Regulation Of Microtubule Function Research Articles

A family of carboxypeptidases catalyzing α- and β-tubulin tail processing and deglutamylation.

Tubulin posttranslational modifications represent an important mechanism involved in the regulation of microtubule functions. The most widespread among them are detyrosination, α∆2-tubulin, and polyglutamylation. Here, we describe a family of tubulin-modifying enzymes composed of two closely related proteins, KIAA0895L and KIAA0895, which have tubulin metallocarboxypeptidase activity and thus were termed TMCP1 and TMCP2, respectively. We show that TMCP1 (also known as MATCAP) acts as α-tubulin detyrosinase that also catalyzes α∆2-tubulin. In contrast, TMCP2 preferentially modifies βI-tubulin by removing three amino acids from its C terminus, generating previously unknown βI∆3 modification. We show that βI∆3-tubulin is mostly found on centrioles and mitotic spindles and in cilia. Moreover, we demonstrate that TMCPs also remove posttranslational polyglutamylation and thus act as tubulin deglutamylases. Together, our study describes the identification and comprehensive biochemical analysis of a previously unknown type of tubulin-modifying enzymes involved in the processing of α- and β-tubulin C-terminal tails and deglutamylation.

A ccpp-6 deletion mutation does not impair gross cilia integrity in C. elegans.

Tubulin glutamylation is a reversible modification that regulates microtubule function in cilia. The removal of glutamylation from microtubules is carried out by a family of cytosolic carboxypeptidase (CCP) enzymes. C. elegans has two deglutamylating enzymes, CCPP-1 and CCPP-6, homologs of mammalian CCP1 and CCP5 respectively. CCPP-1 is required for ciliary stability and function. To determine whether CCPP-6 is similarly required for cilia integrity in C. elegans we analyzed the ccpp-6(ok382) deletion mutant. We find that both dye-filling and male mating are normal, suggesting that CCPP-6 is not required for ciliary integrity in C. elegans.

Circular RNA ZNF609/CKAP5 mRNA interaction regulates microtubule dynamics and tumorigenicity

SummaryCircular RNAs (circRNAs) are widely expressed in eukaryotes and are regulated in many biological processes. Although several studies indicate their activity as microRNA (miRNA) and protein sponges, little is known about their ability to directly control mRNA homeostasis. We show that the widely expressed circZNF609 directly interacts with several mRNAs and increases their stability and/or translation by favoring the recruitment of the RNA-binding protein ELAVL1. Particularly, the interaction with CKAP5 mRNA, which interestingly overlaps the back-splicing junction, enhances CKAP5 translation, regulating microtubule function in cancer cells and sustaining cell-cycle progression. Finally, we show that circZNF609 downregulation increases the sensitivity of several cancer cell lines to different microtubule-targeting chemotherapeutic drugs and that locked nucleic acid (LNA) protectors against the pairing region on circZNF609 phenocopy such effects. These data set an example of how the small effects tuned by circZNF609/CKAP5 mRNA interaction might have a potent output in tumor growth and drug response.

Microtubule Dysfunction: A Common Feature of Neurodegenerative Diseases.

Neurons are particularly susceptible to microtubule (MT) defects and deregulation of the MT cytoskeleton is considered to be a common insult during the pathogenesis of neurodegenerative disorders. Evidence that dysfunctions in the MT system have a direct role in neurodegeneration comes from findings that several forms of neurodegenerative diseases are associated with changes in genes encoding tubulins, the structural units of MTs, MT-associated proteins (MAPs), or additional factors such as MT modifying enzymes which modulating tubulin post-translational modifications (PTMs) regulate MT functions and dynamics. Efforts to use MT-targeting therapeutic agents for the treatment of neurodegenerative diseases are underway. Many of these agents have provided several benefits when tested on both in vitro and in vivo neurodegenerative model systems. Currently, the most frequently addressed therapeutic interventions include drugs that modulate MT stability or that target tubulin PTMs, such as tubulin acetylation. The purpose of this review is to provide an update on the relevance of MT dysfunctions to the process of neurodegeneration and briefly discuss advances in the use of MT-targeting drugs for the treatment of neurodegenerative disorders.

The microtubule-associated histone methyltransferase SET8, facilitated by transcription factor LSF, methylates α-tubulin

Microtubules are cytoskeletal structures critical for mitosis, cell motility, and protein and organelle transport and are a validated target for anticancer drugs. However, how tubulins are regulated and recruited to support these distinct cellular processes is incompletely understood. Posttranslational modifications of tubulins are proposed to regulate microtubule function and dynamics. Although many of these modifications have been investigated, only one prior study reports tubulin methylation and an enzyme responsible for this methylation. Here we used in vitro radiolabeling, MS, and immunoblotting approaches to monitor protein methylation and immunoprecipitation, immunofluorescence, and pulldown approaches to measure protein–protein interactions. We demonstrate that N-lysine methyltransferase 5A (KMT5A or SET8/PR-Set7), which methylates lysine 20 in histone H4, bound α-tubulin and methylated it at a specific lysine residue, Lys311. Furthermore, late SV40 factor (LSF)/CP2, a known transcription factor, bound both α-tubulin and SET8 and enhanced SET8-mediated α-tubulin methylation in vitro. In addition, we found that the ability of LSF to facilitate this methylation is countered by factor quinolinone inhibitor 1 (FQI1), a specific small-molecule inhibitor of LSF. These findings suggest the general model that microtubule-associated proteins, including transcription factors, recruit or stimulate protein-modifying enzymes to target tubulins. Moreover, our results point to dual functions for SET8 and LSF not only in chromatin regulation but also in cytoskeletal modification.

Correction: Alterations in the balance of tubulin glycylation and glutamylation in photoreceptors leads to retinal degeneration.

Tubulin is subject to a wide variety of posttranslational modifications, which, as part of the tubulin code, are involved in the regulation of microtubule functions. Glycylation has so far predominantly been found in motile cilia and flagella, and absence of this modification leads to ciliary disassembly. Here, we demonstrate that the correct functioning of connecting cilia of photoreceptors, which are non-motile sensory cilia, is also dependent on glycylation. In contrast to many other tissues, only one glycylase, TTLL3, is expressed in retina. Ttll3-/- mice lack glycylation in photoreceptors, which results in shortening of connecting cilia and slow retinal degeneration. Moreover, absence of glycylation results in increased levels of tubulin glutamylation in photoreceptors, and inversely, the hyperglutamylation observed in the Purkinje cell degeneration (pcd) mouse abolishes glycylation. This suggests that both posttranslational modifications compete for modification sites, and that unbalancing the glutamylation-glycylation equilibrium on axonemes of connecting cilia, regardless of the enzymatic mechanism, invariably leads to retinal degeneration.

Tubulin polyglutamylation is a general traffic-control mechanism in hippocampal neurons.

Neurons are highly complex cells that heavily rely on intracellular transport to distribute a range of functionally essential cargoes within the cell. Post-translational modifications of tubulin are emerging as mechanisms for regulating microtubule functions, but their impact on neuronal transport is only marginally understood. Here, we have systematically studied the impact of post-translational polyglutamylation on axonal transport. In cultured hippocampal neurons, deletion of a single deglutamylase, CCP1 (also known as AGTPBP1), is sufficient to induce abnormal accumulation of polyglutamylation, i.e. hyperglutamylation. We next investigated how hyperglutamylation affects axonal transport of a range of functionally different neuronal cargoes: mitochondria, lysosomes, LAMP1 endosomes and BDNF vesicles. Strikingly, we found a reduced motility for all these cargoes, suggesting that polyglutamylation could act as a regulator of cargo transport in neurons. This, together with the recent discovery that hyperglutamylation induces neurodegeneration, makes it likely that perturbed neuronal trafficking could be one of the central molecular causes underlying this novel type of degeneration.This article has an associated First Person interview with the first author of the paper.

Microtubules Gate Tau Condensation to Spatially Regulate Microtubule Functions

Microtubules Gate Tau Condensation to Spatially Regulate Microtubule Functions

Microtubules gate tau condensation to spatially regulate microtubule functions.

Tau is an abundant microtubule-associated protein in neurons. Tau aggregation into insoluble fibrils is a hallmark of Alzheimer’s disease and other dementias1, yet the physiological state of tau molecules within cells remains unclear. Using single molecule imaging, we directly observe that the microtubule lattice regulates reversible tau self-association, leading to localized, dynamic condensation of tau molecules on the microtubule surface. Tau condensates form selectively permissible barriers, spatially regulating the activity of microtubule severing enzymes and the movement of molecular motors through their boundaries. We propose that reversible self-association of tau molecules, gated by the microtubule lattice, is an important mechanism of tau’s biological functions, and that oligomerization of tau is a common property shared between the physiological and disease forms of the molecule.

Dynamic Reorganization of Microtubule and Glioma Invasion.

Gliomas are characterized as highly diffuse infiltrating tumors, and currently available treatments such as surgery, radiation and chemotherapy are unfeasible or show limited efficacy against these tumors. Recent genetic and epigenetic analyses of glioma have revealed increasing evidence of the role of driver genetic alterations in glioma development and led to the identification of prognostic factors. Despite these findings, the survival rates of glioma patients remain low, and alternative treatments and novel targets are needed. Recent studies identified neural stem cells as the possible origin of gliomas, and some evidence has revealed shared functions and mechanisms between glioma cells and neurons, also supporting their similarity. The cytoskeleton plays important roles in the migration of normal cells as well as cancer cells. Recent reports have described a role for microtubules, a component of the cytoskeleton, in glioma invasion. Notably, several factors that regulate microtubule functions, such as microtubule-associated proteins, plus-end tracking proteins, or motor proteins, are upregulated in glioma tissues compared with normal tissue, and upregulation of these factors is associated with high invasiveness of glioma cells. In this review, we describe the mechanism of microtubules in glioma invasion and discuss the possibility of microtubule-targeted therapy to inhibit glioma invasion.

Unusual zinc-binding mode of HDAC6-selective hydroxamate inhibitors

Histone deacetylases (HDACs) regulate myriad cellular processes by catalyzing the hydrolysis of acetyl-l-lysine residues in histone and nonhistone proteins. The Zn2+-dependent class IIb enzyme HDAC6 regulates microtubule function by deacetylating α-tubulin, which suppresses microtubule dynamics and leads to cell cycle arrest and apoptosis. Accordingly, HDAC6 is a target for the development of selective inhibitors that might be useful in new therapeutic approaches for the treatment of cancer, neurodegenerative diseases, and other disorders. Here, we present high-resolution structures of catalytic domain 2 from Danio rerio HDAC6 (henceforth simply "HDAC6") complexed with compounds that selectively inhibit HDAC6 while maintaining nanomolar inhibitory potency: N-hydroxy-4-[(N(2-hydroxyethyl)-2-phenylacetamido)methyl)-benzamide)] (HPB), ACY-1215 (Ricolinostat), and ACY-1083. These structures reveal that an unusual monodentate Zn2+ coordination mode is exploited by sterically bulky HDAC6-selective phenylhydroxamate inhibitors. We additionally report the ultrahigh-resolution structure of the HDAC6-trichostatin A complex, which reveals two Zn2+-binding conformers for the inhibitor: a major conformer (70%) with canonical bidentate hydroxamate-Zn2+ coordination geometry and a minor conformer (30%) with monodentate hydroxamate-Zn2+ coordination geometry, reflecting a free energy difference of only 0.5 kcal/mol. The minor conformer is not visible in lower resolution structure determinations. Structural comparisons of HDAC6-inhibitor complexes with class I HDACs suggest active site features that contribute to the isozyme selectivity observed in biochemical assays.

Alterations in the balance of tubulin glycylation and glutamylation in photoreceptors leads to retinal degeneration.

Tubulin is subject to a wide variety of posttranslational modifications, which, as part of the tubulin code, are involved in the regulation of microtubule functions. Glycylation has so far predominantly been found in motile cilia and flagella, and absence of this modification leads to ciliary disassembly. Here, we demonstrate that the correct functioning of connecting cilia of photoreceptors, which are non-motile sensory cilia, is also dependent on glycylation. In contrast to many other tissues, only one glycylase, TTLL3, is expressed in retina. Ttll3-/- mice lack glycylation in photoreceptors, which results in shortening of connecting cilia and slow retinal degeneration. Moreover, absence of glycylation results in increased levels of tubulin glutamylation in photoreceptors, and inversely, the hyperglutamylation observed in the Purkinje cell degeneration (pcd) mouse abolishes glycylation. This suggests that both posttranslational modifications compete for modification sites, and that unbalancing the glutamylation-glycylation equilibrium on axonemes of connecting cilia, regardless of the enzymatic mechanism, invariably leads to retinal degeneration.

The emerging role of the tubulin code: From the tubulin molecule to neuronal function and disease.

Across different cell types and tissues, microtubules are assembled from highly conserved dimers of α- and β-tubulin. Despite their highly similar structures, microtubules have functional heterogeneity, generated either by the expression of different tubulin genes, encoding distinct isotypes, or by posttranslational modifications of tubulin. This genetically encoded and posttranslational generated heterogeneity of tubulin-the "tubulin code"-has the potential to modulate microtubule structure, dynamics, and interactions with associated proteins. The tubulin code is therefore believed to regulate microtubule functions on a cellular and sub-cellular level. This review highlights the importance of the tubulin code for tubulin structure, as well as on microtubule dynamics and functions in neurons. It further summarizes recent developments in the understanding of mutations in tubulin genes, and how they are linked to neurodegenerative and neurodevelopmental disorders. The current advances in the knowledge of the tubulin code on the molecular and the functional level will certainly lead to a better understanding of how complex signaling events control microtubule functions, especially in cells of the nervous system. © 2016 Wiley Periodicals, Inc.

Tau deletion impairs intracellular β-amyloid-42 clearance and leads to more extracellular plaque deposition in gene transfer models.

BackgroundTau is an axonal protein that binds to and regulates microtubule function. Hyper-phosphorylation of Tau reduces its binding to microtubules and it is associated with β-amyloid deposition in Alzheimer’s disease. Paradoxically, Tau reduction may prevent β-amyloid pathology, raising the possibility that Tau mediates intracellular Aβ clearance. The current studies investigated the role of Tau in autophagic and proteasomal intracellular Aβ1-42 clearance and the subsequent effect on plaque deposition.ResultsTau deletion impaired Aβ clearance via autophagy, but not the proteasome, while introduction of wild type human Tau into Tau−/− mice partially restored autophagic clearance of Aβ1-42, suggesting that exogenous Tau expression can support autophagic Aβ1-42 clearance. Tau deletion impaired autophagic flux and resulted in Aβ1-42 accumulation in pre-lysosomal autophagic vacuoles, affecting Aβ1-42 deposition into the lysosome. This autophagic defect was associated with decreased intracellular Aβ1-42 and increased plaque load in Tau−/− mice, which displayed less cell death. Nilotinib, an Abl tyrosine kinase inhibitor that promotes autophagic clearance mechanisms, reduced Aβ1-42 only when exogenous human Tau was expressed in Tau−/− mice.ConclusionsThese studies demonstrate that Tau deletion affects intracellular Aβ1-42 clearance, leading to extracellular plaque.Electronic supplementary materialThe online version of this article (doi:10.1186/1750-1326-9-46) contains supplementary material, which is available to authorized users.

Identification of 526 Conserved Metazoan Genetic Innovations Exposes a New Role for Cofactor E-like in Neuronal Microtubule Homeostasis

The evolution of metazoans from their choanoflagellate-like unicellular ancestor coincided with the acquisition of novel biological functions to support a multicellular lifestyle, and eventually, the unique cellular and physiological demands of differentiated cell types such as those forming the nervous, muscle and immune systems. In an effort to understand the molecular underpinnings of such metazoan innovations, we carried out a comparative genomics analysis for genes found exclusively in, and widely conserved across, metazoans. Using this approach, we identified a set of 526 core metazoan-specific genes (the ‘metazoanome’), approximately 10% of which are largely uncharacterized, 16% of which are associated with known human disease, and 66% of which are conserved in Trichoplax adhaerens, a basal metazoan lacking neurons and other specialized cell types. Global analyses of previously-characterized core metazoan genes suggest a prevalent property, namely that they act as partially redundant modifiers of ancient eukaryotic pathways. Our data also highlights the importance of exaptation of pre-existing genetic tools during metazoan evolution. Expression studies in C. elegans revealed that many metazoan-specific genes, including tubulin folding cofactor E-like (TBCEL/coel-1), are expressed in neurons. We used C. elegans COEL-1 as a representative to experimentally validate the metazoan-specific character of our dataset. We show that coel-1 disruption results in developmental hypersensitivity to the microtubule drug paclitaxel/taxol, and that overexpression of coel-1 has broad effects during embryonic development and perturbs specialized microtubules in the touch receptor neurons (TRNs). In addition, coel-1 influences the migration, neurite outgrowth and mechanosensory function of the TRNs, and functionally interacts with components of the tubulin acetylation/deacetylation pathway. Together, our findings unveil a conserved molecular toolbox fundamental to metazoan biology that contains a number of neuronally expressed and disease-related genes, and reveal a key role for TBCEL/coel-1 in regulating microtubule function during metazoan development and neuronal differentiation.

TIG3 Interaction at the Centrosome Alters Microtubule Distribution and Centrosome Function

TIG3 is an important pro-differentiation regulator that is expressed in the suprabasal epidermis. We have shown that TIG3 activates selective keratinocyte differentiation-associated processes leading to cornified envelope formation. However, TIG3 also suppresses cell proliferation by an unknown mechanism. Our present studies suggest that cessation of growth is mediated through the impact of TIG3 on the centrosome and microtubules. The centrosome regulates microtubule function in interphase cells and microtubule spindle formation in mitotic cells. We show that TIG3 colocalizes with γ-tubulin and pericentrin at the centrosome. Localization of TIG3 at the centrosome alters microtubule nucleation and reduces anterograde microtubule growth, increases acetylation and detyrosination of α-tubulin, increases insoluble tubulin and drives the formation of a peripheral microtubule ring adjacent to the plasma membrane. In addition, TIG3 suppresses centrosome separation, but not duplication, and reduces cell proliferation. We propose that TIG3 regulates the formation of the peripheral microtubule ring observed in keratinocytes of differentiated epidermis and also has a role in the cessation of proliferation in these cells.

Post-translational regulation of the microtubule cytoskeleton: mechanisms and functions

Half a century of biochemical and biophysical experiments has provided attractive models that may explain the diverse functions of microtubules within cells and organisms. However, the notion of functionally distinct microtubule types has not been explored with similar intensity, mostly because mechanisms for generating divergent microtubule species were not yet known. Cells generate distinct microtubule subtypes through expression of different tubulin isotypes and through post-translational modifications, such as detyrosination and further cleavage to Δ2-tubulin, acetylation, polyglutamylation and polyglycylation. The recent discovery of enzymes responsible for many tubulin post-translational modifications has enabled functional studies demonstrating that these post-translational modifications may regulate microtubule functions through an amazing range of mechanisms.

Phosphorylation of the yeast γ-tubulin Tub4 regulates microtubule function.

The yeast γ-tubulin Tub4 is assembled with Spc97 and Spc98 into the small Tub4 complex. The Tub4 complex binds via the receptor proteins Spc72 and Spc110 to the spindle pole body (SPB), the functional equivalent of the mammalian centrosome, where the Tub4 complex organizes cytoplasmic and nuclear microtubules. Little is known about the regulation of the Tub4 complex. Here, we isolated the Tub4 complex with the bound receptors from yeast cells. Analysis of the purified Tub4 complex by mass spectrometry identified more than 50 phosphorylation sites in Spc72, Spc97, Spc98, Spc110 and Tub4. To examine the functional relevance of the phosphorylation sites, phospho-mimicking and non-phosphorylatable mutations in Tub4, Spc97 and Spc98 were analyzed. Three phosphorylation sites in Tub4 were found to be critical for Tub4 stability and microtubule organization. One of the sites is highly conserved in γ-tubulins from yeast to human.

Microtubule End Binding: EBs Sense the Guanine Nucleotide State

EB proteins accumulate at the tips of growing microtubules and recruit to them a multitude of factors to regulate microtubule functions. A new study suggests that EBs recognize microtubule ends by distinguishing between different states of the tubulin-bound guanine nucleotide.

The budding yeast protein Sum1 functions independently of its binding partners Hst1 and Sir2 histone deacetylases to regulate microtubule assembly

The budding yeast protein Sum1 is a transcription factor that associates with the histone deacetylase Hst1p or, in its absence, with Sir2p to form repressed chromatin. In this study, SUM1 has been identified as an allele-specific dosage suppressor of mutations in the major alpha-tubulin-coding gene TUB1. When cloned in a 2mu vector, SUM1 suppressed the cold-sensitive and benomyl-hypersensitive phenotypes associated with the tub1-1 mutation. The suppression was Hst1p- and Sir2p-independent, suggesting that it was not mediated by deacetylation events associated with Sum1p when it functions along with its known partner histone deacetylases. This protein was confined to the nucleus, but did not colocalize with the microtubules nor did it bind to alpha- or beta-tubulin. Cells deleted of SUM1 showed hypersensitivity to benomyl and cold-sensitive growth, phenotypes exhibited by mutants defective in microtubule function and cytoskeletal defects. These observations suggest that Sum1p is a novel regulator of microtubule function. We propose that as a dosage suppressor, Sum1p promotes the formation of microtubules by increasing the availability of the alphabeta-heterodimer containing the mutant alpha-tubulin subunit.