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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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Re-expression of TSC2 changes TSC2−/− cell morphology and dynamics during wound closure. (A) Phase-contrast micrograph of time-lapse analysis of TSC2−/− cell motility during wound closure at 4 h after wound scraping; images are representative of three independent experiments. (B) Phase-contrast and fluorescence micrographs demonstrate the representative phenotypes of GFP- or GFP-TSC2–infected cells. TSC2−/− cells, infected with GFP-TSC2 or control GFP replication-deficient adenovirus constructs, were first serum-deprived, and then subjected to live image analysis of wound closure in the presence of 2% FBS. Short arrows indicate differences in membrane protrusion; long arrows indicate direction of cell movement. Bars, 120 μm. Images were taken using a Leitz Inverted Microscope in both the phase-contrast and green fluorescence channels. Images are representative from three independent experiments. (C) Statistical analysis of the rate of membrane protrusion in TSC2−/− cells infected either with GFP or GFP-TSC2. *, P < 0.0001 for GFP-TSC2-infected cells versus GFP-infected cells by ANOVA (Bonferroni-Dunn test).
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fig1: Re-expression of TSC2 changes TSC2−/− cell morphology and dynamics during wound closure. (A) Phase-contrast micrograph of time-lapse analysis of TSC2−/− cell motility during wound closure at 4 h after wound scraping; images are representative of three independent experiments. (B) Phase-contrast and fluorescence micrographs demonstrate the representative phenotypes of GFP- or GFP-TSC2–infected cells. TSC2−/− cells, infected with GFP-TSC2 or control GFP replication-deficient adenovirus constructs, were first serum-deprived, and then subjected to live image analysis of wound closure in the presence of 2% FBS. Short arrows indicate differences in membrane protrusion; long arrows indicate direction of cell movement. Bars, 120 μm. Images were taken using a Leitz Inverted Microscope in both the phase-contrast and green fluorescence channels. Images are representative from three independent experiments. (C) Statistical analysis of the rate of membrane protrusion in TSC2−/− cells infected either with GFP or GFP-TSC2. *, P < 0.0001 for GFP-TSC2-infected cells versus GFP-infected cells by ANOVA (Bonferroni-Dunn test).

Mentions: To explore whether or not TSC2 regulates cell motility, we performed live imaging of the wound closure of TSC2-deficient smooth muscle ELT3 cells, which were either untreated or transduced with a replication-deficient adenovirus expressing GFP-tagged wild-type TSC2 or control GFP. As shown in Fig. 1 A and Video 1 (available at http://www.jcb.org/cgi/content/full/jcb.200405130/DC1), TSC2−/− cells retained their motile properties (Irani et al., 2002); however, their movement was characterized by the formation of short lamellipodia at the leading edge of migrating cells. Expression of GFP-tagged TSC2 markedly changed the pattern of cell dynamics; during wound closure, moving cells formed dynamic membrane protrusions, and the rate of membrane extension for GFP-TSC2–infected cells was 1.00 ± 0.25 μm min−1 compared with 0.35 ± 0.08 μm min−1 for GFP-infected cells (Fig. 1, B and C; and Videos 2 and 3, available at http://www.jcb.org/cgi/content/full/jcb.200405130/DC1). Expression of control GFP in TSC2−/− cells had little effect on the pattern of cell movement (Fig. 1 B and Videos 4 and 5, available at http://www.jcb.org/cgi/content/full/jcb.200405130/DC1). These data demonstrate that reexpression of TSC2 markedly changes TSC2−/− cell morphology during cell motility and suggest that TSC2 might be important for the formation of membrane protrusions during directional movement.


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)

Re-expression of TSC2 changes TSC2−/− cell morphology and dynamics during wound closure. (A) Phase-contrast micrograph of time-lapse analysis of TSC2−/− cell motility during wound closure at 4 h after wound scraping; images are representative of three independent experiments. (B) Phase-contrast and fluorescence micrographs demonstrate the representative phenotypes of GFP- or GFP-TSC2–infected cells. TSC2−/− cells, infected with GFP-TSC2 or control GFP replication-deficient adenovirus constructs, were first serum-deprived, and then subjected to live image analysis of wound closure in the presence of 2% FBS. Short arrows indicate differences in membrane protrusion; long arrows indicate direction of cell movement. Bars, 120 μm. Images were taken using a Leitz Inverted Microscope in both the phase-contrast and green fluorescence channels. Images are representative from three independent experiments. (C) Statistical analysis of the rate of membrane protrusion in TSC2−/− cells infected either with GFP or GFP-TSC2. *, P < 0.0001 for GFP-TSC2-infected cells versus GFP-infected cells by ANOVA (Bonferroni-Dunn test).
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Related In: Results  -  Collection

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fig1: Re-expression of TSC2 changes TSC2−/− cell morphology and dynamics during wound closure. (A) Phase-contrast micrograph of time-lapse analysis of TSC2−/− cell motility during wound closure at 4 h after wound scraping; images are representative of three independent experiments. (B) Phase-contrast and fluorescence micrographs demonstrate the representative phenotypes of GFP- or GFP-TSC2–infected cells. TSC2−/− cells, infected with GFP-TSC2 or control GFP replication-deficient adenovirus constructs, were first serum-deprived, and then subjected to live image analysis of wound closure in the presence of 2% FBS. Short arrows indicate differences in membrane protrusion; long arrows indicate direction of cell movement. Bars, 120 μm. Images were taken using a Leitz Inverted Microscope in both the phase-contrast and green fluorescence channels. Images are representative from three independent experiments. (C) Statistical analysis of the rate of membrane protrusion in TSC2−/− cells infected either with GFP or GFP-TSC2. *, P < 0.0001 for GFP-TSC2-infected cells versus GFP-infected cells by ANOVA (Bonferroni-Dunn test).
Mentions: To explore whether or not TSC2 regulates cell motility, we performed live imaging of the wound closure of TSC2-deficient smooth muscle ELT3 cells, which were either untreated or transduced with a replication-deficient adenovirus expressing GFP-tagged wild-type TSC2 or control GFP. As shown in Fig. 1 A and Video 1 (available at http://www.jcb.org/cgi/content/full/jcb.200405130/DC1), TSC2−/− cells retained their motile properties (Irani et al., 2002); however, their movement was characterized by the formation of short lamellipodia at the leading edge of migrating cells. Expression of GFP-tagged TSC2 markedly changed the pattern of cell dynamics; during wound closure, moving cells formed dynamic membrane protrusions, and the rate of membrane extension for GFP-TSC2–infected cells was 1.00 ± 0.25 μm min−1 compared with 0.35 ± 0.08 μm min−1 for GFP-infected cells (Fig. 1, B and C; and Videos 2 and 3, available at http://www.jcb.org/cgi/content/full/jcb.200405130/DC1). Expression of control GFP in TSC2−/− cells had little effect on the pattern of cell movement (Fig. 1 B and Videos 4 and 5, available at http://www.jcb.org/cgi/content/full/jcb.200405130/DC1). These data demonstrate that reexpression of TSC2 markedly changes TSC2−/− cell morphology during cell motility and suggest that TSC2 might be important for the formation of membrane protrusions during directional movement.

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