The Tubulin Code, from Molecules to Health and Disease.

Microtubules are essential dynamic polymers composed of α/ß-tubulin heterodimers. They support intracellular trafficking, cell division, cellular motility, and other essential cellular processes. In many species, both α-tubulin and ß-tubulin are encoded by multiple genes with distinct expression profiles and functionality. Microtubules are further diversified through abundant posttranslational modifications, which are added and removed by a suite of enzymes to form complex, stereotyped cellular arrays. The genetic and chemical diversity of tubulin constitute a tubulin code that regulates intrinsic microtubule properties and is read by cellular effectors, such as molecular motors and microtubule-associated proteins, to provide spatial and temporal specificity to microtubules in cells. In this review, we synthesize the rapidly expanding tubulin code literature and highlight limitations and opportunities for the field. As complex microtubule arrays underlie essential physiological processes, a better understanding of how cells employ the tubulin code has important implications for human disease ranging from cancer to neurological disorders.

Regulation of microtubule detyrosination by Ca2+ and conventional calpains.

Detyrosination is a major post-translational modification of microtubules (MTs), which has significant impact on MT function in cell division, differentiation, growth, migration and intracellular trafficking. Detyrosination of α-tubulin occurs mostly via the recently identified complex of vasohibin 1 or 2 (VASH1 and VASH2, respectively) with small vasohibin binding protein (SVBP). However, there is still remaining detyrosinating activity in the absence of VASH1 and/or VASH2 and SVBP, and little is known about the regulation of detyrosination. Here, we found that intracellular Ca2+ is required for efficient MT detyrosination. Furthermore, we show that the Ca2+-dependent proteases calpains 1 and 2 (CAPN1 and CAPN2, respectively) regulate MT detyrosination in VASH1- and SVBP-overexpressing human embryonic kidney (HEK293T) cells. We identified new calpain cleavage sites in the N-terminal disordered region of VASH1. However, this cleavage did not affect the enzymatic activity of vasohibins. In conclusion, we suggest that the regulation of VASH1-mediated MT detyrosination by calpains could occur independently of vasohibin catalytic activity or via another yet unknown tubulin carboxypeptidase. Importantly, the Ca2+ dependency of calpains could allow a fine regulation of MT detyrosination. Thus, identifying the calpain-regulated pathway of MT detyrosination can be of major importance for basic and clinical research.

Vasohibin 1 selectively regulates secondary sprouting and lymphangiogenesis in the zebrafish trunk.

Previous studies have shown that Vasohibin 1 (Vash1) is stimulated by VEGFs in endothelial cells and that its overexpression interferes with angiogenesis in vivo Recently, Vash1 was found to mediate tubulin detyrosination, a post-translational modification that is implicated in many cell functions, such as cell division. Here, we used the zebrafish embryo to investigate the cellular and subcellular mechanisms of Vash1 on endothelial microtubules during formation of the trunk vasculature. We show that microtubules within venous-derived secondary sprouts are strongly and selectively detyrosinated in comparison with other endothelial cells, and that this difference is lost upon vash1 knockdown. Vash1 depletion in zebrafish specifically affected secondary sprouting from the posterior cardinal vein, increasing endothelial cell divisions and cell number in the sprouts. We show that altering secondary sprout numbers and structure upon Vash1 depletion leads to defective lymphatic vessel formation and ectopic lymphatic progenitor specification in the zebrafish trunk.

Tubulin Complexity in Cancer and Metastasis.

Tubulin plays a fundamental role in cellular function and as the subject for microtubule-active agents in the treatment of ovarian cancer. Microtubule-binding proteins (e.g., tau, MAP1/2/4, EB1, CLIP, TOG, survivin, stathmin) and posttranslational modifications (e.g., tyrosination, deglutamylation, acetylation, glycation, phosphorylation, polyamination) further diversify tubulin functionality and may permit additional opportunities to understand microtubule behavior in disease and to develop microtubule-modifying approaches to combat ovarian cancer. Tubulin-based structures that project from suspended ovarian cancer cells known as microtentacles may contribute to metastatic potential of ovarian cancer cells and could represent an exciting novel therapeutic target.

Mechanoactivation of NOX2-generated ROS elicits persistent TRPM8 Ca2+ signals that are inhibited by oncogenic KRas.

Changes in the mechanical microenvironment and mechanical signals are observed during tumor progression, malignant transformation, and metastasis. In this context, understanding the molecular details of mechanotransduction signaling may provide unique therapeutic targets. Here, we report that normal breast epithelial cells are mechanically sensitive, responding to transient mechanical stimuli through a two-part calcium signaling mechanism. We observed an immediate, robust rise in intracellular calcium (within seconds) followed by a persistent extracellular calcium influx (up to 30 min). This persistent calcium was sustained via microtubule-dependent mechanoactivation of NADPH oxidase 2 (NOX2)-generated reactive oxygen species (ROS), which acted on transient receptor potential cation channel subfamily M member 8 (TRPM8) channels to prolong calcium signaling. In contrast, the introduction of a constitutively active oncogenic KRas mutation inhibited the magnitude of initial calcium signaling and severely blunted persistent calcium influx. The identification that oncogenic KRas suppresses mechanically-induced calcium at the level of ROS provides a mechanism for how KRas could alter cell responses to tumor microenvironment mechanics and may reveal chemotherapeutic targets for cancer. Moreover, we find that expression changes in both NOX2 and TRPM8 mRNA predict poor clinical outcome in estrogen receptor (ER)-negative breast cancer patients, a population with limited available treatment options. The clinical and mechanistic data demonstrating disruption of this mechanically-activated calcium pathway in breast cancer patients and by KRas activation reveal signaling alterations that could influence cancer cell responses to the tumor mechanical microenvironment and impact patient survival.

Vasohibin-1 has α-tubulin detyrosinating activity in glomerular podocytes.

Podocytes are highly specialized epithelial cells in glomeruli, with a complex morphology composed of a cell body, primary processes, and foot processes, which maintain barrier function in glomerular filtration. The microtubule-based cytoskeleton is necessary for podocyte morphology. Microtubule structure and function can be affected by post-translational modification of tubulin, including detyrosination. Recent studies have shown that vasohibin-1 (VASH1), an antiangiogenic factor, has tubulin carboxypeptidase activity that causes detyrosination of α-tubulin. We aimed to examine the role of VASH1 in regulating α-tubulin detyrosination in podocytes and the potential involvement of VASH1 deficiency in renal morphology. In normal mouse kidneys, detyrosinated α-tubulin was mainly identified in glomeruli, especially in podocytes; meanwhile, in cultured immortalized podocytes, α-tubulin detyrosination was promoted with cell differentiation. Notably, α-tubulin detyrosination in glomeruli was diminished in Vash1 homozygous knockout (Vash1-/-) mice, and knockdown of VASH1 in cultured podocytes prevented α-tubulin detyrosination. Although VASH1 deficiency-induced downregulation of detyrosination caused no remarkable glomerular lesions, urinary albuminuria excretion and glomerular volume were significantly higher in Vash1-/- mice than in wild-type mice. Furthermore, decreased glomerular nephrin expression and narrower slit diaphragms width were observed in Vash1-/- mice. Taken together, we demonstrated that α-tubulin detyrosination in podocytes was mainly regulated by VASH1 and that VASH1 deficiency-mediated decreases in α-tubulin detyrosination led to minor alterations in podocyte morphology and predisposition to albuminuria. VASH1 expression and α-tubulin detyrosination may be novel targets for maintaining glomerular filtration barrier integrity.

A rice tubulin tyrosine ligase-like 12 protein affects the dynamic and orientation of microtubules.

The detyrosination/retyrosination cycle is the most common post-translational modification of α-tubulin. Removal of the conserved C-terminal tyrosine of α-tubulin by a still elusive tubulin tyrosine carboxypeptidase, and religation of this tyrosine by a tubulin tyrosine ligase (TTL), are probably common to all eukaryotes. Interestingly, for plants, the only candidates qualifying as potential TTL homologs are the tubulin tyrosine ligase-like 12 proteins. To get insight into the biological functions of these potential TTL homologs, we cloned the rice TTL-like 12 protein (OsTTLL12) and generated overexpression OsTTLL12-RFP lines in both rice and tobacco BY-2 cells. We found, unexpectedly, that overexpression of this OsTTLL12-RFP increased the relative abundance of detyrosinated α-tubulin in both coleoptile and seminal root, correlated with more stable microtubules. This was independent of the respective orientation of cortical microtubule, and followed by correspondingly changing growth of coleoptiles and seminal roots. A perturbed organization of phragmoplast microtubules and disoriented cell walls were further characteristics of this phenotype. Thus, the elevated tubulin detyrosination in consequence of OsTTLL12 overexpression affects structural and dynamic features of microtubules, followed by changes in the axiality of cell plate deposition and, consequently, plant growth.

Vasohibin-2 plays an essential role in metastasis of pancreatic ductal adenocarcinoma.

Vasohibin-2 (VASH2) is expressed in various cancers and promotes their progression. We recently reported that pancreatic cancer patients with higher VASH2 expression show poorer prognosis. Herein, we sought to characterize the role of VASH2 in pancreatic cancer. We used LSL-KrasG12D ; LSL-Trp53R172H ; Pdx-1-Cre (KPC) mice, a mouse model of pancreatic ductal adenocarcinoma (PDAC), and cells isolated from them (KPC cells). Knockdown of Vash2 from PDAC cells did not affect their proliferation, but decreased their migration. When Vash2-knockdown PDAC cells were orthotopically inoculated, liver metastasis and peritoneal dissemination were reduced, and the survival period was significantly prolonged. When KPC mice were crossed with Vash2-deficient mice, metastasis was significantly decreased in Vash2-deficient KPC mice. VASH2 was recently identified to have tubulin carboxypeptidase activity. VASH2 knockdown decreased, whereas VASH2 overexpression increased tubulin detyrosination of PDAC cells, and tubulin carboxypeptidase (TCP) inhibitor parthenolide inhibited VASH2-induced cell migration. We next clarified its role in the tumor microenvironment. Tumor angiogenesis was significantly abrogated in vivo when VASH2 was knocked down or deleted. We further examined genes downregulated by Vash2 knockdown in KPC cells, and found chemokines and cytokines that were responsible for the recruitment of myeloid derived suppressor cells (MDSC). Indeed, MDSC were accumulated in PDAC of KPC mice, and they were significantly decreased in Vash2-deficient KPC mice. These findings suggest that VASH2 plays an essential role in the metastasis of PDAC with multiple effects on both cancer cells and the tumor microenvironment, including tubulin detyrosination, tumor angiogenesis and evasion of tumor immunity.

The tubulin code at a glance.

Microtubules are key cytoskeletal elements of all eukaryotic cells and are assembled of evolutionarily conserved α-tubulin-ß-tubulin heterodimers. Despite their uniform structure, microtubules fulfill a large diversity of functions. A regulatory mechanism to control the specialization of the microtubule cytoskeleton is the 'tubulin code', which is generated by (i) expression of different α- and ß-tubulin isotypes, and by (ii) post-translational modifications of tubulin. In this Cell Science at a Glance article and the accompanying poster, we provide a comprehensive overview of the molecular components of the tubulin code, and discuss the mechanisms by which these components contribute to the generation of functionally specialized microtubules.

Spatial pattern formation in microtubule post-translational modifications and the tight localization of motor-driven cargo.

Microtubule (MT) "age" can be interpreted as nucleotide state, lattice defects, or post-translational modification (PTM) such as acetylation and detyrosination. In all three cases, these have been recently shown to have functionally-important effects on the dynamics of MT arrays, and can present spatial and temporal heterogeneity. While mathematical models for MT array densities are well-established, here we present equations describing MT age, defined as the mean time since the MT's building blocks (tubulin) were polymerized from their soluble dimer state. We derive the age equations using a mean first-passage time calculation and two complementary approaches: The continuum limit of spatial discretization model, and an adjoint operator approach. These equations can recapitulate the observation that the oldest (most de-tyrosinated) tubulin in axons is near the middle of axons during neuronal development in chick embryos. Furthermore, PTMs influence motor kinetics up to approximately twofold for off-rates and velocities. Our simulations demonstrate that this relatively weak dependence of motor kinetics is sufficient to target motor cargo to a specific location along the array. This localization is tightly peaked in a way that magnifies the relatively small signal of PTM spatial heterogeneity. Thus, MT age can produce long-range spatial patterning without feedbacks or diffusing signals.

Suppression of tubulin detyrosination by parthenolide recruits the plant-specific kinesin KCH to cortical microtubules.

Detyrosination of α-tubulin seems to be conserved in all eukaryotes. However, its biological function in plants has remained obscure. A conserved C-terminal tyrosine is removed by a still unidentified tubulin-tyrosine carboxypeptidase (TTC) and can be religated by a tubulin-tyrosine ligase (TTL). To obtain insight into the still elusive biological function of this detyrosination-tyrosination cycle, the effects of the TTC inhibitor parthenolide were analysed in BY-2 tobacco cells. Parthenolide caused a depletion of detyrosinated α-tubulin, whereas the level of tyrosinated tubulin was elevated. This biochemical effect was accompanied by growth inhibition in cycling BY-2 cells and alteration of microtubule-dependent events that define division and expansion geometry such as cell plate alignment or axial expansion. Furthermore, parthenolide triggered an apoplastic alkalinization indicative of activation of defence-related calcium influx channels. At the same time, parthenolide promoted the association of the plant-specific kinesin KCH with cortical microtubules. These observations are integrated into a working model, where detyrosination acts as signal to modulate the binding of kinesin motors involved in structural and sensory functions of the microtubular cytoskeleton.

Tubulin perturbation leads to unexpected cell wall modifications and affects stomatal behaviour in Populus.

Cortical microtubules are integral to plant morphogenesis, cell wall synthesis, and stomatal behaviour, presumably by governing cellulose microfibril orientation. Genetic manipulation of tubulins often leads to abnormal plant development, making it difficult to probe additional roles of cortical microtubules in cell wall biogenesis. Here, it is shown that expressing post-translational C-terminal modification mimics of α-tubulin altered cell wall characteristics and guard cell dynamics in transgenic Populus tremula x alba that otherwise appear normal. 35S promoter-driven transgene expression was high in leaves but unusually low in xylem, suggesting high levels of tubulin transgene expression were not tolerated in wood-forming tissues during regeneration of transformants. Cellulose, hemicellulose, and lignin contents were unaffected in transgenic wood, but expression of cell wall-modifying enzymes, and extractability of lignin-bound pectin and xylan polysaccharides were increased in developing xylem. The results suggest that pectin and xylan polysaccharides deposited early during cell wall biogenesis are more sensitive to subtle tubulin perturbation than cellulose and matrix polysaccharides deposited later. Tubulin perturbation also affected guard cell behaviour, delaying drought-induced stomatal closure as well as light-induced stomatal opening in leaves. Pectins have been shown to confer cell wall flexibility critical for reversible stomatal movement, and results presented here are consistent with microtubule involvement in this process. Taken together, the data show the value of growth-compatible tubulin perturbations for discerning microtubule functions, and add to the growing body of evidence for microtubule involvement in non-cellulosic polysaccharide assembly during cell wall biogenesis.

Decoding microtubule detyrosination: enzyme families, structures, and functional implications.

Microtubules are a major component of the cytoskeleton and can accumulate a plethora of modifications. The microtubule detyrosination cycle is one of these modifications; it involves the enzymatic removal of the C-terminal tyrosine of α-tubulin on assembled microtubules and the re-ligation of tyrosine on detyrosinated tubulin dimers. This modification cycle has been implicated in cardiac disease, neuronal development, and mitotic defects. The vasohibin and microtubule-associated tyrosine carboxypeptidase enzyme families are responsible for microtubule detyrosination. Their long-sought discovery allows to review and summarise differences and similarities between the two enzymes families and discuss how they interplay with other modifications and functions of the tubulin code.

Decreasing microtubule detyrosination modulates Nav1.5 subcellular distribution and restores sodium current in mdx cardiomyocytes.

AIMS: The microtubule (MT) network plays a major role in the transport of the cardiac sodium channel Nav1.5 to the membrane, where the latter associates with interacting proteins such as dystrophin. Alterations in MT dynamics are known to impact on ion channel trafficking. Duchenne muscular dystrophy (DMD), caused by dystrophin deficiency, is associated with an increase in MT detyrosination, decreased sodium current (INa), and arrhythmias. Parthenolide (PTL), a compound that decreases MT detyrosination, has shown beneficial effects on cardiac function in DMD. We here investigated its impact on INa and Nav1.5 subcellular distribution. METHODS AND RESULTS: Ventricular cardiomyocytes (CMs) from wild-type (WT) and mdx (DMD) mice were incubated with either 10 µM PTL, 20 µM EpoY, or dimethylsulfoxide (DMSO) for 3-5 h, followed by patch-clamp analysis to assess INa and action potential (AP) characteristics in addition to immunofluorescence and stochastic optical reconstruction microscopy (STORM) to investigate MT detyrosination and Nav1.5 cluster size and density, respectively. In accordance with previous studies, we observed increased MT detyrosination, decreased INa and reduced AP upstroke velocity (Vmax) in mdx CMs compared to WT. PTL decreased MT detyrosination and significantly increased INa magnitude (without affecting INa gating properties) and AP Vmax in mdx CMs, but had no effect in WT CMs. Moreover, STORM analysis showed that in mdx CMs, Nav1.5 clusters were decreased not only in the grooves of the lateral membrane (LM; where dystrophin is localized) but also at the LM crests. PTL restored Nav1.5 clusters at the LM crests (but not at the grooves), indicating a dystrophin-independent trafficking route to this subcellular domain. Interestingly, Nav1.5 cluster density was also reduced at the intercalated disc (ID) region of mdx CMs, which was restored to WT levels by PTL. Treatment of mdx CMs with EpoY, a specific MT detyrosination inhibitor, also increased INa density, while decreasing the amount of detyrosinated MTs, confirming a direct mechanistic link. CONCLUSION: Attenuating MT detyrosination in mdx CMs restored INa and enhanced Nav1.5 localization at the LM crest and ID. Hence, the reduced whole-cell INa density characteristic of mdx CMs is not only the consequence of the lack of dystrophin within the LM grooves but is also due to reduced Nav1.5 at the LM crest and ID secondary to increased baseline MT detyrosination. Overall, our findings identify MT detyrosination as a potential therapeutic target for modulating INa and subcellular Nav1.5 distribution in pathophysiological conditions.

Detyrosinated microtubule arrays drive myofibrillar malformations in mdx muscle fibers.

Altered myofibrillar structure is a consequence of dystrophic pathology that impairs skeletal muscle contractile function and increases susceptibility to contraction injury. In murine Duchenne muscular dystrophy (mdx), myofibrillar alterations are abundant in advanced pathology (>4 months), an age where we formerly established densified microtubule (MT) arrays enriched in detyrosinated (deTyr) tubulin as negative disease modifiers impacting cell mechanics and mechanotransduction. Given the essential role of deTyr-enriched MT arrays in myofibrillar growth, maintenance, and repair, we examined the increased abundance of these arrays as a potential mechanism for these myofibrillar alterations. Here we find an increase in deTyr-tubulin as an early event in dystrophic pathology (4 weeks) with no evidence myofibrillar alterations. At 16 weeks, we show deTyr-enriched MT arrays significantly densified and co-localized to areas of myofibrillar malformation. Profiling the enzyme complexes responsible for deTyr-tubulin, we identify vasohibin 2 (VASH2) and small vasohibin binding protein (SVBP) significantly elevated in the mdx muscle at 4 weeks. Using the genetic increase in VASH2/SVBP expression in 4 weeks wild-type mice we find densified deTyr-enriched MT arrays that co-segregate with myofibrillar malformations similar to those in the 16 weeks mdx. Given that no changes in sarcomere organization were identified in fibers expressing sfGFP as a control, we conclude that disease-dependent densification of deTyr-enriched MT arrays underscores the altered myofibrillar structure in dystrophic skeletal muscle fibers.

A model for generating differences in microtubules between axonal branches depending on the distance from terminals.

In the remodeling of axonal arbor, the growth and retraction of branches are differentially regulated within a single axon. Although cell-autonomously generated differences in microtubule (MT) turnover are thought to be involved in selective branch regulation, the cellular system whereby neurons generate differences of MTs between axonal branches has not been clarified. Because MT turnover tends to be slower in longer branches compared with neighboring shorter branches, feedback regulation depending on branch length is thought to be involved. In the present study, we generated a model of MT lifetime in axonal terminal branches by adapting a length-dependent model in which parameters for MT dynamics were constant in the arbor. The model predicted that differences in MT lifetime between neighboring branches could be generated depending on the distance from terminals. In addition, the following points were predicted. Firstly, destabilization of MTs throughout the arbor decreased the differences in MT lifetime between branches. Secondly, differences of MT lifetime existed even before MTs entered the branch point. In axonal MTs in primary neurons, treatment with a low concentration of nocodazole significantly decreased the differences of detyrosination (deTyr) and tyrosination (Tyr) of tubulins, indicators of MT turnover. Expansion microscopy of the axonal shaft before the branch point revealed differences in deTyr/Tyr modification on MTs. Our model recapitulates the differences in MT turnover between branches and provides a feedback mechanism for MT regulation that depends on the axonal arbor geometry.

Mechanistic Analysis of CCP1 in Generating ΔC2 α-Tubulin in Mammalian Cells and Photoreceptor Neurons.

An important post-translational modification (PTM) of α-tubulin is the removal of amino acids from its C-terminus. Removal of the C-terminal tyrosine residue yields detyrosinated α-tubulin, and subsequent removal of the penultimate glutamate residue produces ΔC2-α-tubulin. These PTMs alter the ability of the α-tubulin C-terminal tail to interact with effector proteins and are thereby thought to change microtubule dynamics, stability, and organization. The peptidase(s) that produces ΔC2-α-tubulin in a physiological context remains unclear. Here, we take advantage of the observation that ΔC2-α-tubulin accumulates to high levels in cells lacking tubulin tyrosine ligase (TTL) to screen for cytosolic carboxypeptidases (CCPs) that generate ΔC2-α-tubulin. We identify CCP1 as the sole peptidase that produces ΔC2-α-tubulin in TTLΔ HeLa cells. Interestingly, we find that the levels of ΔC2-α-tubulin are only modestly reduced in photoreceptors of ccp1-/- mice, indicating that other peptidases act synergistically with CCP1 to produce ΔC2-α-tubulin in post-mitotic cells. Moreover, the production of ΔC2-α-tubulin appears to be under tight spatial control in the photoreceptor cilium: ΔC2-α-tubulin persists in the connecting cilium of ccp1-/- but is depleted in the distal portion of the photoreceptor. This work establishes the groundwork to pinpoint the function of ΔC2-α-tubulin in proliferating and post-mitotic mammalian cells.

Microtubule detyrosination by VASH1/SVBP is regulated by the conformational state of tubulin in the lattice.

Tubulin, a heterodimer of α- and ß-tubulin, is a GTPase that assembles into microtubule (MT) polymers whose dynamic properties are intimately coupled to nucleotide hydrolysis. In cells, the organization and dynamics of MTs are further tuned by post-translational modifications (PTMs), which control the ability of MT-associated proteins (MAPs) and molecular motors to engage MTs. Detyrosination is a PTM of α-tubulin, wherein its C-terminal tyrosine residue is enzymatically removed by either the vasohibin (VASH) or MT-associated tyrosine carboxypeptidase (MATCAP) peptidases. How these enzymes generate specific patterns of MT detyrosination in cells is not known. Here, we use a novel antibody-based probe to visualize the formation of detyrosinated MTs in real time and employ single-molecule imaging of VASH1 bound to its regulatory partner small-vasohibin binding protein (SVBP) to understand the process of MT detyrosination in vitro and in cells. We demonstrate that the activity, but not binding, of VASH1/SVBP is much greater on mimics of guanosine triphosphate (GTP)-MTs than on guanosine diphosphate (GDP)-MTs. Given emerging data showing that tubulin subunits in GTP-MTs are in expanded conformation relative to tubulin subunits in GDP-MTs, we reasoned that the lattice conformation of MTs is a key factor that gates the activity of VASH1/SVBP. We show that Taxol, a drug known to expand the MT lattice, promotes MT detyrosination and that CAMSAP2 and CAMSAP3 are two MAPs that spatially regulate detyrosination in cells. Collectively, our work shows that VASH1/SVBP detyrosination is regulated by the conformational state of tubulin in the MT lattice and that this is spatially determined in cells by the activity of MAPs.

Interplay between stochastic enzyme activity and microtubule stability drives detyrosination enrichment on microtubule subsets.

Microtubules in cells consist of functionally diverse subpopulations carrying distinct post-translational modifications (PTMs). Akin to the histone code, the tubulin code regulates a myriad of microtubule functions, ranging from intracellular transport to chromosome segregation. However, how individual PTMs only occur on subsets of microtubules to contribute to microtubule specialization is not well understood. In particular, microtubule detyrosination, the removal of the C-terminal tyrosine on α-tubulin subunits, marks the stable population of microtubules and modifies how microtubules interact with other microtubule-associated proteins to regulate a wide range of cellular processes. Previously, we found that in certain cell types, only ∼30% of microtubules are highly enriched with the detyrosination mark and that detyrosination spans most of the length of a microtubule, often adjacent to a completely tyrosinated microtubule. How the activity of a cytosolic detyrosinase, vasohibin (VASH), leads to only a small subpopulation of highly detyrosinated microtubules is unclear. Here, using quantitative super-resolution microscopy, we visualized nascent microtubule detyrosination events in cells consisting of 1-3 detyrosinated α-tubulin subunits after nocodazole washout. Microtubule detyrosination accumulates slowly and in a dispersed pattern across the microtubule length. By visualizing single molecules of VASH in live cells, we found that VASH engages with microtubules stochastically on a short timescale, suggesting limited removal of tyrosine per interaction, consistent with the super-resolution results. Combining these quantitative imaging results with simulations incorporating parameters from our experiments, we provide evidence for a stochastic model for cells to establish a subset of detyrosinated microtubules via a detyrosination-stabilization feedback mechanism.

Inhibition of microtubule detyrosination by parthenolide facilitates functional CNS axon regeneration.

Injured axons in the central nervous system (CNS) usually fail to regenerate, causing permanent disabilities. However, the knockdown of Pten knockout or treatment of neurons with hyper-IL-6 (hIL-6) transforms neurons into a regenerative state, allowing them to regenerate axons in the injured optic nerve and spinal cord. Transneuronal delivery of hIL-6 to the injured brain stem neurons enables functional recovery after severe spinal cord injury. Here we demonstrate that the beneficial hIL-6 and Pten knockout effects on axon growth are limited by the induction of tubulin detyrosination in axonal growth cones. Hence, cotreatment with parthenolide, a compound blocking microtubule detyrosination, synergistically accelerates neurite growth of cultured murine CNS neurons and primary RGCs isolated from adult human eyes. Systemic application of the prodrug dimethylamino-parthenolide (DMAPT) facilitates axon regeneration in the injured optic nerve and spinal cord. Moreover, combinatorial treatment further improves hIL-6-induced axon regeneration and locomotor recovery after severe SCI. Thus, DMAPT facilitates functional CNS regeneration and reduces the limiting effects of pro-regenerative treatments, making it a promising drug candidate for treating CNS injuries.