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

siRNATSC1 and siRNATSC2 induce opposite effects on NIH 3T3 fibroblast migration.Cells were transfected with siRNA TSC1 (A), siRNA TSC2 (B), or control siRNA. 48 h post-transfection, protein levels were detected by immunoblot analysis with anti-TSC1 or anti-TSC2 antibodies. C, Upper panel: Representative image of hemacolor-stained membrane with migrated NIH 3T3 fibroblasts for 4 h. 3T3 fibroblasts were transfected with siRNA TSC1, siRNA TSC2, and siGLO RISC-Free siRNA as control cells, serum-deprived followed by migration assay in the presence or absence of 10 ng/ml PDGF performed in triplicate for each experimental condition. C, Lower panel: Statistical analysis of NIH 3T3 cell migration. Data represent mean values ± SE from two independent experiments, six repetitions in each experiment. *P<0.01 for siRNA TSC1 vs. control siRNA, **P<0.001 for siRNA TSC2 vs. control siRNA by ANOVA (Bonferroni-Dunn).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111476-g006: siRNATSC1 and siRNATSC2 induce opposite effects on NIH 3T3 fibroblast migration.Cells were transfected with siRNA TSC1 (A), siRNA TSC2 (B), or control siRNA. 48 h post-transfection, protein levels were detected by immunoblot analysis with anti-TSC1 or anti-TSC2 antibodies. C, Upper panel: Representative image of hemacolor-stained membrane with migrated NIH 3T3 fibroblasts for 4 h. 3T3 fibroblasts were transfected with siRNA TSC1, siRNA TSC2, and siGLO RISC-Free siRNA as control cells, serum-deprived followed by migration assay in the presence or absence of 10 ng/ml PDGF performed in triplicate for each experimental condition. C, Lower panel: Statistical analysis of NIH 3T3 cell migration. Data represent mean values ± SE from two independent experiments, six repetitions in each experiment. *P<0.01 for siRNA TSC1 vs. control siRNA, **P<0.001 for siRNA TSC2 vs. control siRNA by ANOVA (Bonferroni-Dunn).

Mentions: To further validate a differential effect of TSC1 and TSC2 on cell migration, we investigated the effect of siRNA-induced down-regulation of TSC1 and TSC2 on the migration of wild type MEFs and NIH 3T3 fibroblasts. We found that TSC1 depletion in wild type Tsc1+/+ MEFs attenuated cell migration (Fig. 5C). In contrast, siRNA TSC2 promoted Tsc2+/+ MEF migration compared to cells transfected with control siRNA (Fig. 5C). Similarly, siRNA-induced TSC2 knock-down (Fig. 6A) increased basal and PDGF-induced migration of NIH 3T3 fibroblasts (Fig. 6B and 6C) while siRNA TSC1 (Fig. 6A) attenuated both basal and PDGF-induced NIH 3T3 migration compared to cells transfected with control siRNA (Fig. 6B and 6C). Collectively, these data demonstrate that TSC1 and TSC2 differentially modulate cell migration.


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)

siRNATSC1 and siRNATSC2 induce opposite effects on NIH 3T3 fibroblast migration.Cells were transfected with siRNA TSC1 (A), siRNA TSC2 (B), or control siRNA. 48 h post-transfection, protein levels were detected by immunoblot analysis with anti-TSC1 or anti-TSC2 antibodies. C, Upper panel: Representative image of hemacolor-stained membrane with migrated NIH 3T3 fibroblasts for 4 h. 3T3 fibroblasts were transfected with siRNA TSC1, siRNA TSC2, and siGLO RISC-Free siRNA as control cells, serum-deprived followed by migration assay in the presence or absence of 10 ng/ml PDGF performed in triplicate for each experimental condition. C, Lower panel: Statistical analysis of NIH 3T3 cell migration. Data represent mean values ± SE from two independent experiments, six repetitions in each experiment. *P<0.01 for siRNA TSC1 vs. control siRNA, **P<0.001 for siRNA TSC2 vs. control siRNA by ANOVA (Bonferroni-Dunn).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111476-g006: siRNATSC1 and siRNATSC2 induce opposite effects on NIH 3T3 fibroblast migration.Cells were transfected with siRNA TSC1 (A), siRNA TSC2 (B), or control siRNA. 48 h post-transfection, protein levels were detected by immunoblot analysis with anti-TSC1 or anti-TSC2 antibodies. C, Upper panel: Representative image of hemacolor-stained membrane with migrated NIH 3T3 fibroblasts for 4 h. 3T3 fibroblasts were transfected with siRNA TSC1, siRNA TSC2, and siGLO RISC-Free siRNA as control cells, serum-deprived followed by migration assay in the presence or absence of 10 ng/ml PDGF performed in triplicate for each experimental condition. C, Lower panel: Statistical analysis of NIH 3T3 cell migration. Data represent mean values ± SE from two independent experiments, six repetitions in each experiment. *P<0.01 for siRNA TSC1 vs. control siRNA, **P<0.001 for siRNA TSC2 vs. control siRNA by ANOVA (Bonferroni-Dunn).
Mentions: To further validate a differential effect of TSC1 and TSC2 on cell migration, we investigated the effect of siRNA-induced down-regulation of TSC1 and TSC2 on the migration of wild type MEFs and NIH 3T3 fibroblasts. We found that TSC1 depletion in wild type Tsc1+/+ MEFs attenuated cell migration (Fig. 5C). In contrast, siRNA TSC2 promoted Tsc2+/+ MEF migration compared to cells transfected with control siRNA (Fig. 5C). Similarly, siRNA-induced TSC2 knock-down (Fig. 6A) increased basal and PDGF-induced migration of NIH 3T3 fibroblasts (Fig. 6B and 6C) while siRNA TSC1 (Fig. 6A) attenuated both basal and PDGF-induced NIH 3T3 migration compared to cells transfected with control siRNA (Fig. 6B and 6C). Collectively, these data demonstrate that TSC1 and TSC2 differentially modulate cell migration.

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