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.     

The relative contributions of polymer annealing and subunit exchange to microtubule dynamics in vitro

Microtubules that are free of microtubule-associated protein undergo dynamic changes at steady state, becoming longer but fewer in number with time through a process which was previously assumed to be based entirely on mechanisms of subunit exchange at polymer ends. However, we recently demonstrated that brain and erythrocyte microtubules are capable of joining end-to-end and suggested that polymer annealing may also affect the dynamic behavior of microtubules in vitro (Rothwell, S. W., W. A. Grasser, and D. B. Murphy, 1986, J. Cell Biol. 102:619-627). In the present study, we first show that annealing is a general property of cytoplasmic microtubules and is not a specialized characteristic of erythrocyte microtubules by documenting annealing between tryosinolated and detyrosinolated brain microtubules. We then examine the contributions of polymer annealing and subunit exchange to microtubule dynamics by analyzing the composition and length of individual polymers in a mixture of brain and erythrocyte microtubules by immunoelectron microscopy. In concentrated preparations of short-length microtubules at polymer-mass steady state, annealing was observed to be the principal factor responsible for the increase in polymer length, whereas annealing and subunit exchange contributed about equally to the reduction in microtubule number.     

Erythrocyte microtubule assembly in vitro. Tubulin oligomers limit the rate of microtubule self-assembly

Chicken erythrocyte tubulin containing a unique beta tubulin variant polymerizes with greater efficiency (lower critical concentration) but at a slower rate than chicken brain tubulin. In a previous study we demonstrated that the low net rate of assembly is partly due to the presence of large oligomers and rings which reduce the initial rate of subunit elongation on microtubule seeds (Murphy, D.B., and Wallis, K.T. (1985) J. Biol. Chem. 260, 12293-12301). In this study we show that erythrocyte tubulin oligomers also retard the rate of microtubule nucleation and the net rate of self-assembly. The inhibitory effect is most likely to be due to the increased stability of erythrocyte tubulin oligomers, including a novel polymer of coiled rings that forms during the rapid phase of microtubule polymerization. The slow rate of dissociation of rings and coils into dimers and small oligomers appears to limit both the nucleation and elongation steps in the self-assembly of erythrocyte microtubules.     

Direct observation of microtubule treadmilling by electron microscopy

Using an immunoelectron microscopic procedure, we directly observed the concurrent addition and loss of chicken brain tubulin subunits from the opposite ends of microtubules containing erythrocyte tubulin domains. The polarity of growth of the brain tubulin on the ends of erythrocyte microtubules was determined to be similar to growth off the ends of Chlamydomonas axonemes. The flux rate for brain tubulin subunits in vitro was low, approximately 0.9 micron/h. Tubulin subunit flux did not continue through the entire microtubule as expected, but ceased when erythrocyte tubulin domains became exposed, resulting in a metastable configuration that persisted for at least several hours. We attribute this to differences in the critical concentrations of erythrocyte and brain tubulin. The exchange of tubulin subunits into the walls of preformed microtubules other than at their ends was also determined to be insignificant, the exchange rate being less than the sensitivity of the assay, or less than 0.2%/h.     

Isolation of microtubule protein from chicken erythrocytes and determination of the critical concentration for tubulin polymerization in vitro and in vivo

Microtubule protein isolated from nucleated chicken erythrocytes was examined with respect to composition and assembly properties to determine its significance in a microtubule bundle called the marginal band. 1) The protein contains greater than 95% tubulin with small amounts of tau polypeptides and no high molecular weight polypeptides. 2) Microtubule assembly in vitro at 37 degrees C is characterized by low levels of nucleation, despite an abundance of ring oligomers at 5 degrees C, as indicated by long lag times, slow assembly rates, and microtubules that are twice as long as brain microtubules assembled under the same conditions. 3) By radioimmunoassay and sodium dodecyl sulfate gel analysis we determined that 0.6% of erythrocyte protein is tubulin of which three-quarters is in a nonextractable form and is associated with the microtubule bundle and the cell cortex. From these values the in vivo concentrations of total tubulin and tubulin dimer subunits are 2.4 and 0.7 mg/ml, respectively. The value of 0.7 mg/ml is close to the range of values of 0.1-0.6 mg/ml for the critical concentration of erythrocyte microtubule protein in vitro, suggesting that the assembly properties of tubulin in vitro and in vivo are similar.     

Quantitative evaluation of the mode of microtubule transport in Xenopus neurons

Tubulin is synthesized in the cell body and must be delivered to the axon to support axonal growth. However, the exact form in which these proteins, in particular tubulin, move within the axon remains contentious. According to the "polymer transport model", tubulin is transported in the form of microtubules. In an alternative hypothesis, the "short oligomer transport model", tubulin is added to existing, stationary microtubules along the axon. In this study, we measured the translocation of microtubule plus ends in soma segments, the middle of axonal shafts and the growth cone areas, by expressing GFP-EB3 in cultured Xenopus embryonic spinal neurons. We found that none of the microtubules in the three compartments were transported rapidly as would be expected from the polymer transport model. These results suggest that microtubules are stationary in most segments of the axon, thus supporting the model according to which tubulin is transported in non-polymeric form in rapidly growing Xenopus neurons.     

Tubulin subunit sorting: Analysis of the mechanisms involved in the segregation of unique tubulin isotypes within microtubule copolymers

Summary Vertebrate cells contain biochemical and genetic isotypes of tubulin which are expressed in unique combinations in different tissues and cell types. To determine if mixtures of tubulin isotypes assemblein vitro to form different classes of microtubules, we analyzed the composition of microtubule copolymers assembled from mixtures of chicken brain and erythrocyte tubulin. During microtubule elongation brain tubulin assembled onto the ends of microtubules faster than erythrocyte tubulin, resulting in copolymers with continually changing ratios of isotypes along their lengths. Unlike examples of microtubule assembly where the rate of polymerization depends on the association rate constant (k⁺) and the subunit concentration, the rate and extent of sorting in copolymers appear to depend on the dissociation rate constant (k^–), which governs the rate at which subunits are released from tubulin oligomers and microtubules and thereby made available for reassembly into copolymers. The type of microtubule seed used to initiate elongation was also found to influence the composition of copolymers, indicating that polymerization favors association of subunits of the same isotype.     

STOP proteins

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

Increased microtubule assembly in bovine brain tubulin lacking the type III isotype of beta-tubulin

Tubulin, the major constituent protein of microtubules, is a heterodimer of alpha and beta subunits. Both alpha and beta exist in multiple isotypic forms. It is not clear if different isotypes perform different functions. In order to approach this question, we have made a monoclonal antibody specific for the beta III isotype of tubulin. This particular isotype is neuron-specific and appears to be phosphorylated near the C terminus. We have used immunoaffinity depletion chromatography to prepare tubulin lacking the beta III subunit. We find that removal of the beta III isotype results in a tubulin mixture able to assemble much more rapidly than is unfractionated tubulin when reconstituted with either of the two microtubule-associated proteins (MAPs), tau or MAP 2. Our results suggest that the different isotypes of tubulin differ from each other in their ability to polymerize into microtubules. We have also found that the anti-beta III antibody can stimulate microtubule assembly when reconstituted with tubulin and either tau or MAP 2. When reconstituted with tubulin lacking the beta III isotype, the antibody causes the tubulin to polymerize into a polymer that is a microtubule in the presence of MAP 2 and a ribbon in the presence of tau.     

Assembly and turnover of detyrosinated tubulin in vivo

Detyrosinated (Glu) tubulin was prepared from porcine brain and microinjected into human fibroblasts and Chinese hamster ovary (CHO) cells. Glu tubulin assembled onto the ends of preexisting microtubules and directly from the centrosome within minutes of its microinjection. Incorporation into the cytoskeleton continued until almost all of the microtubules were copolymers of Glu and tyrosinated (Tyr) tubulin. However, further incubation resulted in the progressive and ultimately complete loss of Glu-staining microtubules. Glu tubulin injected into nocodazole-treated cells was converted to Tyr tubulin by a putative tubulin/tyrosine ligase activity. The observed decrease in staining with the Glu antibody over time was used to analyze microtubule turnover in microinjected cells. The mode of Glu disappearance was analyzed quantitatively by tabulating the number of Glu-Tyr copolymers and Tyr-only microtubules at fixed times after injection. The proportion of Glu-Tyr copolymers decreased progressively over time and no segmentally labeled microtubules were observed, indicating that microtubules turn over rapidly and individually. Our results are consistent with a closely regulated tyrosination-detyrosination cycle in living cells and suggest that microtubule turnover is mediated by dynamic instability.     

Microtubule protein ADP-ribosylation in vitro leads to assembly inhibition and rapid depolymerization.

Bovine brain microtubule protein, containing both tubulin and microtubule-associated proteins, undergoes ADP-ribosylation in the presence of [14C]NAD+ and a turkey erythrocyte mono-ADP-ribosyltransferase in vitro. The modification reaction could be demonstrated in crude brain tissue extracts where selective ADP-ribosylation of both the alpha and beta chains of tubulin and of the high molecular weight microtubule-associated protein MAP-2 occurred. In experiments with purified microtubule protein, tubulin dimer, the high molecular weight microtubule-associated protein MAP-2, and another high molecular weight mirotubule-associated protein which may be a MAP-1 species were heavily labeled. Tubulin and MAP-2 incorporated [14C]ADP-ribose to an average extent of approximately 2.4 and 30 mol of ADP-ribose/mol of protein, respectively. Assembly of microtubule protein into microtubules in vitro was inhibited by ADP-ribosylation, and incubation of assembled steady-state microtubules with ADP-ribosyltransferase and NAD+ resulted in rapid depolymerization of the microtubules. Thus, the eukaryotic enzyme can ADP-ribosylate tubulin and microtubule-associated proteins to much greater extents than previously observed with cholera and pertussis toxins, and the modification can significantly modulate microtubule assembly and disassembly.     

Regulation of the microtubule nucleating activity of centrosomes in Xenopus egg extracts: role of cyclin A-associated protein kinase

Isolated centrosomes nucleate microtubules when incubated in pure tubulin solutions well below the critical concentration for spontaneous polymer assembly (approximately 15 microM instead of 60 microM). Treatment with urea (2-3 M) does not severely damage the centriole cylinders but inactivates their ability to nucleate microtubules even at high tubulin concentrations. Here we show that centrosomes inactivated by urea are functionally complemented in frog egg extracts. Centrosomes can then be reisolated on sucrose gradients and assayed in different concentrations of pure tubulin to quantify their nucleating activity. We show that the material that complements centrosomes is stored in a soluble form in the egg. Each frog egg contains enough material to complement greater than 6,000 urea-inactivated centrosomes. The material is heat inactivated above 56 degrees C. One can use this in vitro system to study how the microtubule nucleating activity of centrosomes is regulated. Native centrosomes require approximately 15 microM tubulin to begin nucleating microtubules, whereas centrosomes complemented in interphase extracts begin nucleating microtubules around 7-8 microM tubulin. Therefore, the critical tubulin concentrations for polymer assembly off native centrosomes is higher than that observed for the centrosomes first denatured and then complemented in egg extracts. In vivo, the microtubule nucleating activity of centrosomes seems to be regulated by phosphorylation at the onset of mitosis (Centonze, V. E., and G. G. Borisy. 1990. J. Cell Sci. 95:405-411). Since cyclins are major regulators of mitosis, we tested the effect of adding bacterially produced cyclins to interphase egg extracts. Both cyclin A and B activate an H1 kinase in the extracts. Cyclin A-associated kinase causes an increase in the microtubule nucleating activity of centrosomes complemented in the extract but cyclin B does not. The critical tubulin concentration for polymer assembly off centrosomes complemented in cyclin A-treated extracts is similar to that observed for centrosomes complemented in interphase extracts. However, centrosomes complemented in cyclin A treated extracts nucleate much more microtubules at high tubulin concentration. We define this as the "capacity" of centrosomes to nucleate microtubules. It seems that the microtubule nucleating activity of centrosomes can be defined by two distinct parameters: (a) the critical tubulin concentration at which they begin to nucleate microtubules and (b) their capacity to nucleate microtubules at high tubulin concentrations, the latter being modulated by phosphorylation.     

激素引起伊贝母愈伤组织微管蛋白变化的免疫细胞化学研究(简报)

微管蛋白聚合形成微管。微管在维持细胞结构、物质运输、分裂及植物细胞壁的建成等过程中起着重要的作用。70年代后期,在微管生物化学研究取得很大进展的基础上,免疫细胞化学技术与微管研究结合起来,使人们能够从整体水平观察以微管蛋白为主要成份的细胞骨架的动态变化。我们采用免疫酶标技术,对生长在含不同激素培养基上的伊贝母愈伤组织的微管及微管蛋白变化进行了观察和分析,结果表明,激素种类和微管的存在形式是相关的。     

Immunofluorescence examination of beta tubulin expression and marginal band formation in developing chicken erythroblasts

Chicken erythrocyte beta tubulin, a tubulin variant with unique biochemical and assembly properties, is found to be specifically contained in two chicken blood cell types--erythrocytes and thrombocytes. The beta tubulin variant is absent or present in low amounts in a variety of white blood cell types and other body tissues, as determined by immunofluorescence microscopy and a semi-quantitative immunoblotting procedure. During differentiation in the marrow the beta tubulin variant appears suddenly in mid-stage erythroblasts at the onset of hemoglobin synthesis, and forming marginal bands are seen in all subsequent polychromatophilic erythroblast stages. The developmental sequence of events in marginal band formation entails microtubule nucleation at the centrosome, followed by microtubule elongation, consolidation of loose parallel microtubules into a compact bundle, and microtubule association with the cell membrane.     

Microtubule dynamics in interphase cells

The sites of microtubule growth and the kinetics of elongation have been studied in vivo by microinjection of biotin-labeled tubulin and subsequent visualization with immunocytochemical probes. Immunofluorescence and immunoelectron microscopy demonstrate that injected biotin-labeled subunits are incorporated into new segments of growth which are contiguous with unlabeled microtubules. Rapid incorporation occurs by elongation of existing microtubules and new nucleation off the centrosome. The growth rate is 3.6 micron/min and is independent of the concentration of injected labeled tubulin. This rate of incorporation together with turnover of existing microtubules leads to approximately 80% exchange in 15 min. The observed kinetics and pattern of microtubule turnover allow for an evaluation of the relevance of several in vitro models for steady-state dynamics to the in vivo situation. We have also observed a substantial population of quasi-stable microtubules that does not exchange subunits as rapidly as the majority of microtubules and may have specialized functions in the cell.     

Differentiation between dynamic instability and end-to-end annealing models for length changes of steady-state microtubules

Short microtubules can be formed by shearing a sample at polymerization steady state of microtubules formed by glycerol-induced assembly of pure tubulin dimer. Such short microtubules show a rapid increase in mean length. The rate of this increase is too fast to be accounted for by statistical redistribution of subunits between microtubules. We propose that the fast length changes are a result of the end-to-end annealing of microtubules demonstrated by Rothwell et al. (Rothwell, S. W., Grasser, W. A., and Murphy, D. B. (1986) J. Cell Biol. 102, 619-627). This proposal has been tested by measuring the rate of annealing of free microtubules to Tetrahymena axonemes under conditions identical to those used for the lengthening of sheared microtubules. That free microtubules anneal to axonemal microtubules is indicated by the following observations. Axonemes elongate at both ends in the presence of steady state microtubules, as predicted for a symmetrical annealing process; under conditions where the microtubule number concentration is greater than that for axonemes, the initial rate of axoneme elongation is more rapid with a low concentration of long microtubules at steady state than with a high number concentration of short microtubules at steady state. These observations are inconsistent with the predictions of a model based on microtubule dynamic instability (Mitchison, T., and Kirschner, M. (1984) Nature 312, 237-242). The annealing rate observed with axonemes can account for the rate of elongation of sheared steady state microtubules.     

Postpolymerization detyrosination of alpha-tubulin: a mechanism for subcellular differentiation of microtubules

Tyrosinated (Tyr) and detyrosinated (Glu) alpha-tubulin, species interconverted by posttranslational modification, are largely segregated in separate populations of microtubules in interphase cultured cells. We sought to understand how distinct Tyr and Glu microtubules are generated in vivo, by examining time-dependent alterations in Tyr and Glu tubulin levels (by immunoblots probed with antibodies specific for each species) and distributions (by immunofluorescence) after microtubule regrowth and stabilization. When microtubules were allowed to regrow after complete depolymerization by microtubule antagonists, Glu microtubules reappeared with a delay of approximately 25 min after the complete array of Tyr microtubules had regrown. In these experiments, Tyr tubulin immunofluorescence first appeared as an aster of distinct microtubules, while Glu tubulin staining first appeared as a grainy pattern that was not altered by detergent extraction, suggesting that Glu microtubules were created by detyrosination of Tyr microtubules. Treatments with taxol, azide, or vinblastine, to stabilize polymeric tubulin, all resulted in time-dependent increases in polymeric Glu tubulin levels, further supporting the hypothesis of postpolymerization detyrosination. Analysis of monomer and polymer fractions during microtubule regrowth and in microtubule stabilization experiments were also consistent with postpolymerization detyrosination; in each case, Glu polymer levels increased in the absence of detectable Glu monomer. The low level of Glu monomer in untreated or nocodazole-treated cells (we estimate that Glu tubulin comprises less than 2% of the monomer pool) also suggested that Glu tubulin entering the monomer pool is efficiently retyrosinated. Taken together these results demonstrate that microtubules are polymerized from Tyr tubulin and are then rapidly converted to Glu microtubules. When Glu microtubules depolymerize, the resulting Glu monomer is retyrosinated. This cycle generates structurally, and perhaps functionally, distinct microtubules.     

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

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

Autoregulation of tubulin synthesis in hepatocytes and fibroblasts

Microtubule polymer levels in mouse 3T6 fibroblasts and primary cultures of rat hepatocytes can be manipulated by treatment of cells with long term, low doses of colcemid. Such treatment produces a rather uniform population of cells with microtubules of reduced lengths. Using this system, we demonstrate (a) that the rate of tubulin synthesis is sensitive to small changes (10%) in microtubule polymer mass and (b) that the percent of inhibition of synthesis is proportional to the level of soluble tubulin. Experiments with hepatocytes indicate that not only synthesis but the stability of tubulin protein was also regulated to maintain a specific level of tubulin. Treatment of hepatocytes with colcemid or other microtubule-depolymerizing drugs reduced the half-life of tubulin from 50 to 2 h, whereas taxol, which stabilizes microtubules, increased the half-life. To assess the consequences of altering microtubule polymer mass, we have analyzed the effect of controlled depolymerization of microtubules in rat hepatocytes on the processing of endocytosed ligands and found it sensitive to small changes in microtubule polymer levels.