Tubulin constancy during morphological differentiation of mouse neuroblastoma cells

Clonal cell lines N18 and N103 of the mouse neuroblastoma C1300 possess an undifferentiated neuroblast morphology under optimal growth conditions; however, when deprived of serum, N18 can be induced to extend long neurites. Although initial neurite outgrowth is rapid, very long fibers are found only after several days. Both initial outgrowths and established neurites contain microtubules; however, the number and density of these polymerized tubules increase markedly during this time. Optimum conditions have been established for assessing the colchicine-binding activity of neuroblastoma sonicates. A time-decay colchicine-binding assay was used to make a comparative study of the tubulin content of both undifferentiated and differentiated N18 as well as the nondifferentiating N103 and the rat glioma C6. Both morphologies of clone N18 possessed similar concentrations of tubulin (130-140 pmol/10(6) cells). Although cells of clone N103 contain 20% less tubulin than N18 cells, this is considerably more tubulin than is present in the glioma C6 (30 pmol/10(6) cells) which has a similar generation time. Quantitative densitometry of neuroblastoma extracts electrophoresed on SDS-polyacrylamide gels confirmed the constancy of tubulin. Radiolabeled proteins from neuroblastoma cells subjected to both growth conditions show that neurite outgrowth does not create a disproportionate demand for tubulin synthesis. Thus, the morphological differentiation of neuroblastoma cells probably reflects the regulation of tubulin storage and microtubule polymerization.     

Transglutaminase and neuronal differentiation

Summary During mouse brain maturation cellular transglutaminase specific activity increases 2.5 fold from day 3 to adulthood. A more pronounced increase is seen during morphological differentiation of mouse neuroblastoma cells, where serum withdrawal induces neurite outgrowth concomitant with a 10 fold increase in transglutaminase specific activity. In contrast, non-dividing neuroblastoma cells lacking neurites show only a 1.5 fold increase in enzyme specific activity. Transglutaminase activity does not reach maximal levels until extensive neurite formation has occurred. More than 80% of the transglutaminase activity is found in the soluble component of brain and neuroblastoma homogenates. Using [³H]-putrescine as the acyl acceptor, endogenous acyl donor substrates in the neuroblastoma cells included proteins that comigrated on SDS-PAGE with tubulin and actin; however, very high molecular weight crosslinked material is the major reaction product in vitro. When purified brain tubulin, microtubule associated proteins and microtubules were compared as exogenous substrates, only the polymeric microtubules were a good acyl donor substrate. Furthermore, preincubation of purified tubulin with transglutaminase and putrescine stimulated both the rate and extent of microtubule assembly. These findings suggest that transglutaminase may mediate covalent cross-linking of microtubules to other cellular components, or the post-translational modification of tubulin by the formation of -glutamylamines.     

Regulation of a high molecular weight microtubule-associated protein in PC12 cells by nerve growth factor

PC12 rat pheochromocytoma cells respond to nerve growth factor (NGF) protein by shifting from a chromaffin-cell-like phenotype to a neurite-bearing sympathetic-neuron-like phenotype. Comparison of the phosphoprotein patterns of the cells by SDS PAGE after various times of NGF treatment revealed a high molecular weight (Mr greater than or approximately 300,000) band whose relative intensity progressively increased beyond 2 d of NGF exposure. This effect was blocked by inhibitors of RNA synthesis and did not require neurite outgrowth or substrate attachment. The enhancement by NGF occurred in serum-free medium and was not produced by exposure to epidermal growth factor, insulin, dibutyryl cAMP, or dexamethasone. Several different types of experiments indicated that this phosphoprotein corresponds to a high molecular weight (HMW) microtubule-associated protein (MAP). These included cross-reactivity with antiserum against brain HMW MAPs, co-cycling with microtubules and co-assembly with tubulin in the presence of taxol. The affected species also co-migrated in SDS PAGE gels with brain MAP1 and, unlike MAP2, precipitated upon boiling. Studies with [35S]-methionine-labeled PC12 cells indicated that at least a significant proportion of this effect of NGF was due to increased levels of protein rather than to mere enhancement of phosphorylation. On the basis of the apparent effects of MAPs on the formation and stabilization of microtubules and of the importance of microtubules in production and maintenance of neurites, it is proposed that induction of a HMW MAP may be one of the steps in the mechanism whereby NGF promotes neurite outgrowth. Furthermore, these findings may lead to an understanding of the role of MAP1 in the nervous system.     

Characterization of maize microtubule-associated proteins, one of which is immunologically related to tau

Microtubule-associated proteins (MAPs) are identified as proteins that copurify with tubulin, promote tubulin assembly, and bind to microtubules in vitro. Higher plant MAPs remain mostly unknown. One example of non-tubulin carrot proteins, which bind to neural microtubules and induce bundling, has been reported so far [Cyr, R. J., & Palewitz, B. A. (1989) Planta 177, 245-260]. Using taxol, we developed an assay where higher plant microtubules were induced to self-assemble in cytosolic extracts of maize cultured cells and were used as the native matrix to isolate putative plant MAPs. Several polypeptides with an apparent molecular masses between 170 and 32 kDa copolymerized with maize microtubules. These putative maize MAPs also coassembled with pig brain tubulin through two cycles of temperature-dependent assembly-disassembly. They were able to initiate and promote MAP-free tubulin assembly under conditions of nonefficient self-assembly and induced bundling of both plant and neural microtubules. One of these proteins, of about 83 kDa, cross-reacted with affinity-purified antibodies against rat brain tau proteins, suggesting the presence of common epitope(s) between neural tau and maize proteins. This homology might concern the tubulin-binding domain, as plant and neural tubulins are highly conserved and the plant polypeptides coassembled with brain tubulin.     

PC12 cells express juvenile microtubule-associated proteins during nerve growth factor-induced neurite outgrowth

Microtubule-associated proteins (MAPs) are believed to play an important role in regulating the growth of neuronal processes. The nerve growth factor-induced differentiation of PC12 pheochromocytoma cells is a widely used tissue culture model for studying this mechanism. We have found that contrary to previous suggestions, the major MAPs of adult brain, MAP1 and MAP2, are minor components of PC12 cells. Instead two novel MAPs characteristic of developing brain, MAP3 and MAP5, are present and increase more than 10-fold after nerve growth factor treatment; the timing of these increases coinciding with the bundling of microtubules and neurite outgrowth. Immunocytochemical staining showed that MAP3 and MAP5 are initially distributed throughout the cytoplasm. Subsequently MAP5 becomes associated with microtubules in both neurites and growth cones but MAP3 distribution remained diffuse. Thus MAP3 and MAP5, which are characteristic of developing neurons in the juvenile brain, are also induced in PC12 cells during neurite outgrowth in culture. In contrast MAP1, which is characteristic of mature neurons, does not increase during PC12 cell differentiation. These results provide evidence that one set of MAPs is expressed during neurite outgrowth and a different set during the maintenance of neuronal form. It also appears that the PC12 system is an appropriate model for studying the active neurite growth phase of neuronal differentiation but not for neuronal maturation.     

Microtubule-associated proteins from Antarctic fishes

Microtubules and presumptive microtubule-associated proteins (MAPs) were isolated from the brain tissues of four Antarctic fishes (Notothenia gibberifrons, N. coriiceps neglecta, Chaenocephalus aceratus, and a Chionodraco sp.) by means of a taxol-dependent, microtubule-affinity procedure (cf. Vallee: Journal of Cell Biology 92:435-442, 1982). MAPs from these fishes were similar to each other in electrophoretic pattern. Prominent in each preparation were proteins in the molecular weight ranges 410,000-430,000, 220,000-280,000, 140,000-155,000, 85,000-95,000, 40,000-45,000, and 32,000-34,000. The surfaces of MAP-rich microtubules were decorated by numerous filamentous projections. Exposure to elevated ionic strength released the MAPs from the microtubules and also removed the filamentous projections. Addition of fish MAPs to subcritical concentrations of fish tubulins at 0-5 degrees C induced the assembly of microtubules. Both the rate and the extent of this assembly increased with increasing concentrations of the MAPs. Sedimentation revealed that approximately six proteins, with apparent molecular weights between 60,000 and 300,000, became incorporated into the microtubule polymer. Bovine MAPs promoted microtubule formation by fish tubulin at 2-5 degrees C, and proteins corresponding to MAPs 1 and 2 co-sedimented with the polymer. MAPs from C. aceratus also enhanced the polymerization of bovine tubulin at 33 degrees C, but the microtubules depolymerized at 0 degrees C. We conclude that MAPs are part of the microtubules of Antarctic fishes, that these proteins promote microtubule assembly in much the same way as mammalian MAPs, and that they do not possess special capacities to promote microtubule assembly at low temperatures or to prevent cold-induced microtubule depolymerization.     

Characterization and Intracellular Distribution of Microtubule-Associated Protein 2 in Differentiating Human Neuroblastoma Cells

The use of a panel of monoclonal antibodies (mAbs) directed against different determinants of microtubule-associated protein 2 (MAP2) enabled us to identify two distinct high-molecular-mass MAP2 species (270 and 250 kDa) and a substantial amount of MAP2c (70 kDa) in human neuroblastoma cells. The 250-kDa MAP2 species appears to be confined to the human neuroblastoma cells and was not observed in microtubules (MTs) from bovine and rat brain, mouse neuroblastoma, or MTs from human cerebellum. A new overlay method was developed, which demonstrates binding of tubulin to human neuroblastoma high-molecular-mass MAP2 by exposing nitrocellulose-bound MT proteins under polymerization conditions to tubulin. Bound tubulin was detected with a mAb directed against beta-tubulin. The binding of tubulin to MAP2 could be abolished by a peptide homologous to positions 426-445 of the C-terminal region of beta-tubulin. Immunological cross-reactivity with several mAbs directed against bovine brain MAP2, taxol-promoted coassembly into MTs, and immunocytochemical visualization within cells were further criteria utilized to characterize these proteins as true MAPs. Indirect immunofluorescence with anti-MAP2 and anti-beta-tubulin mAbs demonstrated that there is a change in the spatial organization of MTs during induced cell differentiation, as indicated by the appearance of MT bundles and the redistribution of MAP2.     

The removal of the carboxy-terminal region of tubulin favors its vinblastine-induced aggregation into spiral-like structures

Vinblastine induces brain tubulin to assemble into spirals. This process is stimulated by microtubule-associated proteins (MAPs) which copolymerize with brain microtubules assembled in vitro. When the carboxy terminal of tubulin is removed by subtilisin digestion, vinblastine readily induces the aggregation of tubulin into spiral-like or circular structures, even in the absence of MAPs. These results suggest that in the absence of MAPs, the carboxy-terminal domain of tubulin may inhibit vinblastine-induced polymerization of tubulin into spiral-like structures.     

The regulatory effect of Tau protein on polymerization of MCF7 microtubules in vitro

Growing evidence continues to point toward the critical role of beta tubulin isotypes in regulating some intracellular functions. Changes that were observed in the microtubules’ intrinsic dynamics, the way they interact with some chemotherapeutic agents, or differences on translocation specifications of some molecular motors along microtubules, were associated to their structural uniqueness in terms of beta tubulin isotype distributions. These findings suggest that the effects of microtubule associated proteins (MAPs) may also vary on structurally different microtubules. Among different microtubule associated proteins, Tau proteins, which are known as neuronal MAPs, bind to beta tubulin, stabilize microtubules, and consequently promote their polymerizations.In this study, in a set of well controlled experiments, the direct effect of Tau proteins on the polymerization of two structurally different microtubules, porcine brain and breast cancer (MCF7), were tested and compared. Remarkably, we found that in contrast with the promoted effect of Tau proteins on brain microtubules’ polymerization, MCF7 expressed a demoted polymerization while interacting with Tau proteins. This finding can potentially be a novel insight into the mechanism of drug resistance in some breast cancer cells.It has been reported that microtubules show destabilizing behavior in some MCF7 cells with overexpression of Tau protein when treated with a microtubules’ stabilizing agent, Taxol. This behavior has been classified by others as drug resistance, but it may instead be potentially caused by a competition between the destabilizing effect of the Tau protein and the stabilizing effect of the drug on MCF7 microtubules. Also, we quantified the polarization coefficient of MCF7 microtubules in the presence and absence of Tau proteins by the electro-orientation method and compared the values. The two significantly different values obtained can possibly be one factor considered to explain the effect of Tau proteins on the polymerization of MCF7 microtubules.     

A carbamate herbicide causes microtubule and microfilament disruption and nuclear fragmentation in fibroblasts

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.     

Microtubule-associated proteins-dependent colchicine stability of acetylated cold-labile brain microtubules from the Atlantic cod, Gadus morhua

Assembly of brain microtubule proteins isolated from the Atlantic cod, Gadus morhua, was found to be much less sensitive to colchicine than assembly of bovine brain microtubules, which was completely inhibited by low colchicine concentrations (10 microM). The degree of disassembly by colchicine was also less for cod microtubules. The lack of colchicine effect was not caused by a lower affinity of colchicine to cod tubulin, as colchicine bound to cod tubulin with a dissociation constant, Kd, and a binding ratio close to that of bovine tubulin. Cod brain tubulin was highly acetylated and mainly detyrosinated, as opposed to bovine tubulin. When cod tubulin, purified by means of phosphocellulose chromatography, was assembled by addition of DMSO in the absence of microtubule-associated proteins (MAPs), the microtubules became sensitive to low concentrations of colchicine. They were, however, slightly more stable to disassembly, indicating that posttranslational modifications induce a somewhat increased stability to colchicine. The stability was mainly MAPs dependent, as it increased markedly in the presence of MAPs. The stability was not caused by an extremely large amount of cod MAPs, since there were slightly less MAPs in cod than in bovine microtubules. When "hybrid" microtubules were assembled from cod tubulin and bovine MAPs, these microtubules became less sensitive to colchicine. This was not a general effect of MAPs, since bovine MAPs did not induce a colchicine stability of microtubules assembled from bovine tubulin. We can therefore conclude that MAPs can induce colchicine stability of colchicine labile acetylated tubulin.     

Microtubule-associated proteins of HeLa cells: heat stability of the 200,000 mol wt HeLa MAPs and detection of the presence of MAP-2 in HeLa cell extracts and cycled microtubules

One of the major groups of microtubule-associated proteins (MAPs) found associated with the microtubules isolated from HeLa cells has a molecular weight of just over 200,000. Previous work has demonstrated that these heLa MAPs are similar in several properties to MAP-2, one of the major MAPs of mammalian neural microtubules, although the two types of proteins are immunologically distinct. The 200,000 mol wt HeLa MAPs have now been found to remain soluble after incubation in a boiling water bath and to retain the ability to promote tubulin polymerization after this treatment, two unusual properties also shown by neural MAP- 2. This property of heat stability has allowed the development of a simplified procedure for purification of the 200,000 HeLa MAPs and has provided a means for detection of these proteins, even in crude cell extracts. These studies have also led to the detection of a protein in crude extracts of HeLa cells and in cycled HeLa microtubules which has been identified as MAP-2 on the basis of (a) comigration with calf brain MAP-2 on SDS PAGE, (b) presence in purified microtubules, (c) heat stability, and (d) reaction with two types of antibodies prepared against neural high molecular weight-MAPs, one of these a monoclonal antibody against hog brain MAP-2, although present in HeLa cells, is at all stages of microtubule purification a relatively minor component in comparison to the 200,000 HeLa MAP's.     

Microtubules and brain development: The effects of thyroid hormones

The data accumulated during the past twenty years suggest that thyroid hormones have a direct effect on the differentiation of both the neurons and the glial cell during the critical period of brain development. A fast survey of the available data (which is presented in the introduction of this article) on the mechanism of action of thyroid hormones and on their different effects during brain development suggests that the most dramatic effect of hypothyroidism is a hypoplastic neuropile. Both in vivo, during the critical period of nerve cell differentiation and in vitro, when added to primary cultures of embryonic nerve cells thyroid hormones stimulate neurite outgrowth. Since neurite outgrowth requires massive microtubule assembly the assumption was made that thyroid hormones stimulate nerve cell differentiation by changing the concentration and/or activity of the different proteins (tubulin and “microtubule associated proteins”, MAPs) which co-polymerize to form microtubules.

Preliminary information was obtained by following the kinetics of microtubule assembly in crude brain supernatants. The data showed that: (1) the rate of in vitro microtubule assembly increases with age during brain development; (2) hypothyroidism, when produced in the rat at late pregnancy, slows this evolution; (3) early replacement therapy with thyroid hormones restores normal rates of assembly; (4) the addition of purified MAPs to normal young or 15-day-old hypothyroid brain preparations restores normal rates of polymerization. These and other data suggested that thyroid hormones regulate microtubule assembly by changing the concentration and/or activity of one or more of the MAPs.

Further analysis revealed that striking qualitative changes in MAPs composition occur during brain development. For instance, the TAU fraction, a group of 4–5 proteins with a molecular weight of 60–68 K which is present in adult brain, is absent at early stages of postnatal development: two other entities are present, TAU slow and TAU fast, with different molecular weights, lower activity and different peptide mapping. This latter observation suggests that different TAU genes are expressed during brain development; a conclusion which has been confirmed by cell-free translation of the mRNas coding for these proteins. Analysis of the TAU fraction prepared from hypothyroid rat brains also revealed that a group of TAU proteins. “TAU₃”, is almost missing, whereas thyroid hormone administration markedly increases its concentration. Two-dimensional gel electrophoresis showed that the TAU fraction is composed with more than 15 entities, with at least five of them being under thyroid hormone control.

The precise physiological significance of the heterogeneity of MAPs and of the changes in MAPs composition seen during development and in hypothyroid rat brain remains to be determined. The assumption is made that these changes might be of utmost importance to regulate the number and length of the microtubules, and therefore the number and length of the neurites which are formed during the differentiation process of the different neurons. Thyroid hormones would be in these respects one of the epigenic factors required to synchronize sequentially the expression of the genes coding for these proteins in the different nerve cells.     

Two microtubule-associated proteins of the Arabidopsis MAP65 family function differently on microtubules

The organization and dynamics of microtubules are regulated by microtubule-associated proteins, or MAPs. In Arabidopsis (Arabidopsis thaliana), nine genes encode proteins of the evolutionarily conserved MAP65 family. We proposed that different MAP65s might have distinct roles in the interaction with microtubules. In this study, two AtMAP65 proteins, AtMAP65-1 and AtMAP65-6, were chosen to test this hypothesis in vitro. Although both fusion proteins were able to cosediment with microtubules in vitro, different properties on tubulin polymerization and microtubule bundling were observed. AtMAP65-1 was able to promote tubulin polymerization, enhance microtubule nucleation, and decrease the critical concentration for tubulin polymerization. It also induced the formation of large microtubule bundles by forming cross-bridges between microtubules evenly along the whole length of microtubules. In the presence of AtMAP65-1, microtubule bundles were more resistant to cold and dilution treatments. AtMAP65-6, however, demonstrated no activity in promoting tubulin polymerization and stabilizing preformed microtubules. AtMAP65-6 induced microtubules to form a mesh-like network with individual microtubules. Cross-bridge-like interactions were only found at regional sites between microtubules. The microtubule network induced by AtMAP65-6 was more resistant to high concentration of NaCl than the bundles induced by AtMAP65-1. Purified monospecific anti-AtMAP65-6 antibodies revealed that AtMAP65-6 was associated with mitochondria in Arabidopsis cells. It was concluded that these two MAP65 proteins were targeted to distinct sites, thus performing distinct functions in Arabidopsis cells.     

Different assembly properties of cod, bovine, and rat brain microtubules.

Assembly properties of cod, bovine, and rat brain microtubules were compared. Estramustine phosphate, heparin, poly-L-aspartic acid, as well as NaCl, inhibited the assembly and disassembled both bovine and rat microtubules by inhibition of the binding between tubulin and MAPs. The assembly of cod brain microtubules was in contrast only marginally affected by these agents, in spite of a release of the MAPs. The results suggest that cod tubulin has a high intrinsic ability to assemble. This was confirmed by studies on phosphocellulose-purified cod tubulin, since the critical concentration for assembly was independent of the presence or absence of MAPs. The results show therefore that cod brain tubulin has, in contrast to bovine and rat brain tubulins, a high propensity to assembly under conditions which normally require the presence of MAPs. Even if cod MAPs, which have an unusual protein composition, were not needed for the assembly of cod microtubules, they were able to induce assembly of bovine brain tubulin. Both cod and bovine MAPs bound to cod microtubules, and bovine MAP1 and MAP2 bound to, and substituted at least the 400 kDa cod protein. This suggests that the tubulin-binding sites and the assembly-stimulatory ability of MAPs are common properties of MAPs from different species, independent of the tubulin assembly propensity.     

Respective roles of neurofilaments, microtubules, MAP1B, and tau in neurite outgrowth and stabilization.

The respective roles of neurofilaments (NFs), microtubules (MTs), and the microtubule-associated proteins (MAPs) MAP 1B and tau on neurite outgrowth and stabilization were probed by the intracellular delivery of specific antisera into transiently permeabilized NB2a/d1 cells during treatment with dbcAMP. Intracellular delivery of antisera specific for the low (NF-L), middle (NF-M), or extensively phosphorylated high (NF-H) molecular weight subunits did not prevent initial neurite elaboration, nor did it induce retraction of existing neurites elaborated by cells that had been previously treated for 1 d with dbcAMP. By contrast, intracellular delivery of antisera directed against tubulin reduced the percentage of cells with neurites at both these time points. Intracellular delivery of anti-NF-L and anti-NF-M antisera did not induce retraction in cells treated with dbcAMP for 3 d. However, intracellular delivery of antisera directed against extensively phosphorylated NF-H, MAP1B, tau, or tubulin induced similar levels of neurite retraction at this time. Intracellular delivery of monoclonal antibodies (RT97 or SMI-31) directed against phosphorylated NF-H induced neurite retraction in cell treated with dbcAMP for 3 d; a monoclonal antibody (SMI-32) directed against nonphosphorylated NF-H did not induce neurite retraction at this time. By contrast, none of the above antisera induced retraction of neurites in cells treated with dbcAMP for 7 d. Neurites develop resistance to retraction by colchicine, first detectable in some neurites after 3 d and in the majority of neurites after 7 d of dbcAMP treatment. We therefore examined whether or not colchicine resistance was compromised by intracellular delivery of the above antisera. Colchicine treatment resulted in rapid neurite retraction after intracellular delivery of antisera directed against extensively phosphorylated NF-H, MAP1B, or tau into cells that had previously been treated with dbcAMP for 7 d. By contrast, colchicine resistance was not compromised by the intracellular delivery of antisera directed against NF-L, NF-M, or tubulin. These findings support previous studies indicating that MT polymerization mediates certain aspects of axonal neurite outgrowth and suggest that NFs do not directly participate in these events. These findings further suggest that NFs function in stabilization of the axonal cytoskeleton, apparently by interactions among NFs and MTs that are mediated by NF-H and MAPs.     

Nerve growth factor-induced neurite outgrowth in PC12 cells involves the coordinate induction of microtubule assembly and assembly-promoting factors

Nerve growth factor (NGF) regulates the microtubule-dependent extension and maintenance of axons by some peripheral neurons. We show here that one effect of NGF is to promote microtubule assembly during neurite outgrowth in PC12 cells. Though NGF causes an increase in total tubulin levels, the formation of neurites and the assembly of microtubules follow a time course completely distinct from that of the tubulin induction. The increases in microtubule mass and neurite extension closely parallel 10- and 20-fold inductions of tau and MAP1, proteins shown previously to promote microtubule assembly in vitro. When NGF is removed from PC12 cells, neurites disappear, microtubule mass decreases, and both microtubule-associated proteins return to undifferentiated levels. These data suggest that the induction of tau and MAP1 in response to NGF promotes microtubule assembly and that these factors are therefore key regulators of neurite outgrowth.     

A polymer-dependent increase in phosphorylation of beta-tubulin accompanies differentiation of a mouse neuroblastoma cell line

We have examined the phosphorylation of cellular microtubule proteins during differentiation and neurite outgrowth in N115 mouse neuroblastoma cells. N115 differentiation, induced by serum withdrawal, is accompanied by a fourfold increase in phosphorylation of a 54,000-mol-wt protein identified as a specific isoform of beta-tubulin by SDS PAGE, two-dimensional isoelectric focusing/SDS PAGE, and immunoprecipitation with a specific monoclonal antiserum. Isoelectric focusing/SDS PAGE of [35S]methionine-labeled cell extracts revealed that the phosphorylated isoform of beta-tubulin, termed beta 2, is one of three isoforms detected in differentiated N115 cells, and is diminished in amounts in the undifferentiated cells. Taxol, a drug which promotes microtubule assembly, stimulates phosphorylation of beta-tubulin in both differentiated and undifferentiated N115 cells. In contrast, treatment of differentiated cells with either colcemid or nocodazole causes a rapid decrease in beta-tubulin phosphorylation. Thus, the phosphorylation of beta-tubulin in N115 cells is coupled to the levels of cellular microtubules. The observed increase in beta-tubulin phosphorylation during differentiation then reflects developmental regulation of microtubule assembly during neurite outgrowth, rather than developmental regulation of a tubulin kinase activity.     

Traces of brain microtubule-associated proteins affect dynamic properties of microtubules

A method is described for measuring the quantities of stable and dynamic microtubules in a population in vitro. The method exploits the tendency of dynamic microtubules to depolymerize rapidly after being sheared. Stable microtubules, such as those protected by microtubule-associated proteins (MAPs), are broken to a smaller size by shearing, but do not depolymerize into subunits. The usual difficulty with this procedure is that the tubulin released from the dynamic microtubules rapidly repolymerizes before the end point of depolymerization can be measured. This has been overcome by including a small quantity of tubulin-colchicine complex in the mixture to block the repolymerization. For a total of 24 microM tubulin in a polymerization mixture, 10 microM of the sample polymerized originally under the conditions used. When 1.05 microM tubulin-colchicine complex was added at the time of shearing, the dynamic microtubules depolymerized, but the tubulin was released was unable to repolymerize and a small fraction of stable microtubules that resisted shear-induced depolymerization could then be detected. When traces of MAPs (0.23-2.8% by mass) were included in the tubulin mixture, the fraction of stable microtubules increased from 5% in the absence of added MAPs to 41% in the presence of 2.8% MAPs. All the MAPs in the mixture were found in the stable fraction and this stable fraction forms early during microtubule assembly. Calculations on the extent of enrichment of MAPs in the stable fraction indicated that as little as 4% MAPs in a microtubule protected it from shear-induced disassembly. The results suggest that low levels of MAPs may distribute nonrandomly in the microtubule population.