Vallotton Pascal, Gupton Stephanie L, Waterman-Storer Clare M, Danuser Gaudenz
BioMicroMetrics Group, Laboratory for Biomechanics, Swiss Federal Institute of Technology, 8952 Schlieren, Switzerland.
Proc Natl Acad Sci U S A. 2004 Jun 29;101(26):9660-5. doi: 10.1073/pnas.0300552101. Epub 2004 Jun 21.
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 approximately 90% 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.
我们报告了定量荧光斑点显微镜技术的进展，该技术可生成活细胞中细胞骨架流动以及净组装和拆卸速率的同步图谱。我们应用此工具分析迁移细胞前端的丝状肌动蛋白（F-肌动蛋白）动力学。F-肌动蛋白的周转和流动均已知是细胞运动的因素。然而，它们如何协同作用以产生定向细胞运动却知之甚少。我们的数据分析方法使我们能够研究它们的相互依赖性。我们的图谱证实了先前描述的流动组织，即分为片状伪足和片层，两者均呈现逆行流动；以及一个汇聚区，在细胞体腹侧，片层逆行流动与皮质F-肌动蛋白的缓慢顺行流动相遇。周转图谱显示了前缘众所周知的肌动蛋白聚合，但也表明约90%的聚合物在片状伪足-片层交界处解体。在汇聚区也发现了强烈的解聚，此处网络收缩很明显。为了确定收缩和解聚是否为偶联事件，我们用已知可促进肌球蛋白活性的花萼海绵诱癌素A处理细胞。刺激引起的收缩伴随着整个片层中逆行流动加速和解聚增加，而片状伪足-片层交界处的解体不受影响。似乎存在两种不同的解聚机制，其中一种直接依赖于网络收缩。
