Writing and Reading the Tubulin Code

Microtubules give rise to intracellular structures with diverse morphologies and dynamics that are crucial for cell division, motility, and differentiation. They are decorated with abundant and chemically diverse posttranslational modifications that modulate their stability and interactions with cellular regulators. These modifications are important for the biogenesis and maintenance of complex microtubule arrays such as those found in spindles, cilia, neuronal processes, and platelets. Here we discuss the nature and subcellular distribution of these posttranslational marks whose patterns have been proposed to constitute a tubulin code that is interpreted by cellular effectors. We review the enzymes responsible for writing the tubulin code, explore their functional consequences, and identify outstanding challenges in deciphering the tubulin code.     

CLASP Mediates Microtubule Repair by Restricting Lattice Damage and Regulating Tubulin Incorporation

1. Download : Download high-res image (225KB)
2. Download : Download full-size image

     

Tubulin cofactor B plays a role in the neuronal growth cone

Tubulin cofactors, initially identified as alpha-, beta-tubulin folding proteins, are now believed to participate in the complex tubulin biogenesis and degradation routes, and thus to contribute to microtubule functional diversity and dynamics. However, a concrete role of tubulin cofactor B (TBCB) remains to be elucidated because this protein is not required for tubulin biogenesis, and it is apparently not essential for life in any of the organisms studied. In agreement with these data, here we show that TBCB localizes at the transition zone of the growth cones of growing neurites during neurogenesis where it plays a role in microtubule dynamics and plasticity. Gene silencing by means of small interfering RNA segments revealed that TBCB knockdown enhances axonal growth. In contrast, excess TBCB, a feature of giant axonal neuropathy, leads to microtubule depolymerization, growth cone retraction, and axonal damage followed by neuronal degeneration. These results provide an important insight into the understanding of the controlling mechanisms of growth cone microtubule dynamics.     

Differences in Intrinsic Tubulin Dynamic Properties Contribute to Spindle Length Control in Xenopus Species

1. Download : Download high-res image (141KB)
2. Download : Download full-size image

     

Structural Characterization of Tau in Fuzzy Tau:Tubulin Complexes

1. Download : Download high-res image (181KB)
2. Download : Download full-size image

     

Tubulin expression in trypanosomes

Microtubules in trypanosomes are the main component of the flagellar axoneme and of the subpellicular microtubule corset, whose relative positions determine the morphology of each cell stage of the life cycle of these parasites. Microtubules are polymers of tubulin, a protein dimer of two 55-kDa subunits, alpha- and beta-tubulin; in Trypanosoma brucei, the tubulin-coding sequences are clustered in a 40-kb fragment of tandemly repeated alpha- and beta-tubulin genes separated by a 170-bp intergenic zone. This cluster is transcribed in a unique RNA which is rapidly processed into mature mRNAs carrying the 5' 35-nucleotide leader sequence found in all trypanosome mRNAs. Although no heterogeneity has been found at the gene level, tubulin can be post-translationally modified in 2 ways: the C-terminal tyrosine of alpha-tubulin can be selectively cleaved and added again with 2 enzymes, tubulin carboxypeptidase and tubulin-tyrosine ligase; alpha-tubulin can also be acetylated on a lysine residue. Some molecular domains of tubulin are restricted to subpopulations of microtubules; for instance, the beta-tubulin form defined by the monoclonal antibody 1B41 is sequestered into a part of the subpellicular cytoskeleton limited to the flagellar adhesion zone, which might correspond to the group of 4 microtubules associated with a cisterna of the endoplasmic reticulum, forming the so-called "subpellicular microtubule quartet" (SFMQ). The early assembly of this zone in each daughter cell during the cell division of T. brucei, together with the alterations undergone by the domain defined by the monoclonal antitubulin 24E3 during the differentiation of Trypanosoma cruzi, suggest that specific tubulin forms are responsible for dynamic properties of SFMQ possibly involved in trypanosome morphogenesis.     

Functional Conservation of Calreticulin in Euglena gracilis

Calreticulin is the major high capacity, low affinity Ca²⁺ binding protein localized within the endoplasmic reticulum. It functions as a reservoir for triggered release of Ca²⁺ by the endoplasmic reticulum and is thus integral to eukaryotic signal transduction pathways involving Ca²⁺ as a second messenger. The early branching photosynthetic protist Euglena gracilis is shown to possess calreticulin as its major high capacity Ca²⁺ binding protein. The protein was purified, microsequenced and cloned. Like its homologues from higher eukaryotes, calreticulin from Euglena possesses a short signal peptide for endoplasmic reticulum import and the C-terminal retention signal KDEL, indicating that these components of the eukaryotic protein routing apparatus were functional in their present form prior to divergence of the euglenozoan lineage. A gene phytogeny for calreticulin and calnexin sequences in the context of eukaryotic homologues indicates i) that these Ca²⁺ binding endoplasmic reticulum proteins descend from a gene duplication that occurred in the earliest stages of eukaryotic evolution and furthermore iii that Euglenozoa express the calreticulin protein of the kinetoplastid (trypanosomes and their relatives) lineage, rather than that of the eukaryotic chlorophyte which gave rise to Euglena's plastids. Evidence for conservation of endoplasmic reticulum routing and Ca²⁺ binding function of calreticulin from Euglena traces the functional history of Ca²⁺ second messenger signal transduction pathways deep into eukaryotic evolution.     

Tubulin domains for the interaction of microtubule associated protein DMAP-85 from Drosophila melanogaster

The interaction of microtubule associated proteins (MAPs) with the microtubule system has been characterized in depth in neuronal cells from various mammalian species. These proteins interact with well-defined domains within the acidic tubulin carboxyl-terminal regulatory region. However, there is little information on the mechanisms of MAPs-tubulin interactions in nonmammalian systems. Recently, a novel tau-like protein designated as DMAP-85 has been identified in Drosophila melanogaster, and the regulation of its interactions with cytoskeletal elements was analyzed throughout different developmental stages of this organism. In this report, the topographic domains involved in the binding of DMAP-85 with tubulin heterodimer were investigated. Affinity chromatography of DMAP-85 in matrixes of taxol-stabilized microtubules showed the reversible interaction of DMAP-85 with domains on the microtubular surface. Co-sedimentation studies using the subtilisin-treated tubulin (S-tubulin) indicated the lack of association of DMAP-85 to this tubulin moiety. Moreover, studies on affinity chromatography of the purified 4 kDa C-terminal tubulin peptide bound to an affinity column, confirmed that DMAP-85 interacts directly with this regulatory domain on tubulin subunits. Further studies on sequencial affinity chromatography using a calmodulin affinity column followed by the microtubule column confirmed the similarities in the interaction behavior of DMAP-85 with that of tau. DMAP-85 associated to both calmodulin and the microtubular polymer. These studies support the idea that the carboxyl-terminal region on tubulin constitutes a common binding domain for most microtubule-interacting proteins.Abbreviations MAPs microtubule-associated proteins - C-terminal carboxyl-terminal - SDS-PAGE polyacrylamide gel electrophoresis in the presence of SDS - DTT dithiotreitol - BSA bovine serum albumin     

Tubulin glycylases are required for primary cilia,control of cell proliferation and tumor development in colon

TTLL3 and TTLL8 are tubulin glycine ligases catalyzing posttranslational glycylation of microtubules. We show here for the first time that these enzymes are required for robust formation of primary cilia. We further discover the existence of primary cilia in colon and demonstrate that TTLL3 is the only glycylase in this organ. As a consequence, colon epithelium shows a reduced number of primary cilia accompanied by an increased rate of cell division in TTLL3-knockout mice. Strikingly, higher proliferation is compensated by faster tissue turnover in normal colon. In a mouse model for tumorigenesis, lack of TTLL3 strongly promotes tumor development. We further demonstrate that decreased levels of TTLL3 expression are linked to the development of human colorectal carcinomas. Thus, we have uncovered a novel role for tubulin glycylation in primary cilia maintenance, which controls cell proliferation of colon epithelial cells and plays an essential role in colon cancer development.     

Heteroxanthin in Euglena gracilis

Summary 1. From a large scale preparation of Euglena gracilis, strain Z, besides the acetylenic pigments diatoxanthin and diadinoxanthin and the allene neoxanthin, an additional acetylenic xanthophyll has been isolated. 2. Mass and IR spectra and chemical reactions showed typical patterns of heteroxanthin from Vaucheria. 3. The pigment was transformed into diadinochrome-isomers with acidified acetone. 4. A partial synthesis of heteroxanthin from diadinoxanthin by LiAlH₄-reduction is described, confirming the structure proposed by Strain. 5. The identity of heteroxanthin with the trollein—like pigment described for Euglena is discussed.     

Proteolysis in Euglena gracilis

Endoproteolytic activities (EC 3.4.22. and 23.) of cell-free extracts of Euglena gracilis, measured by autolysis and azocaseinolysis, vary considerably during the culture growth cycle. They are high in the lag phase, drop sharply up to the mid-logarithmic phase, and then rise again reaching the initial high levels in the stationary phase. This pattern has been observed for both the soluble and the particulate proteolytic activities of four cell types differing with regard to the developmental state of the chloroplast: dark-grown, light-induced, and light-grown wild-type cells, as well as light-grown apoplastic W₃BUL mutant cells, all on a glucose-based medium. Therefore, the activity of the main intracellular proteinases is neither directly nor indirectly light-regulated, but seems to be controlled by the availability of nutrients. Endogenous inhibitors of proteinases could not be detected. Cysteine proteinase activity has been found in the soluble and the particulate fractions, but aspartic proteinase activity in the latter ones only. Different cysteine proteinases may be present in the two fractions, during the different growth phases, and in the four cell types studied.Abbreviations CBB Coomassie Brilliant Blue G-250 - DFP diisopropyl fluorophosphate - EDTA disodium ethylendiaminetetraacetic acid - E-64 l-transepoxysuccinyl-leucyl-amido(4-guanidino)butane - Iog phase logarithmic growth phase - MET 2-mercaptoethanol - PMSF phenylmethylsulfonyl fluoride - Z benzyloxycarbonyl Paper I of this series is Krauspe and Scheer (1986). A preliminary publication appeared (Krauspe et al. 1982)     

Identification of the Tubulin Gene Family and Sequence Determination of One β-Tubulin Gene in a Cold-Poikilotherm Protozoan, the Antarctic Ciliate Euplotes focardii

ABSTRACT. Four different tubulin genes were identified in the somatic nucleus (macronucleus) of Euplotes focardii , a strictly coldadapted, Antarctic ciliate: one of 1,800 bp for α-tubulin and three of 2,150, 1,900, and 1,600 bp, respectively, for β -tubulin. Preliminarily analysed for restriction fragment length polymorphisms, these genes showed remarkable differences in organisation from tubulin genes of other ciliates which live in temperate areas and were analysed in parallel with E. focardii. The complete coding sequence of the 1,600 bp β -tubulin gene was then determined and shown to contain unique structural features of potential importance for E. focardii microtubule organization and activity. Of eight unique substitutions detected, seven were concentrated in the large amino terminal domain of the molecule that directly interacts with the carboxy terminal region of α-tubulin for heterodimer formation. Sequence analysis of the cloned gene revealed, in addition, a potential new exception in the use of the genetic code by ciliates. A TAG codon was aligned in correspondence with Trp-21 which is strictly conserved in every tubulin sequence so far determined.     

Antimutagenicity in Euglena gracilis

The genotoxic effect of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and furadantine (Fu) was significantly decreased by standard antimutagens (ascorbic acid, α-tocopherol, chlorophyllin and sodium selenite) in the unicellular flagellate Euglena gracilis. The effects of these compounds were verified also by a bacterial test in which three strains of Salmonella typhimurium, TA97, TA100 and TA102, were used. The above compounds were antimutagenic in strains of bacteria used, except for chlorophyllin which had no effect on strain TA102.     

Kinesin-5 Promotes Microtubule Nucleation and Assembly by Stabilizing a Lattice-Competent Conformation of Tubulin

1. Download : Download high-res image (220KB)
2. Download : Download full-size image

     

γ‐Tubulin Ring Complexes and EB1 play antagonistic roles in microtubule dynamics and spindle positioning

γ‐Tubulin is critical for microtubule (MT) assembly and organization. In metazoa, this protein acts in multiprotein complexes called γ‐Tubulin Ring Complexes (γ‐TuRCs). While the subunits that constitute γ‐Tubulin Small Complexes (γ‐TuSCs), the core of the MT nucleation machinery, are essential, mutation of γ‐TuRC‐specific proteins in Drosophila causes sterility and morphological abnormalities via hitherto unidentified mechanisms. Here, we demonstrate a role of γ‐TuRCs in controlling spindle orientation independent of MT nucleation activity, both in cultured cells and in vivo, and examine a potential function for γ‐TuRCs on astral MTs. γ‐TuRCs locate along the length of astral MTs, and depletion of γ‐TuRC‐specific proteins increases MT dynamics and causes the plus‐end tracking protein EB1 to redistribute along MTs. Moreover, suppression of MT dynamics through drug treatment or EB1 down‐regulation rescues spindle orientation defects induced by γ‐TuRC depletion. Therefore, we propose a role for γ‐TuRCs in regulating spindle positioning by controlling the stability of astral MTs.     

Peroxidative Activity in Euglena gracilis

Cell-free homogenates of Euglena gracilis contain very low levels of catalase activity as compared to higher plants and some other algae. Purified Euglena cytochrome c acts catalytically as a peroxidase. The observed catalytic activity of cytochrome c in extracts from heterotrophically grown cells was more than enough to account for the observed rates of hydrogen peroxide destruction. The peroxidative activity of Euglena cytochrome c was completely inhibited by 20 mm 3-amino-1,2,4-triazole.     

Thiamin uptake in Euglena gracilis

Thiamin uptake has been investigated in Euglena gracilis Z. This protozoon possessed an active transport system for thiamin with a Km value of 17 nM and a Vmax value of 7.8 pmol per 10(6) cells per min. Thiamin uptake was dependent on pH and temperature, but not on exogenous glucose as an energy source. Oxythiamin and pyrithiamin were competitive inhibitors with Ki values of 33 nM and 15 nM, respectively. Thiamin monophosphate, thiamin pyrophosphate, thiamin triphosphate, heteropyrithiamin, quinolinothiamin, thiamin chloride and amprolium inhibited uptake. Inhibition of thiamin uptake by various metabolic inhibitors and anaerobiosis suggest that thiamin uptake requires an energy source generated by respiration and glycolysis.