Nuclear tubulin of tissue culture cells

Tubulin has been implicated as a nuclear protein because of the role it plays in mitosis. In this paper we examined the role of tubulin in the nuclei of nonmitotic cells. Tubulin was found distributed throughout the nucleus and particularly in association with the chromatin. It comprised about 6.5% of the nonhistone chromosomal proteins. Nuclear tubulin appeared to be nonmicrotubular in form. Fluorescence microscopy data on metaphase chromosomes revealed that tubulin was present on the outside of the chromosomes. These data suggest a structural role for chromatin-associated tubulin.     

Tubulin-chromatin interactions: evidence for tubulin-binding sites on chromatin and isolated oligonucleosomes

The interaction of tubulin with chromatin has been studied using a radiolabeled tubulin binding assay and velocity sedimentation analysis on isokinetic sucrose gradients. Soluble chromatin was prepared by mild micrococcal nuclease digestion of rat liver nuclei and tubulin was purified from rat brain by temperature-dependent assembly-disassembly and phosphocellulose chromatography. The tubulin-binding assay is based on the ability of chromatin to precipitate quantitatively at physiological ionic strength allowing separation of free tubulin from chromatin-bound tubulin. The binding of tubulin to unfractionated soluble chromatin was rapid, reversible and saturable. Saturation of binding sites was obtained using tubulin concentrations ranging from 0.5 to 400 micrograms/ml, in the presence of a high concentration (2.5 mg/ml) of another acidic protein, bovine serum albumin. The Scatchard and Hill plots showed that tubulin bound to a single class of non-interacting sites and yielded values of (0.5-0.6) X 10(7) M-1 for an apparent Ka and a maximal binding capacity of 0.8 nmol tubulin/mg DNA, i.e. about 1 molecule of tubulin/10 nucleosomes. Similar binding parameters were obtained when binding experiments were performed with insoluble chromatin in 0.15 M NaCl. Velocity sedimentation analysis of tubulin-chromatin complexes revealed that tubulin bound to all classes of chromatin oligomers, irrespective of the length of the nucleosomal chain. Tubulin-trinucleosome complexes formed from isolated trinucleosome in the presence of an excess of tubulin were separated from free reactants. It was found that 10-15% of the starting oligonucleosomal species reacted with tubulin, in a stoichiometry of about 0.8 molecule of tubulin/nucleosome. Given the characteristics of the binding and the expected cellular free tubulin concentration, the tubulin-chromatin interaction could possibly take place in vivo, when the nuclear membrane breaks down during the first steps of mitosis.     

Synthesis and Turnover of Embryonic Sea Urchin Ciliary Proteins during Selective Inhibition of Tubulin Synthesis and Assembly

When ciliogenesis first occurs in sea urchin embryos, the major building block proteins, tubulin and dynein, exist in substantial pools, but most 9+2 architectural proteins must be synthesized de novo. Pulse-chase labeling with [³H]leucine demonstrates that these proteins are coordinately up-regulated in response to deciliation so that regeneration ensues and the tubulin and dynein pools are replenished. Protein labeling and incorporation into already-assembled cilia is high, indicating constitutive ciliary gene expression and steady-state turnover. To determine whether either the synthesis of tubulin or the size of its available pool is coupled to the synthesis or turnover of the other 9+2 proteins in some feedback manner, fully-ciliated mid- or late-gastrula stage Strongylocentrotus droebachiensis embryos were pulse labeled in the presence of colchicine or taxol at concentrations that block ciliary growth. As a consequence of tubulin autoregulation mediated by increased free tubulin, no labeling of ciliary tubulin occurred in colchicine-treated embryos. However, most other proteins were labeled and incorporated into steady-state cilia at near-control levels in the presence of colchicine or taxol. With taxol, tubulin was labeled as well. An axoneme-associated 78 kDa cognate of the molecular chaperone HSP70 correlated with length during regeneration; neither colchicine nor taxol influenced the association of this protein in steady-state cilia. These data indicate that 1) ciliary protein synthesis and turnover is independent of tubulin synthesis or tubulin pool size; 2) steady-state incorporation of labeled proteins cannot be due to formation or elongation of cilia; 3) substantial tubulin exchange takes place in fully-motile cilia; and 4) chaperone presence and association in steady-state cilia is independent of background ciliogenesis, tubulin synthesis, and tubulin assembly state.     

Nucleotide torsional flexibility in solution and the use of the lanthanides as nuclear-magnetic-resonance conformation probes. The case of adenosine 5''-monophosphate

Incubation of brain extracts in the presence of 1 mM CaCl2 results in the permanent loss of tubulin polymerization, even after later addition of ethyleneglycol-bis(beta-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), when assembly conditions are chosen which rely on the presence of microtubule-associated proteins (such as MAP1 and MAP2). Purified microtubular protein, by contrast, recovers readily from calcium inhibition by the later addition of EGTA. Mixing experiments, using purified microtubular protein and brain extract, show that permanent loss of tubulin assembly is always accompanied by proteolysis of high-molecular-weight microtubular-associated proteins. Addition of purified protein MAP2 after chelation of calcium by EGTA, immediately restores microtubule assembly. Furthermore, substitution of guanosine 5'-[alpha, beta-methylene]triphosphate for GTP after EGTA treatment results in the typical tubulin polymerization process, which is independent of the presence of microtubule-associated proteins. Thus, the proteolytic action of a calcium-dependent protease is specific for high-molecular-weight microtubule-associated proteins and not tubulin itself. The protease is soluble and therefore removing during the purification of microtubular protein by cycles of temperature-dependent polymerization and depolymerization. We discuss the potential physiological importance of this calcium-dependent protease.     

Reconstitution of ciliary membranes containing tubulin

Membranes from the gill cilia of the mollusc Aequipecten irradians may be solubilized readily with Nonidet P-40. When the detergent is removed from the solution by adsorption to polystyrene beads, the proteins of the extract remain soluble. However, when the solution is frozen and thawed, nearly all of the proteins reassociate to form membrane vesicles, recruiting lipids from the medium. The membranes equilibrate as a narrow band (d = 1.167 g/cm3) upon sucrose density gradient centrifugation. The lipid composition of reconstituted membranes (1:2 cholesterol:phospholipids) closely resembles that of the original extract, as does the protein content (45%). Ciliary calmodulin is the major extract protein that does not associate with the reconstituted membrane, even in the presence of 1 mM calcium ions, suggesting that it is a soluble matrix component. The major protein of reconstituted vesicles is membrane tubulin, shown previously to differ hydrophobically from axonemal tubulin. The tubulin is tightly associated with the membrane since extraction with 1 mM iodide or thiocyanate leaves a vesicle fraction whose protein composition and bouyant density are unchanged. Subjecting the detergent-free membrane extract to a freeze-thaw cycle in the presence of elasmobranch brain tubulin or forming membranes by warming the extract in the presence of polymerization-competent tubulin yields a membrane fraction with little incorporated brain tubulin. This suggests that ciliary membrane tubulin specifically associates with lipids, whereas brain tubulin preferentially forms microtubules.     

The role of Ppe1/PP6 phosphatase for equal chromosome segregation in fission yeast kinetochore

Mis12 is a kinetochore protein essential for equal chromosome segregation and is evolutionarily conserved from yeast to human. In this study, we report the isolation and characterization of suppressors of the mis12 mutant in fission yeast. Our results indicate that Mis12 is negatively regulated by a highly conserved protein phosphatase Ppe1 (scSit4/dmPPV/hPP6) or its bound partner Ekc1 (scSAP), and it is positively regulated by a counteracting kinase Gsk3. Mass spectrometry analysis shows that at least two sites in Mis12 are phosphorylated. This mechanism of suppression occurs at the level of localization recovery of Mis12 to the kinetochore chromatin. Consistently, Mis12 and a subpopulation of Ppe1/Ekc1 were found to behave like non-histone-type chromatin-associating proteins in the chromatin fractionation assay. Mutant analysis of Ppe1 and Ekc1 revealed that they are important for faithful chromosome segregation, as the mutants exhibited unequal chromosome segregation similar to mis12 in the presence of a low concentration of tubulin poison. Ppe1/PP6 directly or indirectly modulates kinetochore chromatin protein Mis12 to ensure progression into normal anaphase.     

The mechanism of action of Na glutamate, lysine HCl, and piperazine-N,N'-bis(2-ethanesulfonic acid) in the stabilization of tubulin and microtubule formation

Preferential interaction measurements between proteins and monosodium glutamate were carried out to arrive at an understanding of the mechanism of its strong effect on tubulin stability and self-assembly into microtubules. For all proteins studied, i.e. bovine serum albumin, lysozyme, beta-lactoglobulin, and calf brain tubulin, the protein showed a large preferential hydration in the presence of monosodium glutamate. The enhancement of tubulin self-association by monosodium glutamate can be interpreted in terms of the large unfavorable free energy of interaction between the additive and the protein. Preferential interactions were also examined for lysine hydrochloride, which also gave a preferential hydration of the proteins, except for tubulin. The dependence of the preferential hydration parameter on proteins was different for the two additives, suggesting the importance of net electrostatic charges of proteins in their interaction with glutamate anions and lysinium cations. The zero preferential interaction of lysine hydrochloride with tubulin indicates an affinity of the lysine cation for the protein. Both additives increased the transition temperature of proteins. This can be understood in terms of the unfavorable free energy of interaction between the additive and the protein surface, which should be even more unfavorable when the denaturation causes an increase in the surface area.     

Isolation of a subpellicular microtubule protein from Trypanosoma brucei that mediates crosslinking of microtubules

The cell body of Trypanosomatidae is enclosed in densely packed, crosslinked, subpellicular microtubules closely underlying the plasma membrane. We isolated the subpellicular microtubules from bloodstream Trypanosoma brucei parasites by use of a zwitterion detergent. These cold stable structures were solubilized by a high ionic strength salt solution, and the soluble proteins that contained tubulin along with several other proteins were further fractionated by Mono S cation exchange column chromatography. Two distinct peaks were eluted containing one protein each, which had an apparent molecular weight of 52 kDa and 53 kDa. (Mr was determined by SDS-gel electrophoresis). Only the 52 kDa protein showed specific tubulin binding properties, which were demonstrated by exposure of nitrocellulose-bound trypanosome proteins to brain tubulin. When this protein was added to brain tubulin in the presence of taxol and GTP, microtubule bundles were formed with regular crosslinks between the parallel closely packed microtubules. The crosslinks were about 7.2 nm apart (center to center). Under the same conditions, but with the 53 kDA protein or without trypanosome derived proteins, brain tubulin polymerized to single microtubules. It is thus suggested that the unique structural organization of the subpellicular microtubules is dictated by specific parasite proteins and is not an inherent property of the polymerizing tubulin. The in vitro reconstituted microtubule bundles are strikingly similar to the subpellicular microtubule network of the parasite.     

A new tubulin-binding protein

A new brain protein is described which forms an insoluble complex with tubulin, with concomitant stoichiometric hydrolysis of GTP. The complex contains a maximum of one tubulin-binding protein (MW 52,500) per two tubulin dimers. The tubulin-binding protein (TBP) does not compete with colchicine, but in the presence of microtubule-associated proteins tubulin appeared less accessible to it. Proteins such as TBP might sequester tubulin and thereby function either to inhibit indiscriminate polymerization, or to promote ordered nucleation by maintaining high local concentrations.     

Bundling of microtubules in vitro by a high molecular weight protein prepared from the squid axon

A high molecular weight protein has been partially purified from sheaths of squid giant axons. This protein fraction was capable of restoring the membrane excitability of the squid axon which had been destroyed by internal perfusion of microtubule poison, when perfused along with microtubule proteins (Matsumoto et al. (1979) J. Biochem. 86, 1155-1158). This protein, designated as 260 K protein, was purified by gel filtration and Con A-Sepharose affinity chromatography. The apparent molecular weight of the axonal protein was estimated to be 260,000 by electrophoresis in the presence of sodium dodecylsulfate. This protein was revealed to be a glycoprotein. When phosphocellulose-purified tubulin was incubated with 260 K protein at 36 degrees C in the presence of dimethylsulfoxide, turbidity of the solution was much increased. 260 K protein co-sedimented with microtubles assembled from purified tubulin. Light microscopic and electron microscopic observations revealed that the high turbidity was due to bundling of microtubules which was caused by 260 K protein. On the other hand, the effect of this protein on the turbidity increase was not so prominent when microtubules were assembled from microtubule proteins consisting of tubulin and microtubule-associated proteins. High shear and low shear viscometry and co-sedimentation experiments revealed that 260 K protein had little effect on actin polymerization under the same medium conditions as used in tubulin polymerization.     

Polyglutamylation of nucleosome assembly proteins

Polyglutamylation is an original posttranslational modification, discovered on tubulin, consisting in side chains composed of several glutamyl units and leading to a very unusual protein structure. A monoclonal antibody directed against glutamylated tubulin (GT335) was found to react with other proteins present in HeLa cells. After immunopurification on a GT335 affinity column, two prominent proteins of approximately 50 kDa were observed. They were identified by microsequencing and mass spectrometry as NAP-1 and NAP-2, two members of the nucleosome assembly protein family that are implicated in the deposition of core histone complexes onto chromatin. Strikingly, NAP-1 and NAP-2 were found to be substrates of an ATP-dependent glutamylation enzyme co-purifying on the same column. We took advantage of this property to specifically label and purify the polyglutamylated peptides. NAP-1 and NAP-2 are modified in their C-terminal domain by the addition of up to 9 and 10 glutamyl units, respectively. Two putative glutamylation sites were localized for NAP-1 at Glu-356 and Glu-357 and, for NAP-2, at Glu-347 and Glu-348. These results demonstrate for the first time that proteins other than tubulin are polyglutamylated and open new perspectives for studying NAP function.     

The oncoprotein 18/stathmin family of microtubule destabilizers.

The past several years have seen major advances in our understanding of the mechanisms of microtubule destabilization by oncoprotein18/stathmin (Op18/stathmin) and related proteins. New structural information has clearly shown how members of the Op18/stathmin protein family bind tubulin dimers and suggests models for how these proteins stimulate catastrophe, the transition from microtubule growth to shortening. Regulation of Op18/stathmin by phosphorylation continues to capture much attention. Studies suggest that phosphorylation occurs in a localized fashion, resulting in decreased microtubule destabilizing activity near chromatin or microtubule polymer. A spatial gradient of inactive Op18/stathmin associated with chromatin or microtubules could contribute significantly to mitotic spindle assembly.     

Vinblastine-induced aggregation of brine shrimp (Artemia) tubulin

Tubulin from the brine shrimp Artemia readily assembles in vitro in the absence of microtubule-associated proteins under conditions which do not permit assembly of tubulin from brain. Heated microtubule-associated protein preparations from bovine brain do, however, interact with Artemia tubulin, resulting in stimulation of tubulin assembly and formation of morphologically normal cold-sensitive microtubules. Addition of vinblastine to mixtures containing microtubules assembled in the presence of neural microtubule-associated proteins caused a drop and then a rise in turbidity of the solution. The turbidity changes were accompanied by the appearance of coils, presumably derived from the microtubules which disappeared upon addition of vinblastine. Coils also resulted when microtubule-associated proteins and vinblastine were added to tubulin before polymerization was initiated. Vinblastine prevented normal assembly and caused disruption of Artemia microtubules polymerized in the absence of microtubule-associated proteins. Under these conditions clumped or compact coils, different in appearance from those formed in the presence of the microtubule-associated proteins, were observed. The data confirm that tubulin from Artemia, an organism that is phylogenetically far removed from mammals, has retained binding sites for vinblastine and microtubule-associated proteins and that the interrelationship of these sites has been at least partially preserved. The incomplete depolymerization of Artemia microtubules in response to vinblastine when microtubule-associated proteins are absent suggests that the longitudinal tubulin-tubulin interactions involved in microtubule formation are more stable for Artemia than for neural tubulin.     

Effect of tau on the vinblastine-induced aggregation of tubulin

Two microtubule-associated proteins, tau and the high molecular weight microtubule-associated protein 2 (MAP 2), were purified from rat brain microtubules. Addition of either protein to pure tubulin caused microtubule assembly. In the presence of tau and 10 microM vinblastine, tubulin aggregated into spiral structures. If tau was absent, or replaced by MAP 2, little aggregation occurred in the presence of vinblastine. Thus, vinblastine may be a useful probe in elucidating the individual roles of tau and MAP 2 in microtubule assembly.     

Bacterial peptidoglycan binds to tubulin

A search for cellular binding proteins for peptidoglycan (PGN), a CD14- and TLR2-dependent macrophage activator from Gram-positive bacteria, using PGN-affinity chromatography and N-terminal micro-sequencing, revealed that tubulin was a major PGN-binding protein in mouse macrophages. Tubulin also co-eluted with PGN from anti-PGN vancomycin affinity column and bound to PGN coupled to agarose. Tubulin-PGN binding was preferential under the conditions that promote tubulin polymerization, required macromolecular PGN, was competitively inhibited by soluble PGN and tubulin, did not require microtubule-associated proteins, and had an affinity of 100-150 nM. By contrast, binding of tubulin to lipopolysaccharide (LPS) had 2-3 times lower affinity, faster kinetics of binding, and showed positive cooperativity. PGN enhanced tubulin polymerization in the presence of 4 M glycerol, but in the absence of glycerol, both PGN and LPS decreased microtubule polymerization. These results indicate that tubulin is a major PGN-binding protein and that PGN modulates tubulin polymerization.     

The presence of an adenosine-5'-triphosphatase dependent on 6S tubulin and calcium ions in rat brain microtubules.

An ATPase activity was found in rat brain microtubules prepared by successive cycles of polymerization and depolymerization. On phosphocellulose column chromatography, the ATPase activity was recovered in the fraction eluted with 0.6 M KCl and containing the microtubule associated proteins. The ATPase activity was markedly stimulated by the addition of purified brain 6S tubulin, and the stimulation was dependent on the presence of Ca2+ ions. Approximately 50 pmol of purified 6S tubulin was required for the maximal stimulation in the presence of 8 microgram of microtubule associated proteins. The specific activity was 8 to 13 nmol of ATP hydrolyzed per min per mg of protein at 37 degrees C, and the Km value for ATP was 3 X 10(-5) M in the presence of added tubulin.     

Tubulin, actin, and tau protein interactions and the study of their macromolecular assemblies

The intracellular polymerization of cytoskeletal proteins into their supramolecular assemblies raises many questions regarding the regulatory patterns that control this process. Binding experiments using the ELISA solid phase system, together with protein assembly assays and electron microscopical studies provided clues on the protein-protein associations in the polymerization of tubulin and actin networks. In vitro reconstitution experiments of these cytoskeletal filaments using purified tau, tubulin, and actin proteins were carried out. Tau protein association with tubulin immobilized in a solid phase support system was inhibited by actin monomer, and a higher inhibition was attained in the presence of preassembled actin filaments. Conversely, tubulin and assembled microtubules strongly inhibited tau interaction with actin in the solid phase system. Actin filaments decreased the extent of in vitro tau-induced tubulin assembly. Studies on the morphological aspects of microtubules and actin filaments coexisting in vitro, revealed the association between both cytoskeletal filaments, and in some cases, the presence of fine filamentous structures bridging these polymers. Immunogold studies showed the association of tau along polymerized microtubules and actin filaments, even though a preferential localization of labeled tau with microtubules was revealed. The studies provide further evidence for the involvement of tau protein in modulating the interactions of microtubules and actin polymers in the organization of the cytsokeletal network.     

Chaperone-like activity of tubulin. binding and reactivation of unfolded substrate enzymes

The eukaryotic cytoskeletal protein tubulin is a heterodimer of two subunits, alpha and beta, and is a building block unit of microtubules. In a previous communication we demonstrated that tubulin possesses chaperone-like activities by preventing the stress-induced aggregation of various proteins (Guha, S., Manna, T. K., Das, K. P., and Bhattacharyya, B. (1998) J. Biol. Chem. 273, 30077-30080). As an extension of this observation, we explored whether tubulin, like other known chaperones, also protected biological activity of proteins against thermal stress or increased the yields of active proteins during refolding from a denatured state. We show here that tubulin not only prevents the thermal aggregation of alcohol dehydrogenase and malic dehydrogenase but also protects them from loss of activity. We also show that tubulin prevents the aggregation of substrates during their refolding from a denatured state and forms a stable complex with denatured substrate. The activity of malic dehydrogenase, alpha-glucosidase, and lactate dehydrogenase during their refolding from urea or guanidium hydrochloride denatured states increased significantly in presence of tubulin compared with that without tubulin. These results suggest that tubulin, in addition to its role in mitosis, cell motility, and other cellular events, might be implicated in protein folding and protection from stress.     

Nuclear proteins. II. Similarity of nonhistone proteins in nuclear sap and chromatin, and essential absence of contractile proteins from mouse liver nuclei

High resolution SDS slab gel electrophoresis has been used to examine the distribution of nonhistone proteins (NHP) in the saline-EDTA, Tris, and 0.35 M NaCl washes of isolated mouse liver nuclei. These studies led to the following conclusions: (a) all the prominent NHP which remain bound to DNA are also present in somewhat similar proportions in the saline-EDTA, Tris, and 0.35 M NaCl washes of nuclei; (b) a protein comigrating with actin is prominent in the first saline-EDTA wash of nuclei, but present as only a minor band in the subsequent washes and on washed chromatin; (c) the presence of nuclear matrix proteins in all the nuclear washes and cytosol indicates that these proteins are distributed throughout the cell; (d) a histone-binding protein (J2) analogous to the HMG1 protein of K. V. Shooter, G.H. Goodwin, and E.W. Johns (Eur J. Biochem. 47:236-270) is a prominent nucleoplasmic protein; (e) quantitation of the major NHP indicates that they are present in a range of 2.2 X 10(5)-5.2 X 10(6) copies per diploid nucleus. Most of the electrophoretically visible NHP are probably structural rather than regulatory proteins; (f) actin, myosin, tubulin, and tropomyosin, if present at all, constitute a very minor fraction of the nuclear NHP. Contractile proteins constitute a major portion of the NHP only when the chromatin is prepared from crude cell lysates instead of from purified nuclei. These studies support the conclusion that there are no clear differences between many nucleoplasmic and chromatin- bound nonhistone proteins. Except for the histones, many of the intranuclear proteins appear to be in equilibrium between DNA, HnRNA, and the nucleoplasm.     

Divalent cation hinder the solubilization of a tubulin kinase activity from Trypanosoma cruzi epimastigotes

Trypanosoma cruzi epimastigotes were extracted under various conditions in order to examine the role of divalent cations in the solubilization of microtubule proteins. When epimastigotes were homogenized in the presence of 5 mM Mg+2 and 5 mM Ca+2, a protein kinase responsible for phosphorylating tubulin, as well as the tubulin that became phosphorylated, remained tightly associated with the parasite particulate and detergent-resistant fractions. On the contrary, tubulin kinase and its substrate were predominantly released into the parasite cytosolic and detergent-soluble fractions, when epimastigotes were extracted in the presence of 5 mM EDTA and 5 mM EGTA. These evidences demonstrated a divalent cation-dependent solubilization of the enzyme responsible for the phosphorylation of tubulin in T. cruzi epimastigotes and suggested a tight association between tubulin and this kinase. Under all conditions tested, tubulin kinase activity in epimastigote extracts was lower than the addition of the corresponding value in the parasite cytosolic and membranous fractions, suggesting the presence of a kinase inhibitor or regulatory subunit which also seemed to be modulated by divalent cations. Additionally, inhibition experiments in the presence of heparin, 2,3-bisphosphoglycerate and GTP established that the parasite tubulin kinase corresponded to a protein kinase CK2.