Actin-polymerization-driven molecular dynamics of Formin homology proteins in live cells

在之前的研究中，我们发现了mDia1的肌动蛋白盖帽运动（Science 303:2007-10）。2004)。mDia1之前由我与京都大学的shuh Narumiya博士合作确定为GTPase Rho的效应物。目前的研究项目旨在进一步将我们的单分子成像方法扩展到mDia1及其相关分子的天然形式，以阐明Formin同源蛋白在活细胞中的生理功能和调控，无论是空间上还是时间上。mDia1属于formin家族蛋白（formmins），它们共享富含脯氨酸的formin同源1 （FH1）和formin同源2 （FH2）结构域。许多基于肌动蛋白的细胞结构，如酵母肌动蛋白索，细胞动力学裂解沟和哺乳动物细胞中的肌动蛋白束都依赖于形成蛋白。最近的研究表明，FH2或FH1-FH2结构域构成肌动蛋白丝的核，并持续与丝的刺端生长相关。然而，在细胞内，formin介导的肌动蛋白丝形成的生理调控，无论是时间上还是空间上，仍然是未知的。在本项目中，通过单分子活细胞成像，我们发现肌动蛋白单体隔离药物诱导的肌动蛋白单体池的增加迅速激活了mDia1，从而启动了快速的定向运动。非聚合性肌动蛋白的表达足以诱导频繁激活mDia1。Rho活性是激活mDia1所必需的，但FH2区单独可以被latrunculin b激活。这些发现表明，g -肌动蛋白的短暂积累可以作为激活mDia1以执行肌动蛋白细丝快速组装的线索。目前的研究项目因此发现细胞具有一种新的急性肌动蛋白聚合物恢复机制，涉及mDia1（提交）。

In the previous research, we have discovered processive actin capping movement of mDia1 (Science 303:2007-10. 2004). mDia1 was previously identified by myself in collaboration with Dr.Shuh Narumiya in Kyoto University as an effecter of a GTPase, Rho. The current research project aimed to further extend our single-molecule imaging method to a native form of mDia1 and related molecules in order to elucidate the physiological function and regulation of Formin homology proteins, both spatially and temporally, in living cells.mDia1 belongs to the formin family proteins (formins) that share proline-rich formin homology 1 (FH1) and formin homology 2 (FH2) domains. Many actin-based cellular structures such as yeast actin cables, cytokinetic cleavage furrows and actin bundles in mammalian cells are dependent on formins. Recent studies characterized that FH2 or FH1-FH2 domains nucleate actin filaments and they processively remain associated to the growing barbed-end of filaments. However, the physiological regulation of formin-mediated actin filament formation, both temporally and spatially within the cell, is still unknown.In this project, by using single-molecule live-cell imaging, we have found that an increase in the actin monomer pool induced by actin monomer sequestering drugs rapidly activated mDia1 to initiate fast directional movement. The expression of nonpolymerizable actins was sufficient to induce frequent activation of mDia1. Rho activity was required for activation of mDia1, but the FH2 region alone can be activated by latrunculin B. These findings reveal that transient accumulation of G-actin works as a cue to activate mDia1 to execute rapid assembly of actin filaments. The current research project thus discovered that cells possess a novel acute actin polymer restoration mechanism involving mDia1 (submitted).