A microtubule-associated protein antigen unique to mitotic spindle microtubules in PtK1 cells

Microtubule-associated proteins (MAPs) that copurify with tubulin through multiple cycles of in vitro assembly have been implicated as regulatory factors and effectors in the in vivo activity of microtubules. As an approach to the analysis of the functions of these molecules, a collection of lymphocyte hybridoma monoclonal antibodies has been generated using MAPs from HeLa cell microtubule protein as antigen. Two of the hybridoma clones secrete IgGs that bind to distinct sites on what appears to be a 200,000-dalton polypeptide. Both immunoglobulin preparations stain interphase and mitotic apparatus microtubules in cultured human cells. One of the clones (N-3B4.3.10) secretes antibody that reacts only with cells of human origin, while antibody from the other hybridoma (N-2B5.11.2) cross-reacts with BSC and PtK1 cells, but not with 3T3 cells. In PtK1 cells the N-2B5 antigen is associated with the microtubules of the mitotic apparatus, but there is no staining of the interphase microtubule array; rather, the antibody stains an ill-defined juxtanuclear structure. Further, neither antibody stains vinblastine crystals in either human or marsupial cells at any stage of the cell cycle. N-2B5 antibody microinjected into living PtK1 cells binds to the mitotic spindle, but does not cause a rapid dissolution of either mitotic or interphase microtubule structures. When injected before the onset of anaphase, however, the N-2B5 antibody inhibits proper chromosome partition in mitotic PtK1 cells. N-2B5 antibody injected into interphase cells causes a redistribution of MAP antigen onto the microtubule network.     

Intracellular localization of the high molecular weight microtubule accessory protein by indirect immunofluorescence

Microtubule accessory proteins were isolated from porcine brain microtubules by phosphocellulose chromatography, and the high molecular weight protein (HMW protein), purified from this microtubule-associated fraction by electrophoretic elution from SDS gels, was used to raise antisera in rabbits. In agarose double diffusion tests, the antiserum obtained forms precipitin lines with purified HMW protein but not with tau protein or tubulin. When rat glial cells (strain C6) are examined by indirect immunofluorescence, this serum specifically stains a colchicine-sensitive filamentous cytoplasmic network in interphase cells, a network indistinguishable from that seen when cells are treated with antitubulin serum. In dividing cells, specific staining of the mitotic spindle and the stem body is observed with the antiserum to HMW protein. These studies indicate that HMW protein, like tau protein, is associated with microtubules in intact cells.     

Dynamics of microtubules bundled by microtubule associated protein 2C (MAP2C)

MAP2C is a microtubule-associated protein abundant in immature nerve cells. We isolated a cDNA clone encoding whole mouse MAP2C of 467 amino acid residues. In fibroblasts transiently transfected with cDNA of MAP2C, interphase microtubule networks were reorganized into microtubule bundles. To reveal the dynamic properties of microtubule bundles, we analyzed the incorporation sites of exogenously introduced tubulin by microinjection of biotin-labeled tubulin and the turnover rate of microtubule bundles by photoactivation of caged fluorescein- labeled tubulin. The injected biotin-labeled tubulin was rapidly incorporated into distal ends of preexisting microtubule bundles, suggesting a concentration of the available ends of microtubules at this region. Although homogenous staining of microtubule bundles with antibiotin antibody was observed 2 h after injection, the photoactivation study indicated that turnover of microtubule bundles was extremely suppressed and < 10% of tubulin molecules would be exchanged within 1 h. Multiple photoactivation experiments provided evidence that neither catastrophic disassembly at the distal ends of bundles nor concerted disassembly due to treadmilling at the proximal ends could explain the observed rapid incorporation of exogenously introduced tubulin molecules. We conclude that microtubules bundled by MAP2C molecules are very stable while the abrupt increase of free tubulin molecules by microinjection results in rapid assembly from the distal ends within the bundles as well as free nucleation of small microtubules which are progressively associated laterally with preexisting microtubule bundles. This is the first detailed study of the function of MAPs on the dynamics of microtubules in vivo.     

Cell cycle-dependent changes in the dynamics of MAP 2 and MAP 4 in cultured cells

To examine the behavior of microtubule-associated proteins (MAPs) in living cells, MAP 4 and MAP 2 have been derivatized with 6-iodoacetamido-fluorescein, and the distribution of microinjected MAP has been analyzed using a low light level video system and fluorescence redistribution after photobleaching. Within 1 min following microinjection of fluoresceinated MAP 4 or MAP 2, fluorescent microtubule arrays were visible in interphase or mitotic PtK1 cells. After cold treatment of fluorescent MAP 2-containing cells (3 h, 4 degrees C), microtubule fluorescence disappeared, and the only fluorescence above background was located at the centrosomes; microtubule patterns returned upon warming. Loss of microtubule immunofluorescence after nocodozole treatment was similar in MAP-injected and control cells, suggesting that injected fluorescein-labeled MAP 2 did not stabilize microtubules. The dynamics of the MAPs were examined further by FRAP. FRAP analysis of interphase cells demonstrated that MAP 2 redistributed with half-times slightly longer (60 +/- 25 s) than those for MAP 4 (44 +/- 20 s), but both types of MAPs bound to microtubules in vivo exchanged with soluble MAPs at rates exceeding the rate of tubulin turnover. These data imply that microtubules in interphase cells are assembled with constantly exchanging populations of MAP. Metaphase cells at 37 degrees C or 26 degrees C showed similar mean redistribution half-times for both MAP 2 and MAP 4; these were 3-4 fold faster than the interphase rates (MAP 2, t1/2 = 14 +/- 6 s; MAP 4, t1/2 = 17 +/- 5 s). The extent of recovery of spindle fluorescence in MAP-injected cells was to 84-94% at either 26 or 37 degrees C. Although most metaphase tubulin, like the MAPs, turns over rapidly and completely under physiologic conditions, published work shows either reduced rates or extents of turnover at 26 degrees C, suggesting that the fast mitotic MAP exchange is not simply because of fast tubulin turnover. Exchange of MAP 4 bound to telophase midbodies occurred with dynamics comparable to those seen in metaphase spindles (t1/2 = approximately 27 s) whereas midbody tubulin exchange was slow (greater than 300 s). These data demonstrate that the rate of MAP exchange on microtubules is a function of time in the cell cycle.     

A cytoskeletal 50 kDa protein in higher plants that forms intermediate-sized filaments and stabilizes microtubules

Summary Filamentous structures of 7–10 nm in diameter were regenerated in vitro from a soluble 50 kDa protein (p50) that had been isolated from mung bean seedlings and from cultured tobacco cells. The polymerization of p50 in vitro was dependent on the presence of guanosine nucleotides, in particular, guanosine monophosphate (GMP). Unlike tubulin, p50 is a stable basic protein with the ability to polymerize even after heat treatment for 1 min at 70 °C. Furthermore, the freeze-dried powder of p50 retained the ability to regenerate filamentous structures after it had been dissolved in polymerization buffer to which GMP was then added. Two monoclonal antibodies against p50 were obtained. These antibodies stained the filamentous structures that extended from the surface of the nucleus to the cell periphery in interphase tobacco cells. They stained spindles and phragmoplasts as did tubulin-specific antibodies. They also stained fibrillar structures that were present around the spindle poles and the telophase daughter nuclei in which no microtubules were present. These results suggest that a part of the cell's complement of p50 may be associated with microtubules in dividing cells while the rest may itself form unique fibrillar structures. The antibodies against p50 did not stain cortical microtubules or the pre-prophase band of microtubules. The antibody against p50 also stained intermediate filament-like structures in cultured animal cells. The formation of microtubules in vitro was markedly stimulated and the assembled microtubules were greatly stabilized by p50. Further investigation of p50 is indispensable for the understanding of properties and function of intermediate-sized filaments in higher plant cells.Abbreviations EPC Sepharose ethyl N-phenyl-carbamate conjugated Sepharose - p50 50 kDa protein     

STOP proteins

Microtubules assembled from pure tubulin in vitro are labile, rapidly depolymerized upon exposure to the cold. In contrast, in a number of cell types, cytoplasmic microtubules are stable, resistant to prolonged cold exposure. During the past years, the molecular basis of this microtubule stabilization in cells has been elucidated. Cold stability is due to polymer association with different variants of a calmodulin-regulated protein, STOP protein. The dynamic and hence the physiological consequences of STOP association with microtubules vary in different tissues. In neurons, STOP seems almost permanently associated with microtubules. STOP is apparently a major determinant of microtubule turnover in such cells and is required for normal neuronal differentiation. In cycling cells, only minor amounts of STOP are associated with interphase microtubules and STOP does not measurably affects microtubule dynamics. However, STOP is associated with mitotic microtubules in the spindle. Recent results indicate that such an association could be vital for meiosis and for the long-term fidelity of the mitotic process.     

Purification and characterization of a new, ubiquitously distributed class of microtubule-associated protein with molecular mass 250 kDa.

A heat-stable microtubule-associated protein (MAP) with relative molecular mass 250 000, termed 250-kDa MAP, was purified from bovine adrenal cortex. It is classified as a MAP subspecies distinct from MAP1, MAP2, tau, and MAP4, as judged from its electrophoretic mobility, heat stability and immunoreactivity. Purified 250-kDa MAP was able to bind to taxol-stabilized microtubules, although it lacked the ability to polymerize purified tubulin into microtubules. Western-blot analysis showed that this MAP was expressed ubiquitously in mammalian tissues. Immunofluorescence microscopy revealed that polyclonal antibodies raised against 250-kDa MAP stained many punctate structures in the cytoplasm of cultured cells. Blurry cytosolic staining was also observed. Judging from the result of nocodazole treatment, the punctate structures were associated with the microtubule network throughout the cytoplasm, while cytosolic 250-kDa MAP colocalized with free tubulin. Under electron microscopy, 250-kDa MAP has the appearance of a hollow sphere of about 12 nm diameter.     

Decoration of microtubules by fluorescently labeled microtubule-associated protein 2 (MAP2) does not interfere with their spatial organization and progress through mitosis in living fibroblasts

Microtubule-associated protein 2 (MAP2) derivatized with iodoacetamidotetramethylrhodamine or with iodoacetamidofluorescein binds to microtubules after injection into living interphase cells [Scherson et al, 1984]. The binding of derivatized MAP2 stabilized microtubules in vitro; it was therefore important to check if the binding of MAP2 in vivo perturbed the dynamics and organization of the microtubule network. We have addressed these questions by studying the effect of the injection of derivatized MAP2 on mitosis in PtK 1 cells and on the recovery of the microtubule network from low temperature incubation in interphase cells. We found that the presence of derivatized MAP2 did not change the duration of any mitotic stage and that the injected cell normally completed mitosis. We subsequently showed that the injected MAP2 bound to the microtubules within 5 minutes after injection and remained bound throughout the course of mitosis. The reorganization of the microtubule network upon cooling and rewarming was studied in the cytoplasm of human foreskin fibroblasts (356 cells). During the recovery, the distribution of the fluorescent MAP2 in living cells was identical with the microtubule pattern visualized by immunofluorescence in lysed and fixed cells. In these experiments, the fluorescent MAP2 bound to microtubules can be considered as a nonperturbing reporter of the microtubule network. This result is discussed in terms of the role of MAPs in the dynamics and organization of microtubules in living cells.     

Immunofluorescence colocalization of the 90-kDa heat-shock protein and microtubules in interphase and mitotic mammalian cells

A mouse monoclonal antibody (AC88) that was raised against the 88-kDa heat-shock protein of the water mold, Achlya ambisexualis, and that cross-reacts with the 90-kDa mammalian heat-shock protein (hsp90), and an antibody against tubulin were used to localize hsp90 and microtubules, respectively, in the same cultured rat endothelial and PtK1 epithelial cells by indirect immunofluorescence. AC88 and tubulin antibodies labeled the same structures in cells at all stages of the cell cycle, regardless of whether cells were permeabilized before or after fixation. Labeling of cell structures by both AC88 and anti-tubulin antibodies was identically affected by treating cells with colcemid. Double labeling with AC88 and anti-tubulin antibodies in interphase and mitotic cells is consistent with the conclusion that all microtubules are labeled and that no subclass of microtubules is preferentially labeled. Fluorescent labeling by AC88 was prevented by preabsorption of the antibody with purified rat hsp90 but was unaffected by preabsorption with purified 6S tubulin dimer. In contrast to AC88, fluorescent labeling by an anti-tubulin antibody was prevented by preabsorption with tubulin dimer but was unaffected by preabsorption with rat hsp90. Western-blot analysis demonstrated no cross-reactivity of AC88 for tubulin and no cross-reactivity of the anti-tubulin antibody for hsp90. A polyclonal antiserum fraction from a rabbit immunized with the 89-kDa heat-shock protein from chicken also labeled the mitotic apparatus in dividing cells and, somewhat less distinctly, fibrous structures in interphase cells. Labeling by hsp89 anti-serum was prevented by absorption with hsp90. AC88 also labeled microtubules in cultured mouse (L929 and 3T3), rat (endothelium and TRST), hamster (CHO) and primate (BSC, COS-1 and HeLa) cell lines. The demonstration of colocalization of hsp90 with microtubules should provide a valuable clue to eventual understanding of the cellular function of this ubiquitous, conserved and abundant stress-response protein.     

Cell cycle-dependent changes in microtubule dynamics in living cells expressing green fluorescent protein-alpha tubulin

LLCPK-1 cells were transfected with a green fluorescent protein (GFP)-alpha tubulin construct and a cell line permanently expressing GFP-alpha tubulin was established (LLCPK-1alpha). The mitotic index and doubling time for LLCPK-1alpha were not significantly different from parental cells. Quantitative immunoblotting showed that 17% of the tubulin in LLCPK-1alpha cells was GFP-tubulin; the level of unlabeled tubulin was reduced to 82% of that in parental cells. The parameters of microtubule dynamic instability were compared for interphase LLCPK-1alpha and parental cells injected with rhodamine-labeled tubulin. Dynamic instability was very similar in the two cases, demonstrating that LLCPK-1alpha cells are a useful tool for analysis of microtubule dynamics throughout the cell cycle. Comparison of astral microtubule behavior in mitosis with microtubule behavior in interphase demonstrated that the frequency of catastrophe increased twofold and that the frequency of rescue decreased nearly fourfold in mitotic compared with interphase cells. The percentage of time that microtubules spent in an attenuated state, or pause, was also dramatically reduced, from 73.5% in interphase to 11.4% in mitosis. The rates of microtubule elongation and rapid shortening were not changed; overall dynamicity increased 3.6-fold in mitosis. Microtubule release from the centrosome and a subset of differentially stable astral microtubules were also observed. The results provide the first quantitative measurements of mitotic microtubule dynamics in mammalian cells.     

The benzophenanthridine alkaloid sanguinarine perturbs microtubule assembly dynamics through tubulin binding. A possible mechanism for its antiproliferative activity

Sanguinarine has been shown to inhibit proliferation of several types of human cancer cell including multidrug-resistant cells, whereas it has minimal cytotoxicity against normal cells such as neutrophils and keratinocytes. By analyzing the antiproliferative activity of sanguinarine in relation to its effects on mitosis and microtubule assembly, we found that it inhibits cancer cell proliferation by a novel mechanism. It inhibited HeLa cell proliferation with a half-maximal inhibitory concentration of 1.6 +/- 0.1 microM. In its lower effective inhibitory concentration range, sanguinarine depolymerized microtubules of both interphase and mitotic cells and perturbed chromosome organization in mitotic HeLa cells. At concentrations of 2 microM, it induced bundling of interphase microtubules and formation of granular tubulin aggregates. A brief exposure of HeLa cells to sanguinarine caused irreversible depolymerization of the microtubules, inhibited cell proliferation, and induced cell death. However, in contrast with several other microtubule-depolymerizing agents, sanguinarine did not arrest cell cycle progression at mitosis. In vitro, low concentrations of sanguinarine inhibited microtubule assembly. At higher concentrations (> 40 microM), it altered polymer morphology. Further, it induced aggregation of tubulin in the presence of microtubule-associated proteins. The binding of sanguinarine to tubulin induces conformational changes in tubulin. Together, the results suggest that sanguinarine inhibits cell proliferation at least in part by perturbing microtubule assembly dynamics.     

Cytoskeletal architecture of isolated mitotic spindle with special reference to microtubule-associated proteins and cytoplasmic dynein

We have studied cytoskeletal architectures of isolated mitotic apparatus from sea urchin eggs using quick-freeze, deep-etch electron microscopy. This method revealed the existence of an extensive three- dimensional network of straight and branching crossbridges between spindle microtubules. The surface of the spindle microtubules was almost entirely covered with hexagonally packed, small, round button- like structures which were very uniform in shape and size (approximately 8 nm in diameter), and these microtubule buttons frequently provided bases for crossbridges between adjacent microtubules. These structures were removed from the surface of microtubules by high salt (0.6 M NaCl) extraction. Microtubule- associated proteins (MAPs) and microtubules isolated from mitotic spindles which were mainly composed of a large amount of 75-kD protein and some high molecular mass (250 kD, 245 kD) proteins were polymerized in vitro and examined by quick-freeze, deep-etch electron microscopy. The surfaces of microtubules were entirely covered with the same hexagonally packed round buttons, the arrangement of which is intimately related to that of tubulin dimers. Short crossbridges and some longer crossbridges were also observed. High salt treatment (0.6 M NaCl) extracted both 75-kD protein and high molecular weight proteins and removed microtubule buttons and most of crossbridges from the surface of microtubules. Considering the relatively high amount of 75- kD protein among MAPs isolated from mitotic spindles, it is concluded that these microtubule buttons probably consist of 75-kD MAP and that some of the crossbridges in vivo could belong to MAPs. Another kind of granule, larger in size (11-26 nm in diameter), was also on occasion associated with the surface of microtubules of mitotic spindles. A fine sidearm sometimes connected the larger granule to adjacent microtubules. Localization of cytoplasmic dynein ATPase in the mitotic spindle was investigated by electron microscopic immunocytochemistry with a monoclonal antibody (D57) against sea urchin sperm flagellar 21S dynein and colloidal gold-labeled second antibody. Immunogold particles were closely associated with spindle microtubules. 76% of these were within 50 nm and 55% were within 20 nm from the surface of the microtubules. These gold particles were sporadically found on both polar and kinetochore microtubules of half-spindles at both metaphase and anaphase. They localized also on the microtubules between sister chromatids in late anaphase. These data indicate that cytoplasmic dynein is attached to the microtubules in sea urchin mitotic spindles.(ABSTRACT TRUNCATED AT 400 WORDS)     

Analysis of MAP 4 function in living cells using green fluorescent protein (GFP) chimeras

MAP 4 is a ubiquitous microtubule-associated protein thought to play a role in the polymerization and stability of microtubules in interphase and mitotic cells. We have analyzed the behavior of protein domains of MAP 4 in vivo using chimeras constructed from these polypeptides and the green fluorescent protein (GFP). GFP-MAP 4 localizes to microtubules; this is confirmed by colocalization of GFP-MAP 4 with microtubules that have incorporated microinjected rhodamine-tubulin, and by loss of localized fluorescence after treatment of cells with anti-microtubule agents. Different subdomains of MAP 4 have distinct effects on microtubule organization and dynamics. The entire basic domain of MAP 4 reorganizes microtubules into bundles and stabilizes these arrays against depolymerization with nocodazole. Within the basic domain, the PGGG repeats, which are conserved with MAP 2 and tau, have a weak affinity for microtubules and are dispensable for microtubule binding, whereas the MAP 4-unique PSP region can function independently in binding. The projection domain shows no microtubule localization, but does modulate the association of various binding subdomains with microtubules. The acidic carboxy terminus of MAP 4 strongly affects the microtubule binding characteristics of the other domains, despite constituting less than 6% of the protein. These data show that MAP 4 association with microtubules is modulated by sequences both within and outside the basic domain. Further, our work demonstrates that GFP chimeras will allow an in vivo analysis of the effects of MAPs and their variants on microtubule dynamics in real time.     

Regulation of a major microtubule-associated protein by MPF and MAP kinase.

The interphase-M phase transition of microtubule dynamics is thought to be induced by phosphorylation reactions mediated by MPF and by MAP kinase functioning downstream of MPF. We have now identified and purified from Xenopus eggs a major microtubule-associated protein, p220, that may be a target protein for these two M phase-activated kinases. p220, when purified from interphase cells, potently bound to microtubules and stimulated tubulin polymerization, whereas p220 purified from M phase cells showed little or no such activities. Cell staining with a monoclonal anti-p220 antibody revealed that p220 is localized on cytoplasmic microtubule networks during interphase, while it is distributed rather diffusely throughout the cell during M phase. We have further found that p220 is phosphorylated specifically in M phase. Moreover, p220 purified from interphase cells served as a good substrate for MAP kinase and MPF in vitro, and two-dimensional phosphopeptide mapping pattern of the p220 phosphorylated in vitro was very similar to that of p220 phosphorylated at M phase in vivo. These results suggest that the drastic change in p220 activity during the transition from interphase to M phase may be induced by its phosphorylation in M phase probably catalyzed by MAP kinase and MPF.     

Microtubule regulation in mitosis: tubulin phosphorylation by the cyclin-dependent kinase Cdk1

The activation of the cyclin-dependent kinase Cdk1 at the transition from interphase to mitosis induces important changes in microtubule dynamics. Cdk1 phosphorylates a number of microtubule- or tubulin-binding proteins but, hitherto, tubulin itself has not been detected as a Cdk1 substrate. Here we show that Cdk1 phosphorylates beta-tubulin both in vitro and in vivo. Phosphorylation occurs on Ser172 of beta-tubulin, a site that is well conserved in evolution. Using a phosphopeptide antibody, we find that a fraction of the cell tubulin is phosphorylated during mitosis, and this tubulin phosphorylation is inhibited by the Cdk1 inhibitor roscovitine. In mitotic cells, phosphorylated tubulin is excluded from microtubules, being present in the soluble tubulin fraction. Consistent with this distribution in cells, the incorporation of Cdk1-phosphorylated tubulin into growing microtubules is impaired in vitro. Additionally, EGFP-beta3-tubulin(S172D/E) mutants that mimic phosphorylated tubulin are unable to incorporate into microtubules when expressed in cells. Modeling shows that the presence of a phosphoserine at position 172 may impair both GTP binding to beta-tubulin and interactions between tubulin dimers. These data indicate that phosphorylation of tubulin by Cdk1 could be involved in the regulation of microtubule dynamics during mitosis.     

Gamma-tubulin in Chlamydomonas: characterization of the gene and localization of the gene product in cells

In addition to their role in nucleating the assembly of axonemal microtubules, basal bodies often are associated with a microtubule organizing center (MTOC) for cytoplasmic microtubules. In an effort to define molecular components of the basal body apparatus in Chlamydomonas reinhardtii, genomic and cDNA clones encoding gamma-tubulin were isolated and sequenced. The gene, present in a single copy in the Chlamydomonas genome, encodes a protein with a predicted molecular mass of 52,161 D and 73% and 65% conservation with gamma-tubulin from higher plants and humans, respectively. To examine the distribution of gamma-tubulin in cells, a polyclonal antibody was raised against two peptides contained within the protein. Immunoblots of Chlamydomonas proteins show a major cross-reaction with a protein of Mr 53,000. In Chlamydomonas cells, the antibody stains the basal body apparatus as two or four spots at the base of the flagella and proximal to the microtubule rootlets. During cell division, two groups of fluorescent dots separate and localize to opposite ends of the mitotic apparatus. They then migrate during cleavage to positions known to be occupied by basal bodies. Changes in gamma-tubulin localization during the cell cycle are consistent with a role for this protein in the nucleation of microtubules of both the interphase cytoplasmic array and the mitotic spindle. Immunogold labeling of cell sections showed that gamma-tubulin is closely associated with the basal bodies. The flagellar transition region was also labeled, possibly indicating a role for gamma-tubulin in assembly of the central pair microtubules of the axoneme.     

Where and when is microtubule diversity generated inParamecium? Immunological properties of microtubular networks in the interphase and dividing cells

Summary Ciliates are highly differentiated cells which display extensive deployment of microtubular systems. Because genetic diversity of tubulin is extremely reduced in these cells, microtubule diversity is mostly generated at the post-translational level either through direct modification of tubulin or through the binding of associated proteins to microtubules. We have undertaken a systematic exploration of microtubule diversity in ciliates by way of production of monoclonal antibodies. Previously we reported the biochemical characterization of these antibodies. In addition to antibodies directed against primary sequences, we obtained antibodies directed against post-translational modifications. In this paper, we report a detailed analysis of the distribution of the various epitopes on the microtubular networks ofParamecium, both in interphase cells and during division morphogenesis. Each of these antibodies decorates a subset of microtubules. Acetylation, recognized by antibodies TEU 318 and TEU 348, is detected on stable microtubules early after microtubule assembly. Epitopes recognized by two other antibodies (TAP 952 and AXO 58) are found on a subset of stable microtubules; in addition, the TAP 952 antibody is also found on labile microtubules; both epitopes are detected as soon as microtubule assembly occurs. In contrast, the epitope of the antibody, AXO 49, is associated with only a restricted subset of stable microtubules in the interphase cell, and is detected a lag-time after microtubule assembly during division morphogenesis. These data show that microtubule diversity is generated through a time-dependent sequence and according to a definite spatial pattern.     

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.     

Immunoelectron microscopic localization of the 210,000-mol wt microtubule-associated protein in cultured cells of primates

Results from ultrastructural immunocytochemistry on glutaraldehyde- fixed cells confirmed and extended findings previously obtained with immunofluorescence. A microtubule-associated protein (MAP) of 210,000 molecular weight was shown to be specifically associated with all cytoplasmic and mitotic microtubules along their entire length in primate cells. Specific labeling with the anti-MAP antibody could not be detected on any other subcellular structures, notably the centrosomes, kinetochores, microfilaments, and intermediate filaments. Treatment with the microtubule-disrupting drug, nocodazole, induced diffusion of the MAP throughout the cytoplasm. During repolymerization of microtubules following disassembly by nocodazole, the association of the MAP with the microtubules was intermediate and complete. When cells were treated with vinblastine, the tubulin paracrystals formed were heavily stained by the antibody. Neither sodium azide nor taxol affected the association of the MAP with microtubules.     

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.