Over recent decades, considerable effort has been made to understand how mechanical stress applied to the actin network alters actin assembly and disassembly dynamics. However, there are conflicting reports concerning the issue both in vitro and in cells. In this review, we discuss concerns regarding previous quantitative live-cell experiments that have attempted to evaluate myosin regulation of filamentous actin (F-actin) turnover. In particular, we highlight an error-generating mechanism in quantitative live-cell imaging, namely convection-induced misdistribution of actin-binding probes. Direct observation of actin turnover at the single-molecule level using our improved electroporation-based Single-Molecule Speckle (eSiMS) microscopy technique overcomes these concerns. We introduce our recent single-molecule analysis that unambiguously demonstrates myosin-dependent regulation of F-actin stability in live cells. We also discuss the possible application of eSiMS microscopy in the analysis of actin remodeling in striated muscle cells.

中文翻译：

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肌球蛋白依赖性丝状肌动蛋白动力学的定量高精度成像。

在最近的几十年中，人们做出了巨大的努力来了解施加于肌动蛋白网络的机械应力如何改变肌动蛋白的组装和拆卸动力学。但是，关于该问题在体外和在细胞中都有相互矛盾的报道。在这篇综述中，我们讨论了有关以前的定量活细胞实验的担忧，这些实验已经尝试评估肌球蛋白对丝状肌动蛋白（F-肌动蛋白）更新的调节。特别是，我们重点介绍了定量活细胞成像中的错误产生机制，即对流诱导的肌动蛋白结合探针分布不均。使用我们改进的基于电穿孔的单分子散斑（eSiMS）显微镜技术直接观察单分子水平的肌动蛋白周转率可以解决这些问题。我们介绍我们最近的单分子分析，明确地证明了活细胞中肌球蛋白对F-肌动蛋白稳定性的依赖性调节。我们还讨论了eSiMS显微镜在横纹肌细胞肌动蛋白重塑分析中的可能应用。