Bhadriraju Kiran, Yang Michael, Alom Ruiz Sami, Pirone Dana, Tan John, Chen Christopher S
Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA.
Exp Cell Res. 2007 Oct 1;313(16):3616-23. doi: 10.1016/j.yexcr.2007.07.002. Epub 2007 Jul 10.
Adhesion to the extracellular matrix regulates numerous changes in the actin cytoskeleton by regulating the activity of the Rho family of small GTPases. Here, we report that adhesion and the associated changes in cell shape and cytoskeletal tension are all required for GTP-bound RhoA to activate its downstream effector, ROCK. Using an in vitro kinase assay for endogenous ROCK, we found that cells in suspension, attached on substrates coated with low density fibronectin, or on spreading-restrictive micropatterned islands all exhibited low ROCK activity and correspondingly low myosin light chain phosphorylation, in the face of high levels of GTP-bound RhoA. In contrast, allowing cells to spread against substrates rescued ROCK and myosin activity. Interestingly, inhibition of tension with cytochalasin D or blebbistatin also inhibited ROCK activity within 20 min. The abrogation of ROCK activity by cell detachment or inhibition of tension could not be rescued by constitutively active RhoA-V14. These results suggest the existence of a feedback loop between cytoskeletal tension, adhesion maturation, and ROCK signaling that likely contributes to numerous mechanochemical processes.
细胞与细胞外基质的黏附通过调节小GTP酶Rho家族的活性来调控肌动蛋白细胞骨架的众多变化。在此，我们报告，黏附以及细胞形状和细胞骨架张力的相关变化对于结合GTP的RhoA激活其下游效应器ROCK都是必需的。使用针对内源性ROCK的体外激酶测定法，我们发现悬浮细胞、附着在低密度纤连蛋白包被的底物上的细胞或在限制铺展的微图案化岛屿上的细胞，尽管结合GTP的RhoA水平很高，但均表现出低ROCK活性以及相应的低肌球蛋白轻链磷酸化水平。相反，让细胞在底物上铺展可恢复ROCK和肌球蛋白活性。有趣的是，用细胞松弛素D或肌球蛋白轻链激酶抑制剂抑制张力也会在20分钟内抑制ROCK活性。细胞脱离或张力抑制导致的ROCK活性丧失不能通过组成型活性RhoA-V14来挽救。这些结果表明，细胞骨架张力、黏附成熟和ROCK信号传导之间存在反馈回路，这可能对众多机械化学过程有贡献。
