Viscoelastic retraction of single living stress fibers and its impact on cell shape, cytoskeletal organization, and extracellular matrix mechanics
Viscoelastic retraction of single living stress fibers and its impact on cell shape, cytoskeletal organization, and extracellular matrix mechanics
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DOI：
10.1529/biophysj.105.071506
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发表时间：
2006-05-01
影响因子：
3.4
通讯作者：
Ingber, DE
中科院分区：
文献类型：
--
作者：
Kumar, S;Maxwell, IZ;Ingber, DE
细胞通过在其基部组装肌动蛋白应力纤维并对其细胞外基质（ECM）粘附施加牵引力来改变其形式和功能。个别应力纤维被认为是积极紧张的肌动球蛋白马达的行动，并作为弹性电缆，在结构上加强细胞骨架的基础部分的功能;然而，这些原则还没有直接在活细胞中进行测试，其整体细胞形状控制的意义知之甚少。在这里，我们结合联合收割机的激光nanoscissor，牵引力显微镜，和荧光光漂白方法，以确认应力纤维在活细胞中的行为作为粘弹性电缆，通过动作肌球蛋白电机的动作，以量化他们的收缩动力学原位，并探讨其整体的机械稳定性的细胞和相互连接的ECM的贡献。这些研究表明，激光切断后，个别应力纤维的粘弹性回缩部分减缓Rho相关激酶的抑制和肌球蛋白轻链激酶的直接抑制几乎废除。重要的是，在刚性ECM基质上培养的细胞可以耐受多个应力纤维的破坏，而细胞形状的总体变化可以忽略不计，而锚定到顺应性ECM基质上的细胞中的单个应力纤维的破坏损害了整个细胞力平衡，诱导细胞骨架重排，并产生远离切口部位许多微米的ECM缩回;这导致泡孔形状的大规模变化（> 5%的伸长率）。除了揭示对单个应力纤维的机械特性和细胞形状贡献的基本见解并确认ECM有效地是细胞和细胞骨架的物理延伸之外，本文描述的技术提供了一种新的方法来空间映射纳米级活细胞的细胞骨架力学。
Cells change their form and function by assembling actin stress fibers at their base and exerting traction forces on their extracellular matrix (ECM) adhesions. Individual stress fibers are thought to be actively tensed by the action of actomyosin motors and to function as elastic cables that structurally reinforce the basal portion of the cytoskeleton; however, these principles have not been directly tested in living cells, and their significance for overall cell shape control is poorly understood. Here we combine a laser nanoscissor, traction force microscopy, and fluorescence photobleaching methods to confirm that stress fibers in living cells behave as viscoelastic cables that are tensed through the action of actomyosin motors, to quantify their retraction kinetics in situ, and to explore their contribution to overall mechanical stability of the cell and interconnected ECM. These studies reveal that viscoelastic recoil of individual stress fibers after laser severing is partially slowed by inhibition of Rho-associated kinase and virtually abolished by direct inhibition of myosin light chain kinase. Importantly, cells cultured on stiff ECM substrates can tolerate disruption of multiple stress fibers with negligible overall change in cell shape, whereas disruption of a single stress fiber in cells anchored to compliant ECM substrates compromises the entire cellular force balance, induces cytoskeletal rearrangements, and produces ECM retraction many microns away from the site of incision; this results in large-scale changes of cell shape (> 5% elongation). In addition to revealing fundamental insight into the mechanical properties and cell shape contributions of individual stress fibers and confirming that the ECM is effectively a physical extension of the cell and cytoskeleton, the technologies described here offer a novel approach to spatially map the cytoskeletal mechanics of living cells on the nanoscale.