Ballistic intracellular nanorheology reveals ROCK-hard cytoplasmic stiffening response to fluid flow
Ballistic intracellular nanorheology reveals ROCK-hard cytoplasmic stiffening response to fluid flow
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DOI：
10.1242/jcs.02899
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发表时间：
2006-05-01
影响因子：
4
通讯作者：
Wirtz, Denis
中科院分区：
文献类型：
--
作者：
Lee, Jerry S. H.;Panorchan, Porntula;Wirtz, Denis
细胞在体内不断受到机械剪切应力，在各种生理和病理过程中起着重要的调节作用。细胞骨架重组发生在剪切流的响应中已经被广泛研究，但是贴壁细胞的细胞质是否适应其机械特性来响应剪切在很大程度上是未知的。在这里，我们开发了一种新的方法，荧光纳米粒子被弹道注射到细胞中，以高分辨率探测单个细胞细胞质中可能的局部粘弹性变化。这项新的试验，弹道细胞内纳米流变学（BIN），揭示了剪切流诱导血清饥饿的瑞士3T3成纤维细胞的细胞质粘度急剧持续增加25倍。相比之下，肌动蛋白收缩激动剂LPA刺激的细胞，尽管形成了相似的细胞骨架结构，但却表现出高度短暂的低幅度僵硬。剪切诱导的细胞质硬化通过抑制肌动球蛋白相互作用而减弱，并通过特异性rho激酶（ROCK）抑制完全消除。综上所述，这些结果表明，生化和生物物理刺激可能导致细胞骨架结构（即应力纤维和黏附灶）的形成，但诱导的微力学反应在数量上不同。我们的研究结果表明，当贴壁细胞受到剪切应力时，它的第一级作用是通过增强肌动蛋白组装和rho激酶介导的收缩性，使其细胞质大大硬化，以防止其脱离基质。
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.