Graduate Program in Mechanobiology, Mechanobiology Institute, National University of Singapore, Singapore.
Cytoskeleton (Hoboken). 2013 Jul;70(7):394-407. doi: 10.1002/cm.21115. Epub 2013 May 24.
We have developed an optogenetic technique for the activation of diaphanous-related formins. Our approach is based on fusion of the light-oxygen-voltage 2 domain of Avena sativa Phototrophin1 to an isolated Diaphanous Autoregulatory Domain from mDia1. This "caged" diaphanous auto-regulatory domain was inactive in the dark but in the presence of blue light rapidly activated endogenous diaphanous-related formins. Using an F-actin reporter, we observed filopodia and lamellipodia formation as well as a steady increase in F-actin along existing stress fibers, starting within minutes of photo-activation. Interestingly, we did not observe the formation of new stress fibers. Remarkably, a 1.9-fold increase in F-actin was not paralleled by an increase in myosin II along stress fibers and the amount of tension generated by the fibers, as judged by focal adhesion size, appeared unchanged. Our results suggest a decoupling between F-actin accumulation and contractility in stress fibers and demonstrate the utility of photoactivatable diaphanous autoregulatory domain for the study of diaphanous-related formin function in cells.
我们开发了一种光遗传学技术来激活丝状相关formin。我们的方法是将 Avena sativa Phototrophin1 的光-氧-电压 2 结构域融合到 mDia1 的分离的 Diaphanous 自动调节结构域上。这种“笼状”的 Diaphanous 自动调节结构域在黑暗中是无活性的，但在蓝光存在下，它可以快速激活内源性丝状相关formin。使用 F-肌动蛋白报告物，我们观察到了丝状伪足和片状伪足的形成，以及 F-肌动蛋白沿着现有应力纤维的稳定增加，这一过程在光激活后几分钟内开始。有趣的是，我们没有观察到新的应力纤维的形成。值得注意的是，F-肌动蛋白的增加 1.9 倍并没有伴随着肌球蛋白 II 在应力纤维上的增加，而且根据粘着斑的大小判断，纤维产生的张力似乎没有变化。我们的结果表明，应力纤维中的 F-肌动蛋白积累和收缩性之间存在解耦，并且证明了光激活的 Diaphanous 自动调节结构域在研究细胞中丝状相关formin 功能中的实用性。
