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Actin is present in cells in monomeric and polymeric (filamentous) forms. Filamentous actin is distributed in Triton-soluble (cytosolic) and Triton-insoluble (cytoskeletal core) fractions. We have used the DNase 1 inhibition assay and immunofluorescence to investigate the distribution of actin in monomeric and polymeric forms in cloned B16 murine melanoma cell lines of low and high metastatic capacity. The protease trypsin caused rounding up and detachment of both cell lines within 5 min. This was associated with almost complete depolymerization of cytosolic actin filaments but the Triton-insoluble cytoskeleton was not quantitatively affected by trypsin treatment. There were quantitative differences between the clones in their response to incubation in the presence or absence of 10% serum. The highly metastatic cell line contained 35% more actin when incubated in the presence of 10% serum, almost completely distributed to the Triton-insoluble cytoskeleton, an effect not seen in the low metastatic cells.

Solution of thrombosthenin, the contractile protein complex isolated from pig platelets, have been studied by analytical ultracentrifugation and zone sedimentation in sucrose density gradients. Freshly prepared thrombosthenin in 0.6 M KCl shows a prominent peak in the ultracentrifuge with S degrees 20w about 5.5 and higher molecular weight aggregates (greater than 100S) sedimenting quickly to the bottom of the cell. Short term storage of high ionic strength solutions of thrombosthenin induces actomyosin-like gel formation and these gels dissociate with ATP and Mg2+ ions into two components of S degrees 20w 8.0 and S degrees 20w50. The supernatant, after actomyosin gel removal, contains only the S degrees 20w5.5 protein. From results of Ca2+ ATPase activity measurements and SDS polyacrylamide gel electrophoretic mobilities of dissociated thrombosthenin separated into fractions in sucrose density gradients, it is concluded that the S degrees20w5.5 protein species is the myosin-like protein of thrombosthenin. The S degrees 20w8.0 protein is not fibrinogen but also has myosin-like properties and is believed to be myosin dimer. Species of higher S values seen in the presence of ATP and Mg2+ in the analytical ultracentrifuge and located in the higher density zones of the sucrose gradients all gave in SDS polyacrylamide gel electrophoresis a single band of molecular weight 46-47,000 daltons. These subunit proteins appear to be derived from a range of polymeric variants of the F-actin-like protein of the contractile complex. All these higher density F-actin-like proteins readily form superprecipitates and display syneresis when combined with rabbit skeletal muscle myosin or platelet myosin. They are also all capable of conferring upon these two myosins a Mg2+ activated ATPase activity. It is suggested that in thrombosthenin solutions a myosin monomer-dimer equilibrium state exists which can be directionally influenced by a number of factors. The coexistence in the solution of F-actin and Mg2+ ATP, for example, increases the propensity of the myosin-like protein to form the higher molecular weight aggregate. Such aggregation may be the initiating mechanism for the intracellular organization of the thick filaments of the actomyosin complex, preparatory to a contractile event.

Actin has been measured in subcellular fractions from Rat-1 fibroblasts and in Rous sarcoma virus-transformed Rat-1 cells (VIT), using the DNase 1 inhibition assay. The transformed cells showed a significant shift in the actin monomer (G)in equilibrium with polymer (F) equilibrium within the cell cytosol, and a significant increase in actin in the Triton-insoluble cytoskeletal core in comparison with untransformed cells. This incorporation of actin into the cytoskeletal core fraction is associated with a change in filamentous actin assemblies from 'stress fibre' patterns to punctate filament aggregates. These differences have been correlated with changes in morphology, in actin, vinculin and alpha-actinin distribution, in adhesion plaque formation and with the production of pp60v-src-associated protein kinase activity in the transformed cells. Changes in actin distribution and its polymerization in response to src-gene expression may play an important role in the determination of the transformed cell characteristics.

In the analysis of highly purified surface membrane from both resting and phagocytosing neutrophils an increase in the surface membrane associated actin has been demonstrated. This change at the cell periphery is associated with a coincident increase in the F-actin content of the cells following stimulation of the cells by exposure to opsonized Oil Red O droplets. The actin which is newly associated with the surface membrane of the phagocytosing cells was more susceptible to removal by detergent than the membrane-associated actin in resting cells and it was also noted that the F-actin associated with phagosomes was readily disrupted by detergent. A redistribution of the surface membrane glycoprotein 5'-nucleotidase was observed during phagocytosis, but no change in distribution of a 125I-labelled Lens culinaris lectin was observed during the entire phagocytic process.

The contractile proteins actin and myosin have been isolated from the soluble phase of guinea-pig polymorphonuclear leucocytes and partially characterised. Two forms of actin have been identified, designated 'Mg-actin' and 'KCl-actin'. They have different polymerising properties but their propensity to form synthetic homologous and heterologous actomyosins and to inhibit DNAase-1 does not significantly differ. Both show beta and gamma isoelectric forms in focusing gels and the Mg-actin accounts for about 5% of the soluble-phase protein and te KCl-actin around 2%. Leucocyte myosin has been isolated by affinity chromatography on N6-ADP-Sepharose with a good enrichment of both Ca2+-ATPase and the ATPase activity measured in the absence of Ca2+ or Mg2+ and in the presence of EDTA. This protein, too, has the capacity to form synthetic homologous and hybrid actomyosins with enhancement of the basal Mg2+-ATPase activity. The ratio of actin to myosin in the leucocyte calculated on a molar basis is well in excess of 100, a figure consistent with the findings from other non-muscle cells.

Exposure of blood platelets to diamide (azodicarboxylic acid-bis-dimethylamide) results in oxidation of sulphydryl groups present in the cytoskeleton and other proteins. This results in dramatic changes in functional behaviour of the cells. The distribution and level of organization of the major cytoskeletal protein actin has been studied analytically by the DNase-I inhibition assay and morphologically by electron microscopy (EM) of Triton X-100 treated platelets adherent to EM grids. Exposure to diamide results in a redistribution of actin within the cell reflected in an increase in cytoskeletal F-actin and a concomitant decrease in cytosolic actin. The magnitude of these changes depends upon the concentration of diamide and the time of exposure. Diamide also alters platelet aggregatory functions in response to certain stimuli. Treatment of normal human platelets with 0.1 mM diamide proceeds via disaggregation (5 min exposure to diamide), inhibition of aggregation (30 min exposure), to finally a normalization of the aggregation response after 60-120 min incubation with diamide. In parallel with the return to full functional response the distribution of F-actin between the cytoskeleton and cytoplasmic compartments returns to the control pattern. Incubation of the platelets with 0.5 mM diamide for 60 or more minutes leads to total inhibition of the aggregatory ability. In these cells the cytoskeleton associated F-actin remains significantly elevated and the structural organization of the cytoskeleton is markedly altered. In contrast to the network of filaments subadjacent to the surface membrane seen in unstimulated platelets, the cytoskeleton now shows electron dense zones in the more central parts of the cytoplasm. This diamide-induced structural reorganization of platelet cytoskeletal elements, associated with the inhibition of functional responses, emphasizes the dynamic nature of the membrane-cytoskeletal axis and its importance in the expression of shape changes and aggregatory phenomena in response to surface stimuli.