Evidence for regulation of lamellipodial and tail contraction of glycerinated chicken embryonic fibroblasts by myosin light chain kinase

Abstract

Permeabilized cell models of muscle and nonmuscle cells have proven useful for examining the regulation of actin, myosin, and other cytoskeletal proteins during cell contraction. Upon addition of Ca²⁺ and ATP, glycerinated chick embryonic skin fibroblasts retract their tails and lamellipodia. Ca²⁺-independent contractions are obtained by preincubation of cell models in Ca²⁺ ATPγS, followed by EGTA and ATP addition, or by addition of trypsin-treated myosin light chain kinase that no longer requires Ca²⁺ for reactivation. By pretreating cells before glycerination with colchicine, it is possible to study lamellipodial contraction independent of tail contraction. Similar responses to ATPγS pretreatment and unregulated myosin light chain kinase are observed in cells that only contain lamellipodia. SDS-PAGE electrophoresis of glycerinated fibroblasts incubated in ATPγ³⁵S and Ca²⁺ shows that only two major proteins are thiophosphorylated, and that one of them, a band that comigrates with the 20K MW light chain of myosin, is thiophosphorylated in a Ca²⁺-dependent manner. Since the rate of tail contraction is several-fold faster after Ca²⁺ and ATPγS pretreatment or incubation in excess myosin light chain kinase, myosin light chain phosphorylation may be a rate-limiting step during contraction.