In addition, we observed that both the α-actinin and myosin banding patterns stretch in some stress fiber regions upon stimulation of contractility. We have found that whereas some sarcomeres shorten during stress fiber contraction, unexpectedly, others in the same stress fiber elongate. This has allowed us to observe changes along entire stress fibers as well as in individual sarcomeric units demarcated by the GFP-α-actinin. We have used expression of green fluorescent protein (GFP)-tagged α-actinin or GFP-myosin light chain (GFPMLC), to follow the behavior of stress fibers during stimulation of increased actomyosin contractility by treatment with the serine/threonine phosphatase inhibitor, calyculin A or LPA. This has led to the idea that normally stress fibers are under isometric tension and that shortening is opposed by strong adhesion to the underlying rigid substrate mediated by focal adhesions ( Burridge, 1981). Stress fiber shortening in living cells has been observed in quiescent, serum-starved cells stimulated with serum or thrombin ( Giuliano and Taylor, 1990 Giuliano et al., 1992), although under most physiological conditions, shortening is rarely seen. Nevertheless, their organization suggests a contractile function, and isolated stress fibers or those in permeabilized cells will shorten in response to Mg 2+ ATP ( Isenberg et al., 1976 Kreis and Birchmeier, 1980 Katoh et al., 1998). Many stress fiber components display a periodic, “sarcomeric” organization, although they are less ordered than myofibrils at the ultrastructural level ( Gordon, 1978 Byers et al., 1984 Sanger et al., 1986). Like muscle myofibrils, stress fibers are composed of actin filaments ( Lazarides and Weber, 1974 Herman and Pollard, 1979), myosin II ( Weber and Groeschel-Stewart, 1974 Fujiwara and Pollard, 1976), and various actin-binding proteins, including α-actinin, a prominent Z-line component in muscle sarcomeres ( Lazarides and Burridge, 1975). Stress fibers terminate in focal adhesions, transmembrane complexes that mediate cell adhesion to the underlying substrate ( Burridge et al., 1988 Yamada and Geiger, 1997 Peterson and Burridge, 2001). Stress fibers are prominent bundles of actin filaments seen in many cells in culture as well as in cells in situ that are under shear stress conditions ( Gabbiani et al., 1975 White et al., 1983 Wong et al., 1983) or involved in wound healing ( Gabbiani et al., 1972).
In the periphery, the banding patterns for both proteins were shorter, whereas in central regions, where stretching occurred, the bands were wider. Surprisingly, the widths of the myosin and α-actinin bands in stress fibers also varied in different regions. Fluorescence recovery after photobleaching revealed more rapid exchange of myosin and α-actinin in the middle of stress fibers, compared with the periphery.
We detected higher levels of MLC and phosphorylated MLC in the peripheral region of stress fibers. Although peripheral regions shortened, more central regions stretched. We have observed that stress fibers, unlike muscle myofibrils, do not contract uniformly along their lengths. The resulting contraction caused stress fiber shortening and allowed observation of changes in the spacing of stress fiber components. Myosin activation was stimulated by treatment with calyculin A, a serine/threonine phosphatase inhibitor that elevates MLC phosphorylation, or with LPA, another agent that ultimately stimulates phosphorylation of MLC via a RhoA-mediated pathway. To study the dynamics of stress fiber components in cultured fibroblasts, we expressed α-actinin and the myosin II regulatory myosin light chain (MLC) as fusion proteins with green fluorescent protein.
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