Rat Brain Tubulin Research Articles

Differential immunocytochemical localisation of α-tubulin and β-tubulin in cerebellum using monoclonal antibodies

We have utilised monoclonal antibodies against yeast tubulin and rat brain tubulin that specifically recognise α and β subunits of rat brain tubulin when characterised by immunoblotting of brain cytosol onto nitrocellulose sheets. Light microscopic immunocytochemical studies in rat cerebellum demonstrate that while dendrites and glia are labelled with the two antibodies, there is a selective deficiency of α-tubulin compared with β-tubulin immunoreactivity in parallel fibre axons and in myelinated axons in the white matter.

Structural and functional consequences of increased tubulin glycosylation in diabetes mellitus.

The extent of in vitro nonenzymatic glycosylation of purified rat brain tubulin was dependent on time and glucose concentration. Tubulin glycosylation profoundly inhibited GTP-dependent tubulin polymerization. Electron microscopy and NaDodSO4/polyacrylamide gel electrophoresis showed that glycosylated tubulin forms high molecular weight amorphous aggregates that are not disrupted by detergents or reducing agents. The amount of covalently bound NaB3H4-reducible sugars in tubulin recovered from brain of streptozotocin-induced diabetic rats was dramatically increased as compared with tubulin recovered from normal rat brain. Moreover, tubulin recovered from diabetic rat brain exhibited less GTP-induced polymerization than tubulin from nondiabetic controls. The possible implications of these data for diabetic neuropathy are discussed.

ADP-ribosylation of microtubule proteins as catalyzed by cholera toxin.

Incubation of purified rat brain tubulin with cholera toxin and radiolabeled [32P] or [8-3H]NAD results in the labeling of both alpha and beta subunits as revealed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Treatment of these protein bands with snake venom phosphodiesterase resulted in quantitative release of labeled 5'-AMP, respectively labeled with the corresponding isotope. Two-dimensional separation by isoelectric focusing and SDS-PAGE of labeled and native tubulin revealed that labeling occurs at least in four different isotubulins. The isoelectric point of the labeled isotubulins was slightly lower than that of native purified tubulin. This shift in mobility is probably due to additional negative charges involved with the incorporation of ADP-ribosyl residues into the tubulin subunits. SDS-PAGE of peptides derived from [32P]ADP-ribosylated alpha and beta tubulin subunits by Staphylococcus aureus protease cleavage showed a peptide pattern identical with that of native tubulin. Microtubule-associated proteins (MAP1 and MAP2) of high molecular weight were also shown to undergo ADP-ribosylation. Incubation of permeated rat neuroblastoma cells in the presence of [32P]NAD and cholera toxin results in the labeling of only a few cell proteins of which tubulin is one of the major substrates.

A biochemical model for neurite outgrowth during brain development.

Maximal amounts of tubulin in rat brain are observed during the 3 to 10-day postnatal period. The rates of in vitro tubulin polymerization are very low at these stages of development; they increase thereafter during the second postnatal week, reaching a maximum at adulthood. The increased rate of polymerization could depend either on modifications in the concentration and activity of microtubule-associated proteins (MAPs), which play a crucial role in microtubule assembly in vitro, or on changes in their composition. The results show that the composition and activity of TAU proteins (MW: 58-68000) change during development. Analysis of "young" and "adult" TAU protein peptide mapping suggests that their amino acid sequence is different. Our data indicate a good correlation between tubulin capacity to polymerize in vitro and changes in the composition and activity of TAU proteins which occur during the critical period when the neuronal network is constructed.

Translation in vitro of rat brain mRNA coding for a variety of tubulin forms.

Prenatal rat brain tubulin was resolved by isoelectric focusing into five to six components (isotubulins 1--6) while in mature brain nine distinct forms were evident (isotubulins 1--9). Tubulin, isolated from various brain regions, displayed different proportions of these nine isotubulins which may result from the heterogeneity in brain cell population. Mature brain mRNA, when translated in vitro, in the reticulocyte lysate cell-free system, directed the synthesis of five tubulin forms, namely isotubulins 1, 3, 4 (or 5), 6 and 7. The mRNA species coding for isotubulins 3 and 7 could be partially separated on formamide/sucrose gradients, while in the absence of formamide the mRNA species directing the synthesis of isotubulins 1, 4 (or 5) and 6 showed differences in mobility. It therefore appears that brain mRNA may consist of five different species coding for distinct tubulin forms. Moreover, a marked age-dependent enhancement in the relative translation of the mRNA coding for isotubulin 7, which is not apparent among the translation products directed by the prenatal mRNA, was detected. Our results indicate that some of the age-dependent variations in tubulin microheterogeneity may be controlled at the mRNA level.

Experimental phenylketonuria: replacement of carboxyl terminal tyrosine by phenylalanine in infant rat brain tubulin.

In the brains of newborn rats, about half of the tubulin molecules are modified posttranslationally by the addition of an aromatic amino acid at the carboxyl terminus of the alpha chain. Of the added residues, 96 percent are tyrosine and 4 percent are phenylalanine. After induction of hyperphenylalaninemia, the proportion of tubulin molecules containing carboxyl terminal phenylalanine increases up to eightfold and the pool of tyrosine-containing molecules decreases by an equivalent amount.

BIOSYNTHESIS OF HETEROGENEOUS FORMS OF MAMMALIAN BRAIN TUBULIN SUBUNITS BY MULTIPLE MESSENGER RNAs

Abstract—Heterogeneity among the primary translation products of rat brain tubulin messenger RNA was examined. On two‐dimensional gels native cytoplasmic tubulin from randomly bred rats (PB21) consists of two groups of α tubulin subunits among which the most acidic forms, α1 and α2, are most abundant; and β tubulin consists of a minimum of two species, β1 and β2. In the same group of animals the primary translation products of rat brain tubulin mRNA consist of at least these four subunit forms (α1α2, β1 and β2); however, minor basic forms of α subunits were not synthesized. This same result was obtained from a homologous brain protein synthesizing system, a heterologous system prepared from brain polysomes and rabbit reticulocyte initiation factors, and a wheat germ lysate programmed with brain poly A mRNA. A variant form of brain tubulin was found in rats bred monogamously for over 30 generations (MB71 rats). MB71 brain polysomes synthesize overlapping a subunits which migrate in two‐dimensional gels to the α1 position, and the typical PB21 α2 is not present. The addition of PB21 brain mRNA to a protein synthesizing system composed of MB71 polysomes plus reticulocyte initiation factors allowed synthesis of the typical α2 tubulin in addition to the MB71 tubulin subunits. The structural relationship among subunits was examined by radioiodinated peptide mapping. The α subunits are structurally different from the β subunits; however, among the major tyrosine‐containing tryptic peptides no prominent differences were observed between α1 and α2, or between β1 and β2 by the radioiodination procedure. The results provide evidence for heterogeneity among the primary translation products of brain tubulin mRNA, and for the existence of multiple functional tubulin genes in rat brain.

The turnover rate of tubulin in rat brain.

AbstractThe turnover rate of tubulin in rat brain was determined from the decay in specific radioactivity of the protein after pulse‐labeling. When precursors were administered by a parenteral route, the shortest half‐life, 9.8 days, was obtained with [14C]NaHCO3; the longer half‐lives obtained with [U‐14C]glucose or [4,5‐3H]leucine suggest significant reutilization of label. Furthermore, with leucine as precursor maximal specific radioactivity of tubulin was not obtained until eight days after administration of label. Labeling and decay kinetics obtained with [4,5‐3H]leucine were markedly different when the isotope was administered directly into the lateral ventricle. The difference between the turnover rates of the ‐α and β subunits of tubulin purified by means of high resolution polyacrylamide gel electrophoresis was not statistically significant. A half‐life for tubulin of 6.2 days was measured by continuous intravenous infusion of [U‐14C]tyrosine.

Quantification and properties of tubulin polymerization in crude brain extracts and preparations of microtubular and purified tubulin.

Removal of assembled tubulin by centrifugation, followed by measurement in the supernatant of the residual colchicine binding capacity of the non-polymerized, non-precipitable tubulin, is a sensitive and reliable method of measuring tubulin polymerization. This method can be used in both crude and purified preparations of brain tubulin and allows the molar quantification of the total, polymerized and non-polymerized tubulin species in each sample. Only 40--50% of the total tubulin present in crude adult brain extracts is capable of polymerizing when incubated with GTP. The percentage of tubulin polymerizing with GTP is slightly higher in crude foetal brain extracts than in the adult. Incubation of first polymerization supernatants, containing exclusively the GTP-insensitive tubulin, with guanosine 5'-[alpha, beta-methylene]triphosphate (GuoP[CH2]PP) but not with 2.4 M glycerol results in tubulin polymerization. High concentrations of glycerol (2.4 M) promote the polymerization of tubulin in adult but not in crude foetal brain extracts. Both ATP and adenosine 5'-[alpha, beta-methylene]triphosphate (AdoP[CH2]PP) are effective in promoting the polymerization of GDP-free (90%) microtubular protein. The microtubular protein assembled with GuoP[CH2]PP or AdoP[CH2]PP has the unique character of being resistant to calcium concentrations (2mM), which cause complete depolymerization of the tubulin assembled with GTP or ATP. Phosphocellulose-purified tubulin significantly assembles when incubated with GuoP[CH2]PP or AdoP[CH2]PP at protein concentrations at which GTP or ATP do not promote polymerization. 2 micrometer tubulin is the minimal concentration required for polymerization in both crude and purified preparations of adult rat brain tubulin.

Tubulin microheterogeneity increases with rat brain maturation.

MICROTUBULES are present in all eukaryotic cells and have been found to have a variety of structural and dynamic roles in cell shape, division, motility, transport and secretion1. In nervous tissue neurite outgrowth and axoplasmic transport are also thought to depend on microtubule integrity2. The micro-tubule subunit protein, tubulin, is a heterodimer composed of two polypeptides α and β (refs 3,4). The α and β subunits show microheterogeneity and both have been resolved into two or three components5–8. The question therefore arises as to whether changes occur in the relative proportions of the multiple forms of tubulin upon assumption of different roles within the nerve cell. We show here that cytoplasmic rat brain tubulin, as resolved by isoelectric focusing, is highly heterogeneous. Moreover, tubulin microheterogeneity seems to be developmentally determined, increasing from seven to nine distinct components during early postnatal rat brain maturation. However, extensive tubulin microheterogeneity is prominent in the brain, as tubulin from other organs is less heterogeneous.

THE EFFECT OF TRIALKYLTIN COMPOUNDS ON TUBULIN POLYMERISATION

AbstractThe effects of trialkyltin compounds on the colchicine‐binding activity and the in vitro polymerisation of rat brain tubulin have been investigated. Trialkyltins at concentrations between 10 and 100 μM inhibited the colchicine‐binding activity of purified tubulin preparations derived from rat brain. Viscometric studies have shown that the same concentrations of trialkyltins also inhibited the in vitro polymerisation of tubulin. Both the effects were found to be concentration dependent. However, trialkyltins did not interfere with the in vitro preassembled microtubules. The reasons which are believed to explain the inhibitory effects of these compounds are discussed.

Role of nucleotides in tubulin polymerization: effect of guanosine 5'-methylene diphosphonate.

Incubation of purified rat brain tubulin with guanosine 5'-methylene diphosphonate [GMP(CH2)P] (1 mM), a GDP analog resistant to hydrolysis, results in the polymerization of 20-30% of the total tubulin present. Analogous incubations with GDP (1 mM) do not result in tubulin polymerization. Polymerization with GMP(CH2)P occurs in the presence of alkaline phosphatase (EC 3.1.3.1) under conditions that completely hydrolyze the likely phosphate donors (GTP, GDP, and GMP) as well as the potential product [GMP(CH2)PP] of the transphosphorylase activity present in purified tubulin preparations. Tubulin polymerization in vitro thus can occur in the absence of gamma-phosphate and phosphate bond hydrolysis at the exchangeable nucleotide-binding site of tubulin. Polymerization of tubulin by GMP(CH2)P is neither prevented nor reversed by concentrations of calcium (2 mM) that prevent microtubule assembly and disrupt already formed microtubules induced by GTP. However, tubulin polymerized with GMP(CH2)P is readily depolymerized by cold (4 degrees, 30 min). The possible involvement of GTP alpha-beta bond hydrolysis must be considered seriously as playing a role in the process of microtubule depolymerization.

A high molecular weight phosphoprotein in cultured fibroblasts that associates with polymerized tubulin

Microtubule associated proteins (MAPs) are high molecular weight proteins that associate with microtubules during polymerization. This report describes a high molecular weight protein fraction with a molecular weight of approx. 290 000 from cultured mammalian fibroblasts that associates with polymerized rat brain tubulin. This protein(s), which is referred to as f-MAP, is enriched approx. 25-fold in a twice polymerized microtubules when compared with the original cell extract. Polymerization of rat brain extract in the presence of in vivo 32P-labeled fibroblast extract reveals the presence of a 32P-labeled protein in the polymerized pellet with the same electrophoretic mobility as f-MAP. The present study suggests that fibroblasts in culture contain a high molecular weight phosphoprotein with properties and a molecular weight very similar to the MAPs described in mammalian brain.

Immunocytochemical visualization of microtubules and tubulin at the light- and electron-microscopic level.

ABSTRACT An antibody against rat brain tubulin was purified by affinity chromatography and used for the immunocytochemical staining of tubulin-containing structures in tissue-cultured cells at the light- and electron-microscopic level. For this purpose the unlabelled antibody enzyme method (PAP method) was used. Tissue-cultured cells were fixed with glutaraldehyde or formaldehyde, postfixed with acetone and stained with the PAP procedure. With the light microscope, a dense network of darkly stained fibres was visible in non-dividing cells. The fibres converged towards a point in the nuclear vicinity and radiated throughout the cytoplasm towards the cell periphery. Mitotic spindles were also heavily stained in all stages. The cytoplasm of non-dividing and dividing cells contained a variable amount of diffuse staining. The nucleus was unstained. The network could be destroyed by treating the cells with microtubule inhibitors. An intense diffuse staining was visible in cells treated with colchicine or nocodazole. The paracrystalline tubulin precipitates that appeared in cells treated with vinblastine were heavily stained. Preliminary ultrastructural observations were made on sections through cells that were fixed with glutaraldehyde, stained with the PAP procedure and embedded in Epon. The microtubules were seen to be covered with stained PAP complexes. The cortical microfilaments, the 10–nm filaments and the plasma membrane were unstained, as were the contents of nuclei, mitochondria, endoplasmic reticulum, Golgi elements, lysosomes and vacuoles. A variable amount of diffuse staining was visible in the cytoplasm and the membranes of several organelles often showed PAP deposits. This staining was presumed to localize tubulin in non-microtubular form. It is probable that at least part of this staining was immunologically specific, since no PAP complexes were seen in the cells when the antitubulin antibody was omitted from the procedure or when it was replaced by an unrelated antibody solution.

The isolation and characterisation of platelet microtubule proteins

Tubulin, the microtubule subunit protein, has been isolated from a soluble extract of pig platelets, by an in vitro polymerisation process. Several physicochemical properties of platelet tubulin have been investigated and compared with those of mammalian brain tubulins. The molecular weight of the tubulin monomeric subunits was found to be 55 000 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and the platelet protein co-migrated with rat, rabbit, guinea-pig, mouse and calf brain tubulins. The [ 3H]colchicine-binding dimer form of platelet tubulin sedimented in a 5% to 20% linear sucrose gradient with a sedimentation coefficient of 5.7 S, compared with that of 5.9 S for rat brain tubulin. The amino acid compositions of platelet and mammalian brain tubulins were found to be very similar, with glutamate and asparate as the predominant residues. Moreover, the one and two dimensional fingerprints of 125I-labelled tryptic peptides of platelet and brain tubulins showed considerable similarity. Platelet tubulin could be separated into two non-identical (α and β) subunits by Tris/glycine discontinuous alkaline sodium dodecyl sulphate-polyacrylamide gel electrophoresis and the β-tubulin had two or three 125I labelled peptides not seen in the α-tubulin. Two, or perhaps three, high-molecular-weight proteins were always present in the platelet tubulin samples, prepared by in vitro polymerisation and these had molecular weights greater than 200 000 and were probably analogous to the high-molecular weight proteins reported to be present in brain microtubule preparations. Platelet tubulin with its associated high molecular weight proteins will assemble in vitro into tubular structures which in electron micrographs resemble brain microtubules and the microtubules seen in whole platelets. It is believed that this is one of the first mammalian microtubule systems other than that of nervous tissue which has been isolated and studied in detail at the subunit level.

A sensitive method for measuring polymerized and depolymerized forms of tubulin in tissues.

A rapid method for measuring polymerized and depolymerized forms of tubulin in tissues has been developed. The procedure consists of homogenization and centrifugation of the tissue in a microtubule-stabilizing solution and depolymerization of the precipitated microtubules; polymerized and depolymerized forms of tubulin are quantitated by a colchicine-binding assay. The validity of the technique was assessed by electron microscopy and recovery studies with labeled and unlabeled preparations of polymerized and depolymerized forms of rat brain tubulin. The sensitivity of this technique allows quantitation of tubulin in 150 micrograms of tissue, wet weight. The method demonstrated that both the polymerized and depolymerized forms of tubulin were present in rat liver cells, and that in the fed state 31.3 +/-0.7% of the total tubulin pool was in the polymerized form.

The effect of phospholipids on the in vitro polymerization of rat brain tubulin

The association of brain tubulin, as measured by the temperature-dependent development of turbidity at 350 nm, is greatly stimulated by the detergent Nonidet P-40 in crude extracts of rat brain tissue. Stimulation of turbidity development is also obtained with partially purified rat brain tubulin treated with Nonidet or other detergents, or preincubated with phospholipase C or D; treatment with bovine pancreatic phospholipase A 2 produces an inhibition. Exogenous phospholipids, diglycerides, other related derivatives, and lipophilic extracts of tubulin and brain supernatants can also alter the turbidity development. In addition, microtubules arising from tubulin obtained in the presence of Tween-20 or Nonidet P-40 exhibit a 50 and 100% increased specific viscosity, respectively, over that of tubulin prepared in the absence of detergent or in the presence of Kyro or Triton N-101. The effectiveness of these detergents in removing phospholipids from tubulin preparations follows a similar pattern: Nonidet P-40 removes 80%, Tween-20 removes 50%, and Kyro or Triton N-101 removes none. The total mass of microtubule formed, as determined by sedimentation, is the same regardless of the effect of the detergents on the viscosity. The microtubules obtained in the presence of Nonidet P-40 have a normal appearance when examined by electron microscopy, and their composition on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is indistinguishable from that of standard tubulin, especially with regard to the minor protein bands always present in the tubulin preparations. The results obtained suggest that the phospholipids associated to brain tubulin preparations might have a role in determining the association of tubulin and/or the final dimensions of the assembled microtubules.

Rat brain-lymphocyte antigen: Characterization by rabbit antisera to rat brain tubulin and S-100 protein

Antisera to rat brain tubulin and brain S-100 protein were raised in rabbits. Anti-tubulin serum was cyctotoxic and immunofluroescence-positive to rat T lymphoctes, but not B lymphocytes. These activities were removed by absorption with homologous brain tubulin. Anti-S-100 protein serum did not exert cytotoxic effect and positive immunofluorescence on rat T and B lymphoyctes. These results indicate that brain tubulin, but not brain S-100 protein, shares some antigenic determinants with T lymphocytes.

Rat brain-lymphocyte antigen: Characterization by rabbit antisera to rat brain tubulin and S-100 protein

Rat brain-lymphocyte antigen: Characterization by rabbit antisera to rat brain tubulin and S-100 protein

Binding of maytansine to rat brain tubulin

Maytansine and vincristine binding to crude rat brain tubulin is a temperature- and ionic strength-dependent process. The K A values for binding of vincristine and maytansine are 0.5 × 10 6 M −1 and 1.5 × 10 6 M −1, respectively. Maytansine competitively inhibits vincristine binding with a K i for maytansine of 0.4 × 10 −6 M. The binding of both drugs is easily reversible. Unlabeled maytansine rapidly displaces the labeled drug whereas only about 75% of the bound maytansine was displaced by vincristine. Bound labeled vincristine is completely displaced by either vincristine or maytansine. Both drugs appear to share a common binding site, although an additional site specific for maytansine seems to be present. Colchicine did not affect either vincristine or maytansine binding.