Esue Osigwe, Harris Elizabeth S, Higgs Henry N, Wirtz Denis
Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA.
J Mol Biol. 2008 Dec 12;384(2):324-34. doi: 10.1016/j.jmb.2008.09.043. Epub 2008 Sep 26.
Formins are multidomain proteins that regulate actin filament dynamics and are defined by the formin homology 2 domain. Biochemical assays suggest that mammalian formins display actin-filament nucleation, severing, and bundling activities. Whether formins can cross-link actin filaments into viscoelastic arrays and the effectiveness of formins' bundling activity compared with that of important filamentous actin (F-actin) cross-linking/bundling proteins are unknown. Here, we used rigorous in vitro rheologic assays to deconvolve the dynamic cross-linking activity from the bundling activity of formin FRL1 and the closely related mDia1 and mDia2. In addition, we compared these formins with the canonical F-actin bundling protein fascin and cross-linking/bundling proteins alpha-actinin and filamin. We found that FRL1 and mDia2, but not mDia1, can help F-actin form highly elastic networks. FRL1 and mDia2 mediate the formation of highly elastic F-actin networks as effectively and rapidly as alpha-actinin and filamin but only past a relatively high actin-to-formin molar ratio of 50:1. Past that threshold molar ratio, the mechanical properties of F-actin/formin networks are independent of formin concentration, similar to fascin. Moreover, unlike those for alpha-actinin and filamin but similar to those for fascin, F-actin/formin networks show no strain-induced hardening. mDia1 cannot bundle F-actin but can weakly cross-link filaments at high concentrations. Point mutagenesis reveals that reducing the barbed-end binding activity of FRL1 and mDia2 greatly enhances the rate of formation of F-actin gels but does not significantly affect the mechanical properties of the resulting networks at steady state. Together, these results suggest that the mechanical behaviors of FRL1 and mDia2 are fundamentally different from those of cross-linking/bundling proteins alpha-actinin and filamin but qualitatively similar to the mechanical behavior of the bundling protein fascin, albeit with a dramatically increased (>10-fold) threshold concentration for transition to bundling, which nevertheless leads to much stiffer F-actin networks than fascin.
formin是一种多结构域蛋白，可调节肌动蛋白丝动力学，由formin同源性2结构域定义。生化分析表明，哺乳动物formin具有肌动蛋白丝成核、切断和捆绑活性。formin是否能将肌动蛋白丝交联成粘弹性阵列，以及与重要的丝状肌动蛋白（F-肌动蛋白）交联/捆绑蛋白相比，formin的捆绑活性的有效性尚不清楚。在这里，我们使用了严格的体外流变学分析，从formin FRL1以及密切相关的mDia1和mDia2的捆绑活性中解卷积动态交联活性。此外，我们将这些formin与典型的F-肌动蛋白捆绑蛋白fascin以及交联/捆绑蛋白α-辅肌动蛋白和细丝蛋白进行了比较。我们发现，FRL1和mDia2，但不是mDia1，可以帮助F-肌动蛋白形成高弹性网络。FRL1和mDia2介导高弹性F-肌动蛋白网络的形成，其有效性和速度与α-辅肌动蛋白和细丝蛋白相当，但仅在相对较高的肌动蛋白与formin摩尔比为50:1时才会如此。超过该阈值摩尔比后，F-肌动蛋白/formin网络的机械性能与formin浓度无关，类似于fascin。此外，与α-辅肌动蛋白和细丝蛋白不同，但与fascin相似，F-肌动蛋白/formin网络没有应变诱导的硬化。mDia1不能捆绑F-肌动蛋白，但在高浓度下可以弱交联细丝。点突变分析表明，降低FRL1和mDia2的带刺末端结合活性可大大提高F-肌动蛋白凝胶的形成速率，但在稳态下对所得网络的机械性能没有显著影响。总之，这些结果表明，FRL1和mDia2的机械行为与交联/捆绑蛋白α-辅肌动蛋白和细丝蛋白的机械行为有根本不同，但在质量上与捆绑蛋白fascin的机械行为相似，尽管转变为捆绑的阈值浓度显著增加（>10倍），但这仍导致比fascin更硬的F-肌动蛋白网络。
