Department of Cell and Developmental Biology, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.
Biophys J. 2010 Aug 9;99(4):1053-63. doi: 10.1016/j.bpj.2010.06.010.
When microtubules are depolymerized in spreading cells, they experience morphological oscillations characterized by a period of about a minute, indicating that normal interactions between the microfilament and microtubule systems have been significantly altered. This experimental system provides a test bed for the development of both fine- and coarse-grained models of complex motile processes, but such models need to be adequately informed by experiment. Using criteria based on Fourier transform analysis, we detect spontaneous oscillations in spreading cells. However, their amplitude and tendency to operate at a single frequency are greatly enhanced by microtubule depolymerization. Knockdown of RhoA and addition of various inhibitors of the downstream effector of RhoA, Rho kinase, block oscillatory behavior. Inhibiting calcium fluxes from endoplasmic reticulum stores and from the extracellular medium does not significantly affect the ability of cells to oscillate, indicating that calcium plays a subordinate regulatory role compared to Rho. We characterized the dynamic structure of the oscillating cell by light, fluorescence, and electron microscopy, showing how oscillating cells are dynamically polarized in terms of their overall morphology, f-actin and phosphorylated myosin light chain distribution, and nuclear position and shape. Not only will these studies guide future experiments, they will also provide a framework for the development of refined mathematical models of the oscillatory process.
当微管在扩展细胞中解聚时，它们会经历形态学振荡，其特征是周期约为一分钟，表明微丝和微管系统之间的正常相互作用已经发生了重大改变。该实验系统为精细和粗粒度的复杂运动过程模型的发展提供了一个测试平台，但此类模型需要通过实验充分提供信息。我们使用基于傅里叶变换分析的标准，检测到扩展细胞中的自发振荡。然而，通过微管解聚，其振幅和在单一频率下运作的趋势大大增强。RhoA 的敲低和 RhoA 的下游效应物 Rho 激酶的各种抑制剂的添加会阻断振荡行为。抑制内质网库和细胞外介质中钙流的作用不会显著影响细胞振荡的能力，表明与 Rho 相比，钙起次要的调节作用。我们通过光、荧光和电子显微镜来描述振荡细胞的动态结构，展示了振荡细胞在整体形态、肌动蛋白和磷酸化肌球蛋白轻链分布以及核位置和形状方面如何动态极化。这些研究不仅将指导未来的实验，还将为振荡过程的精细数学模型的发展提供框架。
