Abstract
Eukaryotic cells dynamically reorganize the actin cytoskeleton to regulate various cellular activities, such as cell shape change, cell motility, cytokinesis, and vesicular transport. Diaphanous-related formins (DRFs), such as Daam1 and mDia1, play central roles in actin dynamics through assembling linear actin filaments. It has been reported that the GTP-bound active Rho binds directly to DRFs and partially unleashes the intramolecular autoinhibition of DRFs. However, whether proteins other than Rho involve the regulation of the actin assembly activity of DRFs has been unclear. Here, we show that Flightless-I (Fli-I), a gelsolin family protein essential for early development, binds directly to Daam1 and mDia1. Fli-I enhances the intrinsic actin assembly activity of Daam1 and mDia1 in vitro and is required for Daam1-induced actin assembly in living cells. Furthermore, Fli-I promotes the GTP-bound active Rho-mediated relief of the autoinhibition of Daam1 and mDia1. Thus, Fli-I is a novel positive regulator of Rho-induced linear actin assembly mediated by DRFs.
Highlights
Formin proteins regulate the actin dynamics by assembling actin filaments through their formin homology-1 (FH1)7 and EXPERIMENTAL PROCEDURES
Constructs, Recombinant Proteins, Antibodies, and Other Materials—The preparation of cDNAs encoding human Daam1 fragments (Daam1 NT, amino acids 41– 477; Daam1 CT, aa 490 –1078; and Daam1 CT F1046A), human mDia1 fragments (Fig. 1, A and B), and human RhoA were described before [11]. cDNAs encoding Daam1 diaphanous autoinhibitory domain (DAD), mDia1 DAD, Daam1 CT L1040A, and mDia1 CT L1197A were produced by PCR using their respective CT fragments as templates
Daam1 NT containing the diaphanous inhibitory domain (DID) domain inhibited the binding of Fli-I to Daam1 DAD in a concentration-dependent manner (Fig. 6A). These results indicate that Fli-I and the DID domain cannot bind to the DAD segment simultaneously
Summary
Constructs, Recombinant Proteins, Antibodies, and Other Materials—The preparation of cDNAs encoding human Daam fragments (Daam NT, amino acids (aa) 41– 477; Daam CT, aa 490 –1078; and Daam CT F1046A), human mDia fragments (mDia NT, aa 69 – 450; mDia CT, aa 492– 1263; and mDia CT F1203A) (Fig. 1, A and B), and human RhoA were described before [11]. cDNAs encoding Daam DAD (aa 992–1078), mDia DAD (aa 1145–1263), Daam CT L1040A, and mDia CT L1197A were produced by PCR using their respective CT fragments as templates. Glutathione-Sepharose coated with 200 g of GST-Daam CT WT or L1040A was incubated with 20 ml of platelet cytosol (4 mg of proteins/ml) for 2 h at 4 °C and washed five times with buffer A. The WT protein in the purified actin These results suggest that some kind of cytosolic factor(s) may bind to Daam through the conserved leucine residue in the DAD segments and may assist Daam to assemble the actin filaments in the knockdown studies, cells were transfected with siRNA using cytoplasmic environment. Lipofectamine RNAiMAX (Invitrogen) according to the for- Fli-I Interacts with the DAD Segment in the Carboxyl-termiward transfection method of the manufacturer’s instructions nal Region of Daam1—To identify this factor, we performed for 28 h, and cells were transfected with pEGFP-C1 affinity chromatography with platelet cytosol using Daam CT. GST-Daam CT WT- and medium for 20 h and were processed for the fluorescence GST-Daam CT L1040A-coated beads were incubated with microscopy analysis and the immunoblotting analysis
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