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.