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.     

Cytochalasin B inhibits actin-related gelation of HeLa cell extracts

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.     

Actin-binding and dimerization domains of HeLa cell filamin

HeLa cell filamin is a linear, bivalent, homodimer of high molecular weight subunits (Mr 250,000 that may cross-link actin filaments in vivo into supramolecular structures such as networks and bundles. We used millimolar Ca protease from chicken breast muscle to cleave the subunit into smaller fragments that we mapped with respect to the overall structure of the dimer. The enzyme cleaves HeLa filamin into a larger (Mr 192,000) and a smaller (Mr 104,000) fragment; the smaller fragment is the precursor of a still smaller (Mr 92,000) fragment. Only the larger fragment bound to actin in a cosedimentation test, suggesting that it contains the actin-binding region of the subunit. Digestion of HeLa filamin that had been cross-linked with dimethyl adipimidate produced a good yield of the Mr 192,000 fragment but a poor yield of the Mr 104,000/92,000 fragments. Since native filamins are head-to-head dimers, it was expected that cross-linking would proceed most readily at the dimerization site and, therefore, it appears that the Mr 192,000 fragment is cleaved from cross-linked filamin because it is distal to the dimerization region, while the Mr 104,000/92,000 fragments are absent because they lie at the dimerization region and were cross-linked to a form that was not identifiable by sodium dodecyl sulfate electrophoresis.     

Comparison of a major heat-stable microtubule-associated protein in HeLa cells and 190-kDa microtubule-associated protein in bovine adrenal cortex

A heat-stable microtubule-associated protein (MAP) with a molecular weight of 190,000, termed 190-kDa MAP, has been purified from bovine adrenal cortex (Murofushi, H. et al. (1986) J. Cell Biol. 103, 1911-1919). Immunoblotting experiments using an antibody against this MAP revealed that several kinds of culture cells derived from human tissues contain proteins with an apparent molecular weight of 180,000 reacting with the antibody. Indirect immunofluorescence microscopic observation of HeLa cells showed that the immunoreactive protein co-exists with microtubules, indicating that the protein is one of the HeLa MAPs. A heat-stable MAP with a molecular weight of 180,000, termed here HeLa 180-kDa MAP, was purified by the taxol-dependent procedure (Vallee, R.B. (1982) J. Cell Biol. 92, 435-442) and successive co-polymerization with brain tubulin. This protein was the most abundant MAP in HeLa cells, suggesting that the MAP is identical to the major HeLa MAP previously reported by Bulinski and Borisy (Bulinski, J.C. & Borisy, G.G. (1980) J. Biol. Chem. 255, 11570-11576) and Weatherbee et al. [1980) Biochemistry 19, 4116-4123). It was shown that, like bovine adrenal 190-kDa MAP, yet distinct from brain MAP2 and tau, purified HeLa 180-kDa MAP does not interact with actin filaments. This common characteristic of the two MAPs along with the same heat-stability strongly suggests that they are members of the same group of MAPs. The fact that HeLa 180-kDa MAP reacts with an antibody against bovine adrenal 190-kDa MAP means that they share common epitopes, in other words, common local amino acid sequences. However, the limited proteolytic patterns of the two MAPs with S. aureus V8 protease and chymotrypsin were distinct from each other, suggesting the presence of large differences in the overall primary structures between bovine adrenal 190-kDa MAP and HeLa 180-kDa MAP.     

Highly homologous filamin polypeptides have different distributions in avian slow and fast muscle fibers

The high molecular weight actin-binding protein filamin is located at the periphery of the Z disk in the fast adult chicken pectoral muscle (Gomer, R. H., and E. Lazarides, 1981, Cell, 23: 524-532). In contrast, we have found that in the slow anterior latissimus dorsi (ALD) muscle, filamin was additionally located throughout the l band as judged by immunofluorescence with affinity-purified antibodies on myofibrils and cryosections. The Z line proteins desmin and alpha-actinin, however, had the same distribution in ALD as they do in pectoral muscle. Quantitation of filamin and actin from the two muscle types showed that there was approximately 10 times as much filamin per actin in ALD myofibrils as in pectoral myofibrils. Filamin immunoprecipitated from ALD had an electrophoretic mobility in SDS polyacrylamide gels identical to that of pectoral myofibril filamin and slightly greater than that of chicken gizzard filamin. Two-dimensional peptide maps of filamin immunoprecipitated and labeled with 125I showed that ALD myofibril filamin was virtually identical to pectoral myofibril filamin and was distinct from chicken gizzard filamin.     

Immunofluorescence localization of HeLa cell microtubule-associated proteins on microtubules in vitro and in vivo

Rabbit antisera were prepared against the two major groups of microtubule-associated proteins (MAPs) from HeLa cells, proteins of approximately 210,000 molecular weight (210k MAPs), and 125,000 mol wt (125k MAPs). These antisera were characterized by a sensitive antigen detection technique that employs immunofluorescence to localize cross- reactive material in polyacrylamide gels. Antisera prepared against the 210k MAPs showed no cross-reactivity with extract proteins of other molecular weights or with bran MAPs, but did react with proteins of 210,000 mol wt and with a minor HeLa MAP of approximately 255,000 mol wt. Antibodies prepared against the 125k HeLa MAPs, likewise, reacted specifically with proteins of 125,000 mol wt, showing no cross- reactivity with other HeLa extract proteins or porcine brain MAPs. Immunofluorescence with the 210k and 125k MAP antisera was used to demonstrate the association of each of the MAPs with fixed HeLa microtubules in vitro. In addition, immunofluorescence with these antisera revealed a physical association of 210k and 125k MAPs with a Colcemid-sensitive fiber network in fixed interphase and mitotic HeLa cells. Thus, using specific, well-characterized antisera to the two major groups of HeLa MAPs, we have shown that these proteins are components of microtubules in HeLa cells.     

Effect of filamin and controlled linear shear on the microheterogeneity of F-actin/gelsolin gels

We have previously established [Cortese and Frieden, J. Cell Biol. 107:1477-1487, 1988] that actin gels formed under shear are microheterogeneous. In this study, the effect of cross-linking (by chicken gizzard filamin), severing (by plasma gelsolin), and shear on actin microheterogeneity are investigated using fluorescence photobleaching recovery and video microscopy. We find that filamin and shear form microheterogeneous F-actin:gelsolin gels by different mechanisms. Bundling of actin:gelsolin filaments by filamin can be explained by an increase in the apparent length of the filaments due to interfilament binding, resulting in a decrease of the polymer number concentration at which filaments organize into anisotropic phases. Some intrafilament binding of filamin to actin filaments may also be present, and those filaments coated with filamin immobilize more slowly than actin under the same polymerization conditions. The length of F-actin/gelsolin filaments seems to be a major factor in controlling the extent of bundling relative to network formation. In contrast, the effect of shear on the microheterogeneity of actin:gelsolin filaments is consistent with our previous proposal that shear aligns actin filaments, allowing filament-filament interactions and phase formation to occur. Short filaments are unable to organize into branched actin networks, but they can create large aggregates under low shear. Longer actin filaments will exist as networks with variable levels of branching and are less sensitive to shear. The effect of the intensity of a shear field on the spatial distribution of actin may involve a progressively more random orientation of actin molecules and bundles. A regular pattern develops across the sample at low shear rates (0.04-1.39 s-1), and becomes very irregular at higher shear rates (greater than 10 s-1). We suggest here that actin-binding proteins and shear can control the transition between isotropic networks and anisotropic phases by their effect on apparent length and local filament concentration, and also that this transition can have substantial effects on the resistance of cells to mechanical stress.     

Nonerythrocyte spectrins: actin-membrane attachment proteins occurring in many cell types

The properties of brain fodrin have been analyzed and compared with those of erythrocyte spectrin. Both proteins consist of high molecular weight polypeptide doublets on SDS polyacrylamide gels and in solution behave as very large asymmetric molecules. Both proteins show a characteristic increase in sedimentation coefficient in the presence of 20 mM KCl. Antibodies against the brain protein cross-react with erythrocyte spectrin and cross-react with similar high molecular weight doublet polypeptides in SDS polyacrylamide gels of other cell types and plasma membrane preparations. Both proteins bind actin. The brain protein and erythrocyte spectrin show specific and competitive binding to erythrocyte membranes and this binding is inhibited by antibodies against erythrocyte ankyrin. Several of these properties distinguish these proteins from the class of high molecular weight actin-binding proteins that includes filamin and macrophage actin-binding protein. We conclude that together with erythrocyte spectrin, the brain protein and equivalent, immunologically related proteins in other cell types belong to a single class of proteins with the common function of attachment of actin to plasma membranes. Based on the structural and functional similarities, the name spectrin would seem appropriate for this whole class of proteins.     

Purification of mammalian filamin. Similarity to high molecular weight actin-binding protein in macrophages, platelets, fibroblasts, and other tissues

We have purified the high molecular weight actin-binding protein, filamin from guinea pig vas deferens. We find this mammalian filamin is very similar to chicken gizzard filamin in subunit molecular weight, amnio acid composition, actin-binding properties, immunological cross-reactivity, and the ability to be phosphorylated by cyclic AMP-dependent protein kinase. Anti-filamin antibodies cross-react with a high molecular weight macrophage actin-binding protein, and with a high molecular weight protein in platelets and fibroblasts. Furthermore like filamin, these proteins are also phosphorylated and cyclic AMP stimulates their phosphorylation. Anti-filamin antibodies do not cross-react with the erythrocyte membrane protein spectrin or with high molecular weight proteins in brain extracts. We conclude that filamin from avian and mammalian smooth muscle are very similar proteins and furthermore that many, but not all, non-muscle cells contain a protein closely related to filamin.     

Isolation and characterization of a high molecular weight actin-binding protein from Physarum polycephalum plasmodia

A high molecular weight actin-binding protein was isolated from the Physarum polycephalum plasmodia. The protein ( HMWP ) shares many properties with other high molecular weight actin-binding proteins such as spectrin, actin-binding protein from macrophages, and filamin. It has a potent activity to cross-link F-actin into a gel-like structure. Its cross-linking activity does not depend on calcium concentrations. Hydrodynamic studies have revealed that the protein is in the monomeric state of a polypeptide chain with molecular weight of approximately 230,000 in a high ionic strength solvent, while it self-associates into a dimer under physiological ionic conditions. Electron microscopic examinations of HMWP have shown that the monomer particle observed in a high ionic strength solvent is rod shaped with the two-stranded morphology very similar to that of spectrin. On the other hand, under physiological ionic conditions, the HMWP dimer shows the dumb-bell shape with two globular domains connected with a thin flexible strand.     

Chromatin changes related to dedifferentiation and differentiation in tobacco tissue culture (Nicotiana tabacum L.)

Non-histone chromosomal proteins (NHP) were isolated from different stages of Nicotiana tabacum L. pith dedifferentiation to callus and callus redifferentiation. The NHP were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis on slab gels and analyzed by densitometry. Simultaneous histological changes are reported. In both processes, some high molecular weight protein (HMWP) bands increase drastically in an induction period, previous to cell proliferation, and decrease when cell division declines. Some low molecular weight protein bands, intense in pith tissue, decrease early when callus is forming and increase when cells differentiate. chromatin template activity is high when cells proliferate, coinciding with maximum HMWP-bands intensity.Abbreviations HMWP high molecular weight proteins - IAA indole-3-acetic acid - LMWP low molecular weight proteins - NHP non-histone proteins - TA template activity     

Calponin interaction with alpha-actinin-actin: evidence for a structural role for calponin

The purpose of this study was to address the paradox of calponin localization with alpha-actinin and filamin, two proteins with tandem calponin homology (CH) domains, by determining the effect of these proteins on the binding of calponin to actin. The results show that actin can accommodate near-saturating concentrations of either calponin and alpha-actinin or calponin and filamin with little change or no change in ligand affinity. Little direct interaction occurred between alpha-actinin and calponin in the absence of actin, so this effect is not likely to explain the co-distribution of these proteins. Calponin, like alpha-actinin, induced elastic gel formation when added to actin. When alpha-actinin was added to newly formed calponin/actin gels, no change was seen in the mechanical properties of the gel compared to calponin and actin alone. However, when calponin was added to newly formed alpha-actinin/actin gels, the resulting gel was much stronger than the gels formed by either ligand alone. Furthermore, gels formed by the addition of calponin to alpha-actinin/actin exhibited a phenomenon known as strain hardening, a characteristic of mechanically resilient gels. These results add weight to the concept that one of the functions of calponin is to stabilize the actin cytoskeleton.     

Actin deficiency induces cofilin phosphorylation: proteome analysis of HeLa cells after beta-actin gene silencing

Actin-binding proteins regulate the dynamic structure and function of actin filaments in the cell. Much is known about how manipulation of the actin-binding proteins affects the structure and function of actin filaments; however, little is known about how manipulation of actin in the cell affects actin-binding proteins. We addressed this question by utilizing two technologies: RNA interference and 2-dimensional gel electrophoresis. We knocked down beta-actin expression in HeLa cells using short interfering RNA and applied 2-DGE to examine alterations in the HeLa cell proteome. We revealed a 2-5 fold increases of four protein spots on 2-D gels and identified these proteins by mass spectrometry. Three of the four proteins were actin-binding proteins, including cofilin, which promotes both disassembly and assembly of actin filaments but becomes inactivated when phosphorylated. Further examination revealed that the cofilin total protein level barely increased, but the phosphorylated cofilin level increased dramatically in HeLa cells after beta-actin siRNA treatment. These results suggest that in response to siRNA-induced beta-actin deficiency HeLa cells inactivate cofilin by phosphorylation rather than down-regulate its protein expression level. This study also demonstrates that the combination of RNA interference and 2-dimensional gel electrophoresis technologies provides a valuable method to study protein interactions in a specific cellular pathway.     

A 250K-molecular-weight actin-binding protein from actin-based gels formed in sea urchin egg cytoplasmic extract

The actin-based gel formed at 35 degrees C in the cytoplasmic extract from eggs of a sea urchin, Tripneustes gratilla, contains several high-molecular-weight proteins. Among them, the 250K-molecular-weight protein was isolated and characterized. This protein migrated slightly more slowly than filamin from chicken gizzard upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. It reacted only very weakly with antibodies against chicken gizzard filamin or against a high-molecular-weight actin-binding protein from Physarum plasmodia. It did not react with antibodies against chicken erythrocyte alpha-spectrin nor against the 220K protein from the same egg. A chemical crosslinking experiment revealed the presence of dimers in the purified 250K protein preparation. A rotary shadowed specimen of such a preparation showed wavy single-stranded molecules 120-170 nm long, having five to six globular domains, which may represent dimers. The appearance was different from that of spectrin or actin-binding protein from macrophage or chicken gizzard filamin. This protein increased the viscosity of F-actin solution. It bound to F-actin preferably at low KCl concentrations such as 20 mM. The binding ability was not influenced by pH between 6.0 and 7.5, although it was somewhat reduced above pH 8.0. The binding was insensitive to low Ca ion concentrations. Electron microscopy using the negative staining technique supported the idea that this protein crosslinks actin filaments. In addition, a second protein from egg gels, with a reported molecular weight of about 220K (Kane, R.E., J. Cell Biol. 66, 305-315 (1975)), comigrated with human erythrocyte alpha-spectrin on an SDS-gel and reacted with antibodies against chicken erythrocyte alpha-spectrin. This suggests that this protein is a sea urchin egg spectrin. The role of these proteins in the cytoskeleton formation in the sea urchin egg is discussed.     

A complement-resistant HeLa cell line (T638) is blocked at the step of C3 deposition

A complement-resistant line of HeLa cells (T638) was derived by serial passage of complement-susceptible HeLa cells in anti-beta 2-microglobulin (b2m) antiserum and complement. The T638 line maintained stable complement resistance when passed for an additional 1500 generations in the absence of antiserum and complement. T638 cells expressed equivalent levels of cell-associated b2m as did the parent HeLa cell line. Furthermore, T638 cells were resistant to killing by complement and anti-HeLa antiserum with specificity for molecules other than b2m. These results indicate that the resistance of T638 cells does not simply reflect loss of anti-b2m binding antigens. We next investigated the mechanism of resistance of T638 cells to complement-mediated killing. Antibody-sensitized HeLa and T638 cells both consumed CH50 activity completely from normal human serum; cytotoxicity was not mediated via the alternative complement pathway. HeLa and T638 cells caused equivalent utilization of C4 from normal human serum in the presence of antibody. Consumption of C2, greater with T638 than with HeLa cells during incubation in serum, was complete when cells bearing purified C1 and limited C4 were incubated with C2. T638 cells bound more 3H-C4 than HeLa cells during incubation in serum, but binding of 3H-C3 by T638 cells was fourfold to fivefold less than by HeLa cells. Finally, we investigated the rate of decay in the capacity of C142 on HeLa and T638 to cleave and deposit 3H-C3. The T1/2 for decay of C142-mediated binding of 3H-C3 on HeLa was 3.9 min, whereas minimal C3 deposition was detected on T638 cells at all time points. These results show that T638 cells evade complement-mediated lysis despite activating early components of the classical complement pathway. The mechanism of resistance is a failure to form an effective C3 convertase.     

Actin associated with membranes from 3T3 mouse fibroblast and HeLa cells

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 percent of the total membrane protein appears in the actin band on sodium dodecyl sulfate polyacrylamide gels of 3T3 membranes while about 4 percent 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 percent of the actin from 3T3 and HeLa membranes, respectively. Thus, approximately 20 percent of 3T3 membrane actin and 40 percent of HeLa membrane actin remained associated with the membrane even under actin depolymerizing conditions.     

Further characterization of HeLa S3 plasma membrane ghosts

A plasma membrane fraction of HeLa S3 cells, consisting of ghosts, is characterized more fully. A simple procedure is described which permits light and electron microscope study of the plasma membrane fraction through the entire depth of the final product pellet and through large areas parallel to the surface. Contamination by nuclei is 0.14%, too little for DNA detection by the diphenylamine reaction. Contamination by rough endoplasmic reticulum and ribosomes is small, a single ghost containing about 3% of the RNA in a single cell. Mitochondria were not encountered. Electron microscopy also shows (a) small vesicles associated with the outer surface of the ghosts, and (b) a filamentous web at the inner face of the ghost membrane. Sodium dodecyl sulfate (SDS)-polyacrylamide gel analysis shows that of the many Coomassie Blue-stained bands two were prominent. One, 43,000 daltons, co-migrated with purified rabbit muscle actin and constituted about 7.5% of the plasma membrane protein. The other major band, 34,000 daltons, was concentrated in the plasma membrane fraction. Two major glycoproteins detected by autoradiography of [14C]fucose-labeled glycoproteins on the gels, had apparent molecular weights of 35,000 daltons and 32,000 daltons. These major bands did not stain with Coomassie Blue. There were many other minor glycoprotein bands in the 200,000- to 80,000-dalton range. Ouabain-sensitive, Na+, K+-adenosine triphosphatase (ATPase) activity of the ghost fraction is purified 9.1 (+/- 2.2) times over the homogenate; recover of the activity is 12.0 (+/- 3.8%) of the homogenate. Enrichment and recovery of fucosylglycoprotein parallel those for ouabain-sensitive Na+, K+-ATPase activity. Fucosyl glycoprotein is recovered more than the enzyme activity in a smooth membrane vesicle fraction probably containing the bulk of plasma membrane not recovered as ghosts.     

Major oligomeric structural proteins of the HeLa nucleus

The three predominant polypeptides of the insoluble proteinaceous fraction from the HeLa cell nucleus polymerize in vitro upon oxidation of intrinsic sulfhydryl groups. The ease and specificity of this reaction indicate that these polypeptides exist as ordered oligomers in vivo. The comparable insoluble fraction from the rat liver nucleus also contains three predominant polypeptides of the same molecular weights, 65,000 71,000, and 75,000. The insoluble protein of the avian erythrocyte nuclear envelope consists principally of the 71,000- and the 75,000-dalton polypeptides. Indeed, in the avian erythrocyte nucleus these are the predominant polypeptides of the entire nucleus (Shelton, K., Cobbs, C., Povlishock, J. and Burkat, R., 1976, Arch. Biochem. Biophys.174, 177). Further, these avian polypeptides each form homogeneous covalently linked oligomers upon sulfhydryl oxidation (Cochran, D., Cobbs, C. and Shelton, K., 1977, J. Cell Biol.75, 151a). The insolubility, oligomeric disposition, and relative prominence of these polypeptides in a wide variety of cells indicate a fundamental structural role in the nucleus. Morphological features which may reflect this structural or skeletal role could be the nuclear envelope, the fibrous lamina, or perhaps an intrachromatinic matrix. The metabolism of the oligomeric polypeptides has been investigated in HeLa cells. Turnover of the HeLa insoluble nuclear protein is similar to that of the histones which are known to be stable proteins. The insoluble protein, including the oligomeric polypeptides, is synthesized in G1, S, and G2 phases of the cell cycle. This metabolic behavior indicates that the oligomeric polypeptides are reutilized in successive cell cycles and that synthesis accompanies nuclear and cellular expansion rather than deoxyribonucleohistone synthesis. This suggests that neither degradation nor selective synthesis of oligomeric polypeptides at a particular phase of the cell cycle are responsible for the breakdown and reformation of the interphase cell morphological features that occur during mitosis.     

Microtubule-associated proteins of HeLa cells: heat stability of the 200,000 mol wt HeLa MAPs and detection of the presence of MAP-2 in HeLa cell extracts and cycled microtubules

One of the major groups of microtubule-associated proteins (MAPs) found associated with the microtubules isolated from HeLa cells has a molecular weight of just over 200,000. Previous work has demonstrated that these heLa MAPs are similar in several properties to MAP-2, one of the major MAPs of mammalian neural microtubules, although the two types of proteins are immunologically distinct. The 200,000 mol wt HeLa MAPs have now been found to remain soluble after incubation in a boiling water bath and to retain the ability to promote tubulin polymerization after this treatment, two unusual properties also shown by neural MAP- 2. This property of heat stability has allowed the development of a simplified procedure for purification of the 200,000 HeLa MAPs and has provided a means for detection of these proteins, even in crude cell extracts. These studies have also led to the detection of a protein in crude extracts of HeLa cells and in cycled HeLa microtubules which has been identified as MAP-2 on the basis of (a) comigration with calf brain MAP-2 on SDS PAGE, (b) presence in purified microtubules, (c) heat stability, and (d) reaction with two types of antibodies prepared against neural high molecular weight-MAPs, one of these a monoclonal antibody against hog brain MAP-2, although present in HeLa cells, is at all stages of microtubule purification a relatively minor component in comparison to the 200,000 HeLa MAP's.     

Purification and characterization of an F-actin-bundling 55-kilodalton protein from HeLa cells

An F-actin-bundling protein with Mr of 55,000 has been purified from HeLa cells by a simple method using its affinity to F-actin. Briefly, muscle actin was mixed with supernatants of HeLa cell homogenates, and the resultant actin gel was precipitated by low speed centrifugation. The 55-kDa protein in the actin gel was dissociated by depolymerization of F-actin and purified sequentially by chromatography on DEAE-cellulose and hydroxylapatite. The Stokes radius and sedimentation coefficient of the 55-kDa protein were 32 A and 4.35 (S20,w), respectively. These results suggest that the 55-kDa protein is a monomeric globular protein with a native molecular weight of 57,000. The globular form of the protein was confirmed by electron microscopy of rotary shadowed specimens. The binding of the protein to actin was saturated at an approximate stoichiometry of 4 actin monomers to one 55-kDa molecule. The protein made F-actin aggregate side-by-side into bundles as has been reported for other F-actin-bundling proteins such as fimbrin (Mr = 68,000) and fascin (Mr = 58,000). The 55-kDa protein is a new actin-binding protein based on biochemical, morphological, and immunological characterization. Skeletal muscle tropomyosin inhibited the actin-bundling activity of 55-kDa protein by competitive binding to actin, suggesting that the 55-kDa protein binding site on F-actin is in the vicinity of the tropomyosin-binding site.