Actin-based centripetal flow: Phosphatase inhibition by Calyculin-A alters flow pattern, actin organization, and actomyosin distribution
Actin-based centripetal flow: Phosphatase inhibition by Calyculin-A alters flow pattern, actin organization, and actomyosin distribution
复制标题
DOI：
10.1002/cm.10149
复制
发表时间：
2003-12-01
影响因子：
--
通讯作者：
Trabosh, VA
中科院分区：
文献类型：
--
作者：
Henson, JH;Kolnik, SE;Trabosh, VA
先前的研究表明，海胆体腔细胞中基于肌动蛋白的向心流动过程是两部分机制的结果，细胞边缘的肌动蛋白聚合与细胞中心的肌动球蛋白收缩相结合。在本研究中，我们尝试通过用磷酸酶抑制剂 Calyculin A (CalyA) 处理体腔细胞来刺激肌动球蛋白收缩，从而扩展了该两部分模型的测试。使用活细胞数字增强视频显微镜结合免疫荧光定位和扫描电子显微镜研究了该药物的作用。在 CalyA 的影响下，体腔细胞肌动蛋白细胞骨架经历了彻底的重组，从致密网络转变为呈现一系列切向弧和径向小溪的网络，肌动蛋白和 Arp2/3 复合物集中在其中。此外，由于肌动蛋白和膜在该区域的积累以及中央肌动球蛋白环的收缩，细胞中心的结构和动力学发生了改变。实验证明了 CalyA 治疗后基于肌动球蛋白的收缩力增加的生理学证据，其中细胞响应药物而在其细胞中心产生撕裂。使用针对肌球蛋白调节轻链 (MRLC) 磷酸化形式的抗体进行的蛋白质印迹和免疫荧光定位表明，肌球蛋白分布的收缩是 CalyA 诱导的 MRLC 磷酸化的结果。总体而言，结果表明肌动蛋白聚合和肌动球蛋白收缩的两种潜在机制之间存在显着的串扰，并表明肌动球蛋白张力的变化可能转化为肌动蛋白细胞骨架结构组织的改变。
Previous studies have suggested that the actin-based centripetal flow process in sea urchin coelomocytes is the result of a two-part mechanism, actin polymerization at the cell edge coupled with actomyosin contraction at the cell center. In the present study, we have extended the testing of this two-part model by attempting to stimulate actomyosin contraction via treatment of coelomocytes with the phosphatase inhibitor Calyculin A (CalyA). The effects of this drug were studied using digitally-enhanced video microscopy of living cells combined with immunofluorescent localization and scanning electron microscopy. Under the influence of CalyA, the coelomocyte actin cytoskeleton undergoes a radical reorganization from a dense network to one displaying an array of tangential arcs and radial rivulets in which actin and the Arp2/3 complex concentrate. In addition, the structure and dynamics of the cell center are transformed due to the accumulation of actin and membrane in this region and the constriction of the central actomyosin ring. Physiological evidence of an increase in actomyosin-based contractility following CalyA treatment was demonstrated in experiments in which cells generated tears in their cell centers in response to the drug. Western blotting and immunofluorescent localization with antibodies against the phosphorylated form of the myosin regulatory light chain (MRLC) suggested that the demonstrated constriction of actomyosin distribution was the result of CalyA-induced phosphorylation of MRLC. Overall, the results suggest that there is significant cross talk between the two underlying mechanisms of actin polymerization and actomyosin contraction, and indicate that changes in actomyosin tension may be translated into alterations in the structural organization of the actin cytoskeleton.