Changes in K+,Na+-sensitive actin gelation factor during the differentiation of myeloid leukemia cells

A homodimer protein consisting of two 38,000 dalton peptides was isolated from a murine leukemia cell line (M1). The binding molar ratio of the 38K-dimer protein to purified skeletal muscle actin was saturated at 1:3, and when the 38K-dimer/actin ratio exceeded 1:12, gelation occurred. This gelation was completely inhibited by the presence of either 10 mM KCl or 20 mM NaCl. The protein induced actin filament bundling, which required a higher 38K-dimer/actin ratio and was not affected by the presence of monovalent cations. During the differentiation of Ml cells, the sensitivity of the 38K protein to monovalent cations was decreased; that is 20 mM KCl or 50 mM NaCl was required to inhibit the gelation by the 38K protein isolated from differentiated cells. On the other hand, the intracellular K+ content of Ml cells decreased from 70 +/- 5 mM to 18 +/- 3 mM, and Na+ increased from 10 +/- 5 mM to 40 +/- 10 mM during the differentiation. These findings suggest that the differentiation brought about conditions favourable for the 38K protein to induce actin gelation, and in turn, the locomotive and phagocytic activities which were induced only after differentiation in this cell line.     

Changes in contractile proteins during differentiation of myeloid leukemia cells. I. Polymerization of actin

Quantitative and qualitative changes in cellular actin were followed during differentiation of a myeloid leukemia cell line, namely Ml, which was inducible with conditioned medium (CM). During 3 d of incubation with CM, when the Ml cells differentiated to macrophages and lost their mitotic activity, the actin content, F-actin ratio in total actin, and the actin synthesis showed an increase. A greater difference before and after differentiation was found in the ability of G-actin to polymerize. Actin harvested from CM-treated cells showed a greater ability to polymerize, depending on the increased concentration of MgCl2 and/or KCl and proteins, as compared with the actin from untreated Ml cells. Actin harvested from the Mml cell line, a macrophage line, had a particularly high polymerizability with or without CM treatment. In contrast, the actin from the D- subline, which is insensitive to CM, showed almost no polymerization.     

Changes in myosin during differentiation of myeloid leukemia cells

Changes in cellular myosin were followed during the differentiation into macrophages of a myeloid leukemia cell line (Ml) which can be induced by conditioned medium (CM) from a rat embryo culture. To extract the myosin, we used three different procedures, all of which gave a lower yield of myosin for the differentiated than for the undifferentiated Ml cells. This low extractability we attributed to increased binding of the myosin to the plasma membrane. Taking the different extractabilities into consideration, we calculated the myosin contents in the total cellular protein from the densitometry of SDS-polyacrylamide electrophoresis, 0.6% for the untreated Ml cells and 1.0% for the differentiated ones. The three ATPase activities of the Ml cell myosin were in the order, K+-EDTA-=Ca2+- much greater than Mg2+-ATPase in the presence of 0.6 M KCl, whether or not there was treatment with CM. Myosin was purified through fractionation with 25-55% saturated ammonium sulfate, then gel filtration with Sepharose 4B followed by affinity chromatography on F actin-Sepharose 4B. The Ml cell myosin consists of 1 heavy chain (H) and 3 light chains (L1, L2, L3), with molecular ratios of L1 + L2/H not equal to and L3/H not equal to 1. The ratio of L1/L2 was about 1.2 for the untreated Ml cells, but it decreased to about 0.7 after differentiation.     

Changes in protease during differentiation of mouse myeloid leukemia cells.

The protease activities of mouse myeloid leukemia cells Ml were examined using fluorescein isothiocyanate-labeled albumin as substrate. Protease activity in Ml cells was greatest at alkaline pH values with a maximum at pH 11.0, and only slight activity was seen at neutral and acidic pHs. When Ml cells were induced to differentiate into mature cells by lipopolysaccharide, their alkaline protease activity decreased greatly with marked increase in acid protease activity. Moreover, in a variant cell line Mml with the properties of differentiated Ml cells, no protease activity was found at alkaline pH values.     

Preparation and purification of polymerized actin from sea urchin egg extracts

Isotonic extracts of the soluble cytoplasmic proteins of sea urchin eggs, containing sufficient EGTA to reduce the calcium concentration to low levels, form a dense gel on warming to 35-40 degrees C. Although this procedure is similar to that used to polymerize tubulin from mammalian brain, sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows this gel to have actin as a major component and to contain no tubulin. If such extracts are dialyzed against dilute salt solution, they no longer respond to warming, but gelation will occur if they are supplemented with 1 mM ATP and 0.020 M KCl before heating. Gelation is not temperature reversible, but the gelled material can be dissolved in 0.6-1 M KCl and these solutions contain F- actin filaments. These filaments slowly aggregate to microscopic, birefringent fibrils when 1 mM ATP is added to the solution, and this procedure provides a simple method for preparing purified actin. the supernate remaining after actin removal contains the other two components of the gel, proteins of approximately 58,000 and 220,000 mol wt. These two proteins plus actin recombine to form the original gel material when the ionic strength is reduced. This reaction is reversible at 0 degrees C, and no heating is required.     

A monovalent cation-sensitive actin-binding factor in a myeloid leukemia cell line

Cell extracts of a murine leukemia cell line, M1, apparently contain three kinds of actin-gelation factors; a filamin-like protein, and 38K-dimer and 105K-dimer proteins. Unlike gelation by the filamin-like protein, gelation by the latter two proteins is inhibited by low concentrations of KCl. Our study of the 38K protein has been reported elsewhere (Takagi, K. et al., J. Biochem. Tokyo 97, 605-616, 1985). We here describe the purification and characterization of the 105K protein. The 105K protein differs from the alpha-actinin group of proteins in its antigenicity, peptide components and Ca2+-insensitivity. The saturated binding ratio of the protein to purified skeletal muscle actin is 1:8; when this ratio exceeds 1:20, gelation takes place. This gelation is inhibited completely by the presence of 25 mM KCl. Electron microscopy revealed that, in the absence of KCl, the 105K protein/actin mixture forms short actin bundles that are accompanied by a meshwork of short single filaments. The presence of 25 mM KCl did not prevent actin-bundling, but the bundles became longer and the meshwork of short filaments was no longer present.     

Actin polymerization and interaction with other proteins in temperature- induced gelation of sea urchin egg extracts

The gel which forms on warming the extracts of the cytoplasmic proteins of sea urchin eggs has been separated into two fractions, one containing F-actin and the other containing two proteins of 58,000 and 22,000 mol wt. When combined in 0.1 M KCl, even at 0 degrees C, these components will form gel material identical to that formed by warming extracts. This gel is a network of laterally aggregated F-actin filaments which are in register and which display a complex cross-banding pattern generated by the presence of the other two proteins. Low concentrations of calcium block the assembly of these proteins to form this complex structure, which may play some cytoskeletal role in the cytoplasm. This association of F-actin with the other proteins to form a gel is very likely the last step fo the process occurring in warmed extracts. At low temperatures, gelation of extracts is limited by the relative absence of F-actin, as demonstrated by the inability to sediment it at 100,000 g and also by the fact that gelation occurs immediately if exogenous F-actin is added to cold extracts. The transformation of the G-actin present in the extract to the F-form is apparently repressed at low temperatures. This is shown directly by the failure of added G-actin to polymerize at low temperatures in the presence of extract. These observations resemble those which have been reported on preparations from amoeboid cells and may be significant in the involvement of actin and these other proteins in cell division and later developmental processes.     

The role of actin in temperature-dependent gel-sol transformation of extracts of Ehrlich ascites tumor cells

Ehrlich ascites tumor cell extracts form a gel when warmed to 25 degrees C at pH 7.0 in sucrose solution, and the gel rapidly becomes a sol when cooled to 0 degrees C. This gel-sol transformation was studied quantitatively by determining the volume or the total protein of pellets of gel obtained by low-speed centrifugation. The gelation depended on nucleotide triphosphates, Mg2+, KCl, and a reducing agent. Gelation was inhibited reversibly by 0.5 microM free Ca2+, and 25--50 ng/ml of either cytochalasin B or D, but it was not affected by 10 mM colchicine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the gel was composed of six major proteins with mol wt greater than 300,000, 270,000, 89,000, 51,000, 48,000, and 42,000 daltons. The last component was identified as cell actin because it had the same molecular weight as muscle actin and bound with muscle myosin and tropomyosin. The role of actin in gelation was studied by use of actin-inhibitors. Gelation was inhibited by a chemically modified subfragment-1 of myosin, which binds with F-actin even in the presence of ATP, and by bovine pancreatic DNase I, which tightly binds with G-actin. Muscle G-actin neutralized the inhibitory effect of DNase I when added at an equimolar ratio to the latter, and it also restored gelation after its inhibition by DNase I. These findings suggest that gelation depends on actin. However, the extracts showed temperature-dependent, cytochalasin-sensitive, and Ca2+-regulated gelation as did the original extracts when the cell actin in the extracts was replaced by muscle actin, suggesting that components other than cell actin might be responsible for these characteristics of the gelation.     

Induction of either contractile or structural actin-based gels in sea urchin egg cytoplasmic extract

The gel formed by warming the 100,000 g supernate of isotonic extracts of sea urchin eggs to 40 degrees C is made up of actin and two additional proteins of mol wt of 58,000 and 220,000. Actin and 58,000 form a characteristic structural unit which has now been identified in the microvilli of the urchin egg and in the filopods of urchin coelomocytes. However, egg extract gels did not contract as those from other cell types do, and the aim of these experiments was to determine the reason for this lack of contraction. Although the extracts are dialyzed to a low ionic strength, myosin is present in soluble form and makes up approximately 1% of the protein of the extract. It becomes insoluble in the presence of high ATP concentrations at 0 degrees C, and the precipitate formed under these conditions consists almost entirely of myosin. This procedure provides a simple method of isolating relatively pure myosin without affecting other extract components and functions. Contraction will follow gelation in these extracts if the temperature and time of incubation used to induce actin polymerization are reduced to minimize myosin inactivation. At the optimal ATP and KCl concentration for contraction, the contracted material has an additional 250,000 component and contains very little 58,000. The conditions found to provide maximum gel yields favor the formation of the actin-58,000-220,000 structural gel, while reduced temperature and increase in KCl concentration results in a contractile gel whose composition is similar to those reported from amoeboid cell types. Both the structural protein cores found in the egg microvilli and a gel contraction related to the amoeboid motion which is seen in later urchin embryonic development can thus be induced in vitro in the same extract.     

The effect of methylglyoxal on actin

Studying the gelation of Ehrlich ascites tumor cell extracts at various methyglyoxal concentrations, an increase of the gelled protein fraction, composed mainly of actin, was found at 10^?7 M to 10^?5 M. When methylglyoxal was added to intact tumor cells, the filamentous portion of cytoplasmic actin was increased at 10^?7 M to 10^?6 M concentrations. Furthermore, certain functional properties of purified skeletal muscle actin were also affected by μM concentrations of methylglyoxal; the speed of actin polymerization was facilitated and more filamentous actin formed in polymerizing conditions. The possible mode of methylglyoxal action is discussed.     

Differentiation of a cell line of mouse myeloid leukemia : I. Simultaneous induction of lysosomal enzyme activities and phagocytosis

Induction of phagocytic activity in the Ml cell line of mouse myeloid leukemia, on being exposed to a conditioned medium from cultured embryo cells, was accompanied by an increment in the activities of both lysosomal acid phosphatase and acid protease. The activity of these lysosomal enzymes, as well as that of phagocytosis, was not induced when Ml cells were incubated either with the conditioned medium subjected to heat treatment or in the presence of 5-bromodeoxyuridine (BUdR). The levels of these induced enzyme activities in Ml cells were comparable to those in normal mouse peritoneal macrophages. The lysosomal enzyme activity in Mm-1 cells, which were spontaneously differentiated from Ml cells and exhibiting a higher phagocytic activity, were reminiscent of those in peritoneal macrophages. Based on these observations, it was concluded that both phagocytosis and lysosomal enzyme activity occur simultaneously during the course of differentiation. This differentiation, morphological or functional, in Ml cells in the presence of the conditioned medium was further supported by biochemical evidence.     

The contractile basis of amoeboid movement: V. The control of gelation, solation, and contraction in extracts from dictyostelium discoideum

Motile extracts have been prepared from Dictyostelium discoideum by homogenization and differential centrifugation at 4 degrees C in a stabilization solution (60). These extracts gelled on warming to 25 degrees Celsius and contracted in response to micromolar Ca++ or a pH in excess of 7.0. Optimal gelation occurred in a solution containing 2.5 mM ethylene glycol-bis (β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA), 2.5 mM piperazine-N-N'-bis [2-ethane sulfonic acid] (PIPES), 1 mM MgC1(2), 1 mM ATP, and 20 mM KCI at ph 7.0 (relaxation solution), while micromolar levels of Ca++ inhibited gelation. Conditions that solated the gel elicited contraction of extracts containing myosin. This was true regardless of whether chemical (micromolar Ca++, pH >7.0, cytochalasin B, elevated concentrations of KCI, MgC1(2), and sucrose) or physical (pressure, mechanical stress, and cold) means were used to induce solation. Myosin was definitely required for contraction. During Ca++-or pH-elicited contraction: (a) actin, myosin, and a 95,000-dalton polypeptide were concentrated in the contracted extract; (b) the gelation activity was recovered in the material sqeezed out the contracting extract;(c) electron microscopy demonstrated that the number of free, recognizable F-actin filaments increased; (d) the actomyosin MgATPase activity was stimulated by 4- to 10-fold. In the absense of myosin the Dictyostelium extract did not contract, while gelation proceeded normally. During solation of the gel in the absense of myosin: (a) electron microscopy demonstrated that the number of free, recognizable F- actin filaments increased; (b) solation-dependent contraction of the extract and the Ca++-stimulated MgATPase activity were reconstituted by adding puried Dictyostelium myosin. Actin purified from the Dictyostelium extract did not gel (at 2 mg/ml), while low concentrations of actin (0.7-2 mg/ml) that contained several contaminating components underwent rapid Ca++ regulated gelation. These results indicated : (a) gelation in Dictyostelium extracts involves a specific Ca++-sensitive interaction between actin and several other components; (b) myosin is an absolute requirement for contraction of the extract; (c) actin-myosin interactions capable of producing force for movement are prevented in the gel, while solation of the gel by either physical or chemical means results in the release of F-actin capable of interaction with myosin and subsequent contraction. The effectiveness of physical agents in producting contraction suggests that the regulation of contraction by the gel is structural in nature.     

Spectrin plus band 4.1 cross-link actin. Regulation by micromolar calcium

A low-salt extract prepared from human erythrocyte membranes forms a solid gel when purified rabbit muscle G- or F-actin is added to it to give a concentration of approximately 1 mg/ml. This extract contains spectrin, actin, band 4.1, band 4.9, hemoglobin, and several minor components. Pellets obtained by centrifugation of the gelled material at 43,000 g for 10 min contain spectrin, actin, band 4.1, and band 4.9. Although extracts that are diluted severalfold do not gel when actin is added to them, the viscosity of the mixtures increases dramatically over that of G-actin alone, extract alone, or F-actin alone at equivalent concentrations. Heat-denatured extract is completely inactive. Under conditions of physiological ionic strength and pH, information of this supramolecular structure is inhibited by raising the free calcium ion concentration to micromolar levels. Low-salt extracts prepared by initial extraction at 37 degrees C (and stored at 0 degree C) gel after actin is added to them only when warmed, whereas extracts prepared by extraction at 0 degree C are active on ice as well as after warming. Preincubation of the 37 degrees C low-salt extract under conditions that favor conversion of spectrin dimer to tetramer greatly enhances gelation activity at 0 degree C. Conversely, preincubation of the 0 degree C low-salt extract under conditions that favor conversion of spectrin tetramer to dimer greatly diminishes gelation activity at 0 degree C. Spectrin dimers or tetramers are purified from the 37 dgrees or 0 degree C low-salt extract by gel filtration at 4 degrees C over Sepharose 4B. The addition of actin to either purified spectrin dimer (at 32 degrees C) or tetramer (at 0 degree C or 32 degrees C) results in relatively small increases in viscosity, whereas the addition of actin to a high-molecular-weight complex (HMW complex) containing spectrin, actin, band 4.1, and band 4.9 results in dramatic, calcium-sensitive increases in viscosity. These viscosities are comparable to those obtained with the 37 degrees or 0 degree C low-salt extracts. The addition of purified band 4.1 to either purified spectrin dimer (at 32 degrees C) or purified spectrin tetramer (at 0 degree C) plus actin results in large increases in viscosity similar to those observed for the HMW complex and the crude extract, which is in agreement with a recent report by E. Ungewickell, P. M. Bennett, R. Calvert, V. Ohanian, and W. B. Gratzer. 1979 Nature (Lond.) 280:811-814. We suggest that this spectrin-actin-band 4.1 gel represents a major structural component of the erythrocyte cytoskeleton.     

Effects of myosin and heavy meromyosin on actin-related gelation of HeLa cell extracts

The gelation induced by warming (to 25 degrees C) the 100,000 g supernatant fraction (extract) of HeLa cells lysed in a buffer containing sucrose, ATP, DTE, EGTA, imidazole, and Triton X-100 was studied in the presence of myosin and heavy meromyosin (HMM). Myosin mixed with extract induces shrinkage of the gel, but jelled extract or myosin alone does not shrink. In the concentration range, 0.14-1.04 mg/ml of myosin, the degree of shrinkage is roughly proportional to the concentration of myosin. Supplementa MgCl2 also promotes shrinkage. HMM (0.4-0.8 mg/ml) can inhibit gel formation by extract in tubes or floated on a sucrose cushion. Gel electrophoresis of gels shrunken by added myosin or electrophoresis of the proteins which can be sedimented from extract after incubation in the presence of HMM indicate that both myosin and HMM interfere with the changes in sedimentability of the high molecular weight protein (HMWP) thought to participate (together with actin) in gel formation in HeLa cell extracts (R. R. Weihing, 1976. J. Cell Biol. 71:303-307). These results, together with previous results showing that actin is present and that HMWP is enriched in the plasma membrane fraction of HeLa cells (R. R. Weihing, 1976. Cold Spring Harbor Conf. Cell Proliferation. 3:671-684), point to the possibility of dynamic changes in the interactions of HMWP or myosin with actin in processes of movement occurring at the cell surface.     

Proteolytic enhancement of gelation of ascites tumor cell extracts. Relationship to actin binding protein

Proteolysis of cytoplasmic extracts of sarcoma 180 and MAT-C1 adenocarcinoma ascites cells enhances the rate of gelation. Only high molecular weight polypeptides, including actin binding protein and myosin, are cleaved during the process; actin is not cleaved. In MAT-B1 adenocarcinoma extracts the gelation rate was not enhanced by proteolysis and actin binding protein was not readily cleaved. Electrophoretic comparisons of trypsin-treated and untreated extracts of MAT-B1 and MAT-C1 cells show that actin binding protein is the only readily discernible polypeptide which is cleaved in the C1 cells but not in the B1 cells. These results suggest that actin binding protein may act as an inhibitor of gelation.     

Calcium-sensitive, actin-related gelation of cell extracts of thermosensitive Chinese hamster lung cells capable of anchorage-independent growth.

The extent of actin-related gelation of extracts of thermosensitive Chinese hamster lung (CHL) cells capable of anchorage-independent growth was studied quantitatively by monitoring the total protein in the gel obtained by low-speed centrifugation. The gelation depended on the presence of ATP, KCl, MgCl2, and a reducing agent. Micromolar concentrations of Ca2+ and low doses of cytochalasin B inhibited the actin-related gelation of these extracts. The gelation was more sensitive to inhibition by Ca2+ and cytochalasin B when the extracts were prepared from cells cultured at the permissive rather than the nonpermissive temperature. When various ts mutants were examined, the half-maximal inhibitory dose (HMID) of Ca2+ and cytochalasin B for gelation of extracts of cells cultured at the nonpermissive temperature was between 1.25 and 2.19 times higher than that for extracts of the same cells cultured at the permissive temperature. The values of the HMID for Ca2+ and cytochalasin B changed shortly after the shift in temperature of cell cultures from the nonpermissive to permissive temperature. When cell extracts were incubated at the permissive temperature in vitro for only 15 minutes, these changes in values of HMID were also observed. Analysis of polypeptides of cell extracts and gel pellets on polyacrylamide gel electrophoresis suggested that the decrease in amount of a high-molecular-weight actin-binding protein (250 kDa) may play an essential role in the increased sensitivity to inhibition by Ca2+ and cytochalasin B of actin-related gelation in extracts of these ts mutants.     

Plasma membranes purified from myeloid leukemia cells before and after differentiation : I. Characterization of spectrin-like proteins and increased association of actin

Two-step sucrose density gradient centrifugation was used to isolate the plasma membrane of a myeloid leukemia cell line (Ml). Calspectin (or fodrin) was identified in the Triton-insoluble fraction from the plasma membrane, and the molecular size and actin- and calmodulin-binding activity were studied. During differentiation of this cell line, which accompanied the induction of cell motility and phagocytic activity, the membrane-bound actin increased dramatically, whereas calspectin increased only slightly. Therefore, calspectin does not appear to have a major function in the increased binding of actin filaments to the plasma membrane, a requirement for the induction of cell motility.     

Characterization and localization of actinogelin, a Ca2+ - sensitive actin accessory protein, in nonmuscle cells

Actinogelin, which induces gelation of F-actin at Ca2+ concentrations below micromolar concentrations but not at higher concentrations, was isolated in the pure state from Ehrlich tumor cells. The protein consists of subunits of 112,000-115,000 daltons and under physiological conditions is present mostly as a dimer. Up to 1 mol of actinogelin (dimer) binds to 10-12 mol of actin monomer. The binding was slightly decreased by the presence of 50 microM Ca2+ and almost completely inhibited by 300 mM KCl. Antibodies against actinogelin giving a single precipitation line with Ehrlich cell extract and with pure actinogelin were raised in rabbits. Antibody preparations were purified before use in an affinity column containing purified actinogelin. In mouse embryo fibroblasts and 3T3 cells, staining of actin bundles by the antiactinogelin antibody usually was discontinuous or gave a striated appearance. Most of the crossing points of the actin bundles were intensively stained. In epithelial cells from mouse small intestine, actinogelin was distributed throughout the cell, with the exception of the microvilli, which were devoid of staining. In mouse peritoneal cells, the antibody staining patterns were similar to those of tetramethylrhodamine isothiocyanate-labeled heavy meromyosin, but the former usually were sharper than the latter. Intracellular localization of actinogelin was drastically altered by cytochalasin D treatment at 10 microgram/ml. We conclude that actinogelin is present in a wide variety of cell types and discuss the possible participation of actinogelin in the Ca2+-dependent regulation of microfilament distribution.     

Characterization of "abp38" in cytoskeleton of mouse myeloid leukemia cells and distribution of abp38 in various tissues

A specific antibody was prepared against "abp38," a 38 kDa-dimer protein purified from mouse myeloid leukemia cells (M1 cells), that induce gelation of actin filaments in a K+-dependent manner. Immunochemically, the total content of abp38 in undifferentiated M1 cells was found to be 0.89% of the total protein (10.7 micrograms/10(7) cells). This content increased about 8-fold (89.5 micrograms/10(7) cells) after the M1 cells had differentiated into macrophages. When the cells before differentiation were extracted with Triton solution containing 150 mM KCl, almost all abp38 in the cytoskeleton was removed, whereas in cells after differentiation, amount of abp38 remaining in the cytoskeleton was 4.5 micrograms per 10(7) cells. The amount of cytoskeleton-bound abp38 of M1 cells and mouse peritoneal macrophages decreased with increase in K+ concentration in the extraction solution. Immunoreactive molecules against abp38 antibody were present in various tissues and cultured cell lines except for skeletal muscle and erythrocytes. Furthermore, actin binding protein with a molecular size of 38 kDa was found in bovine brain. These data suggest that abp38 is a ubiquitous protein present in various tissues and species.     

Regulation of the microfilament system in normal and polyoma virus transformed cultured (BHK) cells

The content and state of actin in baby hamster kidney (BHK) cells before and after transformation with polyoma virus were examined by deoxyribonuclease assay and gel electrophoresis followed by dye elution. The actin content of the transformed cells, relative to total cell protein, was lower than that of the normal cells by 30-50%. In both the normal and transformed cells the greater part of the total actin was found on lysis to be in the monomeric state. Cytoplasmic and membrane fractions of the two cell lines were, in qualitative terms, very similar in their protein compositions. The plasma membrane isolated from the transformed cells was richer in actin than that from the untransformed, and both membrane fractions contained proteins corresponding to myosin, filamin and alpha-actinin on SDS-polyacrylamide gels. The cell extract from both the normal and transformed lines formed an actin-based gel on incubation at 30 degrees C, although the amount of the cross-linked actin was much smaller in the latter. This was a consequence not only of the lower concentration of total actin in the cell, but also, presumably, of a gross relative deficiency in the concentration or activity of filament cross-linking protein(s) in the cytoplasm. Thus, small aliquots of cytoplasmic fractions from transformed cells, when added to an excess of exogenous F-actin, were able to cross-link the filaments to a much smaller extent than those from the normal cells. A similar range of proteins was found to be associated with the actin gels formed from both cell extracts. One conspicuous difference was that a species migrating in SDS-gel electrophoresis as a doublet with a subunit molecular weight of about 58,000, and tentatively identified as intermediate filament protein, was replaced in the transformed cells by a single band. Filament cross-linking activity of the cytoplasmic fractions was enhanced by addition of Triton extracts of crude membranes, although the latter were not capable of cross-linking exogenous F-actin on their own. The effect of Triton extracts was much greater in the case of membranes from the transformed cells. The cytoplasmic fractions of BHK cells contain capping protein(s) and/or complexes of such proteins with actin; these reveal themselves by the propensity of the extracts to nucleate polymerization of exogenous G-actin. This activity was more abundant in transformed cells, despite their lower actin content. Their membranes were also more effective in nucleating G-actin polymerization, indicating the presence of a greater number of filament ends.(ABSTRACT TRUNCATED AT 400 WORDS)