V.I. Gelfand

The quaternary structure of bovine brain kinesin.

In the present work we have studied the subunit composition of kinesin, the microtubule‐activated, mechanochemical ATPase, isolated from bovine brain. Polypeptides with mol. wts of 120 and 62 kd are the major components of the kinesin preparation. These polypeptides could not be separated by electrophoresis under nondenaturing conditions or by FPLC on a MonoQ column, and are therefore assumed to form a tight complex. As shown by immunoblotting with polyclonal and monoclonal antibodies to the 120‐kd polypeptide and by one‐dimensional peptide mapping, the 62‐kd polypeptide does not appear to be a proteolytic product of the 120‐kd component. Densitometric scanning of polyacrylamide‐SDS gels shows that these polypeptides are present in a complex in a 1:1 molar ratio. The mol. wt of native kinesin was studied by sedimentation equilibrium and was found to be 386 +/‐ 14 kd. A comparison of the mol. wts of individual polypeptides with the mol. wt of the intact molecule indicates that the native molecule contains two 120‐kd subunits and two 62‐kd subunits.

Destruction of microfilament bundles in mouse embryo fibroblasts treated with inhibitors of energy metabolism

Abstract Immunofluorescence with an antiactin antibody and electron microscopy were used to study the distribution of actin in cultured mouse fibroblasts during treatment with inhibitors of energy metabolism. The inhibitors induce gradual disorganization of actin-containing microfilament bundles. At the first stage of the process the bundles degrade into separate fragments; later only small patches of actin can be found in the inhibitor-treated cells. This transformation takes about 90 min and is fully reversible as microfilament bundles are recovered after incubation of the cells in the inhibitor-free growth medium. The inhibitors do not alter actin distribution in the presence of glucose. This shows that their action is due to a reduction of the ATP level in the cells. A 90 min incubation with the inhibitors does not markedly alter either the cell shape or the microtubule system. Inhibitors of the energy metabolism prevent cytochalasin action on cells. Cytochalasin B (CB) or cytochalasin D (CD) rapidly disorganize the microfilament bundles and cause cell arborization. However, microfilament bundle destruction in the cells incubated in the mixture of cytochalasin and any of the inhibitors requires 90 min and is not accompanied by dramatic changes in the cell morphology, so the process is indistinguishable from microfilament bundle destruction in the presence of the inhibitors alone.

HSP70-related 65 kDa protein of beet yellows closterovirus is a microtubule-binding protein

Beet yellows virus (BYV) genome encodes a 65 kDa protein homologous to the HSP70 family of cellular heat‐shock proteins (Agranovsky, A.A., Boyko, V.P., Karasev, A.V., Koonin, E.V. and Dolja, V.V. (1991) J. Mol. Biol. 217, 603–610). The respective gene was cloned and expressed in vitro yielding a product of the expected size (p65). This product was found to bind to the purified microtubules with a binding constant of 4 × 10−7 M. The binding of p65 was stimulated if ATP presented in the translation mixture was hydrolyzed by apyrase. Removal of the short C‐terminal domains of α‐ and β‐tubulin by subtilisin digestion abolished the binding, demonstrating its specificity. The possible role of p65 association with microtubules in the movement of virus within and/or between plant cells is proposed.

Microtubules in mouse embryo fibro blasts extracted with Triton X-100

Treatment of mouse embryo fibroblasts with 1% Triton X-100 at 37 degrees C in the presence of 4M glycerol and 1 mM EGTA results in the extraction of about 80% cellular proteins. Indirect immunofluorescent staining with monospecific antibodies against tubulin showed that extracted cultures contained a well developed system of cytoplasmic microtubules, indistinguishable from a system of control non-extracted cells. Microtubules in extracted cells were sensitive to Ca2+ ions, and to cold or prolonged incubation in a glycerol-free buffer. Sodium dodecylsulphate-polyacrilamide gel electrophoresis revealed proteins co-electrophoresed with tubulin and actin in Triton-treated cultures. Electron microscopy demonstrated the presence of both microtubules and microfilament bundles in the extracted cells, but complete dissolution of plasma and intracellular membranes.

18 kDa microtubule-associated protein: identification as a new light chain (LC-3) of microtubule-associated protein 1 (MAP-1).

SDS gel electrophoresis of microtubule proteins obtained from bovine brain by polymerization cycles revealed a new protein of 18 kDa. This protein was copolymerized with tubulin and its stoichiometry to tubulin remained constant for at least 5 cycles of assembly. Moreover, this protein remained bound to microtubules stabilized with 10 μM taxol and pelleted through a 4 M glycerol cushion. The same 18 kDa protein was found in a purified preparation of the high molecular mass microtubule‐associated protein 1 (MAP‐1). The 18 kDa protein copurified with the MAP‐1 heavy chains during column chromatography on phosphocellulose, DEAE‐cellulose, hydroxyapatite and Bio‐Gel A‐15m. Incubation of the MAP‐1 preparation with a mouse monoclonal antibody to the light chain 1 (LC‐1) of MAP‐1 and with a second precipitating antibody (a rabbit antibody to mouse IgG) immunoprecipitated from the solution all the known components of MAP‐1 (heavy chains, LC‐1, LC‐2), as well as the 18 kDa protein. Immunoblotting showed, however, that this antibody does not interact directly with the 18 kDa protein. These results indicate that the 18 kDa protein forms a complex with all other components of MAP‐1. This polypeptide, therefore, is a new light chain (LC‐3) of M AP‐1.

MICROTUBULE-ASSOCIATED PROTEIN MAP1 PROMOTES MICROTUBULE ASSEMBLY IN VITRO

Several proteins co-purify with tubulin through polymerization-depolymerization cycles. These proteins are called microtubule-associated proteins (MAPS), and some of them have been found to be really associated with cellular microtubules (review [l]). It is MAPS that are thought to be responsible for microtubule functions and interactions with other cellular components. Tubulin was prepared from bovine brain by an assembly-disassembly procedure [IO], modified as in [2], and separated from MAPS by chromatography on phosphocellulose (Whatman P-II) [ 1 l] in a buffer containing 50 mM imidazole-HCl (pH2,, 6.7), 50 mM KCl, 0.5 mM MgC12, 0.1 mM EDTA and 1 mM 2-mercaptoethanol (buffer A). Methods for MAP1 and unheated MAP2 purification were as in [9]. Three MAPS have already been purified. These are MAP2 [2-41 and 7 [5] from mammalian brain and the 210 OOOM, protein from HeLa cells [6,7]. These proteins were found to promote microtubule assembly in vitro. Their assembly-promoting activity is heatresistant. Little has been known about the activities of the heaviest of MAPS, MAP1 , which comprises a significant part of brain MAPS. There was only the report [8], which concluded that MAP1 does not possess any assembly-promoting activity. This conclusion was based on the fact that the removal of MAP1 from microtubule proteins had no effect on their polymerization. Tubulin was polymerized at 37°C in buffer A supplemented with 1 mM GTP and 1 mM EGTA. Polymerization was followed by monitoring the light scattering of the solution at 330 nm [ 121. The morphology of tubulin polymers was studied by electron microscopy after thin-sectioning or negative-staining of the samples with 1% aqueous uranyl acetate. For determination of the protein composition of the polymerization products they were pelleted by centrifugation at 200 000 X g through a cushion of buffer A containing 4 M glycerol and 1 mM EGTA, and the pellets were analysed by SDS gel electrophoresis.

Visualization of cellular focal contacts using a monoclonal antibody to 80 kD serum protein adsorbed on the substratum

We have obtained a monoclonal antibody to 80 kD protein of calf serum; this protein easily and uniformly adsorbs on glass from serum-containing media. Indirect immunofluorescence staining of chick and mouse embryo fibroblasts cultured in the presence of calf serum, fixed with formaldehyde and permeabilized with Triton X-100, revealed black non-fluorescent strips and dots under the ventral cell surface, whereas all other parts of the substratum under and between cells were highly fluorescent. The distribution of non-fluorescent regions coincided with the distributed of focal contacts of cells with the substratum, revealed by interference reflection microscopy, as well as with the distribution of vinculin-containing plaques. The dark regions were also associated with the ends of microfilament bundles revealed by immunofluorescence with an anti-actin antibody. Thus, non-fluorescent regions seen after anti-80 kD staining are parts of the substratum under the focal contacts. Visualization of focal contacts with anti-80 kD provides very contrasting and high resolution pictures. Evidence is presented that 80 kD protein is adsorbed to glass in the areas of focal contacts, but the antibodies used for staining cannot penetrate these contacts.

Purification of high-Mr microtubule proteins MAP1 and MAP2

Microtubules prepared from mammalian brain by polymerization and depolymerlzation cycles consist of tubulin and several minor components, so-called microtubule-associated proteins (MAPS). The most prominent MAPs are two high&fr weight proteins, MAP1 and MAP2, according to the nomenclature of [I]. Microtubule proteins were prepared in buffer A from bovine brain by two cycles of polymerization and depolymerization [9]. Tubulin and MAPS were separated by chromatography on phosphocellulose (Whatman P-II) [IO] in buffer A. Before the elution of MAPs with 0.5 mM KCl in buffer A, the column was washed overnight with buffer A supplemented with KCl to a ‘final concentration of 0.1 M. MAP2 has an Mr of 280 000-350 000, promotes microtubule assembly in vitro and is associated with microtubules in cells [2-81. Methods for MAP2 purification have been developed in several laboratories [2,3,5]. All these use, for removing contaminating proteins, a thermoprecipitation stage at which MAP2 is heated to 85-100°C. So far, purification of native, unheated MAP2 has not been reported. Hydroxyapatite powder (Bio-Gel HTP, Bio-Rad Labs) was hydrated in 50 mM sodium citrate solution and equilibrated with buffer B. DEAE-Sephadex A-50 (Pharmacia) was equilibrated with buffer A. Bio-Gel A-l 5m (Bio-Rad Labs) was equilibrated with buffer A containing 0.1 mM PMSF. Protein concentration was determined as in [ 111, bovine serum albumin being used as a standard. The other proteins, MAP1 , having a still higher M,, has not been isolated and characterized at all. Here we describe a procedure for purification of MAP1 from bovine brain and obtaining unheated purified MAP2. By the peptide mapping of isolated proteins we show also that MAP2 is not a product of proteolytic degradation of MAPl. SDS-gel electrophoresis was performed according to [ 121 on 10% polyacrylamide slab gels at the acrylamide to N,Z#-methylene-bisacrylamide ratio of 100: 1 (w/w).

Immunofluorescent localization of protein synthesis components in mouse embryo fibroblasts

The distribution of initiation factor 2(eIF-2) and elongation factor 2(EF-2) in cultured mouse embryo fibroblasts was studied and compared with the distribution of ribosomes. We used immunofluorescence microscopy with monospecific antibodies to eIF-2, EF-2, and proteins S3a and S7 of the small ribosomal subunit. Ribosomes and factors eIF-2 and EF-2 were found mainly in the vicinity of the cell nucleus. This perinuclear zone coincides with the endoplasm - the central part of the cell containing numerous membraneous organelles and inclusions. Besides the perinuclear zone, small stained regions could be seen at the periphery of some cells. After treatment of the cells with Triton X-100 in a buffer conditions, that stabilizes the major cytoskeletal structures, some of the ribosomes, eIF-2, and EF-2 remained bound to the insoluble material. These components were found near the nucleus and some were located along the microfilament bundles.

Stimulation of actin synthesis in phalloidin-treated cells: Evidence for autoregulatory control

We used the technique of scrape loading to introduce phalloidin into mouse embryo fibroblasts in mass culture. Phalloidin almost completely destroyed actin microfilament bundles, but the amount of polymerized cytoskeleton‐associated actin was increased approximately two‐fold and the amount of monomeric (Triton X‐100 extractable) actin was significantly reduced. The major result of the present study is that the rate of actin synthesis in the phalloidin‐treated cells was 2–3 times higher than in the control cells. Northern blot and translation in a cell‐free system from rabbit reticulocytes showed that the actin mRNA level significantly increased as a result of phalloidin treatment.