Fission yeast Alp14 is a dose-dependent plus end-tracking microtubule polymerase

XMAP215/Dis1 proteins are conserved tubulin-binding TOG-domain proteins that regulate microtubule (MT) plus-end dynamics. Here we show that Alp14, a XMAP215 orthologue in fission yeast, Schizosaccharomyces pombe, has properties of a MT polymerase. In vivo, Alp14 localizes to growing MT plus ends in a manner independent of Mal3 (EB1). alp14-null mutants display short interphase MTs with twofold slower assembly rate and frequent pauses. Alp14 is a homodimer that binds a single tubulin dimer. In vitro, purified Alp14 molecules track growing MT plus ends and accelerate MT assembly threefold. TOG-domain mutants demonstrate that tubulin binding is critical for function and plus end localization. Overexpression of Alp14 or only its TOG domains causes complete MT loss in vivo, and high Alp14 concentration inhibits MT assembly in vitro. These inhibitory effects may arise from Alp14 sequestration of tubulin and effects on the MT. Our studies suggest that Alp14 regulates the polymerization state of tubulin by cycling between a tubulin dimer-bound cytoplasmic state and a MT polymerase state that promotes rapid MT assembly.     

Characterization of a microtubule assembly inhibitor from Xenopus oocytes

The dynamic properties of microtubules (MTs) are important for a wide variety of cellular processes, including cell division and morphogenesis. MT assembly and disassembly in vivo are regulated by cellular factors that influence specific parameters of MT dynamics. Here, we describe the characterization of a previously reported MT assembly inhibitor activity from Xenopus oocytes [Gard and Kirschner, 1987: J. Cell Biol. 105:2191-2201]. Video microscopy measurements reveal that the inhibitor specifically decreases the plus end growth rate of MTs and increases the critical concentration for tubulin. However, catastrophe frequency, rescue frequency, and shrinkage rates are not affected by the activity. Chromatography on Mono Q and hydroxyapatite columns has shown that the activity cofractionates with a subpopulation of tubulin. This tubulin subpopulation and the MT assembly inhibitor activity also co-migrate with a large S value (25-30S) on sucrose gradients. The high molecular weight tubulin complex and the MT assembly inhibitor activity are both developmentally regulated and disappear after oocyte maturation with progesterone.     

Characteristics of the effect of S-100 proteins on the assembly-disassembly of brain microtubule proteins at alkaline pH in vitro

The ability of S-100 proteins to inhibit the assembly of brain microtubule proteins (MTPs) in the presence of microM levels of Ca2+ increases as a function of pH. This seems to be due to an increasingly larger inhibitory effect of S-100 on the nucleation and, probably, on the elongation of microtubules (MTs) as the pH raises. In the presence of microM Ca2+ levels, the ability of S-100 to disassemble MTs also increases linearly with the pH, suggesting that the larger inhibitory effect of S-100 on MTP assembly at alkaline than at acidic pH may depend on both a decrease in the assembly rate and an increase in the disassembly rate. Also, S-100 inhibits the assembly of phosphocellulose-purified tubulin to a larger and larger extent as the pH raises. S-100 brings about its effect on MT assembly-disassembly probably by sequestering soluble tubulin, though additional mechanisms cannot be excluded. The present data are briefly discussed in relation to the role attributed to changes in intracellular pH in the regulation of the state of assembly of cytoplasmic MTs.     

Properties of the kinetochore in vitro. II. Microtubule capture and ATP- dependent translocation

We have studied the interaction of preformed microtubules (MTs) with the kinetochores of isolated chromosomes. This reaction, which we call MT capture, results in MTs becoming tightly bound to the kinetochore, with their ends capped against depolymerization. These observations, combined with MT dynamic instability, suggest a model for spindle morphogenesis. In addition, ATP appears to mobilize dynamic processes at captured MT ends. We used biotin-labeled MT seeds to follow assembly dynamics at the kinetochore. In the presence of ATP and unlabeled tubulin, labeled MT segments translocate away from the kinetochore by polymerization of subunits at the attached end. We have termed this reaction proximal assembly. Further studies demonstrated that translocation could be uncoupled from MT assembly. We suggest that the kinetochore contains an ATPase activity that walks along the MT lattice toward the plus end. This activity may be responsible for the movement of chromosomes away from the pole in prometaphase.     

Growth and shortening of microtubules: a two-state model approach

In this study, a two-state mechanochemical model is presented to describe the dynamic instability of microtubules (MTs) in cells. The MT switches between two states, the assembly and disassembly states. In assembly state, the growth of MTs includes two processes: free GTP-tubulin binding to the tip of protofilament (PF) and conformation change of PF, during which the first tubulin unit that curls outwards is rearranged onto the MT surface, using the energy released from the hydrolysis of GTP in the penultimate tubulin unit. In the disassembly state, the shortening of MTs also includes two processes, the release of GDP-tubulin from the tip of PF and the curling of one new tubulin unit out of the MT surface. Switches between these two states, which are usually called rescue and catastrophe, happen stochastically with external force-dependent rates. Using this two-state model with parameters obtained by fitting the recent experimental data, detailed properties of MT growth are obtained. I find that MT is mainly in the assembly state, its mean growth velocity increases with both the external force and the GTP-tubulin concentration, and an MT will shorten on average without an external force. To know more about the external force and GTP-tubulin concentration-dependent properties of MT growth, and for future experimental verification of this two-state model, 11 critical forces are defined and discussed numerically.     

Root contraction in hyacinth. II. Changes in tubulin levels,microtubule number and orientation associated with differential cell expansion

Root contraction in hyacinth (Hyacinthus orientalis L.) is marked by reoriented cell growth in the cortex of the contractile region. Cellular volume of the inner cortex enlarges fourfold during root contraction. This is associated with large increases in the radial and tangential dimensions and decreases in the longitudinal dimension of the cells. In order to determine the possible role of microtubules (MTs) in these changes we compared tubulin levels and MT numbers and orientation in contracted and non-contracted regions of hyacinth roots. Tubulin content was analysed by a radioimmunoassay; MT numbers and orientation were analyzed by counting profiles in sectioned material using transmission electron microscopy. Contracted tissue was found to have significantly higher levels of tubulin on a per-cell basis than non-contracted tissue, and also increased tubulin levels relative to total protein. The spatial MT frequencies were the same in contracted and non-contracted tissues, indicating a proportional increase in MT numbers in the expanded cells. Although the absolute spatial frequency of MTs was constant, the orientation, as determined by morphometric analysis of MT profiles, was not. While in the longitudinal section plane 42% of the MTs in the non-contracted cells were oblique, in the contracted cells the percentage of MTs presenting oblique profiles increased to 87%. Additionally, a qualitative difference in MTs was observed in contracted cells; electron-opaque material was seen peripherally associated with the MTs of the inner cortex. The changes in tubulin levels and in MT numbers as well as the qualitative differences in the MTs of contracted and non-contracted root regions indicate that, in hyacinth, reoriented cellular enlargement associated with root contraction cannot be explained simply by shifts in the arrangement of preexisting cortical MT arrays, but involves more complex changes in the cytoskeleton.Abbreviations MT(s) microtubule(s) - TEM transmission electron microscopy - RIA radioimmunoassay - M_r apparent molecular mass I=Jernstedt (1984b)     

Rapid assembly and collective behavior of microtubule bundles in the presence of polyamines

Microtubules (MTs) are cylindrical cytoskeleton polymers composed of α-β tubulin heterodimers whose dynamic properties are essential to fulfill their numerous cellular functions. In response to spatial confinement, dynamic MTs, even in the absence of protein partners, were shown to self-organize into higher order structures (spindle or striped structures) which lead to interesting dynamical properties (MT oscillations). In this study, we considered the assembly and sensitivity of dynamic MTs when in bundles. To perform this study, spermine, a natural tetravalent polyamine present at high concentrations in all eukaryote cells, was used to trigger MT bundling while preserving MT dynamics. Interestingly, we first show that, near physiological ionic strengths, spermine promotes the bundling of MTs whereas it does not lead to aggregation of free tubulin, which would have been detrimental to MT polymerization. Experimental and theoretical results also indicate that, to obtain a high rate of bundle assembly, bundling should take place at the beginning of assembly when rapid rotational movements of short and newly nucleated MTs are still possible. On the other hand, the bundling process is significantly slowed down for long MTs. Finally, we found that short MT bundles exhibit a higher sensitivity to cold exposure than do isolated MTs. To account for this phenomenon, we suggest that a collective behavior takes place within MT bundles because an MT entering into a phase of shortening could increase the probability of the other MTs in the same bundle to enter into shortening phase due to their close proximity. We then elaborate on some putative applications of our findings to in vivo conditions including neurons.     

Structural studies of N-terminal mutants of Connexin 32 using (1)H NMR spectroscopy

Microtubules (MTs) control cell replication, material transport and motion in eukaryotic cells, but MT role in several pathologies is still unknown. These functions are related to the MT physico-chemical properties and MT formation mode starting from tubulin molecules. This study describes a new method, based on the computer aided analysis of the electron paramagnetic resonance (EPR) spectra of selected spin probes to obtain structural and dynamical information on tubulins and MTs and the kinetics of MTs formation promoted by guanosine-5'-triphosphate (GTP). It was found that tubulin and MTs avoid radical quenching caused by ethylene glycol tetraacetic acid (EGTA). MT formation showed different kinetics as a function of tubulin concentration. At 5 mg/mL of tubulin, MTs were formed in 8 min. These results are also useful for getting information on MT-drug interactions.     

The interactions between calcium-dependent regulator protein of cyclic nucleotide phosphodiesterase and microtubule proteins. II. Association of calcium-dependent regulator protein with tubulin dimer.

The Ca2+-dependent regulator protein (CDR) of cyclic nucleotide phosphodiesterase (PDE) was reported to be a Ca2+-dependent regulator of microtubule (MT) assembly in the preceding paper. In this paper, the binding of Ca2+-CDR complex to tubulin dimer was investigated in order to elucidate the Ca2+-dependent inhibitory action of CDR on MT assembly. Purified microtubular proteins (PMPs) isolated from porcine brain did not affect the ability of CDR to activate Ca2+-activatable PDE, and did not include any inhibitory protein of Ca2+-activatable PDE. The binding of CDR to the tubulin dimer was observed on Sephadex G-200 gel filtration and ammonium sulfate fractionation in a Ca2+-dependent manner. CDR did not bind to microtubule associated proteins. We now assume that Ca2+-dependent inhibition of MT assembly by CDR is due to the binding of CDR to tubulin dimer in a Ca2+-dependent manner.     

Quantitative analysis of microtubule transport in growing nerve processes

In neurons, tubulin is synthesized primarily in the cell body, whereas the molecular machinery for neurite extension and elaboration of microtubule (MT) array is localized to the growth cone region. This unique functional and biochemical compartmentalization of neuronal cells requires transport mechanisms for the delivery of newly synthesized tubulin and other cytoplasmic components from the cell body to the growing axon. According to the polymer transport model, tubulin is transported along the axon as a polymer. Because the majority of axonal MTs are stationary at any given moment, it has been assumed that only a small fraction of MTs translocates along the axon by saltatory movement reminiscent of the fast axonal transport. Such intermittent "stop and go" MT transport has been difficult to detect or to exclude by using direct video microscopy methods. In this study, we measured the translocation of MT plus ends in the axonal shaft by expressing GFP-EB1 in Xenopus embryo neurons in culture. Formal quantitative analysis of MT assembly/disassembly indicated that none of the MTs in the axonal shaft were rapidly transported. Our results suggest that transport of axonal MTs is not required for delivery of newly synthesized tubulin to the growing nerve processes.     

Microtubule detachment from the microtubule-organizing center as a key event in the complete turnover of microtubules in cells

Microtubule (MT) response to different steady state temperatures and to rapid shifts in temperature was studied quantitatively in large, thin cells (LT-cells) from the goldfish scale. MT number and total tubulin concentration per cell were found to be fairly constant in cells from the same fish, regardless of cell size but between fish, could differ by a factor of two. The total tubulin concentration was similar to that found in mammalian tissue culture cells and the proportion in MT form increased with increasing steady state temperature. Total MT length quickly and exponentially decreased when cells were rapidly chilled to approximately -3 degrees C. In contrast, the average length of the MTs bound to the MT organizing center (MTOC) did not significantly change. Free MTs were generated during chilling and had an average length roughly half that of bound MTs. These observations suggest that 1) there is a functional block to rapid depolymerization at the unattached end of the MTOC bound MTs and 2) depolymerization of the MT occurs from the originally bound end only after its release from the MTOC. The presence of free MTs in a wide variety of cells suggests that these two features may be characteristic of steady state MTs in other cells. When the temperature of the LT-cells was abruptly raised, the number of MTs initiated on the MTOC rapidly increased and reached a brief steady state long before the MTs completely elongated. Many MTs then apparently detached from the MTOC and depolymerized before a final steady state was reached. When cells containing newly polymerized MTs were chilled to approximately -3 degrees C, the MTs detached from the MTOC more rapidly than those starting from steady state. Furthermore, the block to depolymerization at the unattached end was not complete. These observations suggest that newly formed, non-steady state MTs are different from the older, steady state MTs.     

Microinjection of fluorescent tubulin into plant cells provides a representative picture of the cortical microtubule array

By microinjecting rhodamine-labelled tubulin into living plant cells, it is possible to observe microtubules (MTs) directly and to see how the cortical array reorganizes itself. The validity of the conclusions drawn from such observations depends upon the assumption that most, if not all, of the native MTs are dynamic and incorporate labelled tubulin. However, if arrays also contain MTs that are not exchanging tubulin subunits, such MTs will remain unlabelled, and the labelled MT population will be under-representative of the whole array. To address this potential problem, we microinjected pea epidermal cells with rhodamine-labelled tubulin, then fixed the cells and used fluorescein-conjugated antibodies against tubulin to detect the entire MT array. The two fluorescent patterns corresponded well, confirming that the MTs labelled with exogenous tubulin were evenly distributed throughout the entire array. Also, by comparing the MT image before and after aldehyde fixation, we observed that, although some of the MTs were lost in the procedure, the fixation was able to preserve the arrangement of MTs seen in the living cell. We conclude that fluorescence analogue cytochemistry provides a valid representation of the entire cortical MT array.     

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

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

Mutation in the beta-tubulin signature motif suppresses microtubule GTPase activity and dynamics, and slows mitosis.

We introduced a threonine-to-glycine point mutation at position 143 in the "tubulin signature motif" 140Gly-Gly-Gly-Thr-Gly-Ser-Gly146 of Saccharomyces cerevisiae beta-tubulin. In an electron diffraction model of the tubulin dimer, this sequence comes close to the phosphates of a guanine nucleotide bound in the beta-tubulin exchangeable E site. Both the GTP-binding affinity and the microtubule (MT)-dependent GTPase activity of tubulin isolated from haploid tub2-T143G mutant cells were reduced by at least 15-fold, compared to tubulin isolated from control wild-type cells. The growing and shortening dynamics of MTs assembled from alphabeta:Thr143Gly-mutated dimers were also strongly suppressed, compared to control MTs. The in vitro properties of the mutated MTs (slower growing and more stable) are consistent with the effects of the tub2-T143G mutation in haploid cells. The average length of MT spindles in large-budded mutant cells was only 3.7 +/- 0.2 microm, approximately half of the size of MT arrays in large-budded wild-type cells (average length = 7.1 +/- 0.4 microm), suggesting that there is a delay in mitosis in the mutant cells. There was also a higher proportion of large-budded cells with unsegregated nuclei in mutant cultures (30% versus 12% for wild-type cells), again suggesting such a delay. The results show that beta:Thr143 of the tubulin signature motif plays an important role in GTP binding and hydrolysis by the beta-tubulin E site and support the idea that tubulins belong to a family of proteins within the GTPase superfamily that are structurally distinct from the classic GTPases, such as EF-Tu and p21(ras). The data also suggest that MT dynamics are critical for MT function in yeast cells and that spindle MT assembly and disassembly could be coordinated with other cell-cycle events by regulating beta-tubulin GTPase activity.     

The relative roles of centrosomal and kinetochore-driven microtubules in Drosophila spindle formation

Mitotic spindle assembly in centrosome-containing cells relies on two main microtubule (MT) nucleation pathways, one based on centrosomes and the other on chromosomes. However, the relative role of these pathways is not well defined. Here we review the studies on spindle formation in Drosophila centrosome-containing cells. Mutants with impaired centrosome function assemble functional anastral spindles in somatic tissues and survive to adulthood. In contrast, mutants defective in chromosome-driven MT formation form highly aberrant mitotic spindles and die at larval stages. The requirements for spindle assembly in Drosophila male meiotic cells are diametrically opposed to those of somatic cells. Spermatocytes assemble morphologically normal spindles in the complete absence of chromosome-induced MTs, but are unable to organize a functional spindle in the absence of centrosomal MTs. Male meiotic spindles are much larger than mitotic spindles as they contain most of the tubulin needed for sperm tail formation. We suggest that the centrosome-based mechanism of spindle assembly in spermatocytes reflects their need for rapid and efficient polymerization of a particularly large amount of tubulin.     

The human EMAP-like protein-70 (ELP70) is a microtubule destabilizer that localizes to the mitotic apparatus.

In this report, we show that the echinoderm microtubule (MT)-associated protein (EMAP) and related EMAP-like proteins (ELPs) share a similar domain organization with a highly conserved hydrophobic ELP (HELP) domain and a large tryptophan-aspartic acid (WD) repeat domain. To determine the function of mammalian ELPs, we generated antibodies against a 70-kDa human ELP and showed that ELP70 coassembled with MTs in HeLa cell extracts and colocalized with MTs in the mitotic apparatus. To determine whether ELP70 bound to MTs directly, human ELP70 was expressed and purified to homogeneity from baculovirus-infected Sf9 cells. Purified ELP70 bound to purified MTs with a stoichiometry of 0.40 +/- 0.04 mol of ELP70/mol of tubulin dimer and with an intrinsic dissociation constant of 0.44 +/- 0.13 microm. Using a nucleated assembly assay and video-enhanced differential interference contrast microscopy, we demonstrated that ELP70 reduced seeded nucleation, reduced the growth rate, and promoted MT catastrophes in a concentration-dependent manner. As a result, ELP70-containing MTs were significantly shorter than MTs assembled from tubulin alone. These data indicate that ELP70 is a novel MT destabilizer. A lateral destabilization model is presented to describe ELP70's effects on microtubules.     

Detyrosination of tubulin regulates the interaction of intermediate filaments with microtubules in vivo via a kinesin-dependent mechanism

Posttranslationally modified forms of tubulin accumulate in the subset of stabilized microtubules (MTs) in cells but are not themselves involved in generating MT stability. We showed previously that stabilized, detyrosinated (Glu) MTs function to localize vimentin intermediate filaments (IFs) in fibroblasts. To determine whether tubulin detyrosination or MT stability is the critical element in the preferential association of IFs with Glu MTs, we microinjected nonpolymerizable Glu tubulin into cells. If detyrosination is critical, then soluble Glu tubulin should be a competitive inhibitor of the IF-MT interaction. Before microinjection, Glu tubulin was rendered nonpolymerizable and nontyrosinatable by treatment with iodoacetamide (IAA). Microinjected IAA-Glu tubulin disrupted the interaction of IFs with MTs, as assayed by the collapse of IFs to a perinuclear location, and had no detectable effect on the array of Glu or tyrosinated MTs in cells. Conversely, neither IAA-tyrosinated tubulin nor untreated Glu tubulin, which assembled into MTs, caused collapse of IFs when microinjected. The epitope on Glu tubulin responsible for interfering with the Glu MT-IF interaction was mapped by microinjecting tubulin fragments of alpha-tubulin. The 14-kDa C-terminal fragment of Glu tubulin (alpha-C Glu) induced IF collapse, whereas the 36-kDa N-terminal fragment of alpha-tubulin did not alter the IF array. The epitope required more than the detyrosination site at the C terminus, because a short peptide (a 7-mer) mimicking the C terminus of Glu tubulin did not disrupt the IF distribution. We previously showed that kinesin may mediate the interaction of Glu MTs and IFs. In this study we found that kinesin binding to MTs in vitro was inhibited by the same reagents (i.e., IAA-Glu tubulin and alpha-C Glu) that disrupted the IF-Glu MT interaction in vivo. These results demonstrate for the first time that tubulin detyrosination functions as a signal for the recruitment of IFs to MTs via a mechanism that is likely to involve kinesin.     

Microtubule Assembly from Single Flared Protofilaments—Forget the Cozy Corner?

A paradigm shift for models of MT assembly is suggested by a recent cryo-electron microscopy study of microtubules (MTs). Previous assembly models have been based on the two-dimensional lattice of the MT wall, where incoming subunits can add with longitudinal and lateral bonds. The new study of McIntosh et al. concludes that the growing ends of MTs separate into flared single protofilaments. This means that incoming subunits must add onto the end of single protofilaments, forming only a longitudinal bond. How can growth of single-stranded protofilaments exhibit cooperative assembly with a critical concentration? An answer is suggested by FtsZ, the bacterial tubulin homolog, which assembles into single-stranded protofilaments. Cooperative assembly of FtsZ is thought to be based on conformational changes that switch the longitudinal bond from low to high affinity when the subunit is incorporated in a protofilament. This novel mechanism may also apply to tubulin assembly and could be the primary mechanism for assembly onto single flared protofilaments.     

Removal of MAP4 from microtubules in vivo produces no observable phenotype at the cellular level

Microtubule-associated protein 4 (MAP4) promotes MT assembly in vitro and is localized along MTs in vivo. These results and the fact that MAP4 is the major MAP in nonneuronal cells suggest that MAP4's normal functions may include the stabilization of MTs in situ. To understand MAP4 function in vivo, we produced a blocking antibody (Ab) to prevent MAP4 binding to MTs. The COOH-terminal MT binding domain of MAP4 was expressed in Escherichia coli as a glutathione transferase fusion protein and was injected into rabbits to produce an antiserum that was then affinity purified and shown to be monospecific for MAP4. This Ab blocked > 95% of MAP4 binding to MTs in an in vitro assay. Microinjection of the affinity purified Ab into human fibroblasts and monkey epithelial cells abolished MAP4 binding to MTs as assayed with a rat polyclonal antibody against the NH2-terminal projection domain of MAP4. The removal of MAP4 from MTs was accompanied by its sequestration into visible MAP4-Ab immunocomplexes. However, the MT network appeared normal. Tubulin photoactivation and nocodazole sensitivity assays indicated that MT dynamics were not altered detectably by the removal of MAP4 from the MTs. Cells progressed to mitosis with morphologically normal spindles in the absence of MAP4 binding to MTs. Depleting MAP4 from MTs also did not affect the state of posttranslational modifications of tubulin subunits. Further, no perturbations of MT- dependent organelle distribution were detected. We conclude that the association of MAP4 with MTs is not essential for MT assembly or for the MT-based functions in cultured cells that we could assay. A significant role for MAP4 is not excluded by these results, however, as MAP4 may be a component of a functionally redundant system.     

Differential sorting of beta tubulin isotypes into colchicine-stable microtubules during neuronal and muscle differentiation of embryonal carcinoma cells.

Pluripotent P19 embryonal carcinoma (EC) cells were differentiated along the neuronal and muscle pathways. Comparisons of class I, II, III, and IV beta tubulin isotypes in total and colchicine-stable microtubule (MT) arrays from uncommitted EC, neuronal, and muscle cells were made by immunoblotting and by indirect immunofluorescence microscopy. In undifferentiated EC cells the relative amounts of these four isotypes are the same in both the total and stable MT populations. Subcellular sorting of beta tubulin isotypes was demonstrated in both neuronal and muscle differentiated cells. During neuronal differentiation, class II beta tubulin is preferentially incorporated into the colchicine-stable MTs while class III beta tubulin is preferentially found in the colchicine-labile MTs. The subcellular sorting of class II into stable MTs correlates with the increased staining of MAP 1B, and with the expression of MAP 2C and tau. Although muscle differentiated cells express class II beta tubulin, stable MTs in these cells do not preferentially incorporate this isotype but instead show increased incorporation of class IV beta tubulin. Muscle cells do not show high levels of MAP 1B and do not express MAP 2C or tau. These results are consistent with the hypothesis that a subcellular sorting of tubulin isotypes is the result of a complex interaction between tubulin isotypes and MT-associated proteins.