Université Grenoble Alpes, CNRS, LIPhy, F-38000 Grenoble, France.
Institute for Theoretical Physics, Heidelberg University, Heidelberg, Germany; BioQuant-Center for Quantitative Biology, Heidelberg University, Heidelberg, Germany.
Biophys J. 2023 Feb 21;122(4):684-696. doi: 10.1016/j.bpj.2023.01.011. Epub 2023 Jan 12.
Adherent cells use actomyosin contractility to generate mechanical force and to sense the physical properties of their environment, with dramatic consequences for migration, division, differentiation, and fate. However, the organization of the actomyosin system within cells is highly variable, with its assembly and function being controlled by small GTPases from the Rho family. To understand better how activation of these regulators translates into cell-scale force generation in the context of different physical environments, here we combine recent advances in non-neuronal optogenetics with micropatterning and traction force microscopy on soft elastic substrates. We find that, after whole-cell RhoA activation by the CRY2/CIBN optogenetic system with a short pulse of 100 ms, single cells contract on a minute timescale in proportion to their original traction force, before returning to their original tension setpoint with near perfect precision, on a longer timescale of several minutes. To decouple the biochemical and mechanical elements of this response, we introduce a mathematical model that is parametrized by fits to the dynamics of the substrate deformation energy. We find that the RhoA response builds up quickly on a timescale of 20 s, but decays slowly on a timescale of 50 s. The larger the cells and the more polarized their actin cytoskeleton, the more substrate deformation energy is generated. RhoA activation starts to saturate if optogenetic pulse length exceeds 50 ms, revealing the intrinsic limits of biochemical activation. Together our results suggest that adherent cells establish tensional homeostasis by the RhoA system, but that the setpoint and the dynamics around it are strongly determined by cell size and the architecture of the actin cytoskeleton, which both are controlled by the extracellular environment.
黏附细胞利用肌动球蛋白的收缩性产生机械力，并感知其环境的物理特性，这对迁移、分裂、分化和命运都有重大影响。然而，细胞内肌动球蛋白系统的组织高度可变，其组装和功能受 Rho 家族的小 GTPases 控制。为了更好地理解这些调节剂的激活如何在不同物理环境的背景下转化为细胞尺度的力生成，我们在这里将最近在非神经元光遗传学方面的进展与软弹性基底上的微图案化和牵引力显微镜结合起来。我们发现，在用 CRY2/CIBN 光遗传学系统对全细胞 RhoA 进行 100ms 的短脉冲激活后，单个细胞会在微小的时间尺度上收缩，收缩幅度与它们原来的牵引力成正比，然后在几分钟的更长时间尺度上以近乎完美的精度恢复到原来的张力设定点。为了将这种反应的生化和力学元素分开，我们引入了一个数学模型，该模型通过对基底变形能动力学的拟合进行参数化。我们发现，RhoA 的响应在 20 秒的时间尺度上快速建立，但在 50 秒的时间尺度上缓慢衰减。细胞越大，其肌动球蛋白细胞骨架越极化，产生的基底变形能就越多。如果光遗传学脉冲长度超过 50ms，RhoA 的激活就会开始饱和，这揭示了生化激活的内在限制。我们的研究结果表明，黏附细胞通过 RhoA 系统建立张力动态平衡，但设定点及其周围的动力学强烈取决于细胞大小和肌动球蛋白细胞骨架的结构，这两者都受到细胞外环境的控制。
