Limits...
Flightless-I (Fli-I) regulates the actin assembly activity of diaphanous-related formins (DRFs) Daam1 and mDia1 in cooperation with active Rho GTPase.

Higashi T, Ikeda T, Murakami T, Shirakawa R, Kawato M, Okawa K, Furuse M, Kimura T, Kita T, Horiuchi H - J. Biol. Chem. (2010)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan.

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.

Show MeSH
DAD segment of Daam1 enhances the actin assembly in living cells. A and B, NIH 3T3 cells were transiently transfected with GFP-Daam1 CT WT, GFP-Daam1 CT L1040A, or control GFP vector. After 20 h, the cell lysates were analyzed by SDS-PAGE and immunoblotting (IB) with anti-GFP, anti-Fli-I, and anti-tubulin mAbs (A), and cells were stained with Alexa-568-phalloidin (red). GFP signals were shown in green. WT, wild type; LA, L1040A. Scale bars, 20 μm (B). The data shown are representative of at least three independent experiments with similar results.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2871490&req=5

Figure 4: DAD segment of Daam1 enhances the actin assembly in living cells. A and B, NIH 3T3 cells were transiently transfected with GFP-Daam1 CT WT, GFP-Daam1 CT L1040A, or control GFP vector. After 20 h, the cell lysates were analyzed by SDS-PAGE and immunoblotting (IB) with anti-GFP, anti-Fli-I, and anti-tubulin mAbs (A), and cells were stained with Alexa-568-phalloidin (red). GFP signals were shown in green. WT, wild type; LA, L1040A. Scale bars, 20 μm (B). The data shown are representative of at least three independent experiments with similar results.

Mentions: Next, we evaluated whether Fli-I is involved in the formin-induced actin assembly in living cells. It has been reported that the exogenous expression of Daam1 CT induces a stress fiber formation in HeLa cells (24). The overexpression of GFP-tagged Daam1 CT WT induced the formation of stress fiber-like filaments in serum-starved NIH 3T3 cells, whereas GFP alone did not (Fig. 4, A and B). In GFP-Daam1 CT L1040A-expressing cells, such stress fiber formation was scarcely induced compared with Daam1 CT WT (Fig. 4, A and B), which suggests that the Fli-I binding is required for Daam1 CT to exert its actin assembly activity in cells.


Flightless-I (Fli-I) regulates the actin assembly activity of diaphanous-related formins (DRFs) Daam1 and mDia1 in cooperation with active Rho GTPase.

Higashi T, Ikeda T, Murakami T, Shirakawa R, Kawato M, Okawa K, Furuse M, Kimura T, Kita T, Horiuchi H - J. Biol. Chem. (2010)

DAD segment of Daam1 enhances the actin assembly in living cells. A and B, NIH 3T3 cells were transiently transfected with GFP-Daam1 CT WT, GFP-Daam1 CT L1040A, or control GFP vector. After 20 h, the cell lysates were analyzed by SDS-PAGE and immunoblotting (IB) with anti-GFP, anti-Fli-I, and anti-tubulin mAbs (A), and cells were stained with Alexa-568-phalloidin (red). GFP signals were shown in green. WT, wild type; LA, L1040A. Scale bars, 20 μm (B). The data shown are representative of at least three independent experiments with similar results.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2871490&req=5

Figure 4: DAD segment of Daam1 enhances the actin assembly in living cells. A and B, NIH 3T3 cells were transiently transfected with GFP-Daam1 CT WT, GFP-Daam1 CT L1040A, or control GFP vector. After 20 h, the cell lysates were analyzed by SDS-PAGE and immunoblotting (IB) with anti-GFP, anti-Fli-I, and anti-tubulin mAbs (A), and cells were stained with Alexa-568-phalloidin (red). GFP signals were shown in green. WT, wild type; LA, L1040A. Scale bars, 20 μm (B). The data shown are representative of at least three independent experiments with similar results.
Mentions: Next, we evaluated whether Fli-I is involved in the formin-induced actin assembly in living cells. It has been reported that the exogenous expression of Daam1 CT induces a stress fiber formation in HeLa cells (24). The overexpression of GFP-tagged Daam1 CT WT induced the formation of stress fiber-like filaments in serum-starved NIH 3T3 cells, whereas GFP alone did not (Fig. 4, A and B). In GFP-Daam1 CT L1040A-expressing cells, such stress fiber formation was scarcely induced compared with Daam1 CT WT (Fig. 4, A and B), which suggests that the Fli-I binding is required for Daam1 CT to exert its actin assembly activity in cells.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan.

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.

Show MeSH