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First published online 16 January 2007
doi: 10.1242/jcs.03353

Journal of Cell Science 120, 456-467 (2007)
Published by The Company of Biologists 2007
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Filamin links cell shape and cytoskeletal structure to Rho regulation by controlling accumulation of p190RhoGAP in lipid rafts

Akiko Mammoto, Sui Huang and Donald E. Ingber*

Vascular Biology Program, Departments of Pathology and Surgery, Children's Hospital and Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115, USA


Figure 1
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  		Fig. 1. Actin cytoskeleton configuration regulates Rho activity in HMVE cells. (A) Time course of Rho activity in cells plated on fibronectin-coated dishes in the presence or absence of cytoD (upper panel), and comparison of Rho activity in spread cells versus round cells 1.5 hours after replating (lower panel). Rho activity is presented as the ratio of active RhoA (determined using the rhotekin-RBD bead pull down assay) to total RhoA protein in each cell lysate (upper panel, *P<0.05 for Rho activity in spread versus cytoD-treated cells; +P<0.02 for Rho activity at 1 hour after plating versus time zero. Lower panel, *P<0.01). (B) Phase contrast and fluorescence images of cells stained for F-actin showing cell morphology and the actin cytoskeleton of HMVE cells in the presence or absence of cytoD (scale bar, 5 µm).

 

Figure 2
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  		Fig. 2. Actin cytoskeleton configuration regulates p190RhoGAP localization. (A) Levels of active RhoA in p190RhoGAP knockdown HMVE cells in the presence or absence of cytoD 1 hour after replating on fibronectin-coated plates. Quantitative results were normalized to spread control cells without cyto D (*P<0.01). (B) Immunoblots showing lipid raft localization of p190RhoGAP in spread versus cytoD-treated cells based on the detergent insoluble membrane purification method. Data are presented as the ratio of p190RhoGAP in the insoluble TrX fraction to the total protein level (*P<0.01). (C) Localization of p190RhoGAP in spread, suspended, and cytoD-, lat B-, or methyl β-cyclodextrin treated cells based on the detergent-free sucrose gradient floating assay. Data are presented as the ratio of p190RhoGAP in the lipid raft fractions (fractions 4 and 5) to total p190RhoGAP protein distributed throughout the entire gradient (*P<0.01; **P<0.02).

 

Figure 3
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  		Fig. 3. Cell shape effects on the phosphorylation status of p190RhoGAP. (A) Immunoblots showing (a) tyrosine phosphorylated p190RhoGAP detected by phosphotyrosine antibody (4G10) or (b) p190RhoGAP immunoprecipitated with phosphotyrosine antibody (PY20) from total cell lysates of spread, cyto D-, or PP2-treated cells (*P<0.01). (B) Tyrosine phosphorylated p190RhoGAP detected by phosphotyrosine antibody (4G10) in TrX-100-soluble and -insoluble fraction isolated from spread versus cyto D-treated cells. (C) Raft localization of p190RhoGAP and Src tyrosine kinase in spread versus PP2-treated cells, using methods shown in Fig. 2C.

 

Figure 4
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  		Fig. 4. Interactions between p190RhoGAP and filamin. Immunoblots of p190RhoGAP coimmunoprecipitated with anti-filamin antibody (mAb 1678) and vice versa. Mouse IgG was used as a control for immunoprecipitation. Caveolin-1 and TFII-I were used as a positive control for filamin and p190RhoGAP immunoprecipitation respectively.

 

Figure 5
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  		Fig. 5. p190RhoGAP localization in filamin null (M2) and repleted (A7) human melanoma cells. (A) DIC and fluorescence micrographs illustrating cell shape, stress fiber organization and focal adhesion formation in spread or cytoD-treated A7 and M2 cells. F-actin was visualized by staining with Alexa-488-phalloidin; focal adhesions were stained with an anti-paxillin antibody (bar, 5 µm). (B) Raft-localization of p190RhoGAP in spread, suspended, or cytoD-treated A7 and M2 cells. Quantitative results were normalized to spread A7 cells (*P<0.01). (C) Immunoblots showing phosphorylated p190RhoGAP as detected by immunoprecipitation with PY20 antibody in spread, suspended, or cytoD-treated A7 and M2 cells. Quantitative results were normalized to spread A7 cells (*P<0.03, **P<0.05). (D) Levels of active RhoA in spread, suspended, or cyto D-treated A7 and M2 cells 1 hour after replating. Quantitative results were normalized to spread A7 cells (*P<0.01, **P<0.03).

 

Figure 6
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  		Fig. 6. p190RhoGAP localization in filamin knockdown HMVE cells. (A) Filamin expression in siRNA-mediated filamin knockdown HMVE cells. (B,C) Raft-localization of p190RhoGAP and filamin in spread versus cytoD-treated filamin-knockdown HMVE cells. Quantitative results were normalized to spread control cells without cyto D (*P<0.02).

 

Figure 7
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  		Fig. 7. Filamin cleavage controls the localization of p190RhoGAP. (A) Raft-localization and cleavage of filamin in HMVE cells that were cultured in suspension (Susp) or plated on fibronectin in the absence (Spread) or presence of cytochalasin D (CytoD), latrunculinB (LatB), or ALLN. (B) Raft-localization of p190RhoGAP in control versus ALLN-treated filamin knockdown cells. Quantitative results were normalized to spread control cells (*P<0.01). (C) Filamin protein in total cell lysate from M2 cells transfected with intact GFP-filamin (GFP-fil) or a GFP-filamin-{Delta} (GFP-fil-{Delta}) construct that lacks the calpain cleavage site. (D) Raft-localization of p190RhoGAP in total cell population of M2 cells transfected with GFP-fil or GFP-fil-{Delta}. (E) Levels of active RhoA in total cell population of M2 cells transfected with GFP-fil or GFP-fil-{Delta} (*P<0.02).

 

Figure 8
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  		Fig. 8. p190RhoGAP localization in FAK knockdown HMVE cells. (A) FAK expression in HMVE cells transfected with FAK siRNA. (B) Raft-localization of p190RhoGAP in FAK-knockdown cells, as in Fig. 2C. (C) Tyrosine phosphorylation of p190RhoGAP in FAK-knockdown cells.

 

Figure 9
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  		Fig. 9. Model for cell shape-dependent control of Rho activity. (A) Round cells: intact filamin protein binds p190RhoGAP and prevents its accumulation in lipid rafts. p190RhoGAP is inactive and hence Rho activity is high. (B) Spread cells: filamin is cleaved by calpain, thereby releasing p190RhoGAP and allowing it to accumulate in lipid rafts where it becomes activated and suppresses Rho activity.

 

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© The Company of Biologists Ltd 2007