Lee Jerry S H, Panorchan Porntula, Hale Christopher M, Khatau Shyam B, Kole Thomas P, Tseng Yiider, Wirtz Denis
Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA.
J Cell Sci. 2006 May 1;119(Pt 9):1760-8. doi: 10.1242/jcs.02899.
Cells in vivo are constantly subjected to mechanical shear stresses that play important regulatory roles in various physiological and pathological processes. Cytoskeletal reorganizations that occur in response to shear flow have been studied extensively, but whether the cytoplasm of an adherent cell adapts its mechanical properties to respond to shear is largely unknown. Here we develop a new method where fluorescent nanoparticles are ballistically injected into the cells to probe, with high resolution, possible local viscoelastic changes in the cytoplasm of individual cells subjected to fluid flow. This new assay, ballistic intracellular nanorheology (BIN), reveals that shear flow induces a dramatic sustained 25-fold increase in cytoplasmic viscosity in serum-starved Swiss 3T3 fibroblasts. By contrast, cells stimulated with the actin contractile agonist LPA show highly transient stiffening of much lower amplitude, despite the formation of similar cytoskeletal structures. Shear-induced cytoplasmic stiffening is attenuated by inhibiting actomyosin interactions and is entirely eliminated by specific Rho-kinase (ROCK) inhibition. Together, these results show that biochemical and biophysical stimuli may elicit the formation of qualitatively similar cytoskeleton structures (i.e. stress fibers and focal adhesions), but induces quantitatively different micromechanical responses. Our results suggest that when an adherent cell is subjected to shear stresses, its first order of action is to prevent detachment from its substratum by greatly stiffening its cytoplasm through enhanced actin assembly and Rho-kinase mediated contractility.
体内细胞不断受到机械剪切应力的作用，这些应力在各种生理和病理过程中发挥着重要的调节作用。针对剪切流发生的细胞骨架重组已得到广泛研究，但贴壁细胞的细胞质是否会调整其机械特性以响应剪切力，在很大程度上仍不清楚。在此，我们开发了一种新方法，将荧光纳米颗粒通过弹射方式注入细胞，以高分辨率探测受到流体流动作用的单个细胞细胞质中可能的局部粘弹性变化。这种新的检测方法，即弹射式细胞内纳米流变学（BIN），揭示了剪切流会使血清饥饿的瑞士3T3成纤维细胞的细胞质粘度急剧持续增加25倍。相比之下，用肌动蛋白收缩激动剂溶血磷脂酸（LPA）刺激的细胞，尽管形成了相似的细胞骨架结构，但显示出幅度低得多的高度短暂的硬化。抑制肌动球蛋白相互作用可减弱剪切诱导的细胞质硬化，而特异性抑制Rho激酶（ROCK）则可完全消除这种硬化。总之，这些结果表明，生化和生物物理刺激可能引发定性相似的细胞骨架结构（即应力纤维和粘着斑）的形成，但诱导出的微观力学反应在数量上有所不同。我们的结果表明，当贴壁细胞受到剪切应力时，其首要行动是通过增强肌动蛋白组装和Rho激酶介导的收缩性来极大地硬化其细胞质，以防止从其基质上脱离。
