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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.

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Fli-I is required for the actin assembly by Daam1 in living cells. A and B, NIH 3T3 cells were transfected with control or Fli-I siRNA. After 28 h, the cells were transiently transfected with GFP-Daam1 CT and/or hemagglutinin-tagged siRNA-insensitive Fli-I. After 20 h, cells were analyzed by SDS-PAGE and immunoblotting with anti-Fli-I, anti-GFP, anti-hemagglutinin, and anti-tubulin mAbs (A) or were stained with Alexa-568-phalloidin (red). GFP signals are shown in green. B, scale bars, 20 μm. The data shown are representative of at least three independent experiments with similar results.
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Figure 5: Fli-I is required for the actin assembly by Daam1 in living cells. A and B, NIH 3T3 cells were transfected with control or Fli-I siRNA. After 28 h, the cells were transiently transfected with GFP-Daam1 CT and/or hemagglutinin-tagged siRNA-insensitive Fli-I. After 20 h, cells were analyzed by SDS-PAGE and immunoblotting with anti-Fli-I, anti-GFP, anti-hemagglutinin, and anti-tubulin mAbs (A) or were stained with Alexa-568-phalloidin (red). GFP signals are shown in green. B, scale bars, 20 μm. The data shown are representative of at least three independent experiments with similar results.

Mentions: We further examined the significance of Fli-I using a gene knock-down technique. Transfection of two different siRNAs against Fli-I (#1 and #2) almost completely reduced the Fli-I expression level in NIH 3T3 cells (Fig. 5A). Daam1 CT-induced stress fiber formation was significantly reduced in the Fli-I siRNA #1- and #2-treated cells but not in the control siRNA-treated cells (Fig. 5B). Co-expression of siRNA-insensitive human Fli-I with Daam1 CT restored the stress fiber formation in the Fli-I knocked down cells (Fig. 5, A and B). The co-expression of Fli-I and GFP alone did not induce the actin stress fiber formation in the NIH 3T3 cells (supplemental Fig. 2). These results demonstrate that Fli-I is essential for Daam1 FH1-FH2 domains to assemble actin filaments in living 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)

Fli-I is required for the actin assembly by Daam1 in living cells. A and B, NIH 3T3 cells were transfected with control or Fli-I siRNA. After 28 h, the cells were transiently transfected with GFP-Daam1 CT and/or hemagglutinin-tagged siRNA-insensitive Fli-I. After 20 h, cells were analyzed by SDS-PAGE and immunoblotting with anti-Fli-I, anti-GFP, anti-hemagglutinin, and anti-tubulin mAbs (A) or were stained with Alexa-568-phalloidin (red). GFP signals are shown in green. B, scale bars, 20 μm. 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 5: Fli-I is required for the actin assembly by Daam1 in living cells. A and B, NIH 3T3 cells were transfected with control or Fli-I siRNA. After 28 h, the cells were transiently transfected with GFP-Daam1 CT and/or hemagglutinin-tagged siRNA-insensitive Fli-I. After 20 h, cells were analyzed by SDS-PAGE and immunoblotting with anti-Fli-I, anti-GFP, anti-hemagglutinin, and anti-tubulin mAbs (A) or were stained with Alexa-568-phalloidin (red). GFP signals are shown in green. B, scale bars, 20 μm. The data shown are representative of at least three independent experiments with similar results.
Mentions: We further examined the significance of Fli-I using a gene knock-down technique. Transfection of two different siRNAs against Fli-I (#1 and #2) almost completely reduced the Fli-I expression level in NIH 3T3 cells (Fig. 5A). Daam1 CT-induced stress fiber formation was significantly reduced in the Fli-I siRNA #1- and #2-treated cells but not in the control siRNA-treated cells (Fig. 5B). Co-expression of siRNA-insensitive human Fli-I with Daam1 CT restored the stress fiber formation in the Fli-I knocked down cells (Fig. 5, A and B). The co-expression of Fli-I and GFP alone did not induce the actin stress fiber formation in the NIH 3T3 cells (supplemental Fig. 2). These results demonstrate that Fli-I is essential for Daam1 FH1-FH2 domains to assemble actin filaments in living 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