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TSC2 modulates actin cytoskeleton and focal adhesion through TSC1-binding domain and the Rac1 GTPase.

Goncharova E, Goncharov D, Noonan D, Krymskaya VP - J. Cell Biol. (2004)

Bottom Line: Tuberous sclerosis complex (TSC) 1 and TSC2 are thought to be involved in protein translational regulation and cell growth, and loss of their function is a cause of TSC and lymphangioleiomyomatosis (LAM).The down-regulation of TSC1 with TSC1 siRNA in TSC2-/- cells activated Rac1 and induced loss of stress fibers.Our data indicate that TSC1 inhibits Rac1 and TSC2 blocks this activity of TSC1.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

ABSTRACT
Tuberous sclerosis complex (TSC) 1 and TSC2 are thought to be involved in protein translational regulation and cell growth, and loss of their function is a cause of TSC and lymphangioleiomyomatosis (LAM). However, TSC1 also activates Rho and regulates cell adhesion. We found that TSC2 modulates actin dynamics and cell adhesion and the TSC1-binding domain (TSC2-HBD) is essential for this function of TSC2. Expression of TSC2 or TSC2-HBD in TSC2-/- cells promoted Rac1 activation, inhibition of Rho, stress fiber disassembly, and focal adhesion remodeling. The down-regulation of TSC1 with TSC1 siRNA in TSC2-/- cells activated Rac1 and induced loss of stress fibers. Our data indicate that TSC1 inhibits Rac1 and TSC2 blocks this activity of TSC1. Because TSC1 and TSC2 regulate Rho and Rac1, whose activities are interconnected in a reciprocal fashion, loss of either TSC1 or TSC2 function may result in the deregulation of cell motility and adhesion, which are associated with the pathobiology of TSC and LAM.

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TSC2, TSC2-HBD, and siRNA TSC1 promote stress fiber disassembly. (A, left) F-actin staining (red) of TSC2−/− cells transfected with GFP-TSC2 or the indicated GFP-tagged TSC2 constructs identified by immunostaining with anti-GFP antibody (green) or microinjected with siRNA TSC1 and GST to identify injected cells (green). (right) Schematic representation of TSC2 constructs. Bar, 20 μm. (B) Quantitative analysis of F-actin staining. Data represent the percentage of cells with stress fibers per total number of cells transfected with pEGFP, pEGFP-TSC2, pEGFP-TSC2-HBD, or pEGFP-TSC2-ΔHBD taken as 100%. A total of 205 of GFP-TSC2–transfected, 360 of GFP-TSC2-HBD–transfected, 302 of GFP-TSC2-ΔHBD–transfected, 331 of GFP-transfected, and 644 siRNA TSC1-microinjected cells were analyzed from three independent experiments. Data represent the mean ± SE. *, P < 0.0001 for GFP-TSC2–, GFP-TSC2-HBD–transfected cells, or siRNA TSC1 + GST-microinjected cells versus GFP-transfected cells by ANOVA (Bonferroni-Dunn test). (C) Statistical analysis of F-actin staining of LAMD cells transfected with GFP, GFP-TSC2, GFP-TSC2-HBD, and GFP-TSC2-ΔHBD. Data represent the mean ± SE from two independent experiments. *, P < 0.001 for GFP-TSC2– and GFP-TSC2-HBD–transfected cells versus GFP-transfected cells by ANOVA (Bonferroni-Dunn test).
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fig4: TSC2, TSC2-HBD, and siRNA TSC1 promote stress fiber disassembly. (A, left) F-actin staining (red) of TSC2−/− cells transfected with GFP-TSC2 or the indicated GFP-tagged TSC2 constructs identified by immunostaining with anti-GFP antibody (green) or microinjected with siRNA TSC1 and GST to identify injected cells (green). (right) Schematic representation of TSC2 constructs. Bar, 20 μm. (B) Quantitative analysis of F-actin staining. Data represent the percentage of cells with stress fibers per total number of cells transfected with pEGFP, pEGFP-TSC2, pEGFP-TSC2-HBD, or pEGFP-TSC2-ΔHBD taken as 100%. A total of 205 of GFP-TSC2–transfected, 360 of GFP-TSC2-HBD–transfected, 302 of GFP-TSC2-ΔHBD–transfected, 331 of GFP-transfected, and 644 siRNA TSC1-microinjected cells were analyzed from three independent experiments. Data represent the mean ± SE. *, P < 0.0001 for GFP-TSC2–, GFP-TSC2-HBD–transfected cells, or siRNA TSC1 + GST-microinjected cells versus GFP-transfected cells by ANOVA (Bonferroni-Dunn test). (C) Statistical analysis of F-actin staining of LAMD cells transfected with GFP, GFP-TSC2, GFP-TSC2-HBD, and GFP-TSC2-ΔHBD. Data represent the mean ± SE from two independent experiments. *, P < 0.001 for GFP-TSC2– and GFP-TSC2-HBD–transfected cells versus GFP-transfected cells by ANOVA (Bonferroni-Dunn test).

Mentions: To examine whether TSC2 is required for actin remodeling, and to identify which domain of TSC2 is important for stress fiber disassembly, we tested a panel of GFP-tagged deletion constructs of TSC2 (Fig. 3). The reexpression of full-length TSC2 in TSC2−/− cells markedly promoted stress fiber disassembly and the formation of cortical actin compared with cells transfected with control GFP (Fig. 4 A). Interestingly, the expression of TSC2-ΔHBD in TSC2−/− cells containing the Rheb GAP domain (Fig. 4 A) or the expression of C-TSC2 (not depicted) had little effect on stress fiber disassembly. In contrast, expression of N-TSC2 (not depicted) or TSC2-HBD was sufficient to induce stress fiber disassembly (Fig. 4 A).


TSC2 modulates actin cytoskeleton and focal adhesion through TSC1-binding domain and the Rac1 GTPase.

Goncharova E, Goncharov D, Noonan D, Krymskaya VP - J. Cell Biol. (2004)

TSC2, TSC2-HBD, and siRNA TSC1 promote stress fiber disassembly. (A, left) F-actin staining (red) of TSC2−/− cells transfected with GFP-TSC2 or the indicated GFP-tagged TSC2 constructs identified by immunostaining with anti-GFP antibody (green) or microinjected with siRNA TSC1 and GST to identify injected cells (green). (right) Schematic representation of TSC2 constructs. Bar, 20 μm. (B) Quantitative analysis of F-actin staining. Data represent the percentage of cells with stress fibers per total number of cells transfected with pEGFP, pEGFP-TSC2, pEGFP-TSC2-HBD, or pEGFP-TSC2-ΔHBD taken as 100%. A total of 205 of GFP-TSC2–transfected, 360 of GFP-TSC2-HBD–transfected, 302 of GFP-TSC2-ΔHBD–transfected, 331 of GFP-transfected, and 644 siRNA TSC1-microinjected cells were analyzed from three independent experiments. Data represent the mean ± SE. *, P < 0.0001 for GFP-TSC2–, GFP-TSC2-HBD–transfected cells, or siRNA TSC1 + GST-microinjected cells versus GFP-transfected cells by ANOVA (Bonferroni-Dunn test). (C) Statistical analysis of F-actin staining of LAMD cells transfected with GFP, GFP-TSC2, GFP-TSC2-HBD, and GFP-TSC2-ΔHBD. Data represent the mean ± SE from two independent experiments. *, P < 0.001 for GFP-TSC2– and GFP-TSC2-HBD–transfected cells versus GFP-transfected cells by ANOVA (Bonferroni-Dunn test).
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fig4: TSC2, TSC2-HBD, and siRNA TSC1 promote stress fiber disassembly. (A, left) F-actin staining (red) of TSC2−/− cells transfected with GFP-TSC2 or the indicated GFP-tagged TSC2 constructs identified by immunostaining with anti-GFP antibody (green) or microinjected with siRNA TSC1 and GST to identify injected cells (green). (right) Schematic representation of TSC2 constructs. Bar, 20 μm. (B) Quantitative analysis of F-actin staining. Data represent the percentage of cells with stress fibers per total number of cells transfected with pEGFP, pEGFP-TSC2, pEGFP-TSC2-HBD, or pEGFP-TSC2-ΔHBD taken as 100%. A total of 205 of GFP-TSC2–transfected, 360 of GFP-TSC2-HBD–transfected, 302 of GFP-TSC2-ΔHBD–transfected, 331 of GFP-transfected, and 644 siRNA TSC1-microinjected cells were analyzed from three independent experiments. Data represent the mean ± SE. *, P < 0.0001 for GFP-TSC2–, GFP-TSC2-HBD–transfected cells, or siRNA TSC1 + GST-microinjected cells versus GFP-transfected cells by ANOVA (Bonferroni-Dunn test). (C) Statistical analysis of F-actin staining of LAMD cells transfected with GFP, GFP-TSC2, GFP-TSC2-HBD, and GFP-TSC2-ΔHBD. Data represent the mean ± SE from two independent experiments. *, P < 0.001 for GFP-TSC2– and GFP-TSC2-HBD–transfected cells versus GFP-transfected cells by ANOVA (Bonferroni-Dunn test).
Mentions: To examine whether TSC2 is required for actin remodeling, and to identify which domain of TSC2 is important for stress fiber disassembly, we tested a panel of GFP-tagged deletion constructs of TSC2 (Fig. 3). The reexpression of full-length TSC2 in TSC2−/− cells markedly promoted stress fiber disassembly and the formation of cortical actin compared with cells transfected with control GFP (Fig. 4 A). Interestingly, the expression of TSC2-ΔHBD in TSC2−/− cells containing the Rheb GAP domain (Fig. 4 A) or the expression of C-TSC2 (not depicted) had little effect on stress fiber disassembly. In contrast, expression of N-TSC2 (not depicted) or TSC2-HBD was sufficient to induce stress fiber disassembly (Fig. 4 A).

Bottom Line: Tuberous sclerosis complex (TSC) 1 and TSC2 are thought to be involved in protein translational regulation and cell growth, and loss of their function is a cause of TSC and lymphangioleiomyomatosis (LAM).The down-regulation of TSC1 with TSC1 siRNA in TSC2-/- cells activated Rac1 and induced loss of stress fibers.Our data indicate that TSC1 inhibits Rac1 and TSC2 blocks this activity of TSC1.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

ABSTRACT
Tuberous sclerosis complex (TSC) 1 and TSC2 are thought to be involved in protein translational regulation and cell growth, and loss of their function is a cause of TSC and lymphangioleiomyomatosis (LAM). However, TSC1 also activates Rho and regulates cell adhesion. We found that TSC2 modulates actin dynamics and cell adhesion and the TSC1-binding domain (TSC2-HBD) is essential for this function of TSC2. Expression of TSC2 or TSC2-HBD in TSC2-/- cells promoted Rac1 activation, inhibition of Rho, stress fiber disassembly, and focal adhesion remodeling. The down-regulation of TSC1 with TSC1 siRNA in TSC2-/- cells activated Rac1 and induced loss of stress fibers. Our data indicate that TSC1 inhibits Rac1 and TSC2 blocks this activity of TSC1. Because TSC1 and TSC2 regulate Rho and Rac1, whose activities are interconnected in a reciprocal fashion, loss of either TSC1 or TSC2 function may result in the deregulation of cell motility and adhesion, which are associated with the pathobiology of TSC and LAM.

Show MeSH
Related in: MedlinePlus