Using this functional dependence, we find that also the smectic ordering of a simulated array of parallel, striated fibers depends nonmonotonically on substrate stiffness (see Fig. 4). Experiments on interfiber registry Relative sliding of striated acto-myosin bundles into registry has already been reported ZD6474 in McKenna et al. (8). Yet, the mechanism by which striated fibers align in registry to form a smectic-like structure remains unknown. Engler et al. (13) have observed that the formation of striated and fully mature myofibrils in cultured C2C12 cell line depends strongly on substrate stiffness and was most prominent at an intermediate stiffness of 10 kPa, which is characteristic of muscle tissue stiffness, and there has since been several other reports for a variety of primary muscle cells derived from both skeletal and cardiac muscle and from at least four different species, namely chicken, mouse, rat, and man.

To test our theoretical prediction that already the initial registry of striated fibers depends nonmonotonically on substrate stiffness, we examined the interfiber registry in human mesenchymal stem cells that were plated on PA-gels of different stiffness (ranging from 0.3 kPa to 40 kPa). Mesenchymal stem cells have been reported to be capable of committing to a myogenic lineage based on either serum-induced signals (36) or matrix elasticity signals (37). Mesenchymal stem cells express stress-fiber-like acto-myosin bundles with a striated localization of nonmuscle myosin. These fibers resemble the striated fibers found in both nonmuscle cells and developing muscle cells.

Thus mesenchymal stem cells represent a model system to study interfiber registry of striated fibers. Fig. 5 shows the organization of nonmuscle myosin-IIA within three cells that were plated on gels of different stiffness and cultured for 24 h. All three cells display nematically aligned acto-myosin bundles with striated myosin localization that follows ~1 ��m intrafiber periodicity. However, interfiber registry of adjacent striated fibers is observed only for the cell that was cultured on a 10 kPa gel (Fig. 5 B). Myosin bands perpendicular to the axis of nematic fiber organization are clearly visible and most likely connect neighboring acto-myosin bundles in registry. Out of ~20 cells examined per gel, roughly 30�C50% exhibited aligned, striated fibers.

The cells shown in Fig. 5 are representative for these cells with striated fibers. This preliminary experimental evidence is consistent with our prediction of a nonmonotonic relationship between interfiber registry of striated fibers and substrate stiffness, also at very early stages within muscle-related precursors. Figure 5 Mesenchymal stem cells plated on PA-gels of different stiffness AV-951 stained for nonmuscle myosin-IIA. The substrate stiffness is 0.3 kPa, 10 kPa, and 40 kPa in panels A, B, and C, respectively.