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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 modulate focal adhesion formation. (A) Cells were transfected with control pEGFP (top), pEGFP-TSC2 (middle), or pEGFP-TSC2-HBD (bottom) plasmid, and then immunostained with anti-GFP (green) and anti-vinculin (red) antibodies to detect focal adhesions. Images are representative of three separate experiments. (B) Cells were transfected with control GST, GST-tagged V12Rac1, or siRNA TSC1 comicroinjected with GST to identify microinjected cells, followed by immunostaining with anti-GST (green) and anti-vinculin (red) antibodies. Representative images from 138 microinjected cells. Changes in the shape and size of the focal adhesion are represented in enlarged insets (1–3). Bars: (A and B) 30 μm; (insets) 10 μm.
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fig5: TSC2, TSC2-HBD, and siRNA TSC1 modulate focal adhesion formation. (A) Cells were transfected with control pEGFP (top), pEGFP-TSC2 (middle), or pEGFP-TSC2-HBD (bottom) plasmid, and then immunostained with anti-GFP (green) and anti-vinculin (red) antibodies to detect focal adhesions. Images are representative of three separate experiments. (B) Cells were transfected with control GST, GST-tagged V12Rac1, or siRNA TSC1 comicroinjected with GST to identify microinjected cells, followed by immunostaining with anti-GST (green) and anti-vinculin (red) antibodies. Representative images from 138 microinjected cells. Changes in the shape and size of the focal adhesion are represented in enlarged insets (1–3). Bars: (A and B) 30 μm; (insets) 10 μm.

Mentions: Because actin rearrangements are accompanied by focal adhesion remodeling, we examined whether or not TSC2-induced stress fiber disassembly correlates with changes in focal adhesion formation. Immunohistochemical analysis with anti-vinculin antibody showed that in TSC2−/− cells focal adhesions were localized throughout the cell as well as at the cell periphery (Fig. 5 A, top; and Video 6, available at http://www.jcb.org/cgi/content/full/jcb.200405130/DC1). Expression of TSC2 promoted marked changes in the shape and size of the focal adhesions: most focal adhesions in the center of the cells were disassembled, and the quantity of focal adhesions per cell was attenuated by 30.3 ± 5.9% compared with control. At the same time, the size of focal adhesions at the cell periphery was also markedly decreased (Fig. 5 A, middle; and Video 7, available at http://www.jcb.org/cgi/content/full/jcb.200405130/DC1). Importantly, TSC2-HBD, which is involved in TSC1–TSC2 complex formation, also promoted focal adhesion disassembly in the center of the cells (Fig. 5 A, bottom; and Video 8, available at http://www.jcb.org/cgi/content/full/jcb.200405130/DC1). TSC1 regulates cell adhesion, and its inactivation results in the loss of focal adhesion (Lamb et al., 2000). To determine if focal adhesion remodeling in TSC2−/− cells requires TSC1, we microinjected siRNA TSC1 and found that down-regulation of TSC1 promoted focal adhesion disassembly in TSC2−/− cells (Fig. 5 B). These results demonstrate that expression of TSC2-HBD and the down-regulation of TSC1 with siRNA TSC1 are sufficient for focal adhesion disassembly, which suggest that TSC2 involves TSC1 in regulating focal adhesion remodeling.


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 modulate focal adhesion formation. (A) Cells were transfected with control pEGFP (top), pEGFP-TSC2 (middle), or pEGFP-TSC2-HBD (bottom) plasmid, and then immunostained with anti-GFP (green) and anti-vinculin (red) antibodies to detect focal adhesions. Images are representative of three separate experiments. (B) Cells were transfected with control GST, GST-tagged V12Rac1, or siRNA TSC1 comicroinjected with GST to identify microinjected cells, followed by immunostaining with anti-GST (green) and anti-vinculin (red) antibodies. Representative images from 138 microinjected cells. Changes in the shape and size of the focal adhesion are represented in enlarged insets (1–3). Bars: (A and B) 30 μm; (insets) 10 μm.
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Related In: Results  -  Collection

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fig5: TSC2, TSC2-HBD, and siRNA TSC1 modulate focal adhesion formation. (A) Cells were transfected with control pEGFP (top), pEGFP-TSC2 (middle), or pEGFP-TSC2-HBD (bottom) plasmid, and then immunostained with anti-GFP (green) and anti-vinculin (red) antibodies to detect focal adhesions. Images are representative of three separate experiments. (B) Cells were transfected with control GST, GST-tagged V12Rac1, or siRNA TSC1 comicroinjected with GST to identify microinjected cells, followed by immunostaining with anti-GST (green) and anti-vinculin (red) antibodies. Representative images from 138 microinjected cells. Changes in the shape and size of the focal adhesion are represented in enlarged insets (1–3). Bars: (A and B) 30 μm; (insets) 10 μm.
Mentions: Because actin rearrangements are accompanied by focal adhesion remodeling, we examined whether or not TSC2-induced stress fiber disassembly correlates with changes in focal adhesion formation. Immunohistochemical analysis with anti-vinculin antibody showed that in TSC2−/− cells focal adhesions were localized throughout the cell as well as at the cell periphery (Fig. 5 A, top; and Video 6, available at http://www.jcb.org/cgi/content/full/jcb.200405130/DC1). Expression of TSC2 promoted marked changes in the shape and size of the focal adhesions: most focal adhesions in the center of the cells were disassembled, and the quantity of focal adhesions per cell was attenuated by 30.3 ± 5.9% compared with control. At the same time, the size of focal adhesions at the cell periphery was also markedly decreased (Fig. 5 A, middle; and Video 7, available at http://www.jcb.org/cgi/content/full/jcb.200405130/DC1). Importantly, TSC2-HBD, which is involved in TSC1–TSC2 complex formation, also promoted focal adhesion disassembly in the center of the cells (Fig. 5 A, bottom; and Video 8, available at http://www.jcb.org/cgi/content/full/jcb.200405130/DC1). TSC1 regulates cell adhesion, and its inactivation results in the loss of focal adhesion (Lamb et al., 2000). To determine if focal adhesion remodeling in TSC2−/− cells requires TSC1, we microinjected siRNA TSC1 and found that down-regulation of TSC1 promoted focal adhesion disassembly in TSC2−/− cells (Fig. 5 B). These results demonstrate that expression of TSC2-HBD and the down-regulation of TSC1 with siRNA TSC1 are sufficient for focal adhesion disassembly, which suggest that TSC2 involves TSC1 in regulating focal adhesion remodeling.

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