title: Tumor suppressors TSC1 and TSC2 differentially modulate actin cytoskeleton and motility of mouse embryonic fibroblasts.
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-null 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.
authors.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.
authors.forename: Elena A
authors.initials: EA
authors.lastname: Goncharova
authors.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.
authors.forename: Melane L
authors.initials: ML
authors.lastname: James
authors.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.
authors.forename: Tatiana V
authors.initials: TV
authors.lastname: Kudryashova
authors.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.
authors.forename: Dmitry A
authors.initials: DA
authors.lastname: Goncharov
authors.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.
authors.forename: Vera P
authors.initials: VP
authors.lastname: Krymskaya
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D002465:Cell Movement; D004622:Embryo, Mammalian / Q000166:cytology*; D005347:Fibroblasts / Q000166:cytology*; D055786:Gene Knockout Techniques; D000076222:Mechanistic Target of Rapamycin Complex 1; D000076225:Mechanistic Target of Rapamycin Complex 2; D051379:Mice; D046912:Multiprotein Complexes / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D000076226:Rapamycin-Insensitive Companion of mTOR Protein; D022502:Stress Fibers / Q000378:metabolism; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism; D000077004:Tuberous Sclerosis Complex 1 Protein; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D046912:Multiprotein Complexes; D034741:RNA, Small Interfering; D000076226:Rapamycin-Insensitive Companion of mTOR Protein; C000624651:Tsc1 protein, mouse; C000624654:Tsc2 protein, mouse; D000077004:Tuberous Sclerosis Complex 1 Protein; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins; C525637:rictor protein, mouse; D000076222:Mechanistic Target of Rapamycin Complex 1; D000076225:Mechanistic Target of Rapamycin Complex 2; D058570:TOR Serine-Threonine Kinases