Ca2+‐independent myosin II phosphorylation and contraction in chicken embryo fibroblasts

Contraction is an essential component of many fundamental processes in non-muscle cells. Fibroblast contraction mediates orientation of collagen fibres in connective tissue (Harris et al. 1981) and contraction of granulation tissue (Majno et al. 1971). More complex cellular behaviours such as chemotaxis and cytokinesis are mediated by localized contractions within cells (Mabuchi & Okuno, 1977; Jay et al. 1995; Post et al. 1995). Among the myosin superfamily of motor proteins, only myosin II forms bipolar filaments, which can contract cytoplasm in a manner analogous to muscle. Non-muscle myosin II, like smooth muscle myosin II, is activated through phosphorylation of LC20, primarily at Ser19 and to a lesser extent at Thr18 (Sellers & Adelstein, 1987). In smooth muscle, LC20 is phosphorylated predominantly by Ca2+-calmodulin-activated myosin light chain kinase (MLCK) (Somlyo & Somlyo, 1994). However, it has remained unclear if this smooth muscle paradigm of [Ca2+]i-regulated myosin II based contractility also applies to non-muscle cells. Several lines of evidence support the widely held view that non-muscle cell contraction is regulated similarly to the better characterized process of smooth muscle contraction (i.e. through Ca2+-calmodulin stimulated LC20 phosphorylation). First, MLCK activity endogenous to both fibroblasts and endothelial cells is necessary and sufficient to mediate Ca2+-dependent LC20 phosphorylation and contraction in detergent-permeabilized cell models (Cande & Ezzell, 1986; Wysolmerski & Lagunoff, 1991). Second, MLCK inhibitors decrease LC20 phosphorylation and contraction in non-muscle cells (Lamb et al. 1988; Giuliano et al. 1992; Chrzanowska-Wodnicka & Burridge, 1996). Finally, in some cell types, Ca2+ ionophores stimulate structural alterations resembling cell contraction (Winter et al. 1991; Garcia et al. 1997). However, these studies should be interpreted cautiously for several reasons: (1) permeabilization of cells results in the loss of endogenous soluble molecules (Wysolmerski & Lagunoff, 1991) that may have important roles in the regulation of contraction, (2) protein kinase inhibitors thought to have specificity for MLCK may, in fact, exert their effects through inhibition of other kinases, and (3) cell shape reflects a balance between cytoplasmic contraction and resisting forces from cell adhesion and cytoplasmic stiffness (Chicurel et al. 1998). Therefore the morphological alterations caused by Ca2+ ionophores do not necessarily reflect contraction or [Ca2+]i-stimulated activation of myosin. To investigate whether the smooth muscle paradigm for the regulation of contraction also applies to non-muscle cells, we used a model system in which chicken embryo fibroblasts (CEF) contract in response to fetal bovine serum (FBS) stimulation. FBS, like most agonists for non-muscle or smooth muscle contraction, stimulates (1) a rise in [Ca2+]i (McNeil et al. 1985), (2) phosphorylation of LC20 (Giuliano et al. 1992; Kolodney & Elson, 1993), and (3) generation of contractile force (Kolodney & Elson, 1993). We quantified fibroblast isometric force generation by attaching a population of cells, cultured within a collagen matrix, to an isometric force transducer (Kolodney & Elson, 1995), thereby allowing direct measurement of cellular force generation. We examined the putative role of [Ca2+]i as the primary regulator of contractility in CEF by either buffering [Ca2+]i or selectively permeabilizing cells to extracellular Ca2+ and measuring the effects of these interventions on LC20 phosphorylation and force generation.