Endlich Nicole, Otey Carol A, Kriz Wilhelm, Endlich Karlhans
Department of Anatomy and Cell Biology, Ernst Moritz Arndt University, D-17487 Greifswald, Germany.
Cell Motil Cytoskeleton. 2007 Dec;64(12):966-76. doi: 10.1002/cm.20237.
Force generated in contractile actin filament bundles (stress fibers-SFs) is transmitted to the extracellular matrix (ECM) via linker proteins and transmembrane integrins at focal adhesions (FAs). Though it has long been known that actin is rapidly exchanged in FAs, the connection between SFs and FAs has not been studied in detail. We introduced fiduciary marks on SFs by expressing GFP-palladin or GFP-alpha-actinin-1, which are both FA and dense body proteins, and by pattern bleaching of GFP-actin. Following fiduciary marks on SFs over time by time-lapse fluorescence microscopy, we detected assembly of SFs at FAs in stationary cells resulting in movement of SFs away from FAs with a velocity of 0.2-0.4 microm/min. Visualization of FAs in GFP-palladin/DsRed-paxillin double transfected cells showed that SF elongation was not accompanied by a change in FA length. SF elongation at FAs depended on actin polymerization and force as demonstrated by inhibitors of actin polymerization (cytochalasin D, jasplakinolide) and inhibitors of myosin-dependent contraction (blebbistatin, Y-27632), respectively. Our finding of SF assembly at FAs has important implications for SF formation, force transmission, and tension distribution within the actin cytoskeletal network of stationary cells.
收缩性肌动蛋白丝束（应力纤维-SFs）中产生的力通过黏着斑（FAs）处的连接蛋白和跨膜整合素传递至细胞外基质（ECM）。尽管人们早就知道肌动蛋白在黏着斑中会快速交换，但应力纤维与黏着斑之间的联系尚未得到详细研究。我们通过表达GFP-钯蛋白或GFP-α-辅肌动蛋白-1（二者均为黏着斑和致密体蛋白）以及对GFP-肌动蛋白进行图案化漂白，在应力纤维上引入了基准标记。通过延时荧光显微镜随时间追踪应力纤维上的基准标记，我们检测到静止细胞中黏着斑处应力纤维的组装，导致应力纤维以0.2 - 0.4微米/分钟的速度从黏着斑移开。对GFP-钯蛋白/DsRed-桩蛋白双转染细胞中黏着斑的可视化显示，应力纤维的伸长并未伴随着黏着斑长度的变化。黏着斑处应力纤维的伸长分别依赖于肌动蛋白聚合和力，这通过肌动蛋白聚合抑制剂（细胞松弛素D、茉莉酮酸甲酯）和肌球蛋白依赖性收缩抑制剂（blebbistatin、Y-27632）得以证明。我们关于黏着斑处应力纤维组装的发现对于静止细胞肌动蛋白细胞骨架网络中的应力纤维形成、力传递和张力分布具有重要意义。
