Limits...
Tumor suppressors TSC1 and TSC2 differentially modulate actin cytoskeleton and motility of mouse embryonic fibroblasts.

Goncharova EA, James ML, Kudryashova TV, Goncharov DA, Krymskaya VP - PLoS ONE (2014)

Bottom Line: To assess the mechanism(s) by which TSC2 loss promotes actin re-arrangement and cell migration, we explored the role of known downstream effectors of TSC2, mTORC1 and mTORC2.Overexpression of kinase-dead mTOR induced actin stress fiber disassembly and suppressed TSC2-deficient cell migration.Our data demonstrate that TSC1 and TSC2 differentially regulate actin stress fiber formation and cell migration, and that only TSC2 loss promotes mTOR- and mTORC2-dependent pro-migratory cell phenotype.

View Article: PubMed Central - PubMed

Affiliation: Airways Biology Initiative, Pulmonary, Allergy & Critical Care Division, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States of America.

ABSTRACT
TSC1 and TSC2 mutations cause neoplasms in rare disease pulmonary LAM and neuronal pathfinding in hamartoma syndrome TSC. The specific roles of TSC1 and TSC2 in actin remodeling and the modulation of cell motility, however, are not well understood. Previously, we demonstrated that TSC1 and TSC2 regulate the activity of small GTPases RhoA and Rac1, stress fiber formation and cell adhesion in a reciprocal manner. Here, we show that Tsc1(-/-) MEFs have decreased migration compared to littermate-derived Tsc1(+/+) MEFs. Migration of Tsc1(-/-) MEFs with re-expressed TSC1 was comparable to Tsc1(+/+) MEF migration. In contrast, Tsc2(-/-) MEFs showed an increased migration compared to Tsc2(+/+) MEFs that were abrogated by TSC2 re-expression. Depletion of TSC1 and TSC2 using specific siRNAs in wild type MEFs and NIH 3T3 fibroblasts also showed that TSC1 loss attenuates cell migration while TSC2 loss promotes cell migration. Morphological and immunochemical analysis demonstrated that Tsc1(-/-) MEFs have a thin protracted shape with a few stress fibers; in contrast, Tsc2(-/-) MEFs showed a rounded morphology and abundant stress fibers. Expression of TSC1 in either Tsc1(-/-) or Tsc2(-/-) MEFs promoted stress fiber formation, while TSC2 re-expression induced stress fiber disassembly and the formation of cortical actin. To assess the mechanism(s) by which TSC2 loss promotes actin re-arrangement and cell migration, we explored the role of known downstream effectors of TSC2, mTORC1 and mTORC2. Increased migration of Tsc2(-/-) MEFs is inhibited by siRNA mTOR and siRNA Rictor, but not siRNA Raptor. siRNA mTOR or siRNA Rictor promoted stress fiber disassembly in TSC2- cells, while siRNA Raptor had little effect. Overexpression of kinase-dead mTOR induced actin stress fiber disassembly and suppressed TSC2-deficient cell migration. Our data demonstrate that TSC1 and TSC2 differentially regulate actin stress fiber formation and cell migration, and that only TSC2 loss promotes mTOR- and mTORC2-dependent pro-migratory cell phenotype.

Show MeSH

Related in: MedlinePlus

Effects of expression and siRNA-induced down-regulation of TSC1 and TSC2 in NIH 3T3 fibroblasts on actin cytoskeleton.Serum-deprived NIH 3T3 fibroblasts were transfected with GFP-TSC1, GFP-TSC2, or control GFP or microinjected with siRNA TSC1 or siRNA TSC2 and GFP followed by rhodamine phalloidin staining to detect F-actin (red) and immunostaining with anti-GFP antibody (green) to identify transfected or injected cells. Scale bar, 20 µm.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4216017&req=5

pone-0111476-g002: Effects of expression and siRNA-induced down-regulation of TSC1 and TSC2 in NIH 3T3 fibroblasts on actin cytoskeleton.Serum-deprived NIH 3T3 fibroblasts were transfected with GFP-TSC1, GFP-TSC2, or control GFP or microinjected with siRNA TSC1 or siRNA TSC2 and GFP followed by rhodamine phalloidin staining to detect F-actin (red) and immunostaining with anti-GFP antibody (green) to identify transfected or injected cells. Scale bar, 20 µm.

Mentions: To further analyze the effects of TSC1 and TSC2 on the actin cytoskeleton, we performed rhodamine-phalloidin staining of Tsc1−/− and Tsc2−/− MEFs transfected with GFP-TSC1 and GFP-TSC2, respectively. Re-expression of TSC1 in Tsc1−/− MEFs promoted stress fiber formation (Fig. 1B, middle panel); similarly, expression of TSC1 in Tsc2−/− MEFs further promoted stress fiber formation and the attenuation of cortical actin (Fig. 1B, middle panel). Re-expression of GFP-TSC2 in Tsc2−/− MEFs promoted stress fiber disassembly and the enhanced formation of cortical actin (Fig. 1B, lower panel). In Tsc1−/− MEFs transfected with GFP-TSC2, actin was predominantly localized at the edges of cells forming cortical fibers (Fig. 1B, lower panel). As seen in Figure 2, in NIH 3T3 fibroblasts, GFP-TSC1 also promoted stress fiber formation, while siRNA TSC1 promoted stress fiber disassembly. In contrast, the transfection of NIH 3T3 fibroblasts with TSC2 promoted stress fiber disassembly and the formation of cortical actin, while cells with siRNA TSC2 have a thin shape and thin actin fibers (Fig. 2). Collectively, these data demonstrate that TSC1 and TSC2 differentially modulate stress fiber formation and the actin cytoskeleton.


Tumor suppressors TSC1 and TSC2 differentially modulate actin cytoskeleton and motility of mouse embryonic fibroblasts.

Goncharova EA, James ML, Kudryashova TV, Goncharov DA, Krymskaya VP - PLoS ONE (2014)

Effects of expression and siRNA-induced down-regulation of TSC1 and TSC2 in NIH 3T3 fibroblasts on actin cytoskeleton.Serum-deprived NIH 3T3 fibroblasts were transfected with GFP-TSC1, GFP-TSC2, or control GFP or microinjected with siRNA TSC1 or siRNA TSC2 and GFP followed by rhodamine phalloidin staining to detect F-actin (red) and immunostaining with anti-GFP antibody (green) to identify transfected or injected cells. Scale bar, 20 µm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4216017&req=5

pone-0111476-g002: Effects of expression and siRNA-induced down-regulation of TSC1 and TSC2 in NIH 3T3 fibroblasts on actin cytoskeleton.Serum-deprived NIH 3T3 fibroblasts were transfected with GFP-TSC1, GFP-TSC2, or control GFP or microinjected with siRNA TSC1 or siRNA TSC2 and GFP followed by rhodamine phalloidin staining to detect F-actin (red) and immunostaining with anti-GFP antibody (green) to identify transfected or injected cells. Scale bar, 20 µm.
Mentions: To further analyze the effects of TSC1 and TSC2 on the actin cytoskeleton, we performed rhodamine-phalloidin staining of Tsc1−/− and Tsc2−/− MEFs transfected with GFP-TSC1 and GFP-TSC2, respectively. Re-expression of TSC1 in Tsc1−/− MEFs promoted stress fiber formation (Fig. 1B, middle panel); similarly, expression of TSC1 in Tsc2−/− MEFs further promoted stress fiber formation and the attenuation of cortical actin (Fig. 1B, middle panel). Re-expression of GFP-TSC2 in Tsc2−/− MEFs promoted stress fiber disassembly and the enhanced formation of cortical actin (Fig. 1B, lower panel). In Tsc1−/− MEFs transfected with GFP-TSC2, actin was predominantly localized at the edges of cells forming cortical fibers (Fig. 1B, lower panel). As seen in Figure 2, in NIH 3T3 fibroblasts, GFP-TSC1 also promoted stress fiber formation, while siRNA TSC1 promoted stress fiber disassembly. In contrast, the transfection of NIH 3T3 fibroblasts with TSC2 promoted stress fiber disassembly and the formation of cortical actin, while cells with siRNA TSC2 have a thin shape and thin actin fibers (Fig. 2). Collectively, these data demonstrate that TSC1 and TSC2 differentially modulate stress fiber formation and the actin cytoskeleton.

Bottom Line: To assess the mechanism(s) by which TSC2 loss promotes actin re-arrangement and cell migration, we explored the role of known downstream effectors of TSC2, mTORC1 and mTORC2.Overexpression of kinase-dead mTOR induced actin stress fiber disassembly and suppressed TSC2-deficient cell migration.Our data demonstrate that TSC1 and TSC2 differentially regulate actin stress fiber formation and cell migration, and that only TSC2 loss promotes mTOR- and mTORC2-dependent pro-migratory cell phenotype.

View Article: PubMed Central - PubMed

Affiliation: Airways Biology Initiative, Pulmonary, Allergy & Critical Care Division, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States of America.

ABSTRACT
TSC1 and TSC2 mutations cause neoplasms in rare disease pulmonary LAM and neuronal pathfinding in hamartoma syndrome TSC. The specific roles of TSC1 and TSC2 in actin remodeling and the modulation of cell motility, however, are not well understood. Previously, we demonstrated that TSC1 and TSC2 regulate the activity of small GTPases RhoA and Rac1, stress fiber formation and cell adhesion in a reciprocal manner. Here, we show that Tsc1(-/-) MEFs have decreased migration compared to littermate-derived Tsc1(+/+) MEFs. Migration of Tsc1(-/-) MEFs with re-expressed TSC1 was comparable to Tsc1(+/+) MEF migration. In contrast, Tsc2(-/-) MEFs showed an increased migration compared to Tsc2(+/+) MEFs that were abrogated by TSC2 re-expression. Depletion of TSC1 and TSC2 using specific siRNAs in wild type MEFs and NIH 3T3 fibroblasts also showed that TSC1 loss attenuates cell migration while TSC2 loss promotes cell migration. Morphological and immunochemical analysis demonstrated that Tsc1(-/-) MEFs have a thin protracted shape with a few stress fibers; in contrast, Tsc2(-/-) MEFs showed a rounded morphology and abundant stress fibers. Expression of TSC1 in either Tsc1(-/-) or Tsc2(-/-) MEFs promoted stress fiber formation, while TSC2 re-expression induced stress fiber disassembly and the formation of cortical actin. To assess the mechanism(s) by which TSC2 loss promotes actin re-arrangement and cell migration, we explored the role of known downstream effectors of TSC2, mTORC1 and mTORC2. Increased migration of Tsc2(-/-) MEFs is inhibited by siRNA mTOR and siRNA Rictor, but not siRNA Raptor. siRNA mTOR or siRNA Rictor promoted stress fiber disassembly in TSC2- cells, while siRNA Raptor had little effect. Overexpression of kinase-dead mTOR induced actin stress fiber disassembly and suppressed TSC2-deficient cell migration. Our data demonstrate that TSC1 and TSC2 differentially regulate actin stress fiber formation and cell migration, and that only TSC2 loss promotes mTOR- and mTORC2-dependent pro-migratory cell phenotype.

Show MeSH
Related in: MedlinePlus