CORRELATION OF MYOSIN LIGHT-CHAIN PHOSPHORYLATION WITH ISOMETRIC CONTRACTION OF FIBROBLASTS

In vitro studies have indicated that the enzymatic activity of myosin II from non-muscle cells is controlled by phosphorylation of its regul atory light chain (LC20). We have studied one likely functional conseq uence of phosphorylating LC20 in living chick embryo fibroblasts (CEF) by measuring contractile force developed by these cells. Using a rece ntly developed method, we recorded quantitative changes in isometric f orce generated by a population of cells following mitogenic stimulatio n. Fetal bovine serum, thrombin, and lysophosphatidic acid stimulate r apid isometric contraction of CEF. Cells stimulated with thrombin deve lop maximal force within 5-10 min. Force development correlates tempor ally with a 3-5-fold increase in the overall fraction of LC20 phosphor ylated and with the fractions of LC20 in both the monophosphorylated a nd diphosphorylated states. Unloaded shortening velocity also increase s after thrombin stimulation. Although both force and phosphorylation begin to decline 10 min after stimulation, the level of phosphorylatio n declined more rapidly than the force. These results suggest that the role of LC20 phosphorylation in regulating fibroblast contractility i s analogous to its well established role in regulating smooth muscle c ontraction and that quantitative measurements of the force developed b y populations of fibroblasts (or other cultured cells) can be used to study the regulation of non-sarcomeric myosin at the molecular level i n vivo.