Tubulin synthesis during ciliogenesis in the mouse oviduct.

We reported earlier that tubulin levels increase in the developing mouse oviduct during that period after birth when ciliogenesis is at a maximum (Staprans, I., and Dirksen, E. R. (1974) J. Cell Biol., 62, 164). To determine the degree to which de novo synthesis and tubulin pools contribute to this increase, [³H]leucine-incorporation experiments were performed in vivo and in culture. Soluble, particulate and axonemal fractions, obtained from homogenized oviducts of 3-, 5-, 8- and 12-day-old suckling mice, were electrophoresed on sodium dodecyl sulfate gels and the specific activity of the tubulin band determined. The present work shows that more than 90% of the tubulin in 3-day-old and 75% in 5-day-old mouse oviducts is synthesized de novo. From both the in vivo and in culture experiments we conclude that although tubulin pools are present in mouse oviduct, they are continuously being replenished by newly synthesized protein as there is a rapid outflow from the soluble and particulate to the axonemal fraction into structures such as basal bodies and cilia. This burst of de novo tubulin synthesis corresponds to evidence from electron microscopic autoradiography, where label is present to a greater extent over centriole precursors and basal bodies than over other cell organelles. [³H]leucine incorporation into tubulin was inhibited by cycloheximide, demonstrating that we are dealing with synthesis, while colchicine below 10^?3, M concentration had no effect on tubulin assembly into axonemes.     

Rapid tubulin synthesis during ciliated cell differentiation

Using pulse-chase conditions in culture we have investigated the incorporation of 3H-leucine into tubulin of isolated oviducts from 5 day-old mice. Label appears in soluble, particulate and axonemal fractions minutes after incubation. In the latter two fractions, but not in the soluble fraction, this label is rapidly diluted under chase conditions. The data do not fit a simple model of sequential transfer of radioactively labeled, newly synthesized tubulin from a soluble fraction through centriole precursors to assembled ciliary axonemes.     

Colchicine-binding activity in particulate fractions of mouse brain

Both particulate and soluble fractions of brain homogenates bound [³H]colchicine. Approximately one-half of the total colchicine-binding activity in mouse brain was found in the particulate fraction. Of the particulate fractions, the microsomal and nerveending subfractions which sediment at the 1·0–1·2 m interface on sucrose gradients were richest in colchicine-binding activity. Intact microtubules were not found in these fractions, but colchicine-binding activity of these fractions may be related to the presence of microtubular protein.     

Tubulin pools in differentiating neuroblastoma cells

The distribution of tubulin in soluble, reversibly stabilized (assembled) and insoluble forms has been determined in neuroblastoma cells undergoing microtubule-dependent neurite elongation. Procedures were developed to obtain reproducible tubulin fractions and to assay total tubulin. Radioimmunoassays showed that both differentiated and nondifferentiated cell contained approximately 4 pg of tubulin per cell, of which 3-10% was in an insoluble, particulate form. The amount of tubulin assembled in differentiated cells was four to five times greater than in nondifferentiated cells, constituting 48-63% and 11-16% of the total tubulin pool in the respective cell types. Calculation of the concentration of soluble tubulin indifferentiated cells (approximately 0.8 mg/ml) and nondifferentiated cells (approximately 1.6 mg/ml) indicates that a critical concentration of subunits probably does not limit the induction of microtubule formation during neurite elongation.     

QUANTITATION AND CHARACTERIZATION OF BRAIN TUBULIN (COLCHICINE-BINDING ACTIVITY) IN DEVELOPING HYPOTHYROID RATS

The influence of early hypothyroidism on the concentration and biochemical properties of soluble and particulate tubulin from the cerebral cortex and cerebellum was investigated during development in the rat. Cellular soluble tubulin concentration (pmol colchicine bound/μg DNA) was approx 16% lower in both brain areas of hypothyroid animals compared to controls at 25 days of age. No effect of thyroid hormone deficiency was observed when tubulin concentration was expressed in terms of tissue protein or weight. The particulate tubulin concentration was approx 20% lower in the cerebral cortex of 25-day-old hypothyroid rats although the distribution of tubulin between soluble and particulate fractions was similar to controls. The incorporation of [¹⁴C]leucine into cerebral cortical tubulin in vitro (c.p.m. in tubulin/c.p.m. in total protein) was not significantly altered by the hormonal deficiency. Thus there was no apparent evidence of a selective defect in tubulin synthesis. Tubulin from hypothyroid rats behaved similarly to control samples with respect to the effects of pharmacological agents and temperature, lability of binding, chromatographic profile and electrophoretic mobility on sodium dodecyl sulfate polyacrylamide gels.     

Quantitative analysis of tubulin and microtubule compartments in isolated rat hepatocytes

A combined morphometric and biochemical approach has been used to identify and quantitate microtubules and tubulin in isolated hepatocytes. The total soluble pool of microtubule protein was estimated by specific high affinity binding to radiolabeled colchicine. Scatchard analysis of the data identified two populations of binding sites: high affinity-low capacity sites resembling tubulin and low affinity-high capacity sites believed to represent nonspecific colchicine-binding sites. Data from these studies indicate that tubulin represents 1% of the soluble protein of the cell, that 9.0 X 10(-14) dimers of tubulin are present per microgram soluble hepatocyte protein, and that the average hepatocyte contains 3.1 X 10(7) tubulin dimers. Our calculations suggest that this amount of tubulin would form a microtubule 1.9 cm in length if totally assembled. However, stereological measurements indicate that the actual length of microtubules in the cytosolic compartment of the average hepatocyte is only 0.28 cm. Thus, these experiments suggest that only 15% of the available tubulin in hepatocytes of postabsorptive rats is assembled in the form of microtubules.     

Comparison of Axonal Transport of Cytoplasmic- and Particulate-Associated Tubulin in Rat Optic System

Abstract: Adult rats were injected intraocularly with [³⁵S]methionine and killed from 1 to 10 weeks later. Optic nerves, optic tracts, and superior colliculi were dissected and then homogenized and separated into soluble and particulate fractions by centrifugation. Radioactivity coelectrophoresing with tubulin in buffers containing sodium dodecyl sulfate was determined (in cytoplasmic fractions, preliminary enrichment was achieved by vinblastine precipitation). Accumulation of radioactive tubulin along the optic pathway occurred in parallel (and in approximately equal amounts) in cytoplasmic and particulate fractions. Transported tubulin peaked at approximately 2 and 4 weeks in the optic nerve and tract, respectively, corresponding to a transport rate of ～ 0.4 mm/ day. There was little diminution in the amount of transported tubulin between optic nerve and tract, suggesting tubulin was not degraded in the axon. Accumulation in the superior colliculus reached a plateau by 4 weeks at less than 20% of the peak in the optic nerve, indicating turnover of tubulin at the nerve endings. The α/β subunit labeling ratio (radioactivity distribution between the tubulin subunits) was 0.57 for both cytoplasmic- and particulate-transported tubulin. In contrast, this ratio was 0.69 for whole brain tubulin prepared by vinblastine precipitation of soluble material. Isoelectric focusing and two-dimensional gel electrophoresis showed that the subunit compositions (microheterogeneity of the α and β bands) of transported tubulins in the cytoplasmic and particulate fractions were very similar. However, some differences relative to whole brain tubulin were noted; a tubulin subunit not identifiable in whole brain tubulin preparations but present in both soluble- and particulate-transported tubulin was observed. Because of the compositional and metabolic similarities of transported tubulin in the soluble and particulate fractions, we conclude that they form a common metabolic pool. This suggests either that, at least for some membranes, the well-characterized tight association between particulate tubulin and membranes may be artifactual or else that an equilibrium exists between soluble and particulate tubulin.     

Microtubule assembly in developing mammalian brain.

Microtubule protein was measured in mouse brain homogenates by quantitative colchicine binding. Neonatal animals contained more than twice the amount of brain tubulin as adult mice. The percentage of colchicine-binding protein which was polymerized was determined by extracting brain at room temperature into a medium designed to stabilize intact microtubules. Under identical conditions and tubulin concentrations, neonatal brain tubulin (colchicine-binding activity) had a greater proportion of the total extracted in an apparently polymerized state (pelletable by centrifugation) than did adult brain. A slight variation in the ratio of assembled to unassembled tubulin was observed with varying protein concentration (volume of extract), indicating that the values obtained may not reflect exactly the in vivo situation, because a rapid equilibration takes place upon homogenization. At all protein concentrations, the neonatal brain extracts contained a significantly greater proportion of assembled tubulin than did adult brain. This proportion began to fall at 5 days postnatal and reached the adult level at 30 days. The tubulin assembled/not assembled ratios were not altered by addition of nucleoside triphosphates, additional EGTA, or sulfhydryl protecting agents, and did not vary with preparation times of 30–90 min. The colchicine-binding reaction and decay of colchicine-binding activity with time were similar in extracts of different aged mouse brains, with neonatal slightly more stable than adult. Pools of tubulin from any age which were soluble at room temperature (unpolymerized) could not repolymerize well in vitro when incubated with GTP at 37 °C, whereas pools of tubulin which were sedimentable at room temperature (polymerized) could be redissolved at 0 °C and readily reassembled at 37 °C. The neonatal extract tubulin was thus more polymerization competent than the adult extracts; this correlates with a greater proportion of assembled tubulin in extracts at room temperature and possibly in vivo.     

A modified colchicine-binding assay for the measurement of total and microtubule-derived tubulin in rat liver

The usual measurement of liver tubulin by the colchicine-binding assay does not take into account the accelerated decay of the colchicine-binding capacity of tubulin when liver supernatants, especially those containing microtubule-derived tubulin, are incubated at 37°C. This results in marked underestimations. Our findings indicate that this alteration is due to an inhibitor of colchicine-tubulin binding in liver supernatants that is probably extracted from particulate fractions. The inhibitory activity is decreased by dilution of the supernatants and by increasing the concentration of colchicine. However, the former modification decreases the sensitivity of the assay and the latter increases the nonspecific binding of colchicine to liver proteins other than tubulin. Assessment of the decay and correction for it by calculating the initial binding capacity results in complete recovery of brain tubulin from liver supernatants and values for microtubule-derived tubulin that closely correspond to those expected from simultaneous morphometric assessment of liver microtubules by electron microscopy. The modified method also indicates that the fraction of liver tubulin assembled in microtubules is greater than previously reported.     

Membrane-bound tubulin in brain and thyroid tissue.

Brain and thyroid tissue contain membrane-bound colchicine-binding activity that is not due to contamination by loosely bound cytoplasmic tubulin. This activity can be solubilized to the extent of 80 to 90% by treatment with 0.2% Nonidet P-40 with retention of colchicine binding. Extracts so obtained contain a prominent protein band in disc gel electrophoresis that co-migrates with tubulin. Membranes, and the solubilized protein therefrom, exhibit ligand binding properties like tubulin; for colchicine the KA is approximately 1 X 10(6) M-1 in brain and approximately 0.6 X 10(6) M-1 in thyroid; for vinblastine the KA is approximately 8 X 10(6) M-1 for both tissues; and for podophyllotoxin the Ki is approximately 2 X 10(-6) M for both tissues. Displacement by analogues of colchicine is of the same order as for soluble tubulin. Although membrane-bound colchicine-binding activity shows greater thermal stability and a higher optimum binding temperature (54 degrees versus 37 degrees) than soluble tubulin, this appears to be the result of the membrane environment since the solubilized binding activity behaves like the soluble tubulin. Antibody against soluble brain tubulin reacts with membranes and solubulized colchicine-binding activity from both brain and thyroid gland. We conclude that brain and thyroid membrane preparations contain firmly bound tubulin or a very similar protein.     

Subcellular localization of tubulin in chick retina

Tubulin was measured through [3H]colchicine-binding in membrane and soluble components of chick retinal subcellular fractions. Total tubulin content was concentrated in the synaptosomal and rod outer segment fractions. Although in total retinal homogenate only 20% of total tubulin was associated to the membrane, in synaptosomes and photoreceptor outer segments, up to 50% of tubulin was bound to the membrane fraction. Results raise the possibility of tubulin participation in transmembrane phenomena which are common to transmitter release and photoexcitation.     

In vitro polymerization of microtubules into asters and spindles in homogenates of surf clam eggs

The eggs of the surf clam Spisula solidissima were artificially activated, homogenized at various times in cold 0.5 M MES buffer, 1mM EGTA at pH 6.5, and microtubule polymerization was induced by raising the temperature to 28 degrees C. In homogenates of unactivated eggs few microtubules form and no asters are observed. By 2.5 min after activation microtubules polymerize in association with a dense central cylinder, resulting in the formation of small asterlike structures. By 4.5 min after activation the asters formed in vitro contain a distinct centriole, and microtubules now radiate from a larger volume of granular material which surrounds the centriole. By 15 min (metaphase I) the granular material is more disperse and only loosely associated with the centriole. Microtubules are occasionally observed which appear to radiate directly from one end of the centriole. The organizing center can be partially isolated by centrifugation of homogenates of metaphase eggs and will induce aster formation if mixed with tubulin from either activated or unactivated eggs. Pretreatment of the eggs with colchicine does not prevent the formation of a functional organizing center. Complete spindles can also be obtained under polymerizing conditions by either homogenizing the eggs directly into warm buffer or by adding a warm high-speed supernate to spindles which have been isolated in a microtubule stabilizing medium. Extensive addition of new tubulin occurs onto the isolated spindles, resulting primarily in growth of astral fibers, although there occasionally appears to be growth of chromosomal fibers and of pole-to-pole fibers. Negatively stained aster microtubules have a strong tendency to associate side by side, and under some conditions distinct cross bridges can be observed. However, under other conditions large numbers of 300-400-A particles surround the microtubules; the presence of stain between particles can give the appearance of cross bridges.     

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.     

Passive Avoidance Learning Results in Region-Specific Changes in Concentration of and Incorporation into Colchicine-Binding Proteins in the Chick Forebrain

Abstract: Incorporation of [¹⁴T]leucine into trichloracetic acid-precipitable material and tubulin-enriched fractions, and total tubulin levels as determined by colchicine-binding activity and retention on DE81 filter discs, were measured in various regions of the chick brain following training on a one-trial passive avoidance task, suppression of pecking at a chromed bead as a consequence of the aversive taste of methylanthranilate. Radioactive pulse time was 0.5 h. The only brain region in which changes were found was the anterior forebrain roof, the same area in which biochemical changes in response to exposure of the birds to an imprinting stimulus have been observed previously. In the anterior forebrain roof the changes observed as a consequence of training were detectable at 0.5 and 24 h after the 10-s training experience but not 48 h subsequently. One-half hour after training, there were increases of the order of 20 or 30% in [¹⁴T]leucine incorporation into particulate and postmitochondrial TCA-precipitable material and a tubulin-enriched fraction purified as above. There were comparable increases in the total amount of colchicine-binding activity. By 48 h, none of these increases were detectable. Subcellular fractionation of the particulate fraction showed that most of the increase of incorporation into the tubulin-enriched fraction and in colchicine-binding activity was present in the soluble content of the synaptosomes; there were no increases in either measure in the synaptic membrane fraction. The possible role of changed levels and turnover of tubulin in the plastic responses of the brain to learning experiences is discussed.     

Initiation and growth of microtubules from mitotic centers in lysed mammalian cells

Metaphase PtK1 cells, lysed into polymerization-competent microtubule protein, maintain a spindle which will gain or lose birefringence depending on the concentration of disassembled tubulin subunits used in the lysis medium. Concentrations of tubulin subunits greater than the equilibrium monomer value promote a rate and extent of birefringence increase that is proportional to the subunit concentration. Increase in spindle birefringence can be correlated with an increase in tubule number, though the relationship is not strictly linear. Increase in spindle tubule number is due to an vivo-like initiation of tubules at the mitotic centers, as well as tubulin addition onto pre-existing spindle fragments. Colcemid-treated prometaphase cells lysed into polymerization-competent tubulin develop large asters in the region of the centrioles and short tubules at kinetochores, making it unlikely that all microtubule formation in lysed cell preparations is dependent on tubulin addition to short tubule fragments. Asters can also form in colcemid-treated prometaphase cells lysed in tubulin that is incapable of spontaneous tubule initiation, suggesting that the centriolar region serves a tubule-initiator function in our lysed cell preparations. The ability of the centriole to initiate microtubule assembly is a time-dependent process-a ripening effect takes place between prophase and late prometaphase. Ripening is expressed by an increase in the number and length of tubules found associated with the centriolar region.     

Regulation of Microtubule Synthesis and Utilization during Early Embryonic Development of the Sea Urchin

Microtubules deployed during early development of the sea urchinembryo are derived both from a preexisting pool of subunitspresent in the egg and from microtubule protein subunits synthesizedin the embryo. Several aspects of microtubule protein synthesisand utilization are reviewed. Microtubule protein synthesisin early development utilizes oogenetic messenger RNA species.Translation of this mRNA is under regulation. Microtubule proteinsynthesis rises concomitantly with overall protein synthesisat fertilization, but rises at a relatively higher rate laterin cleavage stages. Microtubule protein labeled with [³H]-leucinein early development is incorporated into cilia, indicatingthat newly synthesized protein enters the pool of subunits usedin organelle assembly. The microtubule protein pool comprisesabout 1%of the soluble protein of the egg, and remains constantin size at least until the blastula stage. Direct pool sizeestimates are consistent with results of experiments on recruitmentof microtubule protein subunits into the mitotic apparatus andinto regenerating cilia. Soluble and particulate colchicinebinding fractions, which have been reported from several systems,appear to be present in sea urchin embryos. The possible roleof such fractions are discussed, as are aspects of the regulationof ciliary assembly.     

A sensitive method for determination of 5-fluorouracil and 5-fluoro-2'-deoxyuridine in human plasma by high-pressure liquid chromatography

Polymerization-competent extracts of suspension-cultured HeLa cells and porcine brain tissue were assayed for tubulin content. Five different methods were used to assay identically prepared extracts: two types of sodium dodecyl sulfate-containing acrylamide gels, a DEAE retention assay, a colchicine-binding assay, and a radioimmunoassay. The colchicine-binding and radioimmunoassay results were in close agreement and are therefore considered reliable assays for tubulin content in cell extracts. The DEAE retention assay gave slight overestimates, but the gel methods seriously overestimated tubulin content. Based on data from colchicine binding and radioimmunoassay, the proportion of soluble cell protein which is tubulin is 4.3% for HeLa cell extracts and 12.1% for brain tissue extracts.     

Radioimmune assay of tubulin applied to oligomers, synaptic membranes, and plants

A radioimmune assay for microtubule protein, tubulin, is described, in which unknown amounts of native or denatured tubulin can be quantitated by the ability to compete with pure [¹²⁵I]tubulin for rabbit antibodies produced against purified bovine brain tubulin. The assay is used to demonstrate that crude extracts of mouse brain contain negligible amounts of 30–36S tubulin oligomers under conditions where purified tubulin forms substantial amounts of such structures. Also, the particulate fraction of osmotically shocked and sonicated brain synaptosomes contains negligible tubulin antigenic activity. By contrast, soluble extracts of soybean, especially rapidly dividing regions of the plant, were found to contain significant amounts of cross-reacting material, providing further evidence for the conservative evolutionary nature of this ubiquitous and important protein.     

Colchicine-binding protein of the liver. Its characterization and relation to microtubules

Colchicine-binding activity of mouse liver high-speed supernate has been investigated. It has been found to be time and temperature dependent. Two binding activities with different affinities for colchicine seem to be present in this high-speed supernate, of which only the high-affinity binding site (half maximal binding at 5 x 10(-6) M colchicine) can be attributed to microtubular protein by comparison with purified tubulin. Vinblastine interacted with this binding activity by precipitating it when used at high concentrations (2 x 10(- 3) M), and by stabilizing it at low concentrations (10(-5) M). Lumicolchicine was found not to compete with colchicine. The colchicine-binding activity was purified from liver and compared with that of microtubular protein from brain. The specific binding activity of the resulting preparation, its electrophoretic behavior, and the electron microscope appearance of the paracrystals obtained upon its precipitation with vinblastine permitted its identification as microtubular protein (tubulin). Electrophoretic analysis of the proteins from liver supernate that were precipitated by vinblastine indicated that this drug was not specific for liver tubulin. Preincubation of liver supernate with 5 mM EGTA resulted in a time- dependent decrease of colchicine-binding activity, which was partly reversed by the addition of Ca++. However, an in vitro formation of microtubules upon lowering the Ca++ concentration could not be detected. Finally, a method was developed enabling that portion of microtubular protein which was present as free tubulin to be measured and to be compared with the total amount of this protein in the tissue. This procedure permitted demonstration of the fact that, under normal conditions, only about 40% of the tubulin of the liver was assemled as microtubules. It is suggested that, in the liver, rapid polymerization and depolymerization of microtubules occur and may be an important facet of the functional role of the microtubular system.     

Distribution of tyrosinated and acetylated tubulin in centrioles during mitosis of 3T3 and SV40-3T3 cells

We performed a comparative electron microscopic analysis of centriolar and cytoplasmic microtubules stained with antibodies to acetylated or tyrosinated α-tubulin during the cell cycle of mouse nonmalignant Balb 3T3 (clone A31) and virus-transformed heteroploid SV40-3T3 cell lines. It was shown that the pattern of centriole immunostaining changed during the cell cycle in 3T3 (A31) cells, but not in tumorigenic SV40-3T3 cells. Remarkable changes in the centriole immunostaining pattern were observed during interphase-mitosis or mitosis-interphase transitions when the microtubule system and protein organization of centrosomes underwent drastic rearrangements. A high level of tyrosinated tubulin in centrioles was observed at all stages of the cell cycle except when entering mitosis, whereas a high level of acetylated tubulin was visualized in centrioles at all stages of the cell cycle except at the end of mitosis.