Université de Paris, CNRS, Institut Jacques Monod, Paris, France.
Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay, CEA, CNRS, Gif-sur-Yvette, France.
Biol Cell. 2021 Nov;113(11):441-449. doi: 10.1111/boc.202100014. Epub 2021 Aug 20.
Actin cytoskeleton contractility plays a critical role in morphogenetic processes by generating forces that are then transmitted to cell-cell and cell-ECM adhesion complexes. In turn, mechanical properties of the environment are sensed and transmitted to the cytoskeleton at cell adhesion sites, influencing cellular processes such as cell migration, differentiation and survival. Anchoring of the actomyosin cytoskeleton to adhesion sites is mediated by adaptor proteins such as talin or α-catenin that link F-actin to transmembrane cell adhesion receptors, thereby allowing mechanical coupling between the intracellular and extracellular compartments. Thus, a key issue is to be able to measure the forces generated by actomyosin and transmitted to the adhesion complexes. Approaches developed in cells and those probing single molecule mechanical properties of α-catenin molecules allowed to identify α-catenin, an F-actin binding protein which binds to the cadherin complexes as a major player in cadherin-based mechanotransduction. However, it is still very difficult to bridge intercellular forces measured at cellular levels and those measured at the single-molecule level.
Here, we applied an intermediate approach allowing reconstruction of the actomyosin-α-catenin complex in acellular conditions to probe directly the transmitted forces. For this, we combined micropatterning of purified α-catenin and spontaneous actomyosin network assembly in the presence of G-actin and Myosin II with microforce sensor arrays used so far to measure cell-generated forces.
Using this method, we show that self-organizing actomyosin bundles bound to micrometric α-catenin patches can apply near-nano-Newton forces.
Our results pave the way for future studies on molecular/cellular mechanotransduction and mechanosensing.
肌动蛋白细胞骨架的收缩性在形态发生过程中起着关键作用，它产生的力随后传递到细胞-细胞和细胞-细胞外基质（ECM）黏附复合物。反过来，环境的机械性能在细胞黏附部位被感知并传递到细胞骨架，影响细胞过程，如细胞迁移、分化和存活。肌动球蛋白细胞骨架与黏附位点的锚定是通过衔接蛋白（如塔林或α-连环蛋白）介导的，这些蛋白将 F-肌动蛋白与跨膜细胞黏附受体连接起来，从而允许细胞内和细胞外隔室之间的机械耦联。因此，一个关键问题是能够测量肌动球蛋白产生并传递到黏附复合物的力。在细胞中开发的方法和探测α-连环蛋白单分子机械特性的方法，使我们能够鉴定出α-连环蛋白，它是一种 F-肌动蛋白结合蛋白，作为钙黏蛋白机械转导的主要参与者，与钙黏蛋白复合物结合。然而，仍然很难弥合在细胞水平上测量的细胞间力和在单分子水平上测量的力之间的差距。
在这里，我们应用了一种中间方法，允许在非细胞条件下重建肌动球蛋白-α-连环蛋白复合物，以直接探测传递的力。为此，我们将纯化的α-连环蛋白的微图案化与自发肌动球蛋白网络组装结合起来，在存在 G-肌动蛋白和肌球蛋白 II 的情况下，与迄今为止用于测量细胞产生的力的微力传感器阵列结合使用。
使用这种方法，我们表明，自发组织的肌动球蛋白束与微米级的α-连环蛋白斑块结合，可以施加近纳米牛顿的力。
我们的结果为分子/细胞机械转导和机械传感的未来研究铺平了道路。
