TSC1 Controls Distribution of Actin Fibers through Its Effect on Function of Rho Family of Small GTPases and Regulates Cell Migration and Polarity

<div><p>The tumor-suppressor genes <em>TSC1</em> and <em>TSC2</em> are mutated in tuberous sclerosis, an autosomal dominant multisystem disorder. The gene products of <em>TSC1</em> and <em>TSC2</em> form a protein complex that inhibits the signaling of the mammalian target of rapamycin complex1 (mTORC1) pathway. mTORC1 is a crucial molecule in the regulation of cell growth, proliferation and survival. When the TSC1/TSC2 complex is not functional, uncontrolled mTORC1 activity accelerates the cell cycle and triggers tumorigenesis. Recent studies have suggested that TSC1 and TSC2 also regulate the activities of Rac1 and Rho, members of the Rho family of small GTPases, and thereby influence the ensuing actin cytoskeletal organization at focal adhesions. However, how TSC1 contributes to the establishment of cell polarity is not well understood. Here, the relationship between TSC1 and the formation of the actin cytoskeleton was analyzed in stable TSC1-expressing cell lines originally established from a <em>Tsc1</em>-deficient mouse renal tumor cell line. Our analyses showed that cell proliferation and migration were suppressed when TSC1 was expressed. Rac1 activity in these cells was also decreased as was formation of lamellipodia and filopodia. Furthermore, the number of basal actin stress fibers was reduced; by contrast, apical actin fibers, originating at the level of the tight junction formed a network in TSC1-expressing cells. Treatment with Rho-kinase (ROCK) inhibitor diminished the number of apical actin fibers, but rapamycin had no effect. Thus, the actin fibers were regulated by the Rho-ROCK pathway independently of mTOR. In addition, apical actin fibers appeared in TSC1-deficient cells after inhibition of Rac1 activity. These results suggest that TSC1 regulates cell polarity-associated formation of actin fibers through the spatial regulation of Rho family of small GTPases.</p> </div

The apical actin network associates with TJs in CACL1-TSC1 cells.

<p>(A, B) Apical actin fibers originate more apically of the adherens junction (AJ). The images were analyzed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054503#pone-0054503-g005" target="_blank">Figure 5</a>. Cells in confluent monolayer were stained for F-actin and E-cadherin (A) or β-catenin (B). Open arrowheads show the apical actin network. Scale bars: 10 µm. (C) Apical actin network associated with TJs in CACL1-TSC1 cells. CACL1-TSC1-11 cells in confluent monolayer were stained for F-actin and the TJ component ZO-1. The images were analyzed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054503#pone-0054503-g005" target="_blank">Figure 5</a>. Lower panels show higher magnification photographs. Open arrowheads show the junction of the apical actin network and ZO-1. The shape of the intercellular junctional perimeter was changed as if pulled by apical actin fibers. (D) There was no apparent change in TJ formation in the TSC1- deficient cells compared to the TSC1- expressing cells. ZO-1 in CACL1-Hygro cells was analyzed using the same method as for CACL1-TSC1 cells. Similar observations were obtained from the second clone set (data not shown). Scale bars: 10 µm.</p

TSC1 induces the formation of apical actin network in a RhoA-dependent manner independently of mTOR.

<p>(A) mTORC1 and mTORC2 were inhibited by rapamycin treatment but were not changed by ROCK inhibitor treatment. Cells were harvested and lysed 2 (left panels) or 14 hours (right panels) after inhibitor addition. Total cell lysates were analyzed by immunoblotting with the indicated antibodies. (B) Apical side of cells. Apical actin network remained unchanged by inhibition of mTORC1/2 and was decreased after inhibition of ROCK. Open arrowheads indicate the actin fiber network in CACL1-TSC1 cells. (C) Basal side of cells. Basal actin stress fibers were decreased after inhibition of ROCK regardless of TSC1. Arrows and open arrows show the basal actin stress fibers in CACL1-Hygro and -TSC1 cells, respectively. Confluent cells were treated with rapamycin or Y27632 for 14 hours and stained for F-actin. Images were analyzed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054503#pone-0054503-g005" target="_blank">Figure 5</a>. In (B) and (C), scale bars: 10 µm.</p

TSC1 inhibits cell migration.

<p>(A) TSC1-deficient and TSC1-expressing cells were seeded and grown to 100% confluence. The cell monolayer was scratched with a sterile pipette tip. The wounded cultures were photographed after 14 and 43 hours. Arrows indicate the gap width. Representative images are shown. (B) The rate of wound healing (%) after 14 hours was calculated as described in the Materials and Methods. Data are shown as means ± SE of three independent experiments. Significant differences were determined by one-way ANOVA with Scheffe’s post hoc comparison (*, p<0.05; **, p<0.01).</p

Formation of lamellipodia and filopodia decreases at the leading edge in TSC1-expressing cells.

<p>(A) Low-magnification confocal images of phalloidin stained cells. Boxed areas indicate the leading edge. Scale bars: 100 µm. (B) High-magnification images of the leading edge. Two representative images are shown. In each panel, left side is the scratch edge. Arrows indicate the actin fibers in the filopodia of CACL1-Hygro cells and -YFP cells. Arrowheads show actin fibers in the filopodia of CACL1–TSC1 cells. Scale bars: 10 µm. (C, D) TSC1 inhibited focal adhesion formation without changing its structure. Scratched cells were stained for F-actin and components of focal adhesion. The right panels are merged images of F-actin and paxillin (C) or talin (D). Focal complexes appear as small dot-like structures (arrows). Open arrowheads indicate the focal adhesions. Paxillin (C) and talin (D) were observed to associate with both ends of actin stress fibers. Scale bars: 10 µm.</p

Inhibition of Rac1 led to the formation of apical actin network.

<p>(A) NSC23766, a Rac1 inhibitor, induced TJ-associated apical actin fibers in TSC1-deficient cells. TSC1-deficient cells (CACL1-Hygro), plated 24 hours prior to treatment, were cultured in the absence (upper images) or presence of NSC23766 (lower images) for 3 days. Open arrowheads indicate apical actin fibers at TJ level. Scale bars: 10 µm. Similar results were obtained from the second clone set (data not shown). (B) Model of TSC1 function during actin cytoskeletal change in CACL1-111 cells. Red lines show actin fibers. Stress fibers in the basolateral side decreased and the apical actin network appeared in TSC1-expressing cells. RhoA activity (depicted by the triangle) is suspected to be high in the apical side and low in the basolateral side of TSC1-expressing cells. Thus, spatial regulation of RhoA activity may be regulated by TSC1. Rac1 may inhibit RhoA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054503#pone.0054503-Goncharova1" target="_blank">[29]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054503#pone.0054503-Chauhan1" target="_blank">[52]</a> and downregulation of Rac1 by TSC1 may also contribute to the apical actin network.</p

TSC1 reduces basal actin fibers and induces apical actin fibers in the confluent monolayer.

<p>(A) Low-magnification confocal images of phalloidin stained cells. Boxed areas show the confluent monolayer. Scale bars: 100 µm. (B) TSC1 reduced basal actin fibers and induced apical actin fibers. Images show representative X–Y sections from scans at 0.5 µm steps from the basal (close to the substrate, lower panels) to the apical side (upper panels) of the cell. X–Z (top to bottom) and Y–Z projections (left to right) are shown at the bottom and right side of each panel, respectively. Dotted line indicates the level of X–Y images shown. Arrows denote actin stress fibers in the basal side of cells. Arrowheads indicate the actin fiber network in the apical side of CACL1-TSC1-11cells. Scale bars: 10 µm. (C, D) TSC1 inhibited formation of focal adhesions in the confluent stage. Cells in confluent monolayer were stained for F-actin and paxillin (C) or talin (D). Open arrowheads show focal adhesions connected to stress fibers. Scale bars: 10 µm.</p

Rac1 is downregulated by TSC1 without apparent changes in Rho activity.

<p>(A, B) Cells were subjected to the Rac1 (A) or RhoA (B) activity assay after culturing in normal (FCS+) or serum starvation conditions (FCS−). Western blot analysis was performed to detect Rac1 bound to PAK-1 PBD or rhotekin-PBD beads (top images) and in whole cell lysates (middle images). TSC1 expression was confirmed by immunoblotting of whole cell lysates (bottom images). Representative blots are shown. Quantifications of Rac1 (A) or RhoA (B) activity were performed using Image J (bottom). Levels of active Rac1 (A) or RhoA (B) were normalized to total Rac1 or RhoA, respectively, and expressed as a fold activation relative to CACL1-Hygro cells. Data are shown as means ± SE of three independent experiments. Significant differences were determined by one-way ANOVA with Dunnett’s post hoc comparison (**, p<0.01 vs. CACL1-Hygro).</p

Metabolic abnormalities induced by mitochondrial dysfunction in skeletal muscle of the renal carcinoma Eker (TSC2+/−) rat model

<p>Tuberous sclerosis complex 2 (TSC2) is a mediator of insulin signal transduction, and a loss of function in TSC2 induces hyperactivation of mTORC1 pathway, which leads to tumorigenesis. We have previously demonstrated that Eker rat model, which is heterozygous for a TSC2 mutation, exhibits hyperglycemia and hyperketonemia. The present study was to investigate whether these changes also can affect metabolism in skeletal muscle of the Eker rat. Wild-type (TSC2+/+) and Eker (TSC2+/−) rats underwent an oral glucose tolerance test, and the latter showed decrease in whole-body glucose utilization. Additionally, reductions in the expression of glycolysis-, lipolysis-, and ketone body-related genes in skeletal muscle were observed in Eker rats. Furthermore, ATP content and mitochondrial DNA copy number were lower in skeletal muscle of Eker rats. These data demonstrate that heterozygous to mutation TSC2 not only affects the liver metabolism, but also skeletal muscle metabolism, via mitochondrial dysfunction.</p> <p>Eker rats exhibit glucose intolerance due to the inhibition of glucose uptake at the skeletal muscle, which was explained by low number and dysfunction of mitochondria.</p