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. 2002 May 27;157(5):819-30.
doi: 10.1083/jcb.200112107. Epub 2002 May 20.

ROCK and mDia1 Antagonize in Rho-dependent Rac Activation in Swiss 3T3 Fibroblasts

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Free PMC article

ROCK and mDia1 Antagonize in Rho-dependent Rac Activation in Swiss 3T3 Fibroblasts

Takahiro Tsuji et al. J Cell Biol. .
Free PMC article

Abstract

The small GTPase Rho acts on two effectors, ROCK and mDia1, and induces stress fibers and focal adhesions. However, how ROCK and mDia1 individually regulate signals and dynamics of these structures remains unknown. We stimulated serum-starved Swiss 3T3 fibroblasts with LPA and compared the effects of C3 exoenzyme, a Rho inhibitor, with those of Y-27632, a ROCK inhibitor. Y-27632 treatment suppressed LPA-induced formation of stress fibers and focal adhesions as did C3 exoenzyme but induced membrane ruffles and focal complexes, which were absent in the C3 exoenzyme-treated cells. This phenotype was suppressed by expression of N17Rac. Consistently, the amount of GTP-Rac increased significantly by Y-27632 in LPA-stimulated cells. Biochemically, Y-27632 suppressed tyrosine phosphorylation of paxillin and focal adhesion kinase and not that of Cas. Inhibition of Cas phosphorylation with PP1 or expression of a dominant negative Cas mutant inhibited Y-27632-induced membrane ruffle formation. Moreover, Crk-II mutants lacking in binding to either phosphorylated Cas or DOCK180 suppressed the Y-27632-induced membrane ruffle formation. Finally, expression of a dominant negative mDia1 mutant also inhibited the membrane ruffle formation by Y-27632. Thus, these results have revealed the Rho-dependent Rac activation signaling that is mediated by mDia1 through Cas phosphorylation and antagonized by the action of ROCK.

Figures

Figure 1.
Figure 1.
Effects of C3 exoenzyme and Y-27632 on actin reorganization and localization of tyrosine-phosphorylated proteins in LPA-stimulated Swiss 3T3 cells. (A) Immunofluorescence. Swiss 3T3 cells were maintained in DME containing 10% FBS for 3 d and cultured in serum-free DME for 24 h. During this period, the cells were without any treatment or treated with either 30 μg/ml C3 exoenzyme for 4 d or with 30 μM Y-27632 for 30 min and then exposed to 5 μM LPA for 0 and 5 min. The cells were fixed, permeabilized, and stained with Texas red phalloidin for F-actin (red) and antiphosphotyrosine antibody (green). The top panels show the merged images of the cells without LPA stimulation. The F-actin staining, the phosphotyrosine staining, and the merged images of the LPA-stimulated cells are shown in the second, third, and the bottom rows of the panels, respectively. Note that the cells treated with Y-27632 display a thick rim of F-actin and dot-like phosphotyrosine staining in the cell periphery upon the addition of LPA. (B) Video microscopy. Swiss 3T3 cells transfected with GFP actin were serum starved for 24 h and treated with 30 μM Y-27632 for the last 30 min. LPA was added at 5 μM, and the cell shape change was monitored in the continued presence of Y-27632 by time-lapse confocal microscopy as the image of GFP actin. The number in each image indicates time after the LPA addition in min. See also the video available at http://www.jcb.org/cgi/content/full/jcb.200112107/DC1. Bars, 20 μm.
Figure 1.
Figure 1.
Effects of C3 exoenzyme and Y-27632 on actin reorganization and localization of tyrosine-phosphorylated proteins in LPA-stimulated Swiss 3T3 cells. (A) Immunofluorescence. Swiss 3T3 cells were maintained in DME containing 10% FBS for 3 d and cultured in serum-free DME for 24 h. During this period, the cells were without any treatment or treated with either 30 μg/ml C3 exoenzyme for 4 d or with 30 μM Y-27632 for 30 min and then exposed to 5 μM LPA for 0 and 5 min. The cells were fixed, permeabilized, and stained with Texas red phalloidin for F-actin (red) and antiphosphotyrosine antibody (green). The top panels show the merged images of the cells without LPA stimulation. The F-actin staining, the phosphotyrosine staining, and the merged images of the LPA-stimulated cells are shown in the second, third, and the bottom rows of the panels, respectively. Note that the cells treated with Y-27632 display a thick rim of F-actin and dot-like phosphotyrosine staining in the cell periphery upon the addition of LPA. (B) Video microscopy. Swiss 3T3 cells transfected with GFP actin were serum starved for 24 h and treated with 30 μM Y-27632 for the last 30 min. LPA was added at 5 μM, and the cell shape change was monitored in the continued presence of Y-27632 by time-lapse confocal microscopy as the image of GFP actin. The number in each image indicates time after the LPA addition in min. See also the video available at http://www.jcb.org/cgi/content/full/jcb.200112107/DC1. Bars, 20 μm.
Figure 2.
Figure 2.
LPA-induced Rac activation in Y-27632–treated Swiss 3T3 cells. (A) Inhibition of membrane ruffles by expression of N17Rac in Y-27632–treated cells. Swiss 3T3 cells were transfected with 1 μg of pCMV5–N17Rac or 1 μg of pEGFP–C1. The transfected cells were cultured in DME containing 10% FBS for 16 h and then in serum-free DME for 24 h. The cells were treated with 30 μM Y-27632 for 30 min and then stimulated with LPA for 5 min. The cells were stained for F-actin and phosphotyrosine as described in the legend to Fig. 1. Arrows indicate transfected cells identified with anti-GFP (top) or anti-Flag staining (bottom). Note that membrane ruffles and focal complexes disappeared in the cells overexpressing N17Rac. Bar, 20 μm. (B) Pull-down assay for GTP-Rac. Swiss 3T3 cells were cultured and treated with either C3 exoenzyme or Y-27632 as described in the legend to Fig. 1. The cells were then stimulated with 5 μM LPA for 0 and 5 min and subjected to the pull-down assay as described in Materials and methods. GTP-Rac precipitated from each cell lysates was analyzed by immunoblotting with anti-Rac antibody (top), and the total amounts of Rac present in the cell lysates are shown in the immunoblot in the bottom panels. −, cells without any pretreatment; Y, cells treated with Y-27632; C3, cells treated with C3 exoenzyme. Note that GTP-Rac increased in amount significantly by Y-27632 treatment and remained little in the C3 exoenzyme-treated cells.
Figure 2.
Figure 2.
LPA-induced Rac activation in Y-27632–treated Swiss 3T3 cells. (A) Inhibition of membrane ruffles by expression of N17Rac in Y-27632–treated cells. Swiss 3T3 cells were transfected with 1 μg of pCMV5–N17Rac or 1 μg of pEGFP–C1. The transfected cells were cultured in DME containing 10% FBS for 16 h and then in serum-free DME for 24 h. The cells were treated with 30 μM Y-27632 for 30 min and then stimulated with LPA for 5 min. The cells were stained for F-actin and phosphotyrosine as described in the legend to Fig. 1. Arrows indicate transfected cells identified with anti-GFP (top) or anti-Flag staining (bottom). Note that membrane ruffles and focal complexes disappeared in the cells overexpressing N17Rac. Bar, 20 μm. (B) Pull-down assay for GTP-Rac. Swiss 3T3 cells were cultured and treated with either C3 exoenzyme or Y-27632 as described in the legend to Fig. 1. The cells were then stimulated with 5 μM LPA for 0 and 5 min and subjected to the pull-down assay as described in Materials and methods. GTP-Rac precipitated from each cell lysates was analyzed by immunoblotting with anti-Rac antibody (top), and the total amounts of Rac present in the cell lysates are shown in the immunoblot in the bottom panels. −, cells without any pretreatment; Y, cells treated with Y-27632; C3, cells treated with C3 exoenzyme. Note that GTP-Rac increased in amount significantly by Y-27632 treatment and remained little in the C3 exoenzyme-treated cells.
Figure 3.
Figure 3.
Different effects of C3 exoenzyme and Y-27632 on tyrosine phosphorylation of focal adhesion proteins. (A) Effects of C3 exoenzyme and Y-27632 on LPA-induced tyrosine phosphorylation. Swiss 3T3 cells were cultured and treated either with none, C3 exoenzyme, or Y-27632 as described in the legend to Fig. 1. The cells were then stimulated with 5 μM LPA for 0, 5, and 30 min. The cell lysates were prepared and subjected to SDS-PAGE and immunoblotting using antiphosphotyrosine antibody. A single asterisk and double asterisk indicate the tyrosine-phosphorylated paxillin isoforms and the combined band of phosphorylated FAK and Cas, respectively. (B and C) Inhibition of tyrosine phosphorylation of FAK and paxillin but not that of Cas by Y-27632 treatment. The lysates of cells stimulated with LPA for 5 min were subjected to immunoprecipitation using either antipaxillin, anti-FAK, or anti-p130Cas antibodies (B) or using antiphosphotyrosine antibody (C). The immunoprecipitates were then analyzed by immunoblotting with antiphosphotyrosine antibody (B) or with antipaxillin, anti-FAK, and anti-p130Cas antibodies (C). −, cells without any pretreatment; Y, cells treated with Y-27632; C3, cells treated with C3 exoenzyme. Note that Y-27632 treatment suppressed tyrosine phosphorylation of paxillin and FAK but not that of Cas, whereas the phosphorylation of these three proteins were equally suppressed by C3 exoenzyme treatment.
Figure 3.
Figure 3.
Different effects of C3 exoenzyme and Y-27632 on tyrosine phosphorylation of focal adhesion proteins. (A) Effects of C3 exoenzyme and Y-27632 on LPA-induced tyrosine phosphorylation. Swiss 3T3 cells were cultured and treated either with none, C3 exoenzyme, or Y-27632 as described in the legend to Fig. 1. The cells were then stimulated with 5 μM LPA for 0, 5, and 30 min. The cell lysates were prepared and subjected to SDS-PAGE and immunoblotting using antiphosphotyrosine antibody. A single asterisk and double asterisk indicate the tyrosine-phosphorylated paxillin isoforms and the combined band of phosphorylated FAK and Cas, respectively. (B and C) Inhibition of tyrosine phosphorylation of FAK and paxillin but not that of Cas by Y-27632 treatment. The lysates of cells stimulated with LPA for 5 min were subjected to immunoprecipitation using either antipaxillin, anti-FAK, or anti-p130Cas antibodies (B) or using antiphosphotyrosine antibody (C). The immunoprecipitates were then analyzed by immunoblotting with antiphosphotyrosine antibody (B) or with antipaxillin, anti-FAK, and anti-p130Cas antibodies (C). −, cells without any pretreatment; Y, cells treated with Y-27632; C3, cells treated with C3 exoenzyme. Note that Y-27632 treatment suppressed tyrosine phosphorylation of paxillin and FAK but not that of Cas, whereas the phosphorylation of these three proteins were equally suppressed by C3 exoenzyme treatment.
Figure 3.
Figure 3.
Different effects of C3 exoenzyme and Y-27632 on tyrosine phosphorylation of focal adhesion proteins. (A) Effects of C3 exoenzyme and Y-27632 on LPA-induced tyrosine phosphorylation. Swiss 3T3 cells were cultured and treated either with none, C3 exoenzyme, or Y-27632 as described in the legend to Fig. 1. The cells were then stimulated with 5 μM LPA for 0, 5, and 30 min. The cell lysates were prepared and subjected to SDS-PAGE and immunoblotting using antiphosphotyrosine antibody. A single asterisk and double asterisk indicate the tyrosine-phosphorylated paxillin isoforms and the combined band of phosphorylated FAK and Cas, respectively. (B and C) Inhibition of tyrosine phosphorylation of FAK and paxillin but not that of Cas by Y-27632 treatment. The lysates of cells stimulated with LPA for 5 min were subjected to immunoprecipitation using either antipaxillin, anti-FAK, or anti-p130Cas antibodies (B) or using antiphosphotyrosine antibody (C). The immunoprecipitates were then analyzed by immunoblotting with antiphosphotyrosine antibody (B) or with antipaxillin, anti-FAK, and anti-p130Cas antibodies (C). −, cells without any pretreatment; Y, cells treated with Y-27632; C3, cells treated with C3 exoenzyme. Note that Y-27632 treatment suppressed tyrosine phosphorylation of paxillin and FAK but not that of Cas, whereas the phosphorylation of these three proteins were equally suppressed by C3 exoenzyme treatment.
Figure 4.
Figure 4.
Inhibition of LPA-induced membrane ruffles by inhibiting tyrosine phosphorylation of Cas in Y-27632–treated cells. (A and B) Effects of PP1 on tyrosine phosphorylation of Cas and membrane ruffles induced by LPA. Swiss 3T3 cells were cultured and serum starved as described in the legend to Fig. 1. The serum-starved cells were treated with either DMSO (control) or 50 μM PP1 (PP1) in the presence of 30 μM Y-27632 for 30 min and then stimulated with LPA for 5 min. The cells were then lysed for immunoprecipitation and immunoblotting analysis for tyrosine phosphorylation of Cas (A). Alternatively, the cells were fixed and stained for F-actin and phosphotyrosine as described above (B). Note that the PP1 treatment significantly inhibited Cas phosphorylation as shown by the immunoblot analysis and reduced the number of dot-like structures of tyrosine-phosphorylated proteins as illustrated by antiphosphotyrosine staining. Significant suppression of membrane ruffles is also noted in the PP1-treated cells. (C) Effects of PP1 on the level of GTP-Rac in Y-27632–treated cells. The cells were cultured and treated as described above and then subjected to the pull-down assay for GTP-Rac. Control, cells treated with Y-27632 alone; PP1, cells treated with both Y-27632 and PP1. (D) Inhibition of LPA-induced membrane ruffles by expression of CasΔSD, a tyrosine phosphorylation-defective Cas mutant, in Y-27632–treated cells. Swiss 3T3 cells were transfected with 1 μg of pSSRα–CasΔSD and cultured as described in the legend to Fig. 2 A. The cells were then treated with 30 μM Y-27632 for 30 min and stimulated with 5 μM LPA for 5 min in the continued presence of Y-27632. The cells were fixed, and F-actin and tyrosine-phosphorylated proteins were stained as described above. The cells expressing CasΔSD were identified by HA tag staining and are indicated by arrows. Bars, 20 μm.
Figure 4.
Figure 4.
Inhibition of LPA-induced membrane ruffles by inhibiting tyrosine phosphorylation of Cas in Y-27632–treated cells. (A and B) Effects of PP1 on tyrosine phosphorylation of Cas and membrane ruffles induced by LPA. Swiss 3T3 cells were cultured and serum starved as described in the legend to Fig. 1. The serum-starved cells were treated with either DMSO (control) or 50 μM PP1 (PP1) in the presence of 30 μM Y-27632 for 30 min and then stimulated with LPA for 5 min. The cells were then lysed for immunoprecipitation and immunoblotting analysis for tyrosine phosphorylation of Cas (A). Alternatively, the cells were fixed and stained for F-actin and phosphotyrosine as described above (B). Note that the PP1 treatment significantly inhibited Cas phosphorylation as shown by the immunoblot analysis and reduced the number of dot-like structures of tyrosine-phosphorylated proteins as illustrated by antiphosphotyrosine staining. Significant suppression of membrane ruffles is also noted in the PP1-treated cells. (C) Effects of PP1 on the level of GTP-Rac in Y-27632–treated cells. The cells were cultured and treated as described above and then subjected to the pull-down assay for GTP-Rac. Control, cells treated with Y-27632 alone; PP1, cells treated with both Y-27632 and PP1. (D) Inhibition of LPA-induced membrane ruffles by expression of CasΔSD, a tyrosine phosphorylation-defective Cas mutant, in Y-27632–treated cells. Swiss 3T3 cells were transfected with 1 μg of pSSRα–CasΔSD and cultured as described in the legend to Fig. 2 A. The cells were then treated with 30 μM Y-27632 for 30 min and stimulated with 5 μM LPA for 5 min in the continued presence of Y-27632. The cells were fixed, and F-actin and tyrosine-phosphorylated proteins were stained as described above. The cells expressing CasΔSD were identified by HA tag staining and are indicated by arrows. Bars, 20 μm.
Figure 4.
Figure 4.
Inhibition of LPA-induced membrane ruffles by inhibiting tyrosine phosphorylation of Cas in Y-27632–treated cells. (A and B) Effects of PP1 on tyrosine phosphorylation of Cas and membrane ruffles induced by LPA. Swiss 3T3 cells were cultured and serum starved as described in the legend to Fig. 1. The serum-starved cells were treated with either DMSO (control) or 50 μM PP1 (PP1) in the presence of 30 μM Y-27632 for 30 min and then stimulated with LPA for 5 min. The cells were then lysed for immunoprecipitation and immunoblotting analysis for tyrosine phosphorylation of Cas (A). Alternatively, the cells were fixed and stained for F-actin and phosphotyrosine as described above (B). Note that the PP1 treatment significantly inhibited Cas phosphorylation as shown by the immunoblot analysis and reduced the number of dot-like structures of tyrosine-phosphorylated proteins as illustrated by antiphosphotyrosine staining. Significant suppression of membrane ruffles is also noted in the PP1-treated cells. (C) Effects of PP1 on the level of GTP-Rac in Y-27632–treated cells. The cells were cultured and treated as described above and then subjected to the pull-down assay for GTP-Rac. Control, cells treated with Y-27632 alone; PP1, cells treated with both Y-27632 and PP1. (D) Inhibition of LPA-induced membrane ruffles by expression of CasΔSD, a tyrosine phosphorylation-defective Cas mutant, in Y-27632–treated cells. Swiss 3T3 cells were transfected with 1 μg of pSSRα–CasΔSD and cultured as described in the legend to Fig. 2 A. The cells were then treated with 30 μM Y-27632 for 30 min and stimulated with 5 μM LPA for 5 min in the continued presence of Y-27632. The cells were fixed, and F-actin and tyrosine-phosphorylated proteins were stained as described above. The cells expressing CasΔSD were identified by HA tag staining and are indicated by arrows. Bars, 20 μm.
Figure 4.
Figure 4.
Inhibition of LPA-induced membrane ruffles by inhibiting tyrosine phosphorylation of Cas in Y-27632–treated cells. (A and B) Effects of PP1 on tyrosine phosphorylation of Cas and membrane ruffles induced by LPA. Swiss 3T3 cells were cultured and serum starved as described in the legend to Fig. 1. The serum-starved cells were treated with either DMSO (control) or 50 μM PP1 (PP1) in the presence of 30 μM Y-27632 for 30 min and then stimulated with LPA for 5 min. The cells were then lysed for immunoprecipitation and immunoblotting analysis for tyrosine phosphorylation of Cas (A). Alternatively, the cells were fixed and stained for F-actin and phosphotyrosine as described above (B). Note that the PP1 treatment significantly inhibited Cas phosphorylation as shown by the immunoblot analysis and reduced the number of dot-like structures of tyrosine-phosphorylated proteins as illustrated by antiphosphotyrosine staining. Significant suppression of membrane ruffles is also noted in the PP1-treated cells. (C) Effects of PP1 on the level of GTP-Rac in Y-27632–treated cells. The cells were cultured and treated as described above and then subjected to the pull-down assay for GTP-Rac. Control, cells treated with Y-27632 alone; PP1, cells treated with both Y-27632 and PP1. (D) Inhibition of LPA-induced membrane ruffles by expression of CasΔSD, a tyrosine phosphorylation-defective Cas mutant, in Y-27632–treated cells. Swiss 3T3 cells were transfected with 1 μg of pSSRα–CasΔSD and cultured as described in the legend to Fig. 2 A. The cells were then treated with 30 μM Y-27632 for 30 min and stimulated with 5 μM LPA for 5 min in the continued presence of Y-27632. The cells were fixed, and F-actin and tyrosine-phosphorylated proteins were stained as described above. The cells expressing CasΔSD were identified by HA tag staining and are indicated by arrows. Bars, 20 μm.
Figure 5.
Figure 5.
Cas–Crk interaction in LPA-induced membrane ruffles in Y-27632–treated cells. (A) Inhibition of the ruffle formation by expression of CrkII mutants. The cells were transfected either with 1 μg of pEEB–CrkII(R38K) and 0.1 μg of pEGFP–C1 (top) or with 1 μg of pEEB–CrkII(W170K) and 0.1 μg of pEGFP–C1 (bottom) and cultured as described in the legend to Fig. 2 A. The cells were then treated with 30 μM Y-27632 for 30 min and stimulated with 5 μM LPA for 5 min in the continued presence of Y-27632. The cells were fixed, and F-actin and tyrosine-phosphorylated proteins were stained as described above. The cells expressing each CrkII mutant were identified with anti-GFP staining and are indicated by arrows. Arrowheads indicate nontransfected cells. Note that the cell bodies of the cells expressing these CrkII mutants retracted, and the dot-like structure of tyrosine-phosphorylated proteins disappeared in the cell periphery. Bar, 20 μm. (B) Rho-dependent formation of the Cas–Crk complex in LPA-stimulated cells. Swiss 3T3 cells were cultured and treated with indicated reagents as described in the legends for Figs. 1 and 4. After stimulated with LPA for 0 and 5 min, the cells were lysed and subjected to immunoprecipitation with anti-Crk mAb. The immunoprecipitates were then analyzed by Western blot analysis with anti-Cas antibody.
Figure 5.
Figure 5.
Cas–Crk interaction in LPA-induced membrane ruffles in Y-27632–treated cells. (A) Inhibition of the ruffle formation by expression of CrkII mutants. The cells were transfected either with 1 μg of pEEB–CrkII(R38K) and 0.1 μg of pEGFP–C1 (top) or with 1 μg of pEEB–CrkII(W170K) and 0.1 μg of pEGFP–C1 (bottom) and cultured as described in the legend to Fig. 2 A. The cells were then treated with 30 μM Y-27632 for 30 min and stimulated with 5 μM LPA for 5 min in the continued presence of Y-27632. The cells were fixed, and F-actin and tyrosine-phosphorylated proteins were stained as described above. The cells expressing each CrkII mutant were identified with anti-GFP staining and are indicated by arrows. Arrowheads indicate nontransfected cells. Note that the cell bodies of the cells expressing these CrkII mutants retracted, and the dot-like structure of tyrosine-phosphorylated proteins disappeared in the cell periphery. Bar, 20 μm. (B) Rho-dependent formation of the Cas–Crk complex in LPA-stimulated cells. Swiss 3T3 cells were cultured and treated with indicated reagents as described in the legends for Figs. 1 and 4. After stimulated with LPA for 0 and 5 min, the cells were lysed and subjected to immunoprecipitation with anti-Crk mAb. The immunoprecipitates were then analyzed by Western blot analysis with anti-Cas antibody.
Figure 6.
Figure 6.
Effects of various mDia1 fragments on the mDia1ΔN3 phenotype in HeLa cells. (A) Schematic diagram of the structures of mDia1 and truncation mutants. The domain structure of mDia1 is shown above. The Rho-binding domain and the FH1 and FH2 regions are shown by hatched boxes. The FH3 region that partially overlaps with the Rho-binding domain and the two coiled-coil regions are indicated by a thick line and two-headed arrows, respectively. Truncation mutants, ΔN3, H + P, F2. ΔN3(HindIII) and CC are shown by the lines below. Numbers indicate the amino acid number at the NH2 and COOH terminals of each protein and domain. (B and C) Effects of various fragments on the mDia1ΔN3 phenotype. HeLa cells were transfected with 0.2 μg of pFL–mDia1ΔN3 either alone or with 2 μg of pEGFP plasmid encoding either mDia1 H + P, F2, ΔN3(HindIII), or CC mutant. After the culture for 24 h, the cells were fixed and stained for F-actin (top) and β-tubulin (bottom) (B). Arrows indicate the cells expressing each construct. The ratios of the long versus short axis were measured in the cells expressing each construct and are shown (C). Note that expression of either H + P, F2, or ΔN3(HindIII) inhibits the cell elongation and the parallel alignment of F-actin bundles and MTs induced by mDia1ΔN3.
Figure 6.
Figure 6.
Effects of various mDia1 fragments on the mDia1ΔN3 phenotype in HeLa cells. (A) Schematic diagram of the structures of mDia1 and truncation mutants. The domain structure of mDia1 is shown above. The Rho-binding domain and the FH1 and FH2 regions are shown by hatched boxes. The FH3 region that partially overlaps with the Rho-binding domain and the two coiled-coil regions are indicated by a thick line and two-headed arrows, respectively. Truncation mutants, ΔN3, H + P, F2. ΔN3(HindIII) and CC are shown by the lines below. Numbers indicate the amino acid number at the NH2 and COOH terminals of each protein and domain. (B and C) Effects of various fragments on the mDia1ΔN3 phenotype. HeLa cells were transfected with 0.2 μg of pFL–mDia1ΔN3 either alone or with 2 μg of pEGFP plasmid encoding either mDia1 H + P, F2, ΔN3(HindIII), or CC mutant. After the culture for 24 h, the cells were fixed and stained for F-actin (top) and β-tubulin (bottom) (B). Arrows indicate the cells expressing each construct. The ratios of the long versus short axis were measured in the cells expressing each construct and are shown (C). Note that expression of either H + P, F2, or ΔN3(HindIII) inhibits the cell elongation and the parallel alignment of F-actin bundles and MTs induced by mDia1ΔN3.
Figure 6.
Figure 6.
Effects of various mDia1 fragments on the mDia1ΔN3 phenotype in HeLa cells. (A) Schematic diagram of the structures of mDia1 and truncation mutants. The domain structure of mDia1 is shown above. The Rho-binding domain and the FH1 and FH2 regions are shown by hatched boxes. The FH3 region that partially overlaps with the Rho-binding domain and the two coiled-coil regions are indicated by a thick line and two-headed arrows, respectively. Truncation mutants, ΔN3, H + P, F2. ΔN3(HindIII) and CC are shown by the lines below. Numbers indicate the amino acid number at the NH2 and COOH terminals of each protein and domain. (B and C) Effects of various fragments on the mDia1ΔN3 phenotype. HeLa cells were transfected with 0.2 μg of pFL–mDia1ΔN3 either alone or with 2 μg of pEGFP plasmid encoding either mDia1 H + P, F2, ΔN3(HindIII), or CC mutant. After the culture for 24 h, the cells were fixed and stained for F-actin (top) and β-tubulin (bottom) (B). Arrows indicate the cells expressing each construct. The ratios of the long versus short axis were measured in the cells expressing each construct and are shown (C). Note that expression of either H + P, F2, or ΔN3(HindIII) inhibits the cell elongation and the parallel alignment of F-actin bundles and MTs induced by mDia1ΔN3.
Figure 7.
Figure 7.
Inhibition of LPA-induced membrane ruffles by expression of mDia1ΔN3(HindIII) in Y-27632–treated cells. Swiss 3T3 cells were transfected with 1 μg of pEGFP plasmid encoding ΔN3(HindIII) and cultured as described in the legend to Fig. 2 A. The cells were treated with 30 μM Y-27632 for 30 min and then stimulated with 5 μM LPA for 5 min in the continued presence of Y-27632. Alternatively, the cells were stimulated with 5 ng/ml PDGF for 10 min without Y-27632 or LPA treatment. The cells were fixed, and F-actin and tyrosine-phosphorylated proteins were stained as described above. Left and middle panels show the cells stimulated with Y-27632 and LPA. The right panel shows cells stimulated with PDGF. Arrows indicate the cells expressing GFP–ΔN3–HindIII as identified by anti-GFP antibody staining. Arrowheads indicate nontransfected cells. Note that LPA-induced membrane ruffle formation is significantly suppressed by expression of mDia1–ΔN3(HindIII). Bar, 20 μm.
Figure 8.
Figure 8.
Effects of nocodazole on Y-27632–induced membrane ruffles. Swiss 3T3 cells were cultured in DME containing 10% FBS and treated with or without 30 μM Y-27632 in the presence or absence of 100 ng/ml nocodazole for 30 min. The cells were fixed and then stained for F-actin (top) and β-tubulin (bottom). Note that treatment of Y-27632 induced membrane ruffles at the tips of cells and that the addition of nocodazole inhibited the Y-27632–induced membrane ruffles. Bar, 20 μm.

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