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Long-lived microtubules found in ciliary axonemes, neuronal processes, and migrating cells are marked by [alpha]-tubulin acetylation on lysine 40, a modification that takes place inside the microtubule lumen. The physiological importance of microtubule acetylation remains elusive. Here, we identify a BBSome-associated protein that we name [alpha]TAT1, with a highly specific [alpha]-tubulin K40 acetyltransferase activity and a catalytic preference for microtubules over free tubulin, in mammalian cells, the catalytic activity of [alpha]TAT1 is necessary and sufficient for [alpha]-tubulin K40 acetylation. Remarkably, [alpha]TAT1 is universally and exclusively conserved in ciliated organisms, and is required for the acetylation of axonemal microtubules and for the normal kinetics of primary cilium assembly. In Caenorhabditis elegans, microtubule acetylation is most prominent in touch receptor neurons (TRNs) and MEC-17, a homolog of [alpha]TAT1, and its paralog [alpha]TAT-2 are required for [alpha]-tubulin acetylation and for two distinct types of touch sensation. Furthermore, in animals lacking MEC-17, [alpha]TAT-2, and the sole C. elegans K40[alpha]-tubulin MEC-12, touch sensation can be restored by expression of an acetyl-mimic MEC-12[K40Q]. We conclude that [alpha]TAT1 is the major and possibly the sole [alpha]-tubulin K40 acetyltransferase in mammals and nematodes, and that tubulin acetylation plays a conserved role in several microtubule-based processes. cytoskeleton | posttranslational modification | cilia | Bardet-Biedl Syndrome | ciliopathy doi/ 10.1073/pnas.1013728107

[gamma]-Tubulin is critical for the initiation and regulation of microtubule (MT) assembly. In Drosophila melanogaster, it acts within two main complexes: the [gamma]-tubulin small complex ([gamma]-TuSC) and the [gamma]-tubulin ring complex ([gamma]-TuRC). Proteins specific of the [gamma]-TuRC, although nonessential for viability, are required for efficient mitotic progression. Until now, their role during inter-phase remained poorly understood. Using RNA interference in Drosophila S2 cells, we show that the [gamma]-TuRC is not critical for overall MT organization. However, depletion of any component of this complex results in an increase of MT dynamics. Combined immunofluorescence and live imaging analysis allows us to reveal that the [gamma]-TuRC localizes along interphase MTs and that distal [gamma]-tubulin spots match with sites of pause or rescue events. We propose that, in addition to its role in nucleation, the [gamma]-TuRC associated to MTs may regulate their dynamics by limiting catastrophes. doi/10.1083/jcb.200905060

Changes in cellular microtubule organization often accompany developmental progression. In the Caenorhabditis elegans embryo, the centrosome, which is attached to the nucleus via ZYG-12, organizes the microtubule network. In this study, we investigate ZYG-12 function and microtubule organization before embryo formation in the gonad. Surprisingly, ZYG-12 is dispensable for centrosome attachment in the germline. However, ZYG-12-mediated recruitment of dynein to the nuclear envelope is required to maintain microtubule organization, membrane architecture, and nuclear positioning within the syncytial gonad. We examined [gamma]-tubulin localization and microtubule regrowth after depolymerization to identify sites of nucleation in germ cells. [gamma]-Tubulin localizes to the plasma membrane in addition to the centrosome, and regrowth initiates at both sites. Because we do not observe organized microtubules a round zyg-12(ct350) mutant nuclei with attached centrosomes, we propose that gonad architecture, including membrane and nuclear positioning, is determined by microtubule nucleation at the plasma membrane combined with tension on the microtubules by dynein anchored at the nucleus by ZYG-12.

ABSTRACT Microtubules are one of the three primary constituents of the eukaryotic cytoskeleton and are constructed from the protein tubulin. FtsZ is a close structural homologue of tubulin within prokaryotes, [...]

Tubulin cofactor B (TCoB) plays an important role in microtubule dynamics by facilitating the dimerization of [alpha]- and [beta]-tubulin. Recent evidence suggests that p21-activated kinase 1 (Pak1), a major signaling nodule in eukaryotic cells, phosphorylates TCoB on Ser-65 and Ser-128 and plays an essential role in microtubule regrowth. However, to date, no upstream signaling molecules have been identified to antagonize the functions of TCoB, which might help in maintaining the equilibrium of microtubules. Here, we discovered that TCoB is efficiently nitrated, mainly on Tyr-64 and Tyr-98, and nitrated-TCoB attenuates the synthesis of new microtubules. In addition, we found that nitration of TCoB antagonizes signaling-dependent phosphorylation of TCoB, whereas optimal nitration of TCoB requires the presence of functional Pak1 phosphorylation sites, thus providing a feedback mechanism to regulate phosphorylation-dependent MT regrowth. Together these findings identified TCoB as the third cytoskeleton protein to be nitrated and suggest a previously undescribed mechanism, whereby growth factor signaling may coordinately integrate nitric oxide signaling in the regulation of microtubule dynamics.

Porphyromonas gingivalis, an oral pathogen, can internalize within primary gingival epithelial cells (GECs) through an invasion mechanism mediated by interactions between P. gingivalis fimbriae and integrins on the surface of the GECs. Fimbriae--integrin-based signalling events were studied by fluorescence microscopy, and the subcellular localization of integrin-associated signalling molecules paxillin and focal adhesion kinase (FAK), and the architecture of the actin and microtubule cytoskeleton were examined. GECs infected with P. gingivalis for 30 min demonstrated significant redistribution of paxillin and FAK from the cytosol to cell peripheries and assembly into focal adhesion complexes. In contrast, a fimbriae-deficient mutant of P. gingivalis did not contribute substantially to activation of paxillin or FAK. After 24 h, the majority of paxillin and FAK had returned to the cytoplasm with significant co-localization with P. gingivalis in the perinuclear region. Wild-type P. gingivalis induced nucleation of actin filaments forming microspike-like protrusions and long stable microfilaments distributed throughout the cells. Fimbriae mutants promoted a rich cortical actin meshwork accompanied by membrane ruffling dispersed along the cell membrane. Remarkable disassembly and nucleation of the actin and microtubule filamentous network was observed following 24 h infection with either wild-type or fimbriae-deficient mutants of P. gingivalis. The results show that fimbriated P. gingivalis cells induce formation of integrin-associated focal adhesions with subsequent remodelling of the actin and tubulin cytoskeleton.

Near-simultaneous three-dimensional fluorescence/ differential interference contrast microscopy was used to follow the behavior of microtubules and chromosomes in living a-tubulin/GFP-expressing cells after inhibition of the mitotic kinesin Eg5 with monastrol. Kinetochore fibers (K-fibers) were frequently observed forming in association with chromosomes both during monastrol treatment and after monastrol removal. Surprisingly, these K-fibers were oriented away from, and not directly connected to, centrosomes and incorporated into the spindle by the sliding of their distal ends toward centrosomes via a NuMA-dependent mechanism. Similar preformed K-fibers were also observed during spindle formation in untreated cells. In addition, upon monastrol removal, centrosomes established a transient chromosome-free bipolar array whose orientation specified the axis along which chromosomes segregated. We propose that the capture and incorporation of preformed K-fibers complements the microtubule plus-end capture mechanism and contributes to spindle formation in vertebrates.

Increased flux through the hexosamine biosynthesis pathway has been implicated in the development of glucose-induced insulin resistance and may promote the modification of certain proteins with O-linked N-acetylglucosamine (O-GlcNAc). L6 myotubes (a model of skeletal muscle) were incubated for 18 h in 5 or 25 mM glucose with or without 10 nM insulin. As assessed by immunoblotting with an O-GlcNAc-specific antibody, high glucose and/or insulin enhanced O-GlcNAcylation of numerous proteins, including the transcription factor Sp1, a known substrate for this modification. To identify novel proteins that may be O-GlcNAc modified in a glucose concentration/insulin-responsive manner, total cell membranes were separated by one- or two-dimensional gel electrophoresis. Selected O-GlcNAcylated proteins were identified by mass spectrometry (MS) analysis. MS sequencing of tryptic peptides identified member(s) of the heat shock protein 70 (HSP70) family and rat [alpha]-tubulin. Immunoprecipitation/immunoblot studies demonstrated several HSP70 isoforms and/or posttranslational modifications, some with selectively enhanced O-GlcNAcylation following exposure to high glucose plus insulin. In conclusion, in L6 myotubes, Sp1, membrane-associated HSP70, and [alpha]-tubulin are O-GlcNAcylated; the modification is markedly enhanced by sustained increased glucose flux. insulin resistance; L6 myotubes; O-linked N-acetylglucosamine; heat shock protein 70

Myosin light chain phosphatase (PP1M) is composed of three subunits, i.e., M20, MBS, and a catalytic subunit. Whereas MBS is assigned as a myosin binding subunit, the function of M20 is unknown. In the present study, we found that M20 binds to microtubules. The binding activity was revealed by cosedimentation of M20 with microtubules and binding of tubulin to M20 affinity resin. Green fluorescent protein (GFP)-tagged M20 (M20-GFP) was expressed in chicken primary smooth muscle cells and COS-7 cells and was used as a probe for studying the association between M20 and microtubules in living cells. M20-GFP was localized on filamentous structures in both cell types. Colocalization analysis revealed that M20-GFP colocalized with tubulin. Treatment with nocodazole, but not cytochalasin B, abolished the filamentous structure of M20-GFP. These results indicate that M20-GFP associates with microtubules in cells. Microinjection of rhodamine-tubulin into the M20-expressing cells revealed that incorporation of rhodamine-tubulin into microtubules was significantly facilitated by microtubule-associated M20. Consistent with this result, M20 enhanced the rate of tubulin polymerization in vitro and produced elongated microtubules. These results suggest that M20 has a microtubule binding activity and plays a role in regulating microtubule dynamics. M20; tubulin; myosin light chain phosphatase; protein phosphatase; green fluorescent protein

Tubulins, the protein constituents of the microtubule cytoskeleton, are present in all known eukaryotes but have never been found in the Bacteria or Archaea. Here we report the presence of two tubulin-like genes [bacterial tubulin a (btuba) and bacterial tubulin b (btubb)] in bacteria of the genus Prosthecobacter (Division Verrucomicrobia). In this study, we investigated the organization and expression of these genes and conducted a comparative analysis of the bacterial and eukaryotic protein sequences, focusing on their phylogeny and 3D structures. The btuba and btubb genes are arranged as adjacent loci within the genome along with a kinesin light chain gene homolog. RT-PCR experiments indicate that these three genes are cotranscribed, and a probable promoter was identified upstream of btuba. On the basis of comparative modeling data, we predict that the Prosthecobacter tubulins are monomeric, unlike eukaryotic [alpha] and [beta] tubulins, which form dimers and are therefore unlikely to form microtubule-like structures. Phylogenetic analyses indicate that the Prosthecobacter tubulins are quite divergent and do not support recent horizontal transfer of the genes from a eukaryote. The discovery of genes for tubulin in a bacterial genus may offer new insights into the evolution of the cytoskeleton.

Research has been conducted on the antimitotic agent vinblastine used in cancer chemotherapy. The preparation of vinblastine derivative which retains high affinity for tubulin is described.

Research has been conducted on the dietary flavonoid quercetin. Results demonstrate that quercetin inhibits microtubule polymerization and depolymerizes microtubules made from purified tubulin.

Previously we demonstrated that specific chromatographically resolvable isoforms of rabbit brain GAPDH catalyze either glycolytic flux or membrane fusion activity (but not both). Moreover, GAPDH membrane fusion activity was latent until it was separated from an endogenous cytosolic inhibitor by anion-exchange chromatography. Herein we demonstrate that the cytosolic inhibitor is nondialyzable, heat-labile, and trypsin-sensitive, thereby identifying it as a cytosolic protein constituent. Chromatographic purification of the rabbit-brain cytosolic protein inhibitor of GAPDH isoform-catalyzed membrane fusion identified a predominant 55-kDa doublet that contained an internal 15-aa peptide identical to a sequence present in [alpha]-tubulin (residues 65-79). The identity of the 55-kDa doublet as tubulin was substantiated through Western blot analysis and inhibition of GAPDH-catalyzed membrane fusion by authentic tubulin. Stopped-flow kinetic analysis demonstrated the high-affinity, rapid, and direct modulation of GAPDH-catalyzed fusion activity by tubulin. Because GTP-activated Rab 2 recruits GAPDH to membranes about to undergo fusion [Tisdale, E. J. (2001) J. Biol. Chem. 276, 2480-2486] and protein kinase [C.sub.[iota]]/[lambda] phosphorylates GAPDH modulating its interactions with tubulin [Tisdale, E. J. (2001) J. Biol. Chem. 277, 3334-3341], the present study suggests a coordinated mechanism through which membrane trafficking and cellular signaling can be integrated with glycolytic flux.

Research has been conducted on the cryptophycin-tubulin complexes. The study of these complexes carried out biochemically and via cryoelectron microscopy in investigating the hypothesis that the dimer's curvature is induced in tubulin dimers' linear polymers is presented.

When mammalian somatic cells enter mitosis, a fundamental reorganization of the Mt cytoskeleton occurs that is characterized by the loss of the extensive interphase Mt array and the formation of a bipolar mitotic spindle. Microtubules in cells stably expressing GFP--[alpha]-tubulin were directly observed from prophase to just after nuclear envelope breakdown (NEBD) in early prometaphase. Our results demonstrate a transient stimulation of individual Mt dynamic turnover and the formation and inward motion of microtubule bundles in these cells. Motion of microtubule bundles was inhibited after antibody-mediated inhibition of cytoplasmic dynein/dynactin, but was not inhibited after inhibition of the kinesin-related motor Eg5 or myosin II. In metaphase cells, assembly of small foci of Mts was detected at sites distant from the spindle; these Mts were also moved inward. We propose that cytoplasmic dynein-dependent inward motion of Mts functions to remove Mts from the cytoplasm at prophase and from the peripheral cytoplasm through metaphase. The data demonstrate that dynamic astral Mts search the cytoplasm for other Mts, as well as chromosomes, in mitotic cells.

The female meiotic spindle lacks a centrosome or microtubule-organizing center in many organisms. During cell division, these spindles are organized by the chromosomes and microtubule-associated proteins. Previous studies in Drosophila melanogaster implicated at least one kinesin motor protein, NCD, in tapering the microtubules into a bipolar spindle. We have identified a second Drosophila kinesin-like protein, SUB, that is required for meiotic spindle function. At meiosis I in males and females, sub mutations affect only the segregation of homologous chromosomes. In female meiosis, sub mutations have a similar phenotype to ncd; even though chromosomes are joined by chiasmata they fail to segregate at meiosis I. Cytological analyses have revealed that sub is required for bipolar spindle formation. In sub mutations, we observed spindles that were unipolar, multipolar, or frayed with no defined poles. On the basis of these phenotypes and the observation that sub mutations genetically interact with ncd, we propose that SUB is one member of a group of microtubule-associated proteins required for bipolar spindle assembly in the absence of the centrosomes, sub is also required for the early embryonic divisions but is otherwise dispensable for most mitotic divisions.

Research has been conducted on the antimitotic agents epothilone and paclitaxel which promote microtubule stabilization and mammalian tubulin assembly. Results indicate that epothilone stimulates microtubule formation from the purified yeast tubulin.

Toxoplasma gondii is an obligatory intracellular parasite, an important human pathogen, and a convenient laboratory model for many other human and veterinary pathogens in the phylum Apicomplexa, such as Plasmodium, Eimeria, and Cryptosporidia. 22 subpellicular microtubules form a scaffold that defines the cell shape of T. gondii. Its cytoskeleton also includes an intricate apical structure consisting of the conoid, two intraconoid microtubules, and two polar rings. The conoid is a 380-nm diameter motile organelle, consisting of fibers wound into a spiral like a compressed spring. FRAP analysis of transgenic T. gondii expressing YFP-[alpha]-tubulin reveals that the conoid fibers are assembled by rapid incorporation of tubulin subunits during early, but not late, stages of cell division. Electron microscopic analysis shows that in the mature conoid, tubulin is arranged into a novel polymer form that is quite different from typical microtubules.

The microtubule cytoskeleton plays a fundamental role in cell organization and membrane traffic in higher eukaryotes. It is well established that molecular motors are involved in membrane--microtubule interactions, but it has also been proposed that nonmotor microtubule-binding (MTB) proteins known as CLIPs (cytoplasmic linker proteins) have basic roles in these processes. We report here the characterization of CLIPR-59, a CLIP-170--related protein localized to the trans-most part of the Golgi apparatus. CLIPR-59 contains an acidic region followed by three ankyrin-like repeats and two CLIP-170--related MTB motifs. We show that the 60--amino acid-long carboxy-terminal domain of CLIPR-59 is necessary and sufficient to achieve Golgi targeting, which represents the first identification of a membrane targeting domain in a CLIP-170--related protein. The MTB domain of CLIPR-59 is functional because it localizes to microtubules when expressed as a fragment in HeLa cells. However, our results suggest that this domain is normally inhibited by the presence of adjacent domains, because neither full-length CLIPR-59 nor a CLIPR-59 mutant missing its membrane-targeting region localize to microtubules. Consistent with this observation, over-expression of CLIPR-59 does not affect the microtubule network. However, CLIPR-59 overexpression strongly perturbs early/recycling endosome--TGN dynamics, implicating CLIPR-59 in the regulation of this pathway.

Research has been conducted on the stabilizing cap at microtubule ends. Results indicate that this cap consists of the single layer tubulin GDP-P (sub)i subunits and that the P (sub)i release mechanism destabilizes GDP-tubulin core.

The topography of microtubule assembly events during meiotic maturation of animal oocytes demands tight spatial control and temporal precision. To better understand what regulates the timing and location of microtubule assembly, synchronously maturing mouse oocytes were evaluated with respect to [gamma]-tubulin, pericentrin, and total tubulin polymer fractions at specific stages of meiotic progression. [gamma]-Tubulin remained associated with cytoplasmic centrosomes through diakinesis of meiosis-1. Following chromatin condensation and perinuclear centrosome aggregation, [gamma]-tubulin relocated to a nuclear lamina-bounded compartment in which meiosis-1 spindle assembly occurred. [gamma]-Tubulin was stably associated with the meiotic spindle from prometaphase-1 through to anaphase-2, but also exhibited cell cycle-specific relocalization to cytoplasmic centrosomes. Specifically, anaphase onset of both meiosis-1 and -2 was characterized by the concomitant appearance of [gamma]-tubulin and microtubule nucleation in subcortical centrosomes. Brief pulses of taxol applied at specific cell cycle stages enhanced detection of [gamma]-tubulin compartmentalization, consistent with a [gamma]-tubulin localization-dependent spatial restriction of microtubule assembly during meiotic progression. In addition, a taxol pulse during meiotic resumption impaired subsequent [gamma]-tubulin sorting, resulting in monopolar spindle formation and cell cycle arrest in meiosis-1; despite cell cycle arrest, polar body extrusion occurred roughly on schedule. Therefore, sorting of [gamma]-tubulin is involved in both the timing of location of meiotic spindle assembly as well as the coordination of karyokinesis and cytokinesis in mouse oocytes. Key Words: [gamma]-tubulin; centrosome; spindle; nuclear lamina; oocyte; meiosis; nuclear; cytoplasmic maturation.

Research has been conducted on the synthetic compound of the antitumor agents' cryptophycin family, cryptophycin 52. The ability of cryptophycin 52 to inhibit tubulin self-assembly into microtubules has been investigated and discussed.

Research has been conducted on the tubulin cysteine residues involved in regulating ligand interactions and microtubule formation. Results indicate that oxidized state of tubulin is responsible for the specific chaperone-assisted refolding and that the oxidation or reduction of the cell status can be a critical factor for microtubule assemble regulation.

Research demonstrates that kinetochores comprising of molecular motors and microtubule associated proteins collectively mediate binding of chromosomes to spindle microtubules, imposition of tension to the paired sister kinetochores, and correct chromosome movement during mitosis.

The authors review publications on the ability of the living organisms to place the division site at a specified position within the cell. The topics of interest include the polar zone assembly and disassembly, the MinCDE protein functions and the E-ring formation.

Research has been conducted on the microtubule architecture. The effect of the lateral contacts between tubulin subunits in protofilaments on microtubule stability, rigidity and architecture has been investigated.

Research has been conducted on the D-secopaclitaxel analogues. The synthesis of these analogues from the paclitaxel has been carried out and the results are discussed.

Research has been conducted on the rhazinilam and phenylpyridine analogues. The synthesis of these compounds, their antitubulin activity and atropoisomeric structures have been investigated and are discussed.

Research demonstrates that multiple isoforms of tubulin can form microtubules in the absence of a 'carrier' tubulin. Data show that the microtubules are composed of one each of alpha- and beta-isoforms indicating that no posttranslational modification is involved during their folding into tubulin.

Research reveals that cryptophycin-52 binds rapidly, noncovalently, and mostly irreversibly to tubulin. The complex is denatured by either urea or boiling. The binding results in a conformational change in the tubulin.

Oligomeric tubulin in large transporting complex has been found to be transported via kinesin in squid giant axons. The movement of fluorescence was monitored with confocal laser scanning microscopy and fluorescence correlation spectroscopy after injection of the axons with fluorescence-labeled tubulin

Phosphoproteins of the stathmin family have been studied. The 4-angstrom X-ray structure of a GDP-tubulin with the Escherichia coli-expressed stathmin-like domain of RB3, a stathmin family protein, has been described. The RB3 extensively contacts tubulin through a 91-residue alpha helix.

The binding affinities for binding of four purine nucleoside triphosphates to E-site nucleotide- and nucleoside diphosphate kinase-depleted tubulin have been found. Nucleoside triphosphate specificity of tubulin is discussed. Tubulin shows specificity for the guanine nucleotides.