Identification of intracellular signaling cascade of Rho and mDia1 based on high sensitivity live-cell imaging

在目前的研究项目中，我们发现在活细胞中mDia1的肌动蛋白进行了封顶运动。MDia1属于Forin蛋白家族，是Rho GTP酶的效应因子。福尔明家族在其C末端的一半中共享保守的串联FH1-FH2结构。最近，人们发现FH1-FH2片段(FH1-FH2)在体外具有肌动蛋白成核活性。我们在活细胞中进行了mDia1的单分子成像，发现了mDia1 FH1-FH2的快速定向运动。MDia1 FH1-FH2以每秒2微米的速度行进，最远可达数十微米。3种肌动蛋白干扰药均可阻断mDia1 FH1-FH2的运动，运动速度与肌动蛋白延长率相关。为了排除mDia1运动依赖肌球蛋白的可能性，我们用纯化的肌动蛋白、Profilin和GST-mDia1 FH1-FH2重组了Forin催化的肌动蛋白组装，并在显微镜下观察。MDia1 FH1-FH2与生长的肌动蛋白带刺末端持续相关。这些结果表明，mDia1沿着细胞内肌动蛋白细丝的生长末端进行连续移动。我们的结果也提高了Formins包括不涉及马达蛋白的细胞内运输机制的可能性。这一发现发表在《科学》杂志上。我们还发现，野生型mDia1在显微注射重组Rho蛋白后开始定向移动。这一发现证实了我们之前的模型(NAT。细胞生物。1：136,1999)，其中Rho与mDia1 N-末端的结合通过破坏mDia1内的分子内相互作用来激活mDia1 FH1-FH2。我们通过观察活细胞中野生型mDia1的分子动力学，继续研究mDia1的激活机制。

In the present research project, we have discovered processive actin capping movement of mDia1 in living cells. mDia1, which was previously identified by our group as an effecter of Rho GTPase, belongs to the Formin family of proteins. The Formin family share the conserved tandem FH1-FH2 structure in their C-terminal half. Recently, it was discovered that FH1-FH2 fragments (FH1-FH2) has an actin nucleation activity in vitro. FH1-FH2 is also known to interact with the actin barbed end.We carried out single-molecule imaging of mDia1 in living cells, and discovered fast directional movement of mDia1 FH1-FH2. mDia1 FH1-FH2 traveled at 2 micron per second as far as tens of microns. The movement of mDia1 FH1-FH2 was blocked by three actin-perturbing drugs, and the speed of mDia1 FH1-FH2 movement correlated with actin elongation rates. In order to exclude the possibility that mDia1 movement is myosin-dependent, we reconstituted Formin-catalyzed actin assembly using purified actin, profilin and GST-mDia1 FH1 -FH2, and observed it under the microscope. mDia1 FH1-FH2 associated persistently with the growing actin barbed end. These results suggest that mDia1 moves processively along the growing end of actin filaments in cells. Our results also raise the possibility that Formins comprise intracellular trafficking machinery that does not involve motor proteins. This finding was published in Science.We also found the wild-type mDia1 starts directional movement upon microinjection of recombinant Rho proteins. This finding confirmed our previous model (Nat. Cell Biol. 1:136, 1999) in which Rho-binding to mDia1 N-terminus activates mDia1 FH1-FH2 by disrupting intramolecular interaction within mDia1. We continue to work on the activation mechanism of mDia1 by observing molecular dynamics of wild-type mDia1 in living cells.