Isolation and Characterization of Myosin from Cloned Mouse Fibroblasts

Abstract

Myosin has been isolated from cloned mouse fibroblasts, line L-929. Fibroblast myosin: (i) binds to rabbit muscle actin and is dissociated from it by ATP, (ii) has an ATPase activity that is suppressed by Mg2+ in 0.6 M KCI and is activated by rabbit muscle actin in the presence of Mgg+ in 14 mM KCI, (iii) forms thin bi. polar aggregates in 0.1 M KCI when viewed in the electron microscope, (iv) possesses a heavy chain with the same mobility as muscle myosin (molecular weight 200,000) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In these respects, fibroblast myosin appears to be similar to muscle myosin in structure and function.Since the work of Ishikawa et al. (1) on the presence of actinlike filaments in fibroblasts, the possible existence of a myosin counterpart in these cells has been raised. Recently, Yang and Perdue (2) succeeded in isolating and purifying actin from tertiary cultures of chick embryo cells and found it similar to muscle actin in its molecular properties. Furthermore, the the work of Bray (3) has indicated that cytoplasmic actin can be isolated from several different embryonic sources.cAMP has a marked effect on the motility, adhesion, morphology, and growth of fibroblasts grown in vitro (4-9). It is possible that some of the effects of cAMP could be mediated through one or more of the "contractile" proteins (actin, myosin, and troponin-tropomyosin). To explore this hypothesis, we began by attempting to isolate a myosin-like protein from cloned fibroblasts. Using a modification of a technique developed for the isolation of platelet myosin (10), we isolated myosin from cloned mouse fibroblasts grown in vitro. This protein has been characterized by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, ATPase assay with and without muscle actin, actin binding, and electron microscopy. MATERIALS AND METHODSCloned mouse (L-929) fibroblasts were grown either as monolayers or in spinner bottles. The cells were scraped off the sides of the bottles or sedimented from the suspended culture. They were washed three times in ten volumes of 0.9% NaCl-0.3% sodium citrate-5 mM dithiothreitol (S2threitol)-1 mM EDTA, and sedimented each time at 13,000 X g for 15 min.

Action of Cytochalasin D on Cells of Established Lines

“…However, other more primitive modalities of regulation of the interaction of actin and myosin than the troponin-tropomyosin system (e .g . phosphorylation) may be more important in nonmuscle cells (3,5) .…”

“…The regulatory function of such phosphorylations is probably much more important in nonmuscle cells (3,5) .…”

A comparison of fibroblast and smooth muscle myosins

“…The cells were grown for 3 days and then harvested by scraping off the dishes. The fibroblast myosin was purified by a modification of the method of Adelstein et al [8] using a small amount of 12-day embryo chick brain as a non-muscle carrier as described elsewhere [9] .…”

“…5 and 6). These cytoplasmic myosins have been well characterised from some sources, for example from platelets [7] , and fibroblasts [8] and appear to be similar to muscle myosins by several criteria -in size, enzymatic activity and ability to bind to actin.…”

Localization of [alpha]-actinin and tropomyosin in muscle and nonmuscle systems

“…Because both actin (Bray and Thomas, 1975;Perdue, 1973;Yang and Perdue, 1972) and myosin (Adelstein et , 1972;Ostlund et al , 1974) have been purified from fibroblasts, it seems likely that the 42,000…”