Department of Biochemistry and Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 94158, USA.
Mol Biol Cell. 2011 Nov;22(21):3986-94. doi: 10.1091/mbc.E11-04-0379. Epub 2011 Aug 31.
The actin cytoskeleton is regulated by factors that influence polymer assembly, disassembly, and network rearrangement. Drugs that inhibit these events have been used to test the role of actin dynamics in a wide range of cellular processes. Previous methods of arresting actin rearrangements take minutes to act and work well in some contexts, but can lead to significant actin reorganization in cells with rapid actin dynamics, such as neutrophils. In this paper, we report a pharmacological cocktail that not only arrests actin dynamics but also preserves the structure of the existing actin network in neutrophil-like HL-60 cells, human fibrosarcoma HT1080 cells, and mouse NIH 3T3 fibroblast cells. Our cocktail induces an arrest of actin dynamics that initiates within seconds and persists for longer than 10 min, during which time cells maintain their responsivity to external stimuli. With this cocktail, we demonstrate that actin dynamics, and not simply morphological polarity or actin accumulation at the leading edge, are required for the spatial persistence of Rac activation in HL-60 cells. Our drug combination preserves the structure of the existing cytoskeleton while blocking actin assembly, disassembly, and rearrangement, and should prove useful for investigating the role of actin dynamics in a wide range of cellular signaling contexts.
肌动蛋白细胞骨架受影响聚合组装、解聚和网络重排的因素调节。抑制这些事件的药物已被用于测试肌动蛋白动力学在广泛的细胞过程中的作用。以前的肌动蛋白重排的阻断方法需要几分钟的时间才能起作用，并且在某些情况下效果很好，但在肌动蛋白动力学较快的细胞中，如中性粒细胞，可能会导致显著的肌动蛋白重组。在本文中，我们报告了一种药理学鸡尾酒，它不仅可以阻断肌动蛋白动力学，还可以在类似于中性粒细胞的 HL-60 细胞、人纤维肉瘤 HT1080 细胞和小鼠 NIH 3T3 成纤维细胞中保存现有的肌动蛋白网络结构。我们的鸡尾酒诱导的肌动蛋白动力学阻断在数秒内开始，并持续超过 10 分钟，在此期间细胞保持对外界刺激的反应性。通过这种鸡尾酒，我们证明了肌动蛋白动力学，而不仅仅是形态极性或在前沿处的肌动蛋白积累，对于 HL-60 细胞中 Rac 激活的空间持久性是必需的。我们的药物组合在阻止肌动蛋白组装、解聚和重排的同时，保留了现有细胞骨架的结构，应该有助于研究肌动蛋白动力学在广泛的细胞信号转导背景下的作用。
