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First published online 2 November 2004
doi: 10.1242/jcs.01496

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Adhesion-contractile balance in myocyte differentiation

Biophysical Engineering Laboratory, 112 Towne Building, and Pennsylvania Muscle Institute, University of Pennsylvania, D-700 Richards Building, 3700 Hamilton Walk, Philadelphia, PA 19104, USA

View larger version (51K): [in a new window]   Fig. 1. Nascent C2C12 skeletal myotubes on collagen patterns. (A) Patterns of collagen-coated rectangles, 20 µm wide and 200-1000 µm long, surrounded by interpenetrating polymer network (IPN). Even after 14 days of incubation with fluorescent collagen, the IPN shows no significant adsorption based on intensity. In addition, when measured by AFM, the apparent elastic modulus of the IPN is approximately half that of the collagen-coated glass. Scale bar, 200 µm. (B) Myoblasts plated onto the patterned coverslips were either stained for vinculin (and imaged by TIRF for clarity) or triple-stained for actin (red) myosin (green) and nuclei (blue). The cells invariably fuse to form multinucleated myotubes. The arrow points to the vinculin clustered at the edges of the patterned myotube, indicative of classical focal adhesion structures. The actin and myosin images show no striations and are of the same nascent myotube at day 6. Bars: in A 200 µm; in B 10 µm.

View larger version (37K): [in a new window]   Fig. 2. Myotube striation occurs only on top of nascent myotubes. (A) Schematic showing that myotubes striate on top of other cells but not directly on IPN-patterned coverslips. Myoblasts plated on top of day 1-3 cells generate distinct cell-on-cell arrangements: in the upper myotube, actin and myosin will striate, whereas the lower myocyte bound to glass remains unstriated. (B) Confocal micrographs of the upper cell in a 2-week cell-on-cell system stained for actin and myosin. The actin shows a hint of striation, particularly near the arrow. The lower cell is wider, as in the middle sketch, and shows more isotropically oriented stress fibers. There is a 1.2 µm difference in height between the two actin images. The dashed lines represent the edges of the bottom myotube. Although the bottom cell is a fused myotube, the actin and myosin are striated in the top cell only. Bars, 10 µm. (C) Vinculin staining appears diffuse in both upper (n=15) and lower cells (n=15) in conventional fluorescence microscopy. A nucleus in the lower cell is not visible in the upper cell (dashed oval). The actin and myosin images show striation only in the upper cell (6 µm higher focal plane) and again a nucleus (asterisk) is visible in one cell but not the other. Bars, 10 µm. (D) After 1 week in culture, 68% of cells (n=34) growing on top of other cells exhibit striated myosin compared to 0% (n=25) of cells growing directly on collagen-coated glass. By 4 weeks, more upper cells have striated with no evidence of lower cell striation.

View larger version (55K): [in a new window]   Fig. 3. Cell attachment response to actin depolymerization and myosin II inhibition. Cell-substrate attachment is influenced by lat-A but not by blebbistatin. (A) Before treatment, myotubes are fused and firmly attached to the substrate. (B,C) Control cells and cells incubated in 50 µM blebbistatin for 20 minutes exhibit no visually observable change. (D) After a 20 minute incubation in 5 µM lat-A, however, cells bleb and self-peel off the substrate. The dotted lines indicate the location of the collagen-patterned strip in the IPN. Bars, 20 µm.

View larger version (56K): [in a new window]   Fig. 4. Myotube relaxation after initial detachment. Representative length-relaxation of control cells (A), cells treated with 50 µM blebbistatin (B) and cells relaxing off other cells (C). The data are fitted to the equation L/Linit=1–A(1–e–t /), where A is the amplitude and is the time constant. The respective images in A', B' and C' are snapshots of the relaxation with cells outlined by dotted lines. In the case of untreated cells relaxing off other cells (C, C'), the relaxation is so large that the stage is moved to the right 140 µm. The arrow points to the lower cell that is left unperturbed after relaxation of the upper cell. Bars, 10 µm.

View larger version (26K): [in a new window]   Fig. 5. Cumulative relaxation dynamics of the various myotubes. The results show that cells relax more quickly and to a greater extent when grown on top of other cells; and less so when treated with blebbistatin. (A) No significant difference is seen in the relaxation dynamics of cells exposed to DMSO (three cultures) compared to cells exposed only to media (five cultures). Data are time-binned averages all cells, with error bars representing the standard error of the mean. The bold line is the exponential fit for all 29 cells, R2=0.94. (B) Control cells relax to an average equilibrium length that is A=90% of their initial length. Blebbistatin-treated cells (three cultures) relax significantly less, whereas cells relaxing off other cells (three cultures) relax more. Blebbistatin-treated cells relaxing off other cells (two cultures) also relax more than blebbistatin-treated cells on glass. (C) The amplitudes, A, of each fit, indicate clear differences in the fraction of cell relaxation at equilibrium. (D) In all cases, the time constant for relaxation is greater when the cell is perturbed; but the slowest relaxation occurs in blebbistatin cells-on-glass.

View larger version (24K): [in a new window]   Fig. 6. Myotube adhesion versus prestress. (A) Schematic of a sectioned myotube illustrates the balance between the prestress imposed by the actomyosin cytoskeleton (PA, PM), and the adhesive tension of the focal adhesions (TFA). (B,B') Schematic of the adhesive energetics, contractile stress, and adhesive tension during differentiation. After fusion, myotubes growing on rigid substrates progress to a tightly bound state with high adhesive tension. Cells growing on soft substrates, including other cells, exhibit a reduced adhesive tension and a shallower adhesion energy at this nascent myotube phase. This allows the latter to progress to a fully striated state of high prestress.

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