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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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mTOR and Rictor mediate cell migration.A: siRNA-induced mTOR depletion inhibits Tsc2−/− MEF migration. Tsc2−/− MEFs were transfected with siRNA mTOR or control siRNA followed by migration assay. Migration of control siRNA-transfected cells was taken as 100%. B: Rictor is required for serum-induced MEF migration. Tsc2−/− MEFs were transfected with siRNA Rictor,siRNA Raptor, or control siRNA, and migration assays were subsequently performed. Migration of Tsc2−/− MEFs transfected with control siRNA was taken as 1 fold. C: Rictor is required for cell migration under nutrient-replete conditions but is not sufficient to modulate all migration. Migration assays were performed with Rictor+/+ and Rictor−/− MEFs under basal (unstimulated) and serum-stimulated (10% FBS) conditions. Migration of Rictor+/+ MEFs under basal conditions was taken as 100%.
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pone-0111476-g008: mTOR and Rictor mediate cell migration.A: siRNA-induced mTOR depletion inhibits Tsc2−/− MEF migration. Tsc2−/− MEFs were transfected with siRNA mTOR or control siRNA followed by migration assay. Migration of control siRNA-transfected cells was taken as 100%. B: Rictor is required for serum-induced MEF migration. Tsc2−/− MEFs were transfected with siRNA Rictor,siRNA Raptor, or control siRNA, and migration assays were subsequently performed. Migration of Tsc2−/− MEFs transfected with control siRNA was taken as 1 fold. C: Rictor is required for cell migration under nutrient-replete conditions but is not sufficient to modulate all migration. Migration assays were performed with Rictor+/+ and Rictor−/− MEFs under basal (unstimulated) and serum-stimulated (10% FBS) conditions. Migration of Rictor+/+ MEFs under basal conditions was taken as 100%.

Mentions: Further, siRNA mTOR inhibited the migration of Tsc2−/− MEFs (Fig. 8A) demonstrating that mTOR is required for stress fiber assembly and increased cell migration due to TSC2 loss. siRNA Rictor, but not siRNA Raptor, attenuated migration of Tsc2−/− MEFs (Fig. 8B), showing that Rictor is required for TSC2-dependent MEF migration. Interestingly, the migrations of Rictor−/− and wild type Rictor+/+ MEFs were comparable under basal unstimulated conditions (Fig. 8C, left panel) indicating that loss of Rictor in TSC2-expressing cells is not sufficient to modulate cell migration. However, in serum-replete conditions, migration of Rictor−/− MEFs was significantly decreased compared to wild type cells suggesting a Rictor requirement for cell migration under nutrient-replete conditions. Collectively, our data demonstrate that mTOR and Rictor, components of rapamycin-insensitive mTORC2, but not Raptor, a member rapamycin-sensitive mTORC1, are involved in the regulation of the actin cytoskeleton and cell migration, and mTORC2 is required for TSC2-related phenotypes as seen in TSC2−/− MEFs.


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)

mTOR and Rictor mediate cell migration.A: siRNA-induced mTOR depletion inhibits Tsc2−/− MEF migration. Tsc2−/− MEFs were transfected with siRNA mTOR or control siRNA followed by migration assay. Migration of control siRNA-transfected cells was taken as 100%. B: Rictor is required for serum-induced MEF migration. Tsc2−/− MEFs were transfected with siRNA Rictor,siRNA Raptor, or control siRNA, and migration assays were subsequently performed. Migration of Tsc2−/− MEFs transfected with control siRNA was taken as 1 fold. C: Rictor is required for cell migration under nutrient-replete conditions but is not sufficient to modulate all migration. Migration assays were performed with Rictor+/+ and Rictor−/− MEFs under basal (unstimulated) and serum-stimulated (10% FBS) conditions. Migration of Rictor+/+ MEFs under basal conditions was taken as 100%.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111476-g008: mTOR and Rictor mediate cell migration.A: siRNA-induced mTOR depletion inhibits Tsc2−/− MEF migration. Tsc2−/− MEFs were transfected with siRNA mTOR or control siRNA followed by migration assay. Migration of control siRNA-transfected cells was taken as 100%. B: Rictor is required for serum-induced MEF migration. Tsc2−/− MEFs were transfected with siRNA Rictor,siRNA Raptor, or control siRNA, and migration assays were subsequently performed. Migration of Tsc2−/− MEFs transfected with control siRNA was taken as 1 fold. C: Rictor is required for cell migration under nutrient-replete conditions but is not sufficient to modulate all migration. Migration assays were performed with Rictor+/+ and Rictor−/− MEFs under basal (unstimulated) and serum-stimulated (10% FBS) conditions. Migration of Rictor+/+ MEFs under basal conditions was taken as 100%.
Mentions: Further, siRNA mTOR inhibited the migration of Tsc2−/− MEFs (Fig. 8A) demonstrating that mTOR is required for stress fiber assembly and increased cell migration due to TSC2 loss. siRNA Rictor, but not siRNA Raptor, attenuated migration of Tsc2−/− MEFs (Fig. 8B), showing that Rictor is required for TSC2-dependent MEF migration. Interestingly, the migrations of Rictor−/− and wild type Rictor+/+ MEFs were comparable under basal unstimulated conditions (Fig. 8C, left panel) indicating that loss of Rictor in TSC2-expressing cells is not sufficient to modulate cell migration. However, in serum-replete conditions, migration of Rictor−/− MEFs was significantly decreased compared to wild type cells suggesting a Rictor requirement for cell migration under nutrient-replete conditions. Collectively, our data demonstrate that mTOR and Rictor, components of rapamycin-insensitive mTORC2, but not Raptor, a member rapamycin-sensitive mTORC1, are involved in the regulation of the actin cytoskeleton and cell migration, and mTORC2 is required for TSC2-related phenotypes as seen in TSC2−/− MEFs.

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