而，使用DRIMAPS，我们发现，在异源表达缺少膜结合尾部结构域的截短型肌球蛋白I的成肌细胞中，爬行运动没有改变。这表明，如果内源性肌球蛋白I对细胞运动很重要，那么这种突变体无法干扰其作用。我们得出结论，在脊椎动物细胞中表达细胞骨架的外源或突变蛋白对运动的影响可能很微妙，并且可能被细胞的内在变异性所掩盖。对它们的表征需要自动获取数据的方法，如DRIMAPS，并进行仔细的统计分析，以便考虑其他变异来源。
