Tubulin Isotypes Research Articles

Tubulin Isotypes: Emerging Roles in Defining Cancer Stem Cell Niche.

Although the role of microtubule dynamics in cancer progression is well-established, the roles of tubulin isotypes, their cargos and their specific function in the induction and sustenance of cancer stem cells (CSCs) were poorly explored. But emerging reports urge to focus on the transport function of tubulin isotypes in defining orchestrated expression of functionally critical molecules in establishing a stem cell niche, which is the key for CSC regulation. In this review, we summarize the role of specific tubulin isotypes in the transport of functional molecules that regulate metabolic reprogramming, which leads to the induction of CSCs and immune evasion. Recently, the surface expression of GLUT1 and GRP78 as well as voltage-dependent anion channel (VDAC) permeability, regulated by specific isotypes of β-tubulins have been shown to impart CSC properties to cancer cells, by implementing a metabolic reprogramming. Moreover, βIVb tubulin is shown to be critical in modulating EphrinB1signaling to sustain CSCs in oral carcinoma. These tubulin-interacting molecules, Ephrins, GLUT1 and GRP78, are also important regulators of immune evasion, by evoking PD-L1 mediated T-cell suppression. Thus, the recent advances in the field implicate that tubulins play a role in the controlled transport of molecules involved in CSC niche. The indication of tubulin isotypes in the regulation of CSCs offers a strategy to specifically target those tubulin isotypes to eliminate CSCs, rather than the general inhibition of microtubules, which usually leads to therapy resistance.

Regulation of Tubulin Gene Expression: From Isotype Identity to Functional Specialization.

Genomes of higher eukaryotes encode a large tubulin gene superfamily consisting of at least six α and six β-tubulin isotypes. While some α and β-tubulin isotypes are ubiquitously expressed, others are cell-type specific. The subset of α and β-tubulins that is expressed in a given cell type is defined transcriptionally. But the precise mechanisms of how cells choose which α and β isotypes to express and at what level remain poorly understood. Differential expression of tubulin isotypes is particularly prominent during development and in specialized cells, suggesting that some isotypes are better suited for certain cell type-specific functions. Recent studies begin to rationalize this phenomenon, uncovering important differences in tubulin isotype behavior and their impact on the biomechanical properties of the microtubule cytoskeleton. I summarize our understanding of the regulation of tubulin isotype expression, focusing on the role of these complex regulatory pathways in building a customized microtubule network best suited for cellular needs.

Generation of an induced pluripotent stem cell line (DHMCi008-A) from an individual with TUBA1A tubulinopathy

Variants in different neuronal tubulin isotypes cause severe neurodevelopmental disorders with cerebral malformations accompanied by developmental delay, motor impairment, and epilepsy, known as tubulinopathies. Induced pluripotent stem cells were generated from peripheral blood mononuclear cells from a female subject carrying the heterozygous de novo variant c.[521C > T] (p.[Ala174Val]) in the TUBA1A gene. PBMCs were reprogrammed using the CytoTune™-iPS 2.0 Sendai Reprogramming Kit (Invitrogen) and showed a normal karyotype, expression of pluripotency markers, and spontaneous in vitro differentiation into all three germ layers. The generated iPSCs represent a useful tool to study the pathophysiology of TUBA1A tubulinopathy.

Tubulin isotypes - functional insights from model organisms.

The microtubule cytoskeleton is assembled from the α- and β-tubulin subunits of the canonical tubulin heterodimer, which polymerizes into microtubules, and a small number of other family members, such as γ-tubulin, with specialized functions. Overall, microtubule function involves the collective action of multiple α- and β-tubulin isotypes. However, despite 40 years of awareness that most eukaryotes harbor multiple tubulin isotypes, their role in the microtubule cytoskeleton has remained relatively unclear. Various model organisms offer specific advantages for gaining insight into the role of tubulin isotypes. Whereas simple unicellular organisms such as yeast provide experimental tractability that can facilitate deeper access to mechanistic details, more complex organisms, such as the fruit fly, nematode and mouse, can be used to discern potential specialized functions of tissue- and structure-specific isotypes. Here, we review the role of α- and β-tubulin isotypes in microtubule function and in associated tubulinopathies with an emphasis on the advances gained using model organisms. Overall, we argue that studying tubulin isotypes in a range of organisms can reveal the fundamental mechanisms by which they mediate microtubule function. It will also provide valuable perspectives on how these mechanisms underlie the functional and biological diversity of the cytoskeleton.

Reconstituting Microtubules: A Decades-Long Effort From Building Block Identification to the Generation of Recombinant α/β-Tubulin.

Microtubules are cytoskeletal filaments underlying the morphology and functions of all eukaryotic cells. In higher eukaryotes, the basic building blocks of these non-covalent polymers, ɑ- and β-tubulins, are encoded by expanded tubulin family genes (i.e., isotypes) at distinct loci in the genome. While ɑ/β-tubulin heterodimers have been isolated and examined for more than 50 years, how tubulin isotypes contribute to the microtubule organization and functions that support diverse cellular architectures remains a fundamental question. To address this knowledge gap, in vitro reconstitution of microtubules with purified ɑ/β-tubulin proteins has been employed for biochemical and biophysical characterization. These in vitro assays have provided mechanistic insights into the regulation of microtubule dynamics, stability, and interactions with other associated proteins. Here we survey the evolving strategies of generating purified ɑ/β-tubulin heterodimers and highlight the advances in tubulin protein biochemistry that shed light on the roles of tubulin isotypes in determining microtubule structures and properties.

The Amazing Evolutionary Complexity of Eukaryotic Tubulins: Lessons from Naegleria and the Multi-tubulin Hypothesis.

The multi-tubulin hypothesis proposed in 1976 was motivated by finding that the tubulin to build the flagellar apparatus was synthesized de novo during the optional differentiation of Naegleria from walking amoebae to swimming flagellates. In the next decade, with the tools of cloning and sequencing, we were able to establish that the rate of flagellar tubulin synthesis in Naegleria is determined by the abundance of flagellar α- and β-tubulin mRNAs. These experiments also established that the tubulins for Naegleria mitosis were encoded by separate, divergent genes, candidates for which remain incompletely characterized. Meanwhile an unanticipated abundance of tubulin isotypes has been discovered by other researchers. Together with the surprises of genome complexity, these tubulin isotypes require us to rethink how we might utilize the opportunities and challenges offered by the evolutionary diversity of eukaryotes.

Possible Roles of Specific Amino Acids in β-Tubulin Isotypes in the Growth and Maintenance of Neurons: Novel Insights From Cephalopod Mollusks.

Microtubules, are formed of the protein tubulin, which is a heterodimer of α- and β-tubulin subunits. Both α- and β-tubulin exist as numerous isotypes, differing in amino acid sequence and tissue distribution. Among the vertebrate β isotypes, βIII has a very narrow distribution, being found primarily in neurons and in advanced cancers. The places in the amino acid sequence where βIII differs from the other β isotypes are highly conserved in evolution. βIII appears to be highly resistant to reactive oxygen species and it forms highly dynamic microtubules. The first property would be very useful in neurons, which have high concentrations of free radicals, and the high dynamicity would aid neurite outgrowth. The same properties make βIII useful in cancers. Examination of the amino acid sequences indicates a cysteine cluster at positions 124–129 in βIII (CXXCXC). This occurs in all βIII isotypes but not in βI, βII, or βIV. βIII also lacks the easily oxidized C239. Both features could play roles in free radical resistance. Many aggressive tumors over-express βIII. However, a recent study of breast cancer patients showed that many of them mutated their βI, βII, and βIV at particular places to change the residues to those found at the corresponding sites in βIII; these are all sites that are highly conserved in vertebrate βIII. It is possible that these residues are important, not only in the resistance to free radicals, but also in the high dynamicity of βIII. The cephalopod mollusks are well known to be highly intelligent and can remodel their own brains. Interestingly, several cephalopods contain the cysteine cluster as well as up to 7 of the 17 residues that are highly conserved in vertebrate βIII, but are not found in βI, βII, or βIV. In short, it is possible that we are looking at a case of convergent evolution, that a βIII-like isotype may be required for neuronal growth and function and that a structure-function study of the particular residues conserved between vertebrate βIII and cephalopod tubulin isotypes could greatly increase our understanding of the role of the various tubulin isotypes in neuronal growth and function and could aid in the development of novel anti-tumor drugs.

The Expression and Function of Tubulin Isotypes in Caenorhabditis elegans

Microtubules, made from the polymerization of the highly conserved α/β-tubulin heterodimers, serve as important components of the cytoskeleton in all eukaryotic cells. The existence of multiple tubulin isotypes in metazoan genomes and a dazzling variety of tubulin posttranslational modifications (PTMs) prompted the “tubulin code” hypothesis, which proposed that microtubule structure and functions are determined by the tubulin composition and PTMs. Evidence for the tubulin code has emerged from studies in several organisms with the characterization of specific tubulins for their expression and functions. The studies of tubulin PTMs are accelerated by the discovery of the enzymes that add or remove the PTMs. In tubulin research, the use of simple organisms, such as Caenorhabditis elegans, has been instrumental for understanding the expression and functional specialization of tubulin isotypes and the effects of their PTMs. In this review, we summarize the current understanding of the expression patterns and cellular functions of the nine α-tubulin and six β-tubulin isotypes. Expression studies are greatly facilitated by the CRISPR/Cas9-mediated endogenous GFP knock-in reporters and the organism-wide single cell transcriptomic studies. Meanwhile, functional studies benefit from the ease of genetic manipulation and precise gene replacement in C. elegans. These studies identified both ubiquitously expressed tubulin isotypes and tissue-specific isotypes. The isotypes showed functional redundancy, as well as functional specificity, which is likely caused by the subtle differences in their amino acid sequences. Many of these differences concentrate at the C-terminal tails that are subjected to several PTMs. Indeed, tubulin PTM, such as polyglutamylation, is shown to modulate microtubule organization and properties in both ciliated and non-ciliated neurons. Overall, studies from C. elegans support the distinct expression and function patterns of tubulin isotypes and the importance of their PTMs and offer the promise of cracking the tubulin code at the whole-genome and the whole-organism level.

EP145: Severe Pierre Robin sequence and hydrocephalus in infant with TUBB3 variant

Pathogenic variants in TUBB3 are associated with congenital fibrosis of the extraocular muscles 3 (CFEOM3) and cortical dysplasia, complex, with other brain malformations 1 (CDCBM1). CDCBM1 is considered a tubulinopathy. Tubulinopathies are a group of conditions that include a large spectrum of brain malformations that are caused by pathogenic variants in genes encoding different tubulin isotypes. There are at least nine different alpha and ß tubulin isotypes, which are closely related subunits that are major components of microtubules (MTs).

A dominant tubulin mutation causes cerebellar neurodegeneration in a genetic model of tubulinopathy.

Mutations in tubulins cause distinct neurodevelopmental and degenerative diseases termed “tubulinopathies”; however, little is known about the functional requirements of tubulins or how mutations cause cell-specific pathologies. Here, we identify a mutation in the gene Tubb4a that causes degeneration of cerebellar granule neurons and myelination defects. We show that the neural phenotypes result from a cell type–specific enrichment of a dominant mutant form of Tubb4a relative to the expression other β-tubulin isotypes. Loss of Tubb4a function does not underlie cellular pathology but is compensated by the transcriptional up-regulation of related tubulin genes in a cell type–specific manner. This work establishes that the expression of a primary tubulin mutation in mature neurons is sufficient to promote cell-autonomous cell death, consistent with a causative association of microtubule dysfunction with neurodegenerative diseases. These studies provide evidence that mutations in tubulins cause specific phenotypes based on expression ratios of tubulin isotype genes.

Mutations in the most divergent α‐tubulin isotype, α8‐tubulin, cause defective platelet biogenesis

BackgroundIn the panel of genes commonly associated with inherited macrothrombocytopenia, an important fraction encodes key cytoskeletal proteins such as tubulin isotypes, the building blocks of microtubules. Macrothrombocytopenia‐causing mutations have been identified in the TUBB1 and TUBA4A genes, emphasizing their importance in the formation of platelets and their marginal band, a unique microtubule ring‐like structure that supports the platelet typical disc‐shaped morphology. This raised the hypothesis that other tubulin isotypes normally expressed in platelets could play a similar role in their formation. ObjectivesTo assess whether tubulin isotype genes other than TUBA4A and TUBB1 could be implicated in inherited macrothrombocytopenia. MethodsWe used high throughput sequencing to screen a cohort of 448 French blood donors with mild thrombocytopenia for mutations in a panel of selected genes known or suspected to be involved in platelet biogenesis. ResultsWe identified six distinct novel mutations in TUBA8, which encodes the most‐divergent α‐tubulin, as the causative determinant of macrothrombocytopenia and platelet marginal band defects. Functionally, all TUBA8 mutations were found to fully or partially inhibit the incorporation of the mutated α8‐tubulin in the microtubule network. ConclusionThis study provides strong support for a key role of multiple tubulin genes in platelet biogenesis by discovering variants in a tubulin gene that was previously not known to be important for platelets.

Revisiting the Role of ß-Tubulin in Drosophila Development: β-tubulin60D is not an Essential Gene, and its Novel Pin 1 Allele has a Tissue-Specific Dominant-Negative Impact.

Diversity in cytoskeleton organization and function may be achieved through alternative tubulin isotypes and by a variety of post-translational modifications. The Drosophila genome contains five different β-tubulin paralogs, which may play an isotype tissue-specific function in vivo. One of these genes, the β-tubulin60D gene, which is expressed in a tissue-specific manner, was found to be essential for fly viability and fertility. To further understand the role of the β-tubulin60D gene, we generated new β-tubulin60D null alleles (β-tubulin60D M ) using the CRISPR/Cas9 system and found that the homozygous flies were viable and fertile. Moreover, using a combination of genetic complementation tests, rescue experiments, and cell biology analyses, we identified Pin 1 , an unknown dominant mutant with bristle developmental defects, as a dominant-negative allele of β-tubulin60D. We also found a missense mutation in the Pin1 mutant that results in an amino acid replacement from the highly conserved glutamate at position 75 to lysine (E75K). Analyzing the ß-tubulin structure suggests that this E75K alteration destabilizes the alpha-helix structure and may also alter the GTP-Mg2+ complex binding capabilities. Our results revisited the credence that β-tubulin60D is required for fly viability and revealed for the first time in Drosophila, a novel dominant-negative function of missense β-tubulin60D mutation in bristle morphogenesis.

The Effect of Tau and Taxol on Polymerization of MCF7 Microtubules In Vitro.

Overexpression of Tau protein in breast cancer cells is identified as an indicator for potential resistance to taxane-based therapy. As reported findings have been obtained mostly from clinical studies, the undetermined underlying mechanism of such drug resistance needs to be thoroughly explored through comprehensive in vitro evaluations. Tau and Taxol bind to the beta tubulin site in microtubules’ structure. This is of particular interest in breast cancer, as microtubules of these cancer cells are structurally distinct from some other microtubules, such as neuronal microtubules, due to their unique beta tubulin isotype distribution. The observed changes in the in vitro polymerization of breast cancer microtubules, and the different function of some molecular motors along them, leave open the possibility that the drug resistance mechanism can potentially be associated with different responses of these microtubules to Tau and Taxol. We carried out a series of parallel experiments to allow comparison of the in vitro dual effect of Tau and Taxol on the polymerization of MCF7 microtubules. We observed a concentration-dependent demotion-like alteration in the self-polymerization kinetics of Tau-induced MCF7 microtubules. In contrast, microtubules polymerized under the simultaneous effects of Tau and Taxol showed promoted assembly as compared with those observed in Tau-induced microtubules. The analysis of our data obtained from the length of MCF7 microtubules polymerized under the interaction with Tau and Taxol in vitro suggests that the phenomenon known as drug resistance in microtubule-targeted drugs such as Taxol may not be directly linked to the different responses of microtubules to the drug. The effect of the drug may be mitigated due to the simultaneous interactions with other microtubule-associated proteins such as Tau protein. The observed regulatory effect of Tau and Taxol on the polymerization of breast cancer microtubules in vitro points to additional evidence for the possible role of tubulin isotypes in microtubules’ functions.

Computational study of interactions of anti-cancer drug eribulin with human tubulin isotypes.

Microtubules (MTs) are widely targeted for the treatment of various types of cancer due to their essential role in cell division. MTs are polymers made of αβ-tubulin heterodimers. These α- and β-tubulins have 8 and 10 different isotypes, respectively. It is known that a few tubulin isotypes have anti-cancer drug resistance properties, especially βIII, which shows poor sensitivity to many potent anti-cancer drugs such as eribulin. However, the molecular-level understanding of drug-resistance due to tubulin isotype variation is poorly understood. This paper presents the study of differential binding affinities of different tubulin isotypes with the potent anti-cancer drug eribulin. Eribulin (MT destabilizer) binds at the inter-dimer interface of MTs near the vinca site and induces a lattice deformation, which results in catastrophic events in MT dynamics. In this study, sequence analysis has been done throughway and the binding sites and based on that 2α-tubulin isotypes (αI and αVIII) and 7β tubulin isotypes (βI, βIIa, βIII, βIVa, βVI, βVII and βVIII) were selected. In total, 14 combinations were prepared after building homology models of these selected isotypes. Molecular docking and molecular dynamics simulations were performed to deeply understand the binding mode of eribulin at different MT compositions. RMSD, RMSF, radius of gyration, SASA, ligand-protein interactions, and calculations of binding free energy were performed to investigate the eribulin binding variations to tubulin isotypes and it was found that αIβII showed the maximum binding affinity among all 14 systems to eribulin. The βIII-tubulin isotype, which shows low sensitivity to eribulin in experimental results, had the least binding affinity in the system αVIIIβIII complex and the average binding affinity in the system αIβIII among all 14 systems. Additionally, we performed steered MD simulations and DynDom analysis of the systems with the lowest binding energy (αIβII) and the highest binding energy (αVIIIβIII) and extracted force, displacement, and H-bonding profiles during the pulling simulations to get a better insight.

Kinetically Stabilizing Mutations in Beta Tubulins Create Isotype-Specific Brain Malformations.

Mutations in the family of genes encoding the tubulin subunits of microtubules are associated with a spectrum of human brain malformations known as tubulinopathies. How these mutations impact tubulin activity to give rise to distinct developmental consequences is poorly understood. Here we report two patients exhibiting brain malformations characteristic of tubulinopathies and heterozygous T178M missense mutations in different β-tubulin genes, TUBB2A or TUBB3. RNAseq analysis indicates that both TUBB2A and TUBB3 are expressed in the brain during development, but only TUBB2A maintains high expression in neurons into adulthood. The T178 residue is highly conserved in β-tubulins and located in the exchangeable GTP-binding pocket of β-tubulin. To determine the impact of T178M on β-tubulin function we created an analogous mutation in the β-tubulin of budding yeast and show that the substitution acts dominantly to produce kinetically stabilized microtubules that assemble and disassemble slowly, with fewer transitions between these states. In vitro experiments with purified mutant tubulin demonstrate that T178M decreases the intrinsic assembly activity of β-tubulin and forms microtubules that rarely transition to disassembly. We provide evidence that the T178M substitution disrupts GTPase-dependent conformational changes in tubulin, providing a mechanistic explanation for kinetic stabilization. Our findings demonstrate the importance of tubulin’s GTPase activity during brain development, and indicate that tubulin isotypes play different, important roles during brain development.

Tubulin isotypes optimize distinct spindle positioning mechanisms during yeast mitosis.

Microtubules are dynamic cytoskeleton filaments that are essential for a wide range of cellular processes. They are polymerized from tubulin, a heterodimer of α- and β-subunits. Most eukaryotic organisms express multiple isotypes of α- and β-tubulin, yet their functional relevance in any organism remains largely obscure. The two α-tubulin isotypes in budding yeast, Tub1 and Tub3, are proposed to be functionally interchangeable, yet their individual functions have not been rigorously interrogated. Here, we develop otherwise isogenic yeast strains expressing single tubulin isotypes at levels comparable to total tubulin in WT cells. Using genome-wide screening, we uncover unique interactions between the isotypes and the two major mitotic spindle positioning mechanisms. We further exploit these cells to demonstrate that Tub1 and Tub3 optimize spindle positioning by differentially recruiting key components of the Dyn1- and Kar9-dependent mechanisms, respectively. Our results provide novel mechanistic insights into how tubulin isotypes allow highly conserved microtubules to function in diverse cellular processes.

Modeling a disease-correlated tubulin mutation in budding yeast reveals insight into MAP-mediated dynein function

Mutations in the genes that encode α- and β-tubulin underlie many neurological diseases, most notably malformations in cortical development. In addition to revealing the molecular basis for disease etiology, studying such mutations can provide insight into microtubule function and the role of the large family of microtubule effectors. In this study, we use budding yeast to model one such mutation—Gly436Arg in α-tubulin, which is causative of malformations in cortical development—in order to understand how it impacts microtubule function in a simple eukaryotic system. Using a combination of in vitro and in vivo methodologies, including live cell imaging and electron tomography, we find that the mutant tubulin is incorporated into microtubules, causes a shift in α-tubulin isotype usage, and dramatically enhances dynein activity, which leads to spindle-positioning defects. We find that the basis for the latter phenotype is an impaired interaction between She1—a dynein inhibitor—and the mutant microtubules. In addition to revealing the natural balance of α-tubulin isotype utilization in cells, our results provide evidence of an impaired interaction between microtubules and a dynein regulator as a consequence of a tubulin mutation and sheds light on a mechanism that may be causative of neurodevelopmental diseases.

Insights into the interaction dynamics between volatile anesthetics and tubulin through computational molecular modelling

General anesthetics, able to reversibly suppress all conscious brain activity, have baffled medical science for decades, and little is known about their exact molecular mechanism of action. Given the recent scientific interest in the exploration of microtubules as putative functional targets of anesthetics, and the involvement thereof in neurodegenerative disorders, the present work focuses on the investigation of the interaction between human tubulin and four volatile anesthetics: ethylene, desflurane, halothane and methoxyflurane. Interaction sites on different tubulin isotypes are predicted through docking, along with an estimate of the binding affinity ranking. The analysis is expanded by Molecular Dynamics simulations, where the dimers are allowed to freely interact with anesthetics in the surrounding medium. This allowed for the determination of interaction hotspots on tubulin dimers, which could be linked to different functional consequences on the microtubule architecture, and confirmed the weak, Van der Waals-type interaction, occurring within hydrophobic pockets on the dimer. Both docking and MD simulations highlighted significantly weaker interactions of ethylene, consistent with its far lower potency as a general anesthetic. Overall, simulations suggest a transient interaction between anesthetics and microtubules in general anesthesia, and contact probability analysis shows interaction strengths consistent with the potencies of the four compounds. Communicated by Ramaswamy H. Sarma

Expression Patterns and Levels of All Tubulin Isotypes Analyzed in GFP Knock-In C. elegans Strains.

Most organisms have multiple α- and β-tubulin isotypes that likely contribute to the diversity of microtubule (MT) functions. To understand the functional differences of tubulin isotypes in Caenorhabditis elegans, which has nine α-tubulin isotypes and six β-tubulin isotypes, we systematically constructed null mutants and GFP-fusion strains for all tubulin isotypes with the CRISPR/Cas9 system and analyzed their expression patterns and levels in adult hermaphrodites. Four isotypes-α-tubulins TBA-1 and TBA-2 and β-tubulins TBB-1 and TBB-2-were expressed in virtually all tissues, with a distinct tissue-specific spectrum. Other isotypes were expressed in specific tissues or cell types at significantly lower levels than the broadly expressed isotypes. Four isotypes (TBA-5, TBA-6, TBA-9, and TBB-4) were expressed in different subsets of ciliated sensory neurons, and TBB-4 was inefficiently incorporated into mitotic spindle MTs. Taken together, we propose that MTs in C. elegans are mainly composed of four broadly expressed tubulin isotypes and that incorporation of a small amount of tissue-specific isotypes may contribute to tissue-specific MT properties. These newly constructed strains will be useful for further elucidating the distinct roles of tubulin isotypes.Key words: tubulin isotypes, microtubules, C. elegans.

Purification of Tubulin with Controlled Posttranslational Modifications and Isotypes from Limited Sources by Polymerization-Depolymerization Cycles

One important aspect of studies of the microtubule cytoskeleton is the investigation of microtubule behavior in in vitro reconstitution experiments. They allow the analysis of the intrinsic properties of microtubules, such as dynamics, and their interactions with microtubule-associated proteins (MAPs). The "tubulin code" is an emerging concept that points to different tubulin isotypes and various posttranslational modifications (PTMs) as regulators of microtubule properties and functions. To explore the molecular mechanisms of the tubulin code, it is crucial to perform in vitro reconstitution experiments using purified tubulin with specific isotypes and PTMs. To date, this was technically challenging as brain tubulin, which is widely used in in vitro experiments, harbors many PTMs and has a defined isotype composition. Hence, we developed this protocol to purify tubulin from different sources and with different isotype compositions and controlled PTMs, using the classical approach of polymerization and depolymerization cycles. Compared to existing methods based on affinity purification, this approach yields pure, polymerization-competent tubulin, as tubulin resistant to polymerization or depolymerization is discarded during the successive purification steps. We describe the purification of tubulin from cell lines, grown either in suspension or as adherent cultures, and from single mouse brains. The method first describes the generation of cell mass in both suspension and adherent settings, the lysis step, followed by the successive stages of tubulin purification by polymerization-depolymerization cycles. Our method yields tubulin that can be used in experiments addressing the impact of the tubulin code on the intrinsic properties of microtubules and microtubule interactions with associated proteins.