Erythrocyte microtubule assembly in vitro. Determination of the effects of erythrocyte tau, tubulin isoforms, and tubulin oligomers on erythrocyte tubulin assembly, and comparison with brain microtubule assembly

Two tubulin variants, isolated from chicken brain and erythrocytes and known to have different peptide maps and electrophoretic properties, are demonstrated to exhibit different assembly properties in vitro: 1) erythrocyte tubulin assembles with greater efficiency (lower critical concentration, greater elongation rate) but exhibits a lower nucleation rate than brain tubulin, and 2) erythrocyte tubulin readily forms oligomers whose presence significantly retards the rate of elongation, suggesting that tubulin oligomers may also be important for determining the rate of assembly and the length of microtubules in erythrocytes. Erythrocyte tubulin isolated by cycles of in vitro assembly-disassembly is also demonstrated to contain a 67-kDa tau factor that greatly enhances microtubule nucleation but has little effect on elongation rates or critical concentration. Immunofluorescence microscopy with tau antibody indicates that tau is specifically associated with marginal band microtubules, suggesting that it may be important for determining microtubule function in vivo.     

Myelin-associated glycoprotein inhibits microtubule assembly by a Rho-kinase-dependent mechanism

Myelin-associated glycoprotein (MAG) and Nogo are potent inhibitors of neurite outgrowth from a variety of neurons, and they have been identified as possible components of the central nervous system myelin that prevents axonal regeneration in the adult vertebrate central nervous system. The activation of RhoA and Rho-kinase is reported to be an essential part of the signaling mechanism of these proteins. Here, we report that the collapsing response mediator protein-2 (CRMP-2) is phosphorylated by a Rho-kinase-dependent mechanism downstream of MAG or Nogo-66. The overexpression of the nonphosphorylated form of CRMP-2 at threonine 555, which is the phosphorylation site for Rho-kinase, counteracts the inhibitory effect of MAG on the postnatal cerebellar neurons. Additionally, the expression of the dominant negative form of CRMP-2 or knockdown of the gene using small interference RNA (siRNA) mimics the effect of MAG in vitro. Consistent with the function of CRMP-2, which promotes microtubule assembly, microtubule levels are down-regulated in the cerebellar neurons that are stimulated with MAG in vitro. Reduction in the density of microtubules is also observed in the injured axons following the spinal cord injury, and this effect depends on the Rho-kinase activity. Our data suggest the important roles of CRMP-2 and microtubules in the inhibition of the axon regeneration by the myelin-derived inhibitors.     

Identification of a novel microtubule-destabilizing motif in CPAP that binds to tubulin heterodimers and inhibits microtubule assembly

We have previously identified a new centrosomal protein, centrosomal protein 4.1-associated protein (CPAP), which is associated with the gamma-tubulin complex. Here, we report that CPAP carries a novel microtubule-destabilizing motif that not only inhibits microtubule nucleation from the centrosome but also depolymerizes taxol-stabilized microtubules. Deletion mapping and functional analyses have defined a 112-residue CPAP that is necessary and sufficient for microtubule destabilization. This 112-residue CPAP directly recognizes the plus end of a microtubule and inhibits microtubule nucleation from the centrosome. Biochemical and functional analyses revealed that this 112-residue CPAP also binds to tubulin dimers, resulting in the destabilization of microtubules. Using the tetracycline-controlled system (tet-off), we observed that overexpression of this 112-residue CPAP inhibits cell proliferation and induces apoptosis after G2/M arrest. The possible mechanisms of how this 112-residue motif in CPAP that inhibits microtubule nucleation from the centrosome and disassembles preformed microtubules are discussed.     

Diethylstilbestrol induces metaphase arrest and inhibits microtubule assembly

Diethylstilbestrol produced a dose-dependent increase in the mitotic index of the human prostatic tumour cell line DU 145. This is the result of metaphase arrest which may be induced by the action of diethylstilbestrol on spindle microtubules. Evidence is presented to show that diethylstilbestrol affects microtubules. Diethylstilbestrol completely inhibited the assembly of isolated brain microtubules although only partial disassembly could be induced. In contrast to other microtubule poisons, the inhibitory effect of diethylstilbestrol on taxol-induced self-assembly could be reversed by the addition of GTP.     

Levuglandin E2 inhibits mitosis and microtubule assembly

Levuglandin E2 (LGE2) is a gamma-keto aldehyde produced by rearrangement of the prostaglandin endoperoxide PGH2 under the aqueous conditions of its biosynthesis. We show that exogenous LGE2 enters cells and efficiently inhibits the first synchronous cell division of fertilized sea urchin eggs. We attribute this inhibition to covalent modification of tubulin and thereby to inhibition of microtubule assembly.     

Evidence that S100 proteins regulate microtubule assembly and stability in rat brain extracts

Microtubule re-assembly in rat brain extracts was inhibited by antibodies to S100 proteins. Anti-S100 antibodies caused an increase in the cold-stability of microtubules and this effect was abolished by the presence of short lengths of microtubules formed under control conditions. Anti-S100 antibodies had no effect on the stimulation of assembly or the increase in microtubule stability caused by low zinc concentrations. Addition of exogenous S100a and S100b to brain extracts had different effects on assembly; S100a caused an inhibition of assembly while S100b stimulated the early phase of assembly. The data suggest that endogenous S100b is involved in the regulation of microtubule assembly in brain extracts.     

Posttranslational modification of brain tubulins from the Antarctic fish Notothenia coriiceps: reduced C-terminal glutamylation correlates with efficient microtubule assembly at low temperature

We have shown previously that the tubulins of Antarctic fish assemble into microtubules efficiently at low temperatures (-2 to +2 degrees C) due to adaptations intrinsic to the tubulin subunits. To determine whether changes in posttranslational glutamylation of the fish tubulins may contribute to cold adaptation of microtubule assembly, we have characterized C-terminal peptides from alpha- and beta-tubulin chains from brains of adult specimens of the Antarctic rockcod Notothenia coriiceps by MALDI-TOF mass spectrometry and by Edman degradation amino acid sequencing. Of the four fish beta-tubulin isotypes, nonglutamylated isoforms were more abundant than glutamylated isoforms. In addition, maximal glutamyl side-chain length was shorter than that observed for mammalian brain beta tubulins. For the nine fish alpha-tubulin isotypes, nonglutamylated isoforms were also generally more abundant than glutamylated isoforms. When glutamylated, however, the maximal side-chain lengths of the fish alpha tubulins were generally longer than those of adult rat brain alpha chains. Thus, Antarctic fish adult brain tubulins are glutamylated differently than mammalian brain tubulins, resulting in a more heterogeneous population of alpha isoforms and a reduction in the number of beta isoforms. By contrast, neonatal rat brain tubulin possesses low levels of glutamylation that are similar to that of the adult fish brain tubulins. We suggest that unique residue substitutions in the primary structures of Antarctic fish tubulin isotypes and quantitative changes in isoform glutamylation act synergistically to adapt microtubule assembly to low temperatures.     

A ubiquitous beta-tubulin disrupts microtubule assembly and inhibits cell proliferation

Vertebrate tubulin is encoded by a multigene family that produces distinct gene products, or isotypes, of both the alpha- and beta-tubulin subunits. The isotype sequences are conserved across species supporting the hypothesis that different isotypes subserve different functions. To date, however, most studies have demonstrated that tubulin isotypes are freely interchangeable and coassemble into all classes of microtubules. We now report that, in contrast to other isotypes, overexpression of a mouse class V beta-tubulin cDNA in mammalian cells produces a strong, dose-dependent disruption of microtubule organization, increased microtubule fragmentation, and a concomitant reduction in cellular microtubule polymer levels. These changes also disrupt mitotic spindle assembly and block cell proliferation. Consistent with diminished microtubule assembly, there is an increased tolerance for the microtubule stabilizing drug, paclitaxel, which is able to reverse many of the effects of class V beta-tubulin overexpression. Moreover, transfected cells selected in paclitaxel exhibit increased expression of class V beta-tubulin, indicating that this isotype is responsible for the drug resistance. The results show that class V beta-tubulin is functionally distinct from other tubulin isotypes and imparts unique properties on the microtubules into which it incorporates.     

NEM tubulin inhibits microtubule minus end assembly by a reversible capping mechanism

Although microtubule (MT) dynamic instability is thought to depend on the guanine nucleotide (GTP vs GDP) bound to the beta-tubulin of the terminal subunit(s), the MT minus end exhibits dynamic instability even though the terminal beta-tubulin is always crowned by GTP-alpha-tubulin. As an approach toward understanding how dynamic instability occurs at the minus end, we investigated the effects of N-ethylmaleimide-modified tubulin (NTb) on elongation and rapid shortening of individual MTs. NTb preferentially inhibits minus end assembly when combined with unmodified tubulin (PCTb), but the mechanism of inhibition is unknown. Here, video-enhanced differential interference contrast microscopy was used to observe the effects of NTb on MTs assembled from PCTb onto axoneme fragments. MTs were exposed to mixtures of PCTb (25 microM) and NTb (labeled on approximately 1 Cys per monomer) in which the NTb/PCTb ratio varied from 0.025 to 1. The NTb/PCTb mixture had a slight inhibitory effect on the plus end elongation rate, but significantly inhibited or completely arrested minus end elongation. For the majority of mixtures that were assayed (0.1-1 NTb/PCTb ratio), minus end MT length remained constant until the NTb/PCTb mixture was replaced. Replacement with PCTb allowed elongation to proceed, whereas replacement with buffer or NTb caused minus ends to shorten. Taken together, the results indicate that NTb associates with both plus and minus ends and that NTb acts to reversibly cap minus ends only when PCTb is also present. Low-resolution mapping of labeled Cys residues, along with previous experiments with other Cys-reactive compounds, suggests that modification of beta-tubulin Cys(239) may be associated with the capping action of NTb.     

Levuglandin E2 inhibits mitosis and microtubule assembly

Levuglandin E₂ (LGE₂) is a γ-keto aldehyde produced by rearrangement of the prostaglandin endoperoxide PGH₂ under the aqueous conditions of its biosynthesis. We show that exogenous LGE₂ enters cells and efficiently inhibits the first synchronous cell division of fertilized sea urchin eggs. We attribute this inhibition to covalent modification of tubulin and thereby to inhibition of microtubule assembly.     

Colchicine-binding sites of brain tubulins from an antarctic fish and from a mammal are functionally similar, but not identical: implications for microtubule assembly at low temperature.

The tubulins of Antarctic fishes possess adaptations that favor microtubule formation at low body temperatures (Detrich et al.: Biochemistry 28:10085-10093, 1989). To determine whether some of these adaptations may be present in a domain of tubulin that participates directly or indirectly in lateral contact between microtubule protofilaments, we have examined the energetics of the binding of colchicine, a drug thought to bind to such a site, to pure brain tubulins from an Antarctic fish (Notothenia gibberifrons) and from a mammal (the cow, Bos taurus). At temperatures between 0 and 20 degrees C, the affinity constants for colchicine binding to the fish tubulin were slightly smaller (1.5-2.6-fold) than those for bovine tubulin. van't Hoff analysis showed that the standard enthalpy changes for colchicine binding to the two tubulins were comparable (delta H degrees = +10.6 and +7.4 kcal mol-1 for piscine and bovine tubulins, respectively), as were the standard entropy changes (delta S degrees = +61.3 eu for N. gibberifrons tubulin, +51.2 eu for bovine tubulin). At saturating concentrations of the ligand, the maximal binding stoichiometry for each tubulin was approximately 1 mol colchicine/mol tubulin dimer. The data indicate that the colchicine-binding sites of the two tubulins are similar, but probably not identical, in structure. The apparent absence of major structural modifications at the colchicine site suggests that this region of tubulin is not involved in functional adaptation for low-temperature polymerization. Rather, the colchicine site of tubulin may have been conserved evolutionarily to serve in vivo as a receptor for endogenous molecules (i.e., "colchicine-like" molecules or MAPs) that regulate microtubule assembly.     

Mdp3 is a novel microtubule-binding protein that regulates microtubule assembly and stability

Microtubule-binding proteins are a group of molecules that associate with microtubules, regulate the structural properties of microtubules, and thereby participate in diverse microtubule-mediated cellular activities. A recent mass spectrometry-based proteomic study has identified microtubule-associated protein 7 (MAP7) domain-containing 3 (Mdp3) as a potential microtubule-binding protein. However, its subcellular localization and functional importance are not characterized. In this study, by GST-pulldown assays, we found that Mdp3 interacted with tubulin both in cells and in vitro. Immunofluorescence microscopy and microtubule cosedimentation assays revealed that Mdp3 also associated with microtubules. Serial deletion experiments showed that the two coiled coil motifs of Mdp3 were critical for its interaction with tubulin and microtubules. Cold recovery and nocodazole washout assays further demonstrated an important role for Mdp3 in regulating cellular microtubule assembly. Our data also showed that Mdp3 significantly enhanced the stability of cellular microtubules. By tubulin turbidity assay, we found that Mdp3 could promote microtubule assembly and stability in the purified system. In addition, we found that Mdp3 expression varied during the cell cycle and in primary tissues. These findings thus establish Mdp3 as a novel microtubule-binding protein that regulates microtubule assembly and stability.     

Heterogeneity and structure of brain tubulins from cold-adapted Antarctic fishes. Comparison to brain tubulins from a temperate fish and a mammal

Tubulins purified from brain tissue of Antarctic fishes assemble in vitro to form microtubules at the low temperatures experienced by these extreme psychrophiles (Williams, R. C., Jr., Correia, J. J., and DeVries, A. L. (1985) Biochemistry 24, 2790-2798). We have initiated studies to determine the structural requirements for assembly of Antarctic fish tubulins at low temperatures. As a first step we have compared the heterogeneity, structures, amino acid compositions, and net charge of brain tubulins purified from three Antarctic fishes (Notothenia gibberifrons, Notothenia coriiceps neglecta, and Chaenocephalus aceratus), from the temperate channel catfish (Ictalurus punctatus), and from a mammal (the cow). Each preparation contained the alpha- and beta-tubulins and was free of microtubule-associated proteins. When examined by isoelectric focusing and by two-dimensional electrophoresis, brain tubulins from the Antarctic fishes were found to be highly heterogeneous; each was resolved into approximately 20 isoelectric variants. The distributions of the isotubulins from the cold-adapted fishes were similar but differed significantly from those of tubulins from catfish and cow. The average isoelectric points of the alpha- and beta-tubulins from the Antarctic fishes were more basic than the isoelectric points of the corresponding tubulins from bovine brain. Peptide mapping confirmed that tubulins from the Antarctic fishes and the mammal differed in structure. The amino acid compositions of fish and mammalian tubulins were similar, but Antarctic fish tubulins apparently contained fewer Glx residues than did catfish or bovine tubulins. Finally, native tubulins from an Antarctic fish and the cow differed slightly in net negative charge. Thus, brain tubulins from the cold-adapted fishes differ structurally from the tubulins of a temperate fish and of a mammal.     

Impaired microtubule assembly in brain from zinc-deficient pigs and rats

• 1.1. Microtubule reassembly was studied in brain extracts from pigs and rats deficient in zinc and from zinc-supplemented controls. Tubulin in extracts from zinc-deficient animals showed an impaired ability to repolymerize compared with extracts from control animals.
• 2.2. Crude microtubule protein isolated from zinc-deficient rat brain contained less free SH groups than that isolated from zinc-supplemented animals.
• 3.3. It is concluded that variation in zinc concentration close to the physiological range can influence microtubule assembly, and that zinc may have some function in microtubule polymerization in vivo.

     

Different antigenic reactivities of bovine brain glutamate dehydrogenase isoproteins

The structural differences between two types of glutamate dehydrogenase (GDH) isoproteins (GDH I and GDH II), homogeneously isolated from bovine brain, were investigated using a biosensor technology and monoclonal antibodies. A total of seven monoclonal antibodies raised against GDH II were produced, and the antibodies recognized a single protein band that comigrates with purified GDH II on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot. Of seven anti-GDH II monoclonal antibodies tested in the immunoblot analysis, all seven antibodies interacted with GDH II, whereas only four antibodies recognized the protein band of the other GDH isoprotein, GDH I. When inhibition tests of the GDH isoproteins were performed with the seven anti-GDH II monoclonal antibodies, three antibodies inhibited GDH II activity, whereas only one antibody inhibited GDH I activity. The binding affinity of anti-GDH II monoclonal antibodies for GDH II (K(D) = 1.0 nM) determined using a biosensor technology (Pharmacia BIAcore) was fivefold higher than for GDH I (K(D) = 5.3 nM). These results, together with epitope mapping analysis, suggest that there may be structural differences between the two GDH isoproteins, in addition to their different biochemical properties. Using the anti-GDH II antibodies as probes, we also investigated the cross-reactivities of brain GDHs from some mammalian and an avian species, showing that the mammalian brain GDH enzymes are related immunologically to each other.     

Soluble immune response suppressor (SIRS) inhibits microtubule function in vivo and microtubule assembly in vitro

Soluble immune response suppressor (SIRS) is a product of concanavalin A-stimulated murine T cells that, when activated or oxidized by macrophages or H2O2 (SIRSox), suppresses in vitro immune responses and inhibits cell division by normal and neoplastic cells. SIRSox is inactivated by a variety of electron donors, which suggests that SIRSox may be an oxidizing agent. Incubation of lymphocytes with SIRSox, but not with SIRS, partially reversed concanavalin A-mediated inhibition of capping of membrane immunoglobulin on B cells, and disrupted the cytoplasmic array of microtubules visualized by fluorescence microscopy. SIRSox also inhibited microtubule assembly in vitro in a concentration-dependent manner. Inactivation of SIRSox by dithiothreitol prevented SIRSox-mediated reversal of inhibition of capping and inhibition of microtubule assembly. These results reveal a pattern of SIRSox activity similar to sulfhydryl-dependent cytoskeletal disrupting agents (e.g., N-ethylmaleimide, cytochalasin A, p-benzoquinone), and suggest that SIRSox-mediated suppression of proliferation may involve interference with sulfhydryl-dependent cytoskeletal events critical for cell division.     

Cdk11 is a RanGTP-dependent microtubule stabilization factor that regulates spindle assembly rate

Production of Ran-guanosine triphosphate (GTP) around chromosomes induces local nucleation and plus end stabilization of microtubules (MTs). The nuclear protein TPX2 is required for RanGTP-dependent MT nucleation. To find the MT stabilizer, we affinity purify nuclear localization signal (NLS)-containing proteins from Xenopus laevis egg extracts. This NLS protein fraction contains the MT stabilization activity. After further purification, we used mass spectrometry to identify proteins in active fractions, including cyclin-dependent kinase 11 (Cdk11). Cdk11 localizes on spindle poles and MTs in Xenopus culture cells and egg extracts. Recombinant Cdk11 demonstrates RanGTP-dependent MT stabilization activity, whereas a kinase-dead mutant does not. Inactivation of Cdk11 in egg extracts blocks RanGTP-dependent MT stabilization and dramatically decreases the spindle assembly rate. Simultaneous depletion of TPX2 completely inhibits centrosome-dependent spindle assembly. Our results indicate that Cdk11 is responsible for RanGTP-dependent MT stabilization around chromosomes and that this local stabilization is essential for normal rates of spindle assembly and spindle function.     

Chlorpromazine inhibits the calcium-mediated effects of S-100 protein(s) on assembled brain microtubule proteins, but not those on microtubule protein assembly

We have examined the S-100-chlorpromazine interplay at the level of brain microtubule proteins in vitro. The results indicate that in the presence of 0.12 M KCl and 10 microM free Ca2+ the inhibitory effect of S-100 on microtubule assembly is additive to that of chlorpromazine, but S-100 fails to potentiate the disassembling effect of 0.1 mM Ca2+ if added to assembled microtubule proteins after chlorpromazine and Ca2+, probably because of inhibition of S-100 by the phenothiazine. Chlorpromazine does not compete with S-100 for binding to purified tubulin.     

Calcium/calmodulin-dependent inhibition of microtubule assembly by brain synaptic junction

The effect of synaptic junction (SJ) on microtubule assembly was examined. After preincubation with ATP at 37°C, rat SJ decreased the initial velocity and the extent of the porcine brain microtubule assembly (initiated by the addition of GTP) in a Ca²⁺/calmodulin (CaM)-dependent manner. The degree of the inhibition reached 35% of the control assembly (0-min preincubation) after 20-min preincubation with ATP. There was no inhibition either with heat-treated SJ, at 0°C, or in the presence of EGTA or W-7 (CaM antagonist). The inhibition was due neither to protease(s) nor CaM contaminating the preparations. Free Ca²⁺ concentration level required for the inhibition of microtubule assembly was 10^–6 M. Phosphorylation of microtubule proteins was inhibited by SJ in a Ca²⁺/CaM-dependent manner, and the inhibition occurred in a physiological increase range of intracellular Ca²⁺ concentration (10^–6M) The heat-treated SJ caused no inhibition. The result suggested that the microtubule assembly in the postsynaptic region was regulated by a Ca²⁺/CaM-dependent protein kinase associated with SJ; i. e., major postsynaptic density protein.Abbreviations used CaM calmodulin - DTT dithiothreitol - MAPs microtubule-associated proteins - MES 2-(N-morphorino)ethanesulfonic acid - mPSDp major postsynaptic density protein - PSD postsynaptic density - SDS PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - W-7 N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride