Simultaneous mapping of filamentous actin flow and turnover in migrating cells by quantitative fluorescent speckle microscopy
Simultaneous mapping of filamentous actin flow and turnover in migrating cells by quantitative fluorescent speckle microscopy
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DOI：
10.1073/pnas.0300552101
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发表时间：
2004-06-29
影响因子：
11.1
通讯作者：
Danuser, G
中科院分区：
文献类型：
--
作者：
Vallotton, P;Gupton, SL;Danuser, G
我们报告的进展，在定量荧光斑点显微镜生成的细胞骨架流动和净组装和拆卸率在活细胞的同步地图。我们应用这个工具来分析丝状肌动蛋白（F-actin）的动态迁移细胞的前面。肌动蛋白的更新和流动都是细胞运动的因素。然而，它们是如何被编排以产生定向细胞运动的却知之甚少。我们的数据分析方法使我们能够研究它们的相互依赖性。我们的地图证实了先前描述的组织流到一个lamellipodium和一个lamellum，都表现出逆行流;和一个收敛区，在那里lamellum逆行流满足缓慢顺行流皮质F-肌动蛋白在腹侧的细胞体。周转图显示了在前缘的肌动蛋白聚合，但也表明大约90%的聚合物在板层-板层连接处分解。在辐合区也发现了强烈的解聚，在那里网络收缩是突出的。为了确定收缩和解聚是否是偶联的事件，我们用已知促进肌球蛋白活性的calyculin A处理细胞。刺激收缩伴随着加速逆行流动和增加解聚在整个板层，而在lamellipodium板层交界处的解体保持不受影响。似乎有两种不同的解聚机制，其中之一直接依赖于网络收缩。
We report advances in quantitative fluorescent speckle microscopy to generate simultaneous maps of cytoskeleton flow and rates of net assembly and disassembly in living cells. We apply this tool to analyze the filamentous actin (F-actin) dynamics at the front of migrating cells. F-actin turnover and flow are both known to be factors of cell locomotion. However, how they are orchestrated to produce directed cell movements is poorly understood. Our approach to data analysis allows us to examine their interdependence. Our maps confirm the previously described organization of flow into a lamellipodium and a lamellum, both exhibiting retrograde flow; and a convergence zone, where lamellum retrograde flow meets with slow anterograde flow of cortical F-actin at the ventral side of the cell body. The turnover maps show the well known actin polymerization at the leading edge, but also indicate that approximate to90% of the polymer disassembles at the lamellipodium-lamellum junction. Strong depolymerization is also found in the convergence zone, where meshwork contraction is prominent. To determine whether contraction and depolymerization are coupled events, we have treated cells with calyculin A, which is known to promote myosin activity. Stimulated contraction was accompanied by accelerated retrograde flow and increased depolymerization throughout the lamellum, whereas disassembly at the lamellipodium-lamellum junction remained unaffected. There appear to be two distinct depolymerization mechanisms, of which one depends directly on meshwork contraction.