Specific association of STOP protein with microtubules in vitro and with stable microtubules in mitotic spindles of cultured cells.

STOP (Stable Tubule Only Polypeptide) is a neuronal microtubule associated protein of 145 kd that stabilizes microtubules indefinitely to in vitro disassembly induced by cold temperature, millimolar calcium or by drugs. We have produced monoclonal antibodies against STOP. Using an antibody affinity column, we have produced a homogeneously pure 145 kd protein which has STOP activity as defined by its ability to induce cold stability and resistance to dilution induced disassembly in microtubules in vitro. Western blot analysis, using a specific monoclonal antibody, demonstrates that STOP recycles quantitatively with microtubules through three assembly cycles in vitro. Immunofluorescence analysis demonstrates that STOP is specifically associated with microtubules of mitotic spindles in neuronal cells. Further, and most interestingly, STOP at physiological temperature appears to be preferentially distributed on the distinct microtubule subpopulations that display cold stability; kinetochore-to-pole microtubules and telophase midbody microtubules. The observed distribution suggests that STOP induces the observed cold stability of these microtubule subpopulations in vivo.     

Binding of adenovirus to microtubules. II. Depletion of high-molecular-weight microtubule-associated protein content reduces specificity of in vitro binding.

A specific in vitro association between adenovirus and pruified rat brain microtubules has been previously demonstrated (R. B. Luftig and R. R. Weihing, 1975). When examined by negative-staining electron microscopy, approximately 90% of the virus associated with microtubules was edge bound, i.e., associated within +/-4 nm of the microtubule edge. Similar results are now found for the association of adenovirus with purified chick brain microtubules. When the content of the high-molecular-weight proteins (MAPs) normally present as projections on the surface of microtubules is depleted by fractionation of cold-depolymerized microtubules on agarose A-15M columns or by brief treatment of polymerized microtubules with trypsin, the percentage of edge-bound microtubule-associated viruses is reduced to a level close to that found for particles such as reovirus, coliphage f2, or polystyrene latex spheres, which randomly associate with microtubules (54 to 64% for column-fractionated microtubules; 45 to 68% for trypsin-treated microtubules). Counts of adenovirus particles specifically bound to microtubules, corrected for variations in microtubule and virus concentrations, gave values 2.5 to 3.5 times higher for unfractionated microtubules than for microtubule-associated protein-depleted microtubules. These results are consistent with the suggestion that the specific association between adenovirus and microtubules is mediated by microtubule-associated proteins.     

Tubulin assembly protein: immunochemical and immunofluorescent studies on its function and distribution in microtubules and cultured cells.

Cytoplasmic microtubule assembly from tubulin monomers requires an accessory protein or proteins present is isolated microtubules. These proteins have been designated "tau" factors. One such factor, tubulin assembly protein (TAP), has been purified to homogeneity from calf brain microtubules. A precipitating, monospecific antibody against the protein has been prepared. The antibody has been used to investigate the mechanism of TAP action in microtubule assembly and the distribution of TAP in cellular microtubules. Immunochemical, immunofluorescent and electron microscopic studies indicate that TAP functions stoichiometrically by binding physically to tubulin to form a complex active in microtubule assembly. TAP is an elongation protein which is required throughout the growth of a microtubule and which is actually present along the entire microtubule. Immunofluorescence microscopy has been used to demonstrate that TAP is distributed throughout the cytoplasmic microtubule network of cultured human, hamster and rat cells-both normal and virally transformed. Immunofluorescence of cells in mitosis shows that TAP is present in the mitotic spindle. These results demonstrate the biological importance of tubulin assembly protein and suggest that it or immunologically related "tau" proteins represent ubiquitous cofactors in cytoplasmic microtubule assembly.     

Induction of cold stability of microtubules in cultured tobacco cells

In suspension-cultured tobacco (Nicotiana tabacum) cells, we have often encountered cold-stable microtubules (MTs). The cold-stable MTs were found in the pelleted fraction of tobacco cell homogenates. These cold-stable MTs were shown to be accompanied by unidentified filamentous structures that extended along part of their length. However, during the early hours in culture such cold-stable MTs were never observed. They were detectable from 120 h after the beginning of subculture and then their numbers increased gradually. The number of cells with cold-stable MTs eventually accounted for more than 95% of the total population of cells at the stationary phase of culture. The rapid loss of cold stability of MTs occurred when such cells were transferred to fresh medium for subculture. However, if the fresh medium was supplemented with once-used medium, the cold stability of MTs was retained. The active agent in the medium appeared to be of low molecular weight and to be heat resistant. A similar activity was detected in a pectin hydrolyzate. When an inhibitor of protein kinase, either 6-dimethylaminopurine or staurosporin, was added to the cells at an early stage of culture, when cold-stable MTs were normally completely absent, most cells acquired cold-stable MTs. It appears that acquisition or loss of cold stability of MTs in tobacco cells is regulated by the action of a kinase/phosphatase or a phosphorylation/dephosphorylation system on some MT protein(s), such as a cold stabilizer of MTs, some unidentified MT-associated filamentous structure, or even tubulin itself.     

Scanning microfluorometric measurement of immunofluorescently labelled microtubules in cultured cells

Summary A method for evaluation of microtubule content in cultured cells has been developed. The method is based on scanning microfluorometric measurement of immunofluorescently labelled microtubules. The method has been applied to the comparison of microtubule content in epithelial XTH-2 cells grown in culture at various cell densities. The results have shown that the microtubule content in the cells is not dependent on their proliferative state rather than it depends on cellular contacts.     

Tubulin assembly sites and the organization of cytoplasmic microtubules in cultured mammalian cells

The number, distribution, and nucleating capacity of microtubule- organizing centers (MTOCs) has been investigated in a variety of cultured mammalian cells. Most interphase cells contain a single MTOC that is localized at the centrosome region and corresponds to the centriole and pericentriolar material. MTOCs, like centrioles, become duplicated during the S phase of the cell cycle and are equationally distributed to daughter cells in mitosis. Multiple MTOCs were rarely observed in cultured cells except in one cell line (neuroblastoma), which also displayed an equally large number of centrioles in the cytoplasm. The kinetics of microtubule assembly and the tubulin nucleating capacity of MTOCs was assayed by incubating tubulin- depleted, permeabilized 3T3 and simian virus 40-transformed 3T3 cells with phosphocellulose-purified 65 brain tubulin and microtubule assembly buffer. Initiation and assembly of 65 tubulin occurred in association with the cells' endogenous MTOCs, and the length, number, and distribution of microtubules generated about the organizing centers were regulated and cell specific. Our results are consistent with the notion that the specification of microtubule length, number, and spatial arrangement resides largely in the MTOCs and surrounding cytoplasm and not in the tubulin subunits.     

Stabilization of cortical microtubules by the cell wall in cultured tobacco cells

Cortical microtubules (MTs) in protoplasts prepared from tobacco (Nicotiana tabacum L.) BY-2 cells were found to be sensitive to cold. However, as the protoplasts regenerated cell walls they became resistant to cold, indicating that the cell wall stabilizes cortical MTs against the effects of cold. Since poly-l-lysine was found to stabilize MTs in protoplasts, we examined extensin, an important polycationic component of the cell wall, and found it also to be effective in stabilizing the MTs of protoplasts. Both extensin isolated from culture filtrates of tobacco BY-2 cells and extensin isolated in a similar way from cultures of tobacco XD-6S cells rendered the cortical MTs in protoplasts resistant to cold. Extensin at 0.1 mg·ml⁻¹ was as effective as the cell wall in this respect. It is probable that extensin in the cell wall plays an important role in stabilizing cortical MTs in tobacco BY-2 cells.     

Association of microtubule-associated protein 2 (MAP 2) with microtubules and intermediate filaments in cultured brain cells

The classification of MAP 2 as a microtubule-associated protein is based on its affinity for microtubules in vitro and its filamentous distribution in cultured cells. We sought to determine whether MAP 2 is also able to bind in situ to organelles other than microtubules. For this purpose, primary cultures of rat brain cells were stained for immunofluorescence microscopy with a rabbit anti-MAP 2 antibody prepared in our laboratory, as well as with antibodies to vimentin, an intermediate filament protein, and to tubulin, the major subunit of microtubules. MAP 2 was present on cytoplasmic fibers in neurons and in a subpopulation of the flat cells present in the cultures. Our observations were concentrated on the flat cells because of their suitability for high-resolution immunofluorescence microscopy. Double antibody staining revealed co-localization of MAP 2 with both tubulin and vimentin in the flat cells. Pretreatment of the cultures with vinblastine resulted in the redistribution of MAP 2 into perinuclear cables that contained vimentin. Tubulin paracrystals were not stained by anti-MAP 2. In cells extracted with digitonin, the normal fibrillar distribution of MAP 2 was resistant to several treatments (PIPES buffer plus 10 mM Ca++, phosphate buffer at pH 7 or 9) that induced depolymerization of microtubules, but not intermediate filaments. Staining of the primary brain cells was not observed with preimmune serum nor with immune serum adsorbed prior to use with pure MAP 2. We detected MAP 2 on intermediate filaments not only with anti-MAP 2 serum, but also with affinity purified anti-MAP 2 and with a monoclonal anti-MAP 2 prepared in another laboratory. We conclude from these experiments that material recognized by anti-MAP 2 antibodies associates with both microtubules and intermediate filaments. We propose that one function of MAP 2 is to cross-link the two types of cellular filaments.     

Identification of a 100 kD protein associated with microtubules, intermediate filaments and coated vesicles in cultured cells

We have obtained several hybridoma clones producing antibodies to microtubule-associated proteins (MAPs) from bovine brain. Interaction of one of these antibodies, named RN 17, with cultured cells was studied by indirect immunofluorescence and immunoelectron microscopy. RN 17 antibody recognized both high molecular weight (HMW) MAPs, MAP 1 and MAP 2, in immunoblotting reaction with brain microtubules. In lysates of cultured cells, it bound to a protein doublet with a molecular weight of 100 kD. By immunofluorescence microscopy we showed that RN 17 antibody stained cytoplasmic fibrils, mitotic spindles and small particles in the cytoplasm of various cultured cells. The cytoplasmic fibrils were identified as both microtubules and intermediate filaments by double fluorescence microscopy and by their response to colcemid and 0.6 M KCl. This identification was confirmed by immunoelectron microscopy which also showed that the particles stained by RN 17 antibody are coated vesicles. Thus, cultured non-neural cells may contain a novel protein that binds to microtubules, intermediate filaments, and coated vesicles.     

Effects of strychnopentamine on cells cultured in vitro

This paper describes the powerful cytotoxic action exerted by strychnopentamine (SP), a dimeric indole alkaloid extracted from Strychnos usambarensis Gilg, on B16 melanoma cells and on non-cancer human fibroblasts cultured in vitro. SP strongly inhibits cell proliferation and induces cell death at a relatively low concentration (less than 1 microgram/ml) after 72 h of treatment in the two lines. Incorporation of [3H]thymidine and [3H]leucine by B16 cells significantly decreases after only 1 h of treatment at 0.5 microgram/ml. SP induces the formation of dense lamellar bodies and vacuolization in the cytoplasm, intense blebbing at the cell surface and various cytological alterations leading to cell death.     

In vitro polymerization of microtubules from 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 degrees C and depolymerization at 4 degrees C 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.     

Effect of elevated temperatures on spindle microtubules and chromosome movements in cultured newt lung cells

The effect of elevated temperature shocks (ETS) on anaphase chromosome movements and spindle structure was studied in lung epithelial cells cultured from the newt Taricha granulosa granulosa. Mitosis proceeds normally up to temperatures of 31--32 degrees C. At slightly higher temperatures (33--34 degrees C) anaphase is desynchronized while still higher temperatures (35--36 degrees C) arrest chromosome movements. The desynchronization of chromosome movement is related on the ultrastructural level to the re-arrangement of kinetochore fibre microtubules (Mts) into hexagonally packed structures, and with the disappearance of non-kinetochore Mts. During desynchronized movement the distance a chromosome travels depends on the structure of its kinetochore fibre: those which have divergent kinetochore fibre Mts approach the poles, while those with hexagonally packed kinetochore fibre Mts do not. The data provide evidence concerning the anchorage of Mts and demonstrate that normal anaphase chromosome movements are functionally related both to the rearrangement of kinetochore Mts and to Mt disassembly.     

High molecular weight protein MAP 2 promoting microtubuleassembly in vitro is associated with microtubules in cells

Brain high molecular weight (HMW) protein promoting microtubule assembly in vitro and identical to MAP 2 (one of the proteins which copurify with tubulin through microtubule assembly-disassembly cycles), is shown to be associated with microtubules in interphase and mitotic cells. This HMW protein was purified earlier (Kuznetsov et al., 1978), directly from bovine brain without previous obtaining total microtubule protein. Now we have obtained a monospecific antibody against it. Identity of the HMW protein with MAP 2 is inferred from SDS-electrophoresis and immunological tests; its intra-cellular localization is determined by indirect immunofluorescent staining of cultured bovine tracheal epithelium.The anti-MAP 2 antibody stains the same structures in the cells as the tubulin antibody: it stains the fibrillar network in interphase cells, mitotic spindle, and the stem body. No fibrillar structures in the cells treated with colchicine or vinblastine were stained with the antibody against MAP 2. Anti-MAP 2 also stains tubulin-containing paracrystals which have been formed in the vinblastine-treated cells. Therefore HMW protein MAP 2 which promotes tubulin polymerization in vitro is associated with microtubules in vivo.     

Evaluation of techniques for immunofluorescent staining of microtubules in cultured plant cells

Various modifications to the immunofluorescent labeling procedures for microtubules in plant cells have been compared using cell cultures of Vicia hajastana Grossh. Using serial section electron microscopic reconstructions as a reference, we have chosen as our standard procedure a method that maximizes both the preservation of the cytoskeleton and the proportion of cells staining, while minimizing the degree of nonspecific staining. The critical steps of the procedure include stabilization of the cytoskeleton, cell wall permeabilization, and cell extraction. To maintain structural integrity during the procedure, it is necessary to stabilize the cytoskeleton with paraformaldehyde. To facilitate antibody penetration into the cell, it was necessary that the walls be made permeable via partial enzymatic digestion. Detergent extraction of cells increased the proportion of cells staining and decreased the level of nonspecific binding of the antibodies. The procedures detailed in this article provide a good starting point for the application of immunofluorescent labeling techniques to other plant systems.     

Effect of agents disrupting microtubules on the distribution of receptors on the surface of cultured cells]

Substrate-attached normal mouse fibroblasts, transformed mouse fibroblasts (L-strain) and epithelial cells (MPTR strain) were incubated with two ligands that are cross-linking different group of the surface receptors: concanavalin A and cationic ferritin. Surface-attached ligands were revealed by the indirect immunofluorescent methods. The incubation of control cells with these ligands induced a patching of corresponding surface receptors, and a clearing of these receptors from the surface zones located on the lamellar cytoplasm near the cell edges actively protruding pseudopodia. Effects of three antitubulins (colcemid, colchicine and vinblastin) on the ligand-induced redistribution of receptors were examined and compared with the previously described effects of these drugs on the distribution of active cell edges.