Conventional myosin has two different light chains bound to the neck region of the molecule. It has been suggested that the light chains contribute to myosin function by providing structural support to the neck region, therefore amplifying the conformational changes in the head following ATP hydrolysis (Rayment et al., 1993). The regulatory light chain is also believed to be important in regulating the actin-activated ATPase and myosin motor function as assayed by an in vitro motility assay (Griffith et al., 1987). Despite extensive in vitro biochemical study, little is known regarding RMLC function and its regulatory role in vivo. To better understand the importance and contribution of RMLC in vivo, we engineered Dictyostelium cell lines with a disrupted RMLC gene. Homologous recombination between the introduced gene disruption vector and the chromosomal RMLC locus (mlcR) resulted in disruption of the RMLC-coding region, leading to cells devoid of both the RMLC transcript and the 18-kD RMLC polypeptide. RMLC-deficient cells failed to divide in suspension, becoming large and multinucleate, and could not complete development following starvation. These results, similar to those from myosin heavy chain mutants (DeLozanne et al., 1987; Manstein et al., 1989), suggest the RMLC subunit is required for normal cytokinesis and cell motility. In contrast to the myosin heavy chain mutants, however, the mlcR cells are able to cap cell surface receptors following concanavilin A treatment. By immunofluorescence microscopy, RMLC null cells exhibited myosin localization patterns different from that of wild-type cells. The myosin localization in RMLC null cells also varied depending upon whether the cells were cultured in suspension or on a solid substrate. In vitro, purified RMLC- myosin assembled to form thick filaments comparable to wild-type myosin, but the filaments then exhibit abnormal disassembly properties. These results indicate that in vivo RMLC is necessary for myosin function.
In Dictyostelium amebas, myosin appears to be organized into filaments that relocalize during cell division and in response to stimulation by cAMP. To better understand the regulation of myosin assembly, we have studied the polymerization properties of purified Dictyostelium myosin. In 150 mM KCl, the myosin remained in the supernate following centrifugation at 100,000 g. Rotary shadowing showed that this soluble myosin was monomeric and that approximately 80% of the molecules had a single bend 98 nm from the head-tail junction. In very low concentrations of KCl (less than 10 mM) the Dictyostelium myosin was also soluble at 100,000 g. But rather than being monomeric, most of the molecules were associated into dimers or tetramers. At pH 7.5 in 50 mM KCl, dephosphorylated myosin polymerized into filaments whereas myosin phosphorylated to a level of 0.85 mol Pi/mol heavy chain failed to form filaments. The phosphorylated myosin could be induced to form filaments by lowering the pH or by increasing the magnesium concentration to 10 mM. The resulting filaments were bipolar, had blunt ends, and had a uniform length of approximately 0.43 micron. In contrast, filaments formed from fully dephosphorylated myosin were longer, had tapered ends, and aggregated to form very long, threadlike structures. The Dictyostelium myosin had a very low critical concentration for assembly of approximately 5 micrograms/ml, and this value did not appear to be affected by the level of heavy chain phosphorylation. The concentration of polymer at equilibrium, however, was significantly reduced, indicating that heavy chain phosphorylation inhibited the affinity of subunits for each other. Detailed assembly curves revealed that small changes in the concentration of KCl, magnesium, ATP, or H+ strongly influenced the degree of assembly. Thus, changes in both the intracellular milieu and the level of heavy chain phosphorylation may control the location and state of assembly of myosin in response to physiological stimuli.