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First published online 13 July 2004
doi: 10.1242/jcs.01197

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Modulation of acto-myosin contractility in skeletal muscle myoblasts uncouples growth arrest from differentiation

Jyotsna Dhawan^1,* and David M. Helfman^2,

¹ Center for Cellular and Molecular Biology, Uppal Road, Hyderabad, 500 007, India
² Cold Spring Harbor Laboratory, PO Box 100, 1 Bungtown Road, Cold Spring Harbor, NY 11074, USA

View larger version (61K): [in a new window]   Fig. 1. Myosin inhibitors affect cell shape and stress fiber organization. Asynchronous cultures of myoblasts were treated for 24 hours with low serum medium alone (a), or in the presence of DMSO (b), 30 mM BDM (c) or 15 µM ML7 (d). Actin stress fibers (Oregon Green-phalloidin) and cell spreading are markedly reduced in drug-treated cells. Scale bar: 20 µm.

View larger version (35K): [in a new window]   Fig. 2. Myosin inhibitors reversibly arrest myoblast proliferation. (A) Dose response of DNA synthesis in asynchronous myoblasts in low serum. Asynchronous myoblasts were treated for 24 hours with medium containing 2% HS alone (C), or 2% HS + vehicle (D) or BDM at 3, 10 or 30 mM (B3, B10, B30 respectively) or ML7 at 1.5, 5 or 15 µM (M1.5, M5, M15, respectively). Cells were pulsed with BrdU and labeled nuclei detected by immunostaining. Both BDM and ML7 elicited a dose-dependent decrease in the frequency of S phase cells (mean ± s.e.m. n=2). (B) Dose response of DNA synthesis in asynchronous myoblasts in high serum. Asynchronous myoblasts were treated with MI in high serum (20% FBS) and DNA synthesis measured as described in A. Whereas BDM effectively inhibited DNA synthesis, ML7's action was not evident in the presence of serum (mean ± s.e.m. n=2). (C) MIs prevent progression to S phase. Myoblasts were synchronized in G0 by suspension culture, replated in either 2% HS (black bars) or 20% FBS (gray bars) for 24 hours, and DNA synthesis measured as described in Fig. 2A. Vehicle-treated cells (D) entered S phase in a serum-dependent manner. BDM (but not ML7) inhibited S phase entry in a dose-dependent manner in 20% FBS. (mean ± s.d. n=4). (D) Growth arrest by MI is rapid. The kinetics of arrest by MI were measured by treating asynchronous myoblasts for 2-30 hours with 2% HS + DMSO (D), 30 mM BDM (B) or 15 µM ML7 (M). Inhibition of DNA synthesis was evident within 6 hours of MI addition. (mean ± s.e.m. n=2). (E) Arrest is reversible. Myoblasts were arrested by 24 hours treatment (0 hours after drug removal) in 15 µM ML7 or 30 mM BDM compared to vehicle alone. Cumulative BrdU-labeling of cells entering S from 5-28 hours after addition of growth medium showed that 90% were labeled by 22-28 hours. MI-treated cells took 4-8 hours longer to achieve 90% labeling. (F) MI treatment arrests cells in G0. Asynchronous myoblasts were treated for 24 hours with vehicle, 30 mM BDM or 15 µM ML7 and DNA content measured by FACS. Whereas control cells were distributed in all phases of the cell cycle, 90% of MI-treated cells possessed a G1 DNA content consistent with arrest in G0. (mean ± s.e.m. n=2).

View larger version (72K): [in a new window]   Fig. 3. Disruption of focal adhesions in myoblasts treated with MIs. Asynchronous myoblasts were incubated for 24 hours in 2% HS + DMSO, 30 mM BDM or 15 µM ML7. (A) Cells were triple-stained to reveal actin (green), the focal adhesion component vinculin (red) and DNA (blue). Vinculin-positive focal adhesions were located at the ends of stress fibers in control cells. Stress fibers and focal adhesions were disrupted in MI-treated cells, accompanied by an increase in diffuse cytoplasmic staining. Scale bar: 20 µm. (B) Immunodetection of the focal adhesion component paxillin (red). Both the number and size of paxillin+ focal contacts were reduced by MI treatment. Scale bar: 10 µm.

View larger version (58K): [in a new window]   Fig. 4. MIs do not induce myogenin and p21. Asynchronous myoblasts were incubated in control medium or 30 mM BDM or 15 µM ML7 for 24 hours. Myogenin (green) and p21 (red) were simultaneously detected using antibodies. (A) Staining of both early myogenic markers was readily detected in control cells but not in MI-treated cells. Scale bar: 10 µm. (B) The frequency of myogenin and p21 expression (mean ± s.e.m. n=2). The low level of myogenin and p21 expression in growth medium (1), rises after 24 hours in differentiation medium alone (2) or with DMSO vehicle (3). Both markers are suppressed by BDM and ML7 (4,5). (C,D) Induction of quiescence-related protein p27kip1 after BDM and ML7 treatment. Serum withdrawal caused some induction of p27 as expected (compare 1 with 2 or 3; numbers indicate conditions as in panel B), but MI treatment (4,5) led to increased frequency and intensity of p27 staining (mean ± s.e.m. n=2). Scale bar in C: 25 µm.

View larger version (21K): [in a new window]   Fig. 5. MIs reversibly inhibit MyoD expression. Asynchronous myoblasts were incubated in 20% serum alone (1), 2% HS alone (2), 2% HS + vehicle (3), 30 mM BDM (4) or 15 µM ML7 (5) for 24 hours. (A) MyoD (red) was detected in 50% of control cells, but was reduced in both BDM and ML7 treated cells (Scale bar: 10 µm). (B) The frequency of MyoD expression (mean ± s.e.m., n=2). (C) Replacement of MIs with growth medium leads to a rapid return of MyoD expression (mean ± s.e.m., n=2). The differences in the kinetics and plateau of the recovery curves may reflect differences in extent of cell cycle synchrony, the enzymatic properties of the two target enzymes or their effects on upstream regulators of MyoD.

View larger version (103K): [in a new window]   Fig. 6. MI treatment inhibits differentiation. Dense asynchronous cultures were treated with 2% HS containing DMSO (control), 15 mM BDM or 15 µM ML7 for 3 days, and stained for muscle-specific myosin expression. Control cultures fused into large multinucleated myotubes, but BDM and ML7 inhibited fusion. Scale bar: 25 µm.

View larger version (27K): [in a new window]   Fig. 7. Transfection of wild-type or dominant-interfering RhoA, or C3 transferase leads to arrest. Asynchronous myoblasts were co-transfected with plasmids encoding nlsGFP alone (GFP), WT RhoHA alone (RhoA/WT Rho), GFP + RhoAN19 (DN Rho) or GFP + C3 transferase (C3 Tr). After 24 hours, cells were pulsed with BrdU. (A) Co-detection of transfection marker and BrdU. WTRhoHA transfectants were detected using an anti-HA tag antibody (red); co-transfected nlsGFP was used to detect DN Rho transfectants (green). Anti-BrdU was detected using streptavidin-Alexa Fluor 350 (blue). Arrows indicate BrdU-negative untransfected cells (top) and transfected cells (middle and bottom). Scale bar: 10 µm. (B) Compared to control transfected cells, expression of WT Rho, DN Rho or C3 Tr led to suppression of DNA synthesis (mean ± s.d., n=3; *P<0.004, **P<0.001). (C) Wild-type RhoA and dominant-negative RhoA have divergent effects on MRFs. Asynchronously growing myoblasts were co-transfected as in A. Endogenous regulators were detected using immunofluorescence. Expression of WT Rho did not significantly affect MyoD expression, but increased the frequency of cells expressing myogenin and p21. By contrast, expression of DN Rho led to suppression of MyoD, and no induction of myogenin or p21 (mean ± s.d. n=3; *P<0.004, **P<0.001).

View larger version (110K): [in a new window]   Fig. 8. ROCK inhibitor Y27632 blocks proliferation but not MRF expression. (A) Asynchronous myoblasts were treated with control medium (a-c, g-i) or 10 µM Y27632 (d-f, j-l) for 24 hours. Oregon Green-phalloidin staining reveals a distinct morphology in Y27632-treated cells (d) marked by elongated processes (compare to control in a). Pulse labeling with BrdU is inhibited by ROCK inhibitor (compare e with b); (c,f) phase contrast + DNA stain of the same fields as in b and e, respectively. MyoD (j) and myogenin (k) staining are undiminished in Y27632-treated cells compared to control cells (g) and (h) respectively, (i,l) phase contrast + DNA stain of the same fields as in g,h and j,k, respectively. Scale bar: 25 µm.

View larger version (104K): [in a new window]   Fig. 9. Over-expression of RhoA overrides the effect of BDM on cytoskeletal organization. Asynchronous cultures were transfected as in Fig. 7 and 12 hours later were treated with vehicle (a-f) or 30 mM BDM (g-l) for a further 12 hours, (d-f) phase contrast + DNA stain of a-c. Control GFP cells were well spread with distributed paxillin staining; WT Rho-transfected cells were smaller with increased paxillin-positive focal contacts. Insets in b and c show cells expressing low levels of GFP marker, not visible in the merged images. DNRho transfection suggested a reciprocal effect to WTRho, with dispersed focal contacts. BDM treatment led to cell elongation and reduction of paxillin in GFP transfected cells (g), but did not reverse Rho-mediated cell shrinkage (h), (j-l) phase contrast + DNA stain of g-i. Scale bar: 10 µm.

View larger version (51K): [in a new window]   Fig. 10. (A) BDM does not decrease actin polymer levels. Actin polymer status was measured using FACS analysis of rhodamine-phalloidin binding in myoblasts treated with vehicle, latB (0.5 µM) or BDM (15 mM). (Expressed as a percentage of vehicle control, mean ± s.e.m.; n=2). (B) MyoD expression responds differentially to actin depolymerizing drugs. Myoblasts were treated with vehicle (a,b), 15 mM BDM (c,d), 0.5 µM latB (e,f) or 0.5 µM cytoD (g,h) for 18 hours before staining for MyoD. Numbers represent the percentage of MyoD-positive cells. Scale bar: 10 µm.