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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.

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Effects of TSC1 and TSC2 on actin cytoskeleton.A: F-actin staining of serum-deprived NIH 3T3 fibroblasts and matched Tsc1+/+, Tsc1โˆ’/โˆ’ and Tsc2+/+, and Tsc2โˆ’/โˆ’ MEFs. B: F-actin staining (red) of Tsc1โˆ’/โˆ’ and Tsc2โˆ’/โˆ’ MEFs transfected with either GFP-TSC1, GFP-TSC2, or GFP. Representative images of two separate experiments were taken using a Nikon Eclipse TE-2000E microscope at 200x magnification. Scale bar, 20 ยตm.
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pone-0111476-g001: Effects of TSC1 and TSC2 on actin cytoskeleton.A: F-actin staining of serum-deprived NIH 3T3 fibroblasts and matched Tsc1+/+, Tsc1โˆ’/โˆ’ and Tsc2+/+, and Tsc2โˆ’/โˆ’ MEFs. B: F-actin staining (red) of Tsc1โˆ’/โˆ’ and Tsc2โˆ’/โˆ’ MEFs transfected with either GFP-TSC1, GFP-TSC2, or GFP. Representative images of two separate experiments were taken using a Nikon Eclipse TE-2000E microscope at 200x magnification. Scale bar, 20 ยตm.

Mentions: Aberrant migration and invasiveness are characteristics of tumor cells with increased metastatic potential. Given the pivotal role of the actin cytoskeleton in the morphology, motility and invasiveness of cells, we performed rhodamine-phalloidin staining of serum-deprived NIH 3T3 fibroblasts, Tsc1โˆ’/โˆ’ and Tsc2โˆ’/โˆ’ MEFs, and littermate-derived Tsc1+/+ and Tsc2+/+ MEFs to determine the effects of TSC1 and TSC2 deficiency on the actin cytoskeleton. In contrast to stress fibers seen in NIH 3T3 fibroblasts and wild type MEFs, which are typical for mesenchymal cells, Tsc1โˆ’/โˆ’ MEFs had thin-shaped bodies and thin extended filopodia protrusions with linear thin stress fibers (Fig. 1A). In contrast, Tsc2โˆ’/โˆ’ MEFs have round or ellipsoid shapes and showed thick stress fibers predominantly attached to cortical actin at lamellipodia (Fig. 1A).


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 TSC1 and TSC2 on actin cytoskeleton.A: F-actin staining of serum-deprived NIH 3T3 fibroblasts and matched Tsc1+/+, Tsc1โˆ’/โˆ’ and Tsc2+/+, and Tsc2โˆ’/โˆ’ MEFs. B: F-actin staining (red) of Tsc1โˆ’/โˆ’ and Tsc2โˆ’/โˆ’ MEFs transfected with either GFP-TSC1, GFP-TSC2, or GFP. Representative images of two separate experiments were taken using a Nikon Eclipse TE-2000E microscope at 200x magnification. Scale bar, 20 ยตm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111476-g001: Effects of TSC1 and TSC2 on actin cytoskeleton.A: F-actin staining of serum-deprived NIH 3T3 fibroblasts and matched Tsc1+/+, Tsc1โˆ’/โˆ’ and Tsc2+/+, and Tsc2โˆ’/โˆ’ MEFs. B: F-actin staining (red) of Tsc1โˆ’/โˆ’ and Tsc2โˆ’/โˆ’ MEFs transfected with either GFP-TSC1, GFP-TSC2, or GFP. Representative images of two separate experiments were taken using a Nikon Eclipse TE-2000E microscope at 200x magnification. Scale bar, 20 ยตm.
Mentions: Aberrant migration and invasiveness are characteristics of tumor cells with increased metastatic potential. Given the pivotal role of the actin cytoskeleton in the morphology, motility and invasiveness of cells, we performed rhodamine-phalloidin staining of serum-deprived NIH 3T3 fibroblasts, Tsc1โˆ’/โˆ’ and Tsc2โˆ’/โˆ’ MEFs, and littermate-derived Tsc1+/+ and Tsc2+/+ MEFs to determine the effects of TSC1 and TSC2 deficiency on the actin cytoskeleton. In contrast to stress fibers seen in NIH 3T3 fibroblasts and wild type MEFs, which are typical for mesenchymal cells, Tsc1โˆ’/โˆ’ MEFs had thin-shaped bodies and thin extended filopodia protrusions with linear thin stress fibers (Fig. 1A). In contrast, Tsc2โˆ’/โˆ’ MEFs have round or ellipsoid shapes and showed thick stress fibers predominantly attached to cortical actin at lamellipodia (Fig. 1A).

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