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

Morphology and dynamics of Tsc1−/− and Tsc2−/− MEFs during wound closure.Phase-contrast micrographs of cell motility during wound closure at 4 h after wound scraping. Arrows indicate direction of cell movement. Images were taken using a Nikon Eclipse TE2000-E microscope at 100X magnification in the phase contrast channel. Images are representative from three independent experiments. Scale bar, 100 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111476-g003: Morphology and dynamics of Tsc1−/− and Tsc2−/− MEFs during wound closure.Phase-contrast micrographs of cell motility during wound closure at 4 h after wound scraping. Arrows indicate direction of cell movement. Images were taken using a Nikon Eclipse TE2000-E microscope at 100X magnification in the phase contrast channel. Images are representative from three independent experiments. Scale bar, 100 µm.

Mentions: The differences in the actin cytoskeleton are indicative of dynamic changes in cell morphology. Thus, we compared the morphology of Tsc1−/− and Tsc2−/− MEFs with littermate-matched wild type MEFs and NIH 3T3 fibroblasts during wound closure. Cell monolayers were serum-deprived and a wound assay was subsequently performed for 4 h in the presence of 2% FBS. As seen in Figure 3A, the morphology of migrating Tsc1−/− MEFs and Tsc2−/− MEFs were markedly different not only compared to those of wild type MEFs (Fig. 3B) and NIH 3T3 fibroblasts (Fig. 3C), but also between each other. In contrast to wild type MEFs and 3T3 fibroblasts that showed typical fibroblast morphology having irregular shape with a few protrusions towards wound closure, Tsc1−/− MEFs had a stretched shape with multiple filopodia-like extensions at the leading edge and thin extended cell shape of retraction in the rear (Fig. 3A, left panel). In contrast, motile Tsc2−/− MEFs showed broad lamellipodium at the front and round-like shape at the rear (Fig. 3A, right panel), as seen in the larger images of Tsc2−/− cells at higher magnification shown in Figure 1A and Figure 1B. Additionally, Tsc2−/− MEFs show phenotypic differences in apparent large cell size compared to Tsc2+/+ MEFs (Figure 3A). Indeed, increased cell size due to activation of mTOR signaling was demonstrated in [28]. These data show that loss of TSC1 or TSC2 differentially modulate dynamic changes in cell morphology during motility.


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)

Morphology and dynamics of Tsc1−/− and Tsc2−/− MEFs during wound closure.Phase-contrast micrographs of cell motility during wound closure at 4 h after wound scraping. Arrows indicate direction of cell movement. Images were taken using a Nikon Eclipse TE2000-E microscope at 100X magnification in the phase contrast channel. Images are representative from three independent experiments. Scale bar, 100 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111476-g003: Morphology and dynamics of Tsc1−/− and Tsc2−/− MEFs during wound closure.Phase-contrast micrographs of cell motility during wound closure at 4 h after wound scraping. Arrows indicate direction of cell movement. Images were taken using a Nikon Eclipse TE2000-E microscope at 100X magnification in the phase contrast channel. Images are representative from three independent experiments. Scale bar, 100 µm.
Mentions: The differences in the actin cytoskeleton are indicative of dynamic changes in cell morphology. Thus, we compared the morphology of Tsc1−/− and Tsc2−/− MEFs with littermate-matched wild type MEFs and NIH 3T3 fibroblasts during wound closure. Cell monolayers were serum-deprived and a wound assay was subsequently performed for 4 h in the presence of 2% FBS. As seen in Figure 3A, the morphology of migrating Tsc1−/− MEFs and Tsc2−/− MEFs were markedly different not only compared to those of wild type MEFs (Fig. 3B) and NIH 3T3 fibroblasts (Fig. 3C), but also between each other. In contrast to wild type MEFs and 3T3 fibroblasts that showed typical fibroblast morphology having irregular shape with a few protrusions towards wound closure, Tsc1−/− MEFs had a stretched shape with multiple filopodia-like extensions at the leading edge and thin extended cell shape of retraction in the rear (Fig. 3A, left panel). In contrast, motile Tsc2−/− MEFs showed broad lamellipodium at the front and round-like shape at the rear (Fig. 3A, right panel), as seen in the larger images of Tsc2−/− cells at higher magnification shown in Figure 1A and Figure 1B. Additionally, Tsc2−/− MEFs show phenotypic differences in apparent large cell size compared to Tsc2+/+ MEFs (Figure 3A). Indeed, increased cell size due to activation of mTOR signaling was demonstrated in [28]. These data show that loss of TSC1 or TSC2 differentially modulate dynamic changes in cell morphology during motility.

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