Purification and characterization of actinogelin, a calcium-sensitive actin-accessory protein, from rat liver

Although cell-free extracts prepared from several types of free-living cells, including Ehrlich tumor cells, macrophages and sea-urchin eggs, readily form gels under low Ca2+ conditions, no such ability to induce actin-related gel has been detected in tissue-cell extracts. Ca2+ -insensitive gelation activity was discovered, however, in several tissue-cell extracts, including liver and brain, provided that the extracts were supplemented with skeletal muscle actin. Based on sodium dodecylsulfate/polyacrylamide gel electrophoretic analysis of the gel, these extracts seem to contain both a Ca2+ -insensitive gelation factor and Ca2+ -sensitive one, actinogelin. A procedure for purification of actinogelin from rat liver was developed, and the properties of actinogelin thus purified were compared with those of Ehrlich tumor cell actinogelin. No appreciable difference was found in these two proteins, and Ca2+ sensitivity (50% inhibition of gelation at 1 microM) was very similar. Some of the molecular characteristics are described, and the importance of the presence of actinogelin in tissue cells is discussed.     

Characteristic structures of actin gels induced with hepatic actinogelin or with chicken gizzard alpha-actinin: implication for their function

We studied the properties of actinogelin, a Ca2+-regulated actin cross-linking protein isolated from Ehrlich tumor cells or rat liver. Chicken gizzard alpha-actinin was used as a Ca2+-insensitive control. Actinogelin, which has very high gelation activity under low Ca2+ conditions, was found using electron microscopic or fluorescence studies to induce formation of a characteristic structure in which actin filaments and bundles radiate to (or converge from) all directions from spot-like core structures. A similar structure was induced with actinogelin, even in the presence of 0.7 saturation of tropomyosin. No such structure was detected with actinogelin under high Ca2+ conditions, and only a few were found with gizzard alpha-actinin. Because reconstituted structures are similar to those observed intracellularly, actinogelin may be important in the formation of similar microfilament organization in the cells. It seems also important that these structures are reconstituted with only two purified protein components, i.e., actinogelin and actin. Immunocompetition studies showed that actinogelin and gizzard alpha-actinin partially shared antigenicity, and their molecular shape and peptide maps were similar. Their amino acid compositions [Kuo et al., 1982], subunit and domain structures, and binding sites on actin [Mimura and Asano, 1987] are also very similar. Therefore, it is concluded that actinogelin belongs to alpha-actinin superfamily proteins. Furthermore, the presence of functionally different subfamilies concerned with Ca2+ sensitivity, gelation-efficiency, and others is discussed. Actinogelin, which induces networks of actin filaments, may be classified as high gelation type.     

Interaction of actinogelin with actin. No nucleation but high gelation activity

Nucleation activity of actin polymerization of actinogelin, a calcium-sensitive F-actin cross-linking protein from rat liver, was measured by a fluorescence enhancement method using pyrenyl-actin and by high shear viscometry. No stimulation of nucleation by the addition of actinogelin was observed under several ionic conditions using the fluorescent method. Similar results were also obtained by viscometry. Therefore, it can be concluded that actinogelin has no nucleation activity for actin polymerization. By electron microscopy, it was found that actinogelin molecule has a dumbbell shape, binds to side of F-actin through its end(s), and cross-links actin filaments by binding with its two ends. It was also found that meshwork formation occurred in low Ca2+ conditions from F-actin and actinogelin. Under non-gelling high Ca2+ conditions, binding of actinogelin along the side of F-actin with its one end was still detected in accordance with the binding assay using ultracentrifugation and protein determination. Under low Ca2+ conditions, the critical gelling concentration of actinogelin measured by low shear viscometry at 20 degrees C was 6 micrograms/ml for 250 micrograms/ml of actin. Comparing this value with those of the other actin cross-linking proteins, it was found that actinogelin was one of proteins with the highest gelation activity. On the other hand, gelation activity of actinogelin in high Ca2+ conditions was one order of magnitude lower; more than 50 micrograms/ml of the protein was required for gelation. At 37 degrees C, gelation activity of actinogelin at low Ca2+ concentration was decreased to about a quarter of that at 20 degrees C, but this was still higher than that of gizzard alpha-actinin at 20 degrees C. Thus, role of actinogelin as an efficient and Ca2+-regulated cross-linker of microfilaments was substantiated.     

Isolation and characterization of a conserved actin-binding domain from rat hepatic actinogelin, rat skeletal muscle, and chicken gizzard alpha-actinins

A common protease-resistant fragment (Mr = 27,000) was generated from purified rat hepatic actinogelin, and rat skeletal muscle and chicken gizzard alpha-actinins by limited proteolysis using thermolysin. A monoclonal antibody (A-1) which was raised against the rat hepatic actinogelin and has a cross-reactivity with rat skeletal muscle and chicken gizzard alpha-actinins was found to bind to all of the 27-kDa fragments selectively. Furthermore, one-dimensional peptide maps of the 27-kDa fragments showed a close similarity indicating the presence of some conservation in primary structure of the fragments. The 27-kDa fragments were purified to homogeneity by the same procedure using ammonium sulfate fractionation and hydrophobic chromatography. As the fragments were easily separated from other peptides during purification, they might be present as an independent structural domain. Purified 27-kDa fragments were found to bind to F-actin in a Ca2+-insensitive manner. The fragments failed to affect the low-shear viscosity of F-actin up to a molar ratio to actin monomer of 1:3.2, indicating that gelation activity of the parental molecules was lost and the fragments have only a single binding site on F-actin. Binding of the fragments to F-actin was almost completely inhibited by respective parental molecules, while binding of the whole molecules was blocked partly by their 27-kDa fragments. Thus, the interaction of the fragments with F-actin seemed to be specific, although apparent affinity was lower than the parental molecules. Considering these results, we concluded that the 27-kDa fragments are a conserved, monovalent, and Ca2+-insensitive actin-binding domain of the actinogelin and muscle alpha-actinins.     

Rheological studies on calcium-sensitive gelation of actin filaments by actinogelin

Actinogelin, a regulatory protein of cell motility, enhanced gelation of actin filaments in the absence of calcium ions, only on standing still or with very low velocity gradients ( less than 0.1 s-1). The Ca2+-sensitive action of actinogelin on action filaments was dependent on a weak external force. In the presence of a micromolar level of Ca2+, actinogelin did not affect the network formation of actin filaments at all.     

Myosin and Ca2+-sensitive streaming in the alga Chara: detection of two polypeptides reacting with a monoclonal anti-myosin and their localization in the streaming endoplasm

A monoclonal antibody to the heavy chain of myosin from mouse 3T3 cells was used to detect and localize related proteins in the green alga Chara. Proteins of 200,000 and 110,000 Mr reacted on immunoblots of proteins precipitated rapidly with trichloroacetic acid to minimize proteolysis. Immunofluorescence of whole cells localized these proteins to organelles of the streaming endoplasm, to a system of endoplasmic strands and to the subcortical actin bundles. Except that fewer endoplasmic strands and organelles were found and the strands were tangled, the localization pattern was similar in cells rapidly perfused to remove the bulk of the streaming endoplasm. Actin was confined almost entirely to the system of subcortical actin bundles in both whole and perfused cells. Myosin that was associated with the tangled endoplasmic strands but not that associated with the organelles or actin bundles was removed by concentrations of Ca2+ inhibiting ATP-dependent streaming in perfused cells. ATP extracted both organelles and endoplasmic strands but left a continuous pattern of myosin immunostaining along the actin bundles. The findings are discussed in relation to the possible existence of two forms of myosin and of separate mechanisms moving the bulk endoplasm and individual organelles.     

Purification and properties of mouse thymus thymidylate synthase. Comparison of the enzyme from mammalian normal and tumour tissues

Mouse thymus thymidylate synthase has been purified to apparent electrophoretic homogeneity and compared with the enzyme from mouse tumour L1210 and Ehrlich ascites carcinoma cells. The enzyme is a dimer composed of 35,000 mol. wt monomers. Mouse thymus and tumour enzymes exhibit allosteric properties reflected by cooperative binding of both dUMP and 5-fluoro-dUMP. Activation energy for the reaction, catalyzed by thymidylate synthase from mouse tumour but not from mouse thymus, lowers at temperatures above 34 degrees C, reflecting a change of rate-limiting step in dTMP formation. MgATP at millimolar concentrations inhibits mouse thymus enzyme.     

Studies on the organization and localization of actin and myosin in neurons

The organization of actin in mouse neuroblastoma and chicken dorsal root ganglion (DRG) nerve cells was investigated by means of a variety of electron microscope techniques. Microspikes of neuroblastoma cells contained bundles of 7- to 8-nm actin filaments which originated in the interior of the neurite. In the presence of high concentrations of Mg++ ion, filaments in these bundles became highly ordered to form paracrystals. Actin filaments, but not bundles, were observed in growth cones of DRG cells. Actin was localized in the cell body, neurites, and microspikes of both DRG and neuroblastoma nerve cells by fluorescein-labeled S1. Myosin was localized primarily in the neurites of chick DRG nerve cells with fluorescein-labeled anti-brain myosin antibody. This antibody also stained stress fibers in fibroblasts and myoblasts but did not stain muscle myofibrils.     

Calcyclin is a calcium and zinc binding protein

Calcyclin, a cell cycle regulated protein, was recently purified from Ehrlich ascites tumour (EAT) cells and shown to be a calcium binding protein. Here we show that calcyclin monomer and dimer also bind zinc ions. Zinc binding sites seem to be different from calcium binding sites since: preincubation with Ca2+ lacks effect on the binding of Zn2+, and Ca2+ (but not Zn2+) increases tyrosine fluorescence intensity. Binding of Zn2+ reduces the extent of the conformational changes induced by Ca2+, and seems to affect Ca2(+)-binding. The data suggest that Ca2+ and Zn2+ might trigger the biological activity of calcyclin.     

Microfilament bundles in cultured cells : Correlation with anchorage independence and tumorigenicity in nude mice

The distribution of actin microfilament bundles in cell lines 3T3B, CHO, HeLa and CLID extracted with 0.1% Triton X-100 was examined by indirect immunofluorescence using human actin antibodies and by electron microscopy of whole cells grown directly on support grids. Anchorage dependence as determined by growth in soft agar and tumorigenicity in nude mice was also investigated. Immunofluorescent staining showed that CHO and HeLa cells have normal numbers and distributions of actin microfilament bundles as compared with similarly spread control 3T3B cells. A significant fraction of the mouse CLID cells showed comparable numbers of microfilament bundles as 3T3B cells but their distribution was markedly different. In many cases the bundles radiated from a region close to the cell's centre or near its projections and usually penetrated the projections. The presence of diffuse staining in 4% of the cell population also indicated the existence in these cells of disorganized actin. Electron microscope studies of well spread regions of negatively-stained, Triton-extracted cells corroborated the observations made with the immunofluorescence technique. In 3T3B, CHO and CLID cells abundant microtubules were found, colinearly arranged with actin filaments in the thin cytoplasmic extensions. While CLID, CHO and HeLa cells showed the capacity to grow in soft agar, only CLID and HeLa cells produced tumours in athymic nude mice. The observations suggest that a reduction or disorganisation of the actin microfilament bundles may not in itself be essential at least for the non-virally transformed cells studied to show anchorage independence or to produce tumours in nude mice.     

Identification of a new 84/82 kDa calmodulin-binding protein, which also interacts with actin filaments, tubulin and spectrin, as synapsin I

A new 84/82 kDa calmodulin-binding protein, which also interacts with actin filaments, tubulin and spectrin, was purified from the bovine synaptosomal membrane. The binding of calmodulin to this protein was Ca2+-dependent, and was inhibited by trifluoperazine, the association constant being calculated to be 2.2 X 10(6) M-1. Maximally, 1 mol of calmodulin bound to 1 mol of the purified protein. This protein was phosphorylated by both kinase II (Ca2+- and calmodulin-dependent kinase) and cyclic AMP-dependent kinase. In addition, antibody against this protein was demonstrated to have an immunological crossreactivity with synapsin I in the synaptosomal membrane.     

The Dictyostelium discoideum 30,000-dalton protein is an actin filament- bundling protein that is selectively present in filopodia

The interaction with actin and intracellular localization of the 30,000-D actin-binding protein from the cellular slime mold Dictyostelium discoideum have been investigated to analyze the potential contributions of this protein to cell structure and movement. The formation of anisotropic cross-linked filament networks (bundles) containing actin and the 30,000-D protein has been observed by electron microscopy, light scattering, viscometry, and polarization microscopy. Cosedimentation experiments indicate that a maximum of one molecule of the 30,000-D protein can bind to 10 actin monomers in filaments with an apparent association constant of 1 X 10(7) liters/mol. Inhibition of the interaction of the 30,000-D protein with actin by either magnesium or calcium was observed by viscometry, light scattering, polarization microscopy, and direct binding assays. However, the concentration of magnesium required to diminish the interaction is greater than 100 times greater than that of calcium. The association constant of the 30,000-D protein for actin is 4.2 X 10(6) liters/mol, or less than 1 X 10(5) liters/mol in the presence of increased concentrations of either Mg2+ or Ca2+, respectively. Enzyme-linked immunoassays indicate that the 30,000-D protein comprises 0.04% of the protein in D. discoideum. Extensive interaction of the 30,000-D protein with actin in cytoplasm is predicted from these measurements of the concentration of this protein and its affinity for actin. The distribution of the 30,000-D protein was analyzed by immunofluorescence microscopy using mono-specific affinity-purified polyclonal antibody. The 30,000-D protein exhibits a diffuse distribution in cytoplasm, is excluded from prominent organelles, and is quite prominent in fine extensions protruding from the cell surface. The number, length, and distribution of these extensions containing the 30,000-D protein are similar to those of filopodia observed by scanning electron microscopy. To analyze the effects of cell thickness and the distribution of organelles on the immunofluorescence localization, fluorescein-labeled BSA was incorporated into the cytoplasm of living cells before fixation and staining using a sonication loading technique. The results indicate that the 30,000-D protein is selectively incorporated into filopodia. These results provide a clear distinction between the multiple actin-cross-linking proteins present in D. discoideum, and suggest that the 30,000-D protein contributes to organization of bundles of actin filaments in filopodia.     

Reversible induction of actin rods in mouse C3H-2K cells by incubation in salt buffers and by treatment with non-ionic detergents

Incubating conditions which induced actin paracrystal-like intracellular structures (actin rods) were investigated by using several cell lines. We have found that an incubation of cells of a mouse fibroblastic cell line, C3H-2K, in an isotonic solution of NaCl containing 1 mM MgCl2, 1 mM CaCl2 and 10 mM MES, pH 6.5, induced disintegration of stress fibers and formation of actin rods in the cytoplasm. Actin rods were induced also by incubating in salt buffers in which Na+ of the above solution was substituted by most cations except K+ or Rb+. When the actin rod-forming cells were transferred back to DMEM containing 10% FBS, actin rods disappeared and stress fibers subsequently re-formed within 1 h at 37 degrees C. Although the induction was observed in NaCl buffer at a wide range of pH values (5.5-10), the optimal pH was 6.5. Formation of actin rods is dependent upon cellular metabolism, as it was inhibited at 4 degrees C, or by metabolic inhibitors. Incubation in NaCl buffer induced actin rods in HeLa, L, NRK, BALB/c 3T3 and Swiss 3T3 cells, but not in CEF or MEF cells. A decrease in cell volume was observed parallel with the induction of actin rods, except for CEF and MEF cells. Alterations in intracellular concentrations of Na, K or Ca were not correlated with the induction, however. Actin rods were also induced in C3H-2K cells by a brief treatment with non-ionic detergents. Tween 80 at concentrations as low as 0.003% was effective for the induction, but did not increase the passive membrane transport of p-nitrophenylphosphate. In contrast to the induction by NaCl buffer, treatment with Tween 80 induced numerous tiny actin rods at 4 degrees C, which became larger when further incubated at 37 degrees C. Double immunofluorescence staining with anti-actin antibody and anti-vinculin antibody showed that vinculin plaques remained at least in an early stage of the actin rod formation. We discuss the mechanism for the induction of actin rods based upon the present findings.     

Smooth muscle gelsolin and a Ca2+-sensitive contractile cell model

Smooth muscle gelsolin, termed smooth muscle 90-kDa protein in our previous paper (Kanno et al. FEBS Lett. 1985; 184:202-206), was purified from bovine aorta. Antibody prepared against smooth muscle gelsolin was used to detect the presence of gelsolin in human lung fibroblast MRC-5 cells permeabilized with Triton X-100 (MRC-5 cell models). These cells contracted in the presence of MgATP and Ca2+ in doses over 1 microM. Immunofluorescence microscopy using phalloidin and antigelsolin antibody showed that gelsolin was distributed along the stress fibers, except for a marginal bundle of cells, when MRC-5 cells were growth-arrested in serum-depleted medium. Making use of immunoblotting and indirect immunofluorescence techniques, we demonstrated that gelsolin is not retained in the MRC-5 cell models. We used purified smooth muscle gelsolin as a specific agent to sever the actin filaments. Preincubation of MRC-5 cell models with gelsolin led to a destruction of stress fibers, in a dose- and Ca2+ -dependent manner. The contractility was also lost, in the same manner described above, thereby indicating that a continuous distribution of actin filaments within the stress fibers is required for cell contraction. Treatment of MRC-5 cells with the Ca2+ ionophore A23187 induced an extracellular Ca2+ -dependent contraction but not a massive destruction of stress fibers, thereby indicating that most of the endogenous gelsolin was inactive under these conditions. Our interpretation of these results is that increases in cytoplasmic Ca2+ concentrations are sufficient for the contraction but may be too transient to activate endogenous gelsolin and thereby disrupt the stress fibers. Indeed, the inhibition of contraction of the MRC-5 cell, as induced by smooth muscle gelsolin, required preincubation in the presence of Ca2+, before the addition of MgATP. These results suggest that destruction of the stress fibers by endogenous gelsolin, which leads to inhibition of cell contraction, may occur if the cytoplasmic Ca2+ is maintained at high concentrations for a few minutes.     

The role of extracellular calcium ions in HVJ (Sendai virus)-induced cell fusion

The biochemical and biophysical roles of extracellular calcium ions in HVJ (Sendai virus)-induced cell fusion were studied. (1) Various kinds of cell, such as Ehrlich ascites tumor cells, mouse melanoma cells (B16-CW1 cells) and human epidermoid carcinoma cells (KB cells), could fuse in Ca2+-free medium containing a cheletor, glycoletherdiaminetetraacetic acid, in the same way as in Ca2+-containing medium. (2) The ATP content in Ehrlich ascites tumor cells decreased rapidly when the cells were treated with the virus in Ca2+-free medium but not in Ca2+-containing medium. (3) Intracellular adenine nucleotides leaked out into the reaction medium when the cells were treated with the virus in Ca2+-free medium but not in Ca2+-containing medium. (4) On addition of the virus, O2 consumption of Ehrlich ascites tumor cells decreased in Ca2+-free medium, but not in Ca2+-containing medium. (5) HVJ (Sendai virus) did not affect production of lactate by Ehrlich ascites tumor cells in both Ca2+-free medium and Ca2+-containing medium. These observations suggest that the role of extracellular Ca2+ in virus-induced cell fusion is to maintain the ATP and other intracellular metabolite contents at normal levels instead of triggering the fusion reaction itself.     

A re-evaluation of cytoplasmic gelsolin localization

Gelsolin is a 90,000-mol-wt Ca2+-binding, actin-associated protein that can nucleate actin filament growth, sever filaments, and cap barbed filament ends. Brevin is a closely related 92,000-mol-wt plasma protein with similar properties. Gelsolin has been reported to be localized on actin filaments in stress fibers, in cardiac and skeletal muscle I-bands, and in cellular regions where actin filaments are known to be concentrated. Previous localization studies have used sera or antibody preparations that contain brevin. Using purified brevin-free IgG and IgA monoclonal antibodies or affinity-purified polyclonal antibodies for gelsolin and brevin, we find no preferential stress fiber staining in cultured human fibroblasts or I-band staining in isolated rabbit skeletal muscle sarcomeres. Cardiac muscle frozen sections show no pronounced I-band staining, except in local areas where brevin may have penetrated from adjacent blood vessels. Spreading platelets show endogenous gelsolin localized at the cell periphery, in the central cytoplasmic mass and on thin fibers that radiate from the central cytoplasm. Addition of 3-30 micrograms/ml of brevin to the antibodies restores intense stress fiber and I-band staining. We see no evidence for large-scale severing and removal of filaments in stress fibers in formaldehyde-fixed, acetone-permeabilized cells even at brevin concentrations of 30 micrograms/ml. The added brevin or brevin antibody complex binds to actin filaments and is detected by the fluorescently tagged secondary antibody. Brevin binding occurs in either Ca2+ or EGTA, but is slightly more intense in EGTA suggesting some severing and filament removal may occur in Ca2+. The I-band staining is limited to the region where actin and myosin do not overlap. In addition, brevin does not appear to bind at the Z-line. A comparison of cells double-labeled with fluorescein-phallotoxin, exogenous brevin, and a monoclonal antibody, detected with a rhodamine-labeled secondary antibody, shows almost complete co-localization of F-actin with the brevin-gelsolin-binding sites. A major exception is in the area of the adhesion plaque. A quantitative comparison of the fluorescein-rhodamine fluorescence intensities along a stress fiber and into the adhesion plaque shows that the fluorescein signal, associated with F-actin, increases while the rhodamine signal decreases. We infer that exogenous brevin or endogenous gelsolin can bind to and potentially sever most actin filaments, but that actin-associated proteins in the adhesion plaque can prevent binding and severing.(ABSTRACT TRUNCATED AT 400 WORDS)     

The relationship between intermediate filaments and microfilaments before and during the formation of desmosomes and adherens-type junctions in mouse epidermal keratinocytes

Actin, keratin, vinculin and desmoplakin organization were studied in primary mouse keratinocytes before and during Ca2+-induced cell contact formation. Double-label fluorescence shows that in cells cultured in low Ca2+ medium, keratin-containing intermediate filament bundles (IFB) and desmoplakin-containing spots are both concentrated towards the cell center in a region bounded by a series of concentric microfilament bundles (MFB). Within 5-30 min after raising Ca2+ levels, a discontinuous actin/vinculin-rich, submembranous zone of fluorescence appears at cell-cell interfaces. This zone is usually associated with short, perpendicular MFB, which become wider and longer with time. Later, IFB and the desmoplakin spots are seen aligned along the perpendicular MFB as they become redistributed to cell-cell interfaces where desmosomes form. Ultrastructural analysis confirms that before the Ca2+ switch, IFB and desmosomal components are found predominantly within the perimeter defined by the outermost of the concentric MFB. Individual IF often splay out, becoming interwoven into these MFB in the region of cell-substrate contact. In the first 30 min after the Ca2+ switch, areas of submembranous dense material (identified as adherens junctions), which are associated with the perpendicular MFB, can be seen at newly formed cell-cell contact sites. By 1-2 h, IFB-desmosomal component complexes are aligned with the perpendicular MFB as the complexes become redistributed to cell-cell interfaces. Cytochalasin D treatment causes the redistribution of actin into numerous patches; keratin-containing IFB undergo a concomitant redistribution, forming foci that coincide with the actin-containing aggregates. These results are consistent with an IF-MF association before and during desmosome formation in the primary mouse epidermal keratinocyte culture system, and with the temporal and spatial coordination of desmosome and adherens junction formation.     

Transfection of chicken skeletal muscle alpha-actinin cDNA into nonmuscle and myogenic cells: dimerization is not essential for alpha-actinin to bind to microfilaments.

alpha-Actinins from striated muscle, smooth muscle, and nonmuscle cells are distinctive in their primary structure and Ca2+ sensitivity for the binding to F-actin. We isolated alpha-actinin cDNA clones from a cDNA library constructed from poly(A)+ RNA of embryonic chicken skeletal muscle. The amino acid sequence deduced from the nucleotide sequence of these cDNAs was identical to that of adult chicken skeletal muscle alpha-actinin. To examine whether the differences in the structure and Ca2+ sensitivity of alpha-actinin molecules from various tissues are responsible for their tissue-specific localization, the cDNA cloned into a mammarian expression vector was transfected into cell lines of mouse fibroblasts and skeletal muscle myoblasts. Immunofluorescence microscopy located the exogenous alpha-actinin by use of an antibody specific for skeletal muscle alpha-actinin. When the protein was expressed at moderate levels, it coexisted with endogenous alpha-actinin in microfilament bundles in the fibroblasts or myoblasts and in Z-bands of sarcomeres in the myotubes. These results indicate that Ca2+ sensitivity or insensitivity of the molecules does not determine the tissue-specific localization. In the cells expressing high levels of the exogenous protein, however, the protein was diffusely present and few microfilament bundles were found. Transfection with cDNAs deleted in their 3' portions showed that the expressed truncated proteins, which contained the actin-binding domain but lacked the domain responsible for dimerization, were able to localize, though less efficiently in microfilament bundles. Thus, dimer formation is not essential for alpha-actinin molecules to bind to microfilaments.     

Distribution among tissues and intracellular localization of cofilin, a 21kDa actin-binding protein

Cofilin, a 21kDa actin-binding protein, binds to F-actin in a 1:1 molar ratio of cofilin to actin molecule (Nishida, E., S. Maekawa, and H. Sakai, Biochemistry, 23, 5307-5313, 1984) and is capable of controlling actin polymerization and depolymerization in vitro in a pH-sensitive manner (Yonezawa, N., E. Nishida, and H. Sakai, J. Biol. Chem., 260, 14410-14412, 1985). In this study, immunoblot analysis using monospecific antibodies against cofilin showed that cofilin is ubiquitously distributed in a variety of bovine and rat organs and tissues. Cofilin is also present in various cultured cell lines. Indirect immunofluorescence staining of mouse fibroblastic cells and human epidermoid carcinoma cells indicated that cofilin is distributed nearly uniformly in the cytoplasm and is concentrated in ruffling membranes where F-actin is also concentrated as revealed by staining with rhodamine-phalloin. Stress fiber structures were not strongly stained with the anti-cofilin antibody, although stress fiber staining was sometimes observed near the cell periphery in mouse 3T3 cells. These results suggest that the bulk of cofilin may not be associated with F-actin bundles in vivo.     

Chromaffin cell scinderin, a novel calcium-dependent actin filament-severing protein.

Scinderin, a novel Ca2+-activated actin filament-severing protein, has been purified to homogeneity from bovine adrenal medulla using a combination of several chromatographic procedures. The protein has an apparent mol. wt of 79,600 +/- 450 daltons, three isoforms (pIs 6.0, 6.1 and 6.2) and two Ca2+ binding sites (Kd 5.85 x 10(-7) M, Bmax 0.81 mol Ca2+/mol protein and Kd 2.85 x 10(-6) M, Bmax 1.87 mol Ca2+/mol protein). Scinderin interacts with F-actin in the presence of Ca2+ and produces a decrease in the viscosity of actin gels as a result of F-actin filament severing as demonstrated by electron microscopy. Scinderin is a structurally different protein from chromaffin cell gelsolin, another actin filament-severing protein described. Scinderin and gelsolin have different mol. wts, isoelectric points, amino acid composition and yield different peptide maps after limited proteolytic digestion by either Staphylococcus V8 protease or chymotrypsin. Moreover, scinderin antibodies do not cross-react with gelsolin and gelsolin antibodies fail to recognize scinderin. Immunofluorescence with anti-scinderin demonstrated that this protein is mainly localized in the subplasmalemma region of the chromaffin cell. Immunoblotting tests with the same antibodies indicated that scinderin is also expressed in brain and anterior as well as posterior pituitary. Presence of scinderin and gelsolin, two Ca2+-dependent actin filament-severing proteins in the same tissue, suggests the possibility of synergistic functions by the two proteins in the control of cellular actin filament networks. Alternatively, the actin filament-severing activity of the two proteins might be under the control of different transduction and modulating influences.