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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 enhances the actin assembly activity of mDia1 as well as that of Daam1. A, cytoplasmic actin assembly assay of mDia1 CT mutants. WT, wild type; LA, L1197A; FA, F1203A. B, direct binding of mDia1 to Fli-I. Beads coated with 50 pmol of GST-tagged proteins were incubated with 12.5 pmol of His6-tagged proteins as indicated. Bound proteins were analyzed by SDS-PAGE and immunoblotting (IB). C, cytoplasmic actin assembly was measured for 1 pmol of mDia1 CT in Fli-I-depleted cytosol used in Fig. 3C in the absence or presence of 125 nm purified Fli-I. pre, preimmune IgG-treated cytosol; ID, Fli-I-immunodepleted cytosol. D, concentration-dependent disruption of DID-DAD interaction of mDia1 by GTP-RhoA. Beads coated with 50 pmol of GST-mDia1 DAD were incubated with 125 nm His6-mDia1 NT in the presence of various concentrations of His6-RhoA G14V. Bead-associated His6-mDia1 NT was analyzed by immunoblotting with anti-His6 mAb. E, Fli-I enhances the DID-DAD dissociation of mDia1 by GTP-RhoA. Beads coated with 100 nm GST-mDia1 NT were incubated with 20 pmol of His6-mDia1 CT in the absence or presence of 0.1 μm His6-RhoA G14V and various concentrations of His6-Fli-I G4–6. Bead-bound His6-mDia1 CT was detected by immunoblotting. F, Fli-I promotes the mDia1 activation by GTP-RhoA. The actin nucleation activity of 5 nm mDia1 CT in the absence or presence of 20 nm mDia1 NT, 400 nm Fli-I G4–6, and 100 nm RhoA G14V was evaluated by the pyrene-actin assembly assay, as indicated. The data shown in A and C are represented as means ± S.E. of five independent experiments, and the data shown in B and D–F are representative of three independent experiments with similar results. a.u., arbitrary unit.
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Figure 7: Fli-I enhances the actin assembly activity of mDia1 as well as that of Daam1. A, cytoplasmic actin assembly assay of mDia1 CT mutants. WT, wild type; LA, L1197A; FA, F1203A. B, direct binding of mDia1 to Fli-I. Beads coated with 50 pmol of GST-tagged proteins were incubated with 12.5 pmol of His6-tagged proteins as indicated. Bound proteins were analyzed by SDS-PAGE and immunoblotting (IB). C, cytoplasmic actin assembly was measured for 1 pmol of mDia1 CT in Fli-I-depleted cytosol used in Fig. 3C in the absence or presence of 125 nm purified Fli-I. pre, preimmune IgG-treated cytosol; ID, Fli-I-immunodepleted cytosol. D, concentration-dependent disruption of DID-DAD interaction of mDia1 by GTP-RhoA. Beads coated with 50 pmol of GST-mDia1 DAD were incubated with 125 nm His6-mDia1 NT in the presence of various concentrations of His6-RhoA G14V. Bead-associated His6-mDia1 NT was analyzed by immunoblotting with anti-His6 mAb. E, Fli-I enhances the DID-DAD dissociation of mDia1 by GTP-RhoA. Beads coated with 100 nm GST-mDia1 NT were incubated with 20 pmol of His6-mDia1 CT in the absence or presence of 0.1 μm His6-RhoA G14V and various concentrations of His6-Fli-I G4–6. Bead-bound His6-mDia1 CT was detected by immunoblotting. F, Fli-I promotes the mDia1 activation by GTP-RhoA. The actin nucleation activity of 5 nm mDia1 CT in the absence or presence of 20 nm mDia1 NT, 400 nm Fli-I G4–6, and 100 nm RhoA G14V was evaluated by the pyrene-actin assembly assay, as indicated. The data shown in A and C are represented as means ± S.E. of five independent experiments, and the data shown in B and D–F are representative of three independent experiments with similar results. a.u., arbitrary unit.

Mentions: Finally, we evaluated whether the regulatory mechanism of Daam1 by Fli-I is conserved in other DRFs. mDia1 is closely related to Daam1 and has a domain structure similar to that of Daam1 (Fig. 1B). We generated a carboxyl-terminal fragment protein of mDia1 (mDia1 CT) harboring L1197A mutation, which corresponds to L1040A mutation in Daam1, and we evaluated its actin assembly activity in the cytoplasmic actin assembly assay. mDia1 L1197A exhibited reduced actin assembly activity compared with mDia1 CT WT or F1203A, which corresponds to F1046A in Daam1 (Fig. 7A). The actin nucleation activity of mDia1 CT L1197A was comparable with mDia1 CT WT or F1203A in the pyrene-actin assembly assay (supplemental Fig. 1A). mDia1 CT interacted directly with Fli-I G4–6 region (Fig. 7B) dependent on the Leu-1197 residue (supplemental Fig. 1B). The immunodepletion of Fli-I from the cytosol reduced the actin assembly activity of mDia1 CT in the cytoplasmic actin assembly assay, and the activity was rescued by the addition of recombinant Fli-I (Fig. 7C). As observed for Daam1, mDia1 NT competes with Fli-I on the binding to mDia1 DAD (supplemental Fig. 1C). Although a relatively higher concentration of RhoA G14V (0.5 μm) was required for the disruption of the DID-DAD interaction of mDia1 in the absence of Fli-I (Fig. 7D), a lower concentration of RhoA G14V (0.1 μm) induced the DID-DAD dissociation in the presence of Fli-I (Fig. 7E). Finally, the addition of Fli-I G4–6 enhanced the Rho-mediated activation of the actin nucleation activity of mDia1 CT in the presence of mDia1 NT in the pyrene-actin assembly assay (Fig. 7F). These results suggest that Fli-I binds to mDia1 through the G4–6 region and that Fli-I assists GTP-Rho to release the actin assembly activity of mDia1 by disrupting the autoinhibitory interaction of mDia1. Thus, Fli-I is a common regulator of two DRF members, Daam1 and mDia1.


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 enhances the actin assembly activity of mDia1 as well as that of Daam1. A, cytoplasmic actin assembly assay of mDia1 CT mutants. WT, wild type; LA, L1197A; FA, F1203A. B, direct binding of mDia1 to Fli-I. Beads coated with 50 pmol of GST-tagged proteins were incubated with 12.5 pmol of His6-tagged proteins as indicated. Bound proteins were analyzed by SDS-PAGE and immunoblotting (IB). C, cytoplasmic actin assembly was measured for 1 pmol of mDia1 CT in Fli-I-depleted cytosol used in Fig. 3C in the absence or presence of 125 nm purified Fli-I. pre, preimmune IgG-treated cytosol; ID, Fli-I-immunodepleted cytosol. D, concentration-dependent disruption of DID-DAD interaction of mDia1 by GTP-RhoA. Beads coated with 50 pmol of GST-mDia1 DAD were incubated with 125 nm His6-mDia1 NT in the presence of various concentrations of His6-RhoA G14V. Bead-associated His6-mDia1 NT was analyzed by immunoblotting with anti-His6 mAb. E, Fli-I enhances the DID-DAD dissociation of mDia1 by GTP-RhoA. Beads coated with 100 nm GST-mDia1 NT were incubated with 20 pmol of His6-mDia1 CT in the absence or presence of 0.1 μm His6-RhoA G14V and various concentrations of His6-Fli-I G4–6. Bead-bound His6-mDia1 CT was detected by immunoblotting. F, Fli-I promotes the mDia1 activation by GTP-RhoA. The actin nucleation activity of 5 nm mDia1 CT in the absence or presence of 20 nm mDia1 NT, 400 nm Fli-I G4–6, and 100 nm RhoA G14V was evaluated by the pyrene-actin assembly assay, as indicated. The data shown in A and C are represented as means ± S.E. of five independent experiments, and the data shown in B and D–F are representative of three independent experiments with similar results. a.u., arbitrary unit.
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Figure 7: Fli-I enhances the actin assembly activity of mDia1 as well as that of Daam1. A, cytoplasmic actin assembly assay of mDia1 CT mutants. WT, wild type; LA, L1197A; FA, F1203A. B, direct binding of mDia1 to Fli-I. Beads coated with 50 pmol of GST-tagged proteins were incubated with 12.5 pmol of His6-tagged proteins as indicated. Bound proteins were analyzed by SDS-PAGE and immunoblotting (IB). C, cytoplasmic actin assembly was measured for 1 pmol of mDia1 CT in Fli-I-depleted cytosol used in Fig. 3C in the absence or presence of 125 nm purified Fli-I. pre, preimmune IgG-treated cytosol; ID, Fli-I-immunodepleted cytosol. D, concentration-dependent disruption of DID-DAD interaction of mDia1 by GTP-RhoA. Beads coated with 50 pmol of GST-mDia1 DAD were incubated with 125 nm His6-mDia1 NT in the presence of various concentrations of His6-RhoA G14V. Bead-associated His6-mDia1 NT was analyzed by immunoblotting with anti-His6 mAb. E, Fli-I enhances the DID-DAD dissociation of mDia1 by GTP-RhoA. Beads coated with 100 nm GST-mDia1 NT were incubated with 20 pmol of His6-mDia1 CT in the absence or presence of 0.1 μm His6-RhoA G14V and various concentrations of His6-Fli-I G4–6. Bead-bound His6-mDia1 CT was detected by immunoblotting. F, Fli-I promotes the mDia1 activation by GTP-RhoA. The actin nucleation activity of 5 nm mDia1 CT in the absence or presence of 20 nm mDia1 NT, 400 nm Fli-I G4–6, and 100 nm RhoA G14V was evaluated by the pyrene-actin assembly assay, as indicated. The data shown in A and C are represented as means ± S.E. of five independent experiments, and the data shown in B and D–F are representative of three independent experiments with similar results. a.u., arbitrary unit.
Mentions: Finally, we evaluated whether the regulatory mechanism of Daam1 by Fli-I is conserved in other DRFs. mDia1 is closely related to Daam1 and has a domain structure similar to that of Daam1 (Fig. 1B). We generated a carboxyl-terminal fragment protein of mDia1 (mDia1 CT) harboring L1197A mutation, which corresponds to L1040A mutation in Daam1, and we evaluated its actin assembly activity in the cytoplasmic actin assembly assay. mDia1 L1197A exhibited reduced actin assembly activity compared with mDia1 CT WT or F1203A, which corresponds to F1046A in Daam1 (Fig. 7A). The actin nucleation activity of mDia1 CT L1197A was comparable with mDia1 CT WT or F1203A in the pyrene-actin assembly assay (supplemental Fig. 1A). mDia1 CT interacted directly with Fli-I G4–6 region (Fig. 7B) dependent on the Leu-1197 residue (supplemental Fig. 1B). The immunodepletion of Fli-I from the cytosol reduced the actin assembly activity of mDia1 CT in the cytoplasmic actin assembly assay, and the activity was rescued by the addition of recombinant Fli-I (Fig. 7C). As observed for Daam1, mDia1 NT competes with Fli-I on the binding to mDia1 DAD (supplemental Fig. 1C). Although a relatively higher concentration of RhoA G14V (0.5 μm) was required for the disruption of the DID-DAD interaction of mDia1 in the absence of Fli-I (Fig. 7D), a lower concentration of RhoA G14V (0.1 μm) induced the DID-DAD dissociation in the presence of Fli-I (Fig. 7E). Finally, the addition of Fli-I G4–6 enhanced the Rho-mediated activation of the actin nucleation activity of mDia1 CT in the presence of mDia1 NT in the pyrene-actin assembly assay (Fig. 7F). These results suggest that Fli-I binds to mDia1 through the G4–6 region and that Fli-I assists GTP-Rho to release the actin assembly activity of mDia1 by disrupting the autoinhibitory interaction of mDia1. Thus, Fli-I is a common regulator of two DRF members, Daam1 and mDia1.

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
Related in: MedlinePlus