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

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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.