Microtubules from suspension cultures of HeLa cells have been purified by carrying them through four complete cycles of polymerization at 37 degrees C and depolymerization at 4 degrees C. These microtubules show, in addition to the major alpha- and beta-tubulin components, major proteins with molecular weights of 201 000-206 000 (comprising 4.5% of the total protein), proteins with molecular weights of 97 000, 100 000, 104 000, and 114 000 (together comprising approximately 2% of the total protein), and minor components with molecular weights of 68 000 and 151 000. HeLa microtubules have also been reconstituted from purified HeLa tubulin and proteins from HeLa microtubules separated from tubulin by DEAE-cellulose column chromatography. Experiments on the fractionation and reconstitution of both two- and four-cycle microtubules suggest that the 201 000-206 000-dalton proteins are incorporated into microtubules and promote tubulin polymerization. Microtubules formed by fractionationand reconstitution of two-cycle microtubules also contain several other proteins with molecular weights of 132 000, 146 000, 151 000, 160 000, and 284 000, although these are not present in microtubules carried through four assembly-disassembly cycles. Evidence is also presented which shows that a 68 000-dalton protein which is a prominent component of HeLa microtubules after two polymerization-depolymerization cycles does not stoichiometrically copurify with tubulin through repeated assembly--disassembly cycles and does not stimulate tubulin polymerization. On the other hand, the sedimentation of this 68 000-dalton protein is apparently influenced by the presence of polymerized microtubules, suggesting that this protein may be a component of a system whjich interacts weakly with microtubules. Finally, evidence is presented suggesting that two-cycle microtubules contain a proteolytic activity that can digest the 201 000-206 000-dalton proteins.

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When the 100,000 g supernatant fraction (extract) of HeLa cells lysed in a buffer containing sucrose, ATP, DTE, EGTA, imidazole, and Triton X-100 is incubated at 25 degrees C, it gels, and actin and a HMWP are progressively enriched in the extract and in gel isolated from extract. CB (greater than or equal to 0.25 muM) inhibits gelation and specifically lowers the concentrations of actin and the HMWP in the fraction which sediments at 100,000 g after incubation. These results indicate that actin and HMWP are partly disaggregated by cytochalasin treatment, and thus that their aggregation is related gelation. Inasmuch as previous results showed that actin is present and HMWP is enriched in the plasma membrane fraction of HeLa cells, the results also point to a possible relation between plasma membrane-associated gel and in vivo effects of CB.

The filamins are a group of homologous proteins defined by their high native molecular weight (500,000), their amino acid compositions, their cross-reactivity to antibodies to heterologous filamins, their localization to actin networks and bundles in situ, and their ability to cross-link actin filaments in vitro into three-dimensional networks and bundles. Native filamins contain two subunits (relative mass = 250 000). Each subunit carries at least one actin-binding site and formation of bivalent dimers is therefore believed to explain filamin's ability to cross-link actin filaments. Formation of networks in vitro (corresponding to formation of macroscopic gels) has been analyzed using the theory of Flory. As predicted, a sharp transition to gel (at the critical gelation concentration of filamin) is observed when actin is mixed with increasing concentrations of filamin and the critical gelation concentration is found to vary inversely with the length of actin filaments. However, the measured values of the critical gelation concentration are all higher (2- to 14-fold) than predicted by the theory and the prediction that the critical concentration varies directly with the actin concentration was verified with only one of two techniques used. Filamin's length (160-190 nm) and flexibility (1000-fold greater than actin filaments) may make it especially well fitted to cross-link actin filaments into three-dimensional networks when present in low molar ratios (1:200 to 1:50) relative to actin. At higher molar ratios (greater than 1:20) it also cross-links actin filaments into bundles. Assuming that filamin actually helps organize supramolecular structures inside cells (not yet tested directly), then its concentration relative to actin may help determine whether networks or bundles are formed. Other factors that may influence its localization and function inside cells include competition with other actin-binding proteins (such as myosin and tropomyosin) for binding sites on actin and phosphorylation, which may alter its ability to bind to actin.

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A protein component of membranes isolated from 3T3 mouse fibroblasts and HeLa cells has been identified as actin by peptide mapping. Extensive but apparently not total coincidence was found between the peptide maps of these two nonmuscle membrane-associated actins compared to chick skeletal muscle actin. Between 2 and 4% of the total membrane protein appears in the actin band on sodium dodecyl sulfate polyacrylamide gels of 3T3 membranes while about 4% of the membrane protein appears as the actin band from HeLa membranes. These values represent approximately the same proportion of actin to total protein found in the cell homogenates. Treatment of intact cells with levels of cytochalasin B sufficient to cause pronounced morphological changes did not change the amount of actin associated with the membrane in either 3T3 or HeLa cells. However, incubation of isolated membranes under conditions favoring conversion of actin from filamentous to monomeric form resulted in dissociation of approximately 80 and 60% of the actin from 3T3 and HeLa membranes, respectively. Thus, approximately 20% of 3T3 membrane actin and 40% of HeLa membrane actin remained associated with the membrane even under actin depolymerizing conditions.Actin has now been identified as a component of essentially all eukaryotic animal cells in which it has been sought. In addition, myosin-and tropomyosin-like proteins have been identified in many of these nonmuscle cells. (An extensive review on the subject of contractile proteins of nonmuscle cells has recently appeared [29].) In several electron microscope preparations, apparent contact between plasma membrane and actin filaments (or thin filaments with the dimensions of actin) has been observed in situ (24,36,42,44,45) or in isolated plasma membranes (28). Biochemical evidence has also been presented that actin is associated with membranes of synaptosomes (3). It is, therefore, likely that actin filaments function at least in part via attachment to the plasma membrane of cells. Furthermore, control of the membrane-actin association seems a likely point for regulation of actin function as an effector of diverse cellular movements and morphological changes. We have, therefore, chosen as the initial phase of studies on the mechanisms of cellular movements to try to identify actin in membranes from 3T3 mouse fibroblasts and HeLa cells.

Although the purification of microtubules from brain by alternate cycles of polymerization and depolymerization in vitro has become routine, the application of this method to non-neural, cultured cells has been less successful. Previous investigations have suggested that it was necessary to use substrate-grown cells and 4 M glycerol to obtain microtubules from cultured cells. We have developed a method for preparing microtubules from HeLa cells in spinner cultures without the use of glycerol. Microtubules can be readily carried through two complete cycles of polymerization at 37~ and depolymerization at 4~ in vitro. The microtubules obtained are morphologically similar to brain microtubules in electron micrographs, and the tubulin subunits have mobilities similar to those of brain tubulins on polyacrylamide gels. Typical yields in the second polymerization pellet are about 1 mg protein/ml of packed cells or 2.5-3.0% of the total protein in the soluble cell extract. The major nontubulin protein present after two cycles of polymerization and depolymerization has an apparent mol wt of 68,000 daltons. If glycerol is used during polymerization, this band is virtually absent.

Actin has been isolated from the soil amoeba Acanthamoeba castellanii (Neff strain) and shown to resemble rabbit muscle actin in several ways: it is a globular 3S monomer at low ionic strength and a fibrous 30S polymer at high ionic strength; the fibers form viscous complexes with muscle myosin which are dissociated by ATP; the fibrous amoeba actin activates Mg-ATPase of rabbit heavy meromyosin at low ionic strength. The amino acid analysis of Acanthamoeba actin, including the presence of 3-methylhistidine, resembles that of var-