Tubulin Isoforms Research Articles

Changes in αB-crystallin, tubulin, and MHC isoforms by hindlimb unloading show different expression patterns in various hindlimb muscles.

[Purpose]αB-crystallin is a small heat shock protein that acts as a molecular chaperone under various stress conditions. Microtubules, which consist of tubulin, are related to maintain the intracellular organelles and cellular morphology. These two proteins have been shown to be related to the properties of different types of myofibers based on their contractile properties. The response of these proteins during muscular atrophy, which induces a myofibril component change, is not clearly understood.[Methods]We performed 15 days of hindlimb unloading on rats to investigate the transitions of these proteins by analyzing their absolute quantities. Protein contents were analyzed in the soleus, plantaris, and gastrocnemius muscles of the unloading and control groups (N = 6).[Results]All three muscles were significantly atrophied by hindlimb unloading (P < 0.01): soleus (47.5%), plantaris (16.3%), and gastrocnemius (21.3%) compared to each control group. αB-crystallin was significantly reduced in all three examined unloaded hindlimb muscles compared to controls (P < 0.01) during the transition of the myosin heavy chain to fast twitch muscles. α-Tubulin responded only in the unloaded soleus muscle. Muscle atrophy induced the reduction of αB-crystallin and α-tubulin expressions in plantar flexor muscles with a shift to the fast muscle fiber compared to the control.[Conclusion]The novel finding of this study is that both proteins, αB-crystallin and α-tubulin, were downregulated in slow muscles (P < 0.01); However, α-tubulin was not significantly reduced compared to the control in fast muscles (P < 0.01).

Multiple mechanisms underlying acquired resistance to taxanes in selected docetaxel-resistant MCF-7 breast cancer cells

BackgroundChemoresistance is a major factor involved in a poor response and reduced overall survival in patients with advanced breast cancer. Although extensive studies have been carried out to understand the mechanisms of chemoresistance, many questions remain unanswered.MethodsIn this research, we used two isogenic MCF-7 breast cancer cell lines selected for resistance to doxorubicin (MCF-7DOX) or docetaxel (MCF-7TXT) and the wild type parental cell line (MCF-7CC) to study mechanisms underlying acquired resistance to taxanes in MCF-7TXT cells. Cytotoxicity assay, immunoblotting, indirect immunofluorescence and live imaging were used to study the drug resistance, the expression levels of drug transporters and various tubulin isoforms, apoptosis, microtubule formation, and microtubule dynamics.ResultsMCF-7TXT cells were cross resistant to paclitaxel, but not to doxorubicin. MCF-7DOX cells were not cross-resistant to taxanes. We also showed that multiple mechanisms are involved in the resistance to taxanes in MCF-7TXT cells. Firstly, MCF-7TXT cells express higher level of ABCB1. Secondly, the microtubule dynamics of MCF-7TXT cells are weak and insensitive to the docetaxel treatment, which may partially explain why docetaxel is less effective in inducing M-phase arrest and apoptosis in MCF-7TXT cells in comparison with MCF-7CC cells. Moreover, MCF-7TXT cells express relatively higher levels of β2- and β4-tubulin and relatively lower levels of β3-tubulin than both MCF-7CC and MCF-7DOX cells. The subcellular localization of various β-tubulin isoforms in MCF-7TXT cells is also different from that in MCF-7CC and MCF-7DOX cells.ConclusionMultiple mechanisms are involved in the resistance to taxanes in MCF-7TXT cells. The high expression level of ABCB1, the specific composition and localization of β-tubulin isoforms, the weak microtubule dynamics and its insensitivity to docetaxel may all contribute to the acquired resistance of MCF-7TXT cells to taxanes.

N-Formyl-7-amino-11-cycloamphilectene, a marine sponge metabolite, binds to tubulin and modulates microtubule depolymerization

The importance of protein-small molecule interaction in drug discovery, medicinal chemistry and biology has driven the development of new analytical methods to disclose the whole interactome of bioactive compounds. To accelerate targets discovery of N-formyl-7-amino-11-cycloamphilectene (CALe), a marine bioactive diterpene isolated from the Vanuatu sponge Axinella sp., a chemoproteomic-based approach has been successfully developed. CALe is a potent anti-inflammatory agent, modulating NO and prostaglandin E2 overproduction by dual inhibition of the enhanced inducible NO synthase expression and cyclo-oxygenase-2 activity, without any evidence of cytotoxic effects. In this paper, several isoforms of tubulin have been identified as CALe off-targets by chemical proteomics combined with bio-physical orthogonal approaches. In the following biological analysis of its cellular effect, CALe was found to protect microtubules against the colcemid depolymerizing effect.

A cellular genome-wide association study reveals human variation in microtubule stability and a role in inflammatory cell death

Pyroptosis is proinflammatory cell death that occurs in response to certain microbes. Activation of the protease caspase-1 by molecular platforms called inflammasomes is required for pyroptosis. We performed a cellular genome-wide association study (GWAS) using Salmonella typhimurium infection of human lymphoblastoid cell lines as a means of dissecting the genetic architecture of susceptibility to pyroptosis and identifying unknown regulatory mechanisms. Cellular GWAS revealed that a common human genetic difference that regulates pyroptosis also alters microtubule stability. An intergenic single-nucleotide polymorphism on chromosome 18 is associated with decreased pyroptosis and increased expression of TUBB6 (tubulin, β 6 class V). TUBB6 is unique among tubulin isoforms in that its overexpression can completely disrupt the microtubule network. Cells from individuals with higher levels of TUBB6 expression have lower microtubule stability and less pyroptosis. Reducing TUBB6 expression or stabilizing microtubules pharmacologically with paclitaxel (Taxol) increases pyroptosis without affecting the other major readout of caspase-1 activation, interleukin-1β secretion. The results reveal a new role for microtubules and possibly specific tubulin isoforms in the execution of pyroptosis. Furthermore, the finding that there is common diversity in TUBB6 expression and microtubule stability could have broad consequences for other microtubule-dependent phenotypes, diseases, and pharmacological responses.

Beta tubulin isoforms are not interchangeable for rescuing impaired radial migration due to Tubb3 knockdown

Over the last years, the critical role of cytoskeletal proteins in cortical development including neuronal migration as well as in neuronal morphology has been well established. Inputs from genetic studies were provided through the identification of several mutated genes encoding either proteins associated with microtubules (DCX, LIS1, KIF2A, KIF5C, DYNC1H1) or tubulin subunits (TUBA1A, TUBB2B, TUBB5 and TUBG1), in malformations of cortical development (MCD). We also reported the identification of missense mutations in TUBB3, the postmitotic neuronal specific tubulin, in six different families presenting either polymicrogyria or gyral disorganization in combination with cerebellar and basal ganglial abnormalities. Here, we investigate further the association between TUBB3 mutations and MCDs by analyzing the consequences of Tubb3 knockdown on cortical development in mice. Using the in utero-electroporation approach, we demonstrate that Tubb3 knockdown leads to delayed bipolar morphology and radial migration with evidence, suggesting that the neuronal arrest is a transient phenomenon overcome after birth. Silenced blocked cells display a round-shape and decreased number of processes and a delay in the acquisition of the bipolar morphology. Also, more Tbr2 positive cells are observed, although less cells express the proliferation marker Ki67, suggesting that Tubb3 inactivation might have an indirect effect on intermediate progenitor proliferation. Furthermore, we show by rescue experiments the non-interchangeability of other beta-tubulins which are unable to rescue the phenotype. Our study highlights the critical and specific role of Tubb3 on the stereotyped morphological changes and polarization processes that are required for initiating radial migration to the cortical plate.

MAPping out distribution routes for kinesin couriers.

In the crowded environment of eukaryotic cells, diffusion is an inefficient distribution mechanism for cellular components. Long-distance active transport is required and is performed by molecular motors including kinesins. Furthermore, in highly polarised, compartmentalised and plastic cells such as neurons, regulatory mechanisms are required to ensure appropriate spatio-temporal delivery of neuronal components. The kinesin machinery has diversified into a large number of kinesin motor proteins as well as adaptor proteins that are associated with subsets of cargo. However, many mechanisms contribute to the correct delivery of these cargos to their target domains. One mechanism is through motor recognition of sub-domain-specific microtubule (MT) tracks, sign-posted by different tubulin isoforms, tubulin post-translational modifications, tubulin GTPase activity and MT-associated proteins (MAPs). With neurons as a model system, a critical review of these regulatory mechanisms is presented here, with a particular focus on the emerging contribution of compartmentalised MAPs. Overall, we conclude that - especially for axonal cargo - alterations to the MT track can influence transport, although in vivo, it is likely that multiple track-based effects act synergistically to ensure accurate cargo distribution.

PHLP2 is essential and plays a role in ciliogenesis and microtubule assembly in Tetrahymena thermophila

Recent studies have implicated the phosducin-like protein-2 (PHLP2) in regulation of CCT, a chaperonin whose activity is essential for folding of tubulin and actin. However, the exact molecular function of PHLP2 is unclear. Here we investigate the significance of PHLP2 in a ciliated unicellular model, Tetrahymena thermophila, by deleting its single homolog, Phlp2p. Cells lacking Phlp2p became larger and died within 96 h. Overexpressed Phlp2p-HA localized to cilia, basal bodies, and cytosol without an obvious change in the phenotype. Despite similar localization, overexpressed GFP-Phlp2p caused a dominant-negative effect. Cells overproducing GFP-Phlp2p had decreased rates of proliferation, motility and phagocytosis, as compared to wild type cells or cells overproducing a non-tagged Phlp2p. Growing GFP-Phlp2p-overexpressing cells had fewer cilia and, when deciliated, failed to regenerate cilia, indicating defects in cilia assembly. Paclitaxel-treated GFP-Phlp2p cells failed to elongate cilia, indicating a change in the microtubules dynamics. The pattern of ciliary and cytosolic tubulin isoforms on 2D gels differed between wild type and GFP-Phlp2p-overexpressing cells. Thus, in Tetrahymena, PhLP2 is essential and under specific experimental conditions its activity affects tubulin and microtubule-dependent functions including cilia assembly.

Difficulties in Generating Specific Antibodies for Immunohistochemical Detection of Nitrosylated Tubulins

Protein S-nitrosylation, the covalent attachment of a nitroso moiety to thiol groups of specific cysteine residues, is one of the major pathways of nitric oxide signaling. Hundreds of proteins are subject to this transient post-translational modification and for some the functional consequences have been identified. Biochemical assays for the analysis of protein S-nitrosylation have been established and can be used to study if and under what conditions a given protein is S-nitrosylated. In contrast, the equally desirable subcellular localization of specific S-nitrosylated protein isoforms has not been achieved to date. In the current study we attempted to specifically localize S-nitrosylated α- and β-tubulin isoforms in primary neurons after fixation. The approach was based on in situ replacement of the labile cysteine nitroso modification with a stable tag and the subsequent use of antibodies which recognize the tag in the context of the tubulin polypeptide sequence flanking the cysteine residue of interest. We established a procedure for tagging S-nitrosylated proteins in cultured primary neurons and obtained polyclonal anti-tag antibodies capable of specifically detecting tagged proteins on immunoblots and in fixed cells. However, the antibodies were not specific for tubulin isoforms. We suggest that different tagging strategies or alternative methods such as fluorescence resonance energy transfer techniques might be more successful.

Tubulin posttranslational modifications induced by cadmium in the sponge Clathrina clathrus

As sessile filter feeders, sponges are exposed to environmental stress due to pollutants of both anthropogenic and natural origins and are able to accumulate harmful substances. Thus, sponges are considered a good tool for the biomonitoring of coastal areas. In this study, we used biochemical and immunocytochemical analyses to provide new data on the cadmium-related changes in sponge cells. In particular, we analyzed the effects of different concentrations of cadmium on the microtubule network in the calcisponge Clathrina clathrus. Quantitative densitometry of the immunoblots showed that, while the levels of α- and β-tubulin remained relatively constant in C. clathrus when exposed to 1 and 5μM CdCl2, there were progressive shifts in the levels of some tubulin isoforms. Exposure for 24h to sublethal concentrations of cadmium reduced the level of tyrosinated α-tubulin and enhanced the levels of acetylated and detyrosinated α-tubulin relative to the levels in controls. Confocal microscopy analysis of immunolabeled tissue sections showed that the inhibitory effect of cadmium was associated with a decrease in the labeling of the cells with a monoclonal antibody that recognizes tyrosinated α-tubulin. By contrast, the reactivity with a monoclonal antibody that recognizes acetylated α-tubulin and with a polyclonal antibody specific for detyrosinated α-tubulin was enhanced at the same time points. Because the acetylation and detyrosination of α-tubulin occur on stable microtubules, the marked enhancement of α-tubulin acetylation and detyrosination in Cd2+-treated cells indicates that divalent Cd ions stabilize microtubules. The possibility that Cd2+ may increase the stability of cytoplasmic microtubules was tested by exposing Cd2+-treated cells to a cold temperature (0°C). As shown, the microtubule bundles induced by Cd2+, which were labeled by the monoclonal antibodies against acetylated and detyrosinated α-tubulin, were resistant to cold.

New Model of Action for Mood Stabilizers: Phosphoproteome from Rat Pre-Frontal Cortex Synaptoneurosomal Preparations

BackgroundMitochondrial short and long-range movements are necessary to generate the energy needed for synaptic signaling and plasticity. Therefore, an effective mechanism to transport and anchor mitochondria to pre- and post-synaptic terminals is as important as functional mitochondria in neuronal firing. Mitochondrial movement range is regulated by phosphorylation of cytoskeletal and motor proteins in addition to changes in mitochondrial membrane potential. Movement direction is regulated by serotonin and dopamine levels. However, data on mitochondrial movement defects and their involvement in defective signaling and neuroplasticity in relationship with mood disorders is scarce. We have previously reported the effects of lithium, valproate and a new antipsychotic, paliperidone on protein expression levels at the synaptic level.HypothesisMitochondrial function defects have recently been implicated in schizophrenia and bipolar disorder. We postulate that mood stabilizer treatment has a profound effect on mitochondrial function, synaptic plasticity, mitochondrial migration and direction of movement.MethodsSynaptoneurosomal preparations from rat pre-frontal cortex were obtained after 28 daily intraperitoneal injections of lithium, valproate and paliperidone. Phosphorylated proteins were identified using 2D-DIGE and nano LC-ESI tandem mass spectrometry.ResultsLithium, valproate and paliperidone had a substantial and common effect on the phosphorylation state of specific actin, tubulin and myosin isoforms as well as other proteins associated with neurofilaments. Furthermore, different subunits from complex III and V of the electron transfer chain were heavily phosphorylated by treatment with these drugs indicating selective phosphorylation.ConclusionsMood stabilizers have an effect on mitochondrial function, mitochondrial movement and the direction of this movement. The implications of these findings will contribute to novel insights regarding clinical treatment and the mode of action of these drugs.

Randomized Phase II Study of Ixabepilone or Paclitaxel Plus Carboplatin in Patients With Non–Small-Cell Lung Cancer Prospectively Stratified by Beta-3 Tubulin Status

Retrospective studies have reported that tumor expression of the beta-3 tubulin (β3T) isoform is an unfavorable prognostic factor in non-small-cell lung cancer (NSCLC) treated with tubulin-inhibiting chemotherapy. Ixabepilone is a tubulin-inhibiting agent with low susceptibility to multiple resistance mechanisms including β3T isoform expression in several tumor models. This randomized phase II study evaluated ixabepilone-based chemotherapy in stage IIIb/IV NSCLC, compared with paclitaxel-based chemotherapy. Tumor specimens were prospectively evaluated for β3T expression. Patients were stratified by β3T status (positive v negative) and randomly assigned at a ratio of 1:1 to receive ixabepilone (32 mg/m(2)) and carboplatin (area under concentration-time curve [AUC], 6) or paclitaxel (200 mg/m(2)) and carboplatin (AUC, 6) for up to six cycles. The primary end point was progression-free survival (PFS) in the β3T-positive subgroup. Ninety-five patients (β3T positive, 52; β3T negative, 43) received ixabepilone plus carboplatin; 96 patients (β3T positive, 49; β3T negative, 47) received paclitaxel plus carboplatin. No significant differences in median PFS were observed between arms for either subgroup (β3T positive, 4.3 months in both arms; β3T negative, 5.8 v 5.3 months). Ixabepilone did not significantly improve overall survival (OS) for the β3T-positive subset or the overall population. Adverse events were similar between the two arms and comparable with those in previous studies. There was no predictive value of β3T in differentiating clinical activity of ixabepilone- or paclitaxel-containing regimens. Ixabepilone did not improve PFS or OS in patients with β3T-positive tumors. β3T-positive patients had worse PFS relative to β3T-negative patients, regardless of treatment; hence, β3T expression seems to be a negative prognostic factor, but not a predictive factor, in advanced NSCLC treated with either ixabepilone or paclitaxel platinum-based doublets.

RCB20, an experimental benzimidazole derivative, affects tubulin expression and induces gross anatomical changes in Taenia crassiceps cysticerci

Helminth β-tubulins are the targets of benzimidazole (BZM) carbamate compounds. The specificity of the interactions between such compounds and their in vivo targets depends on the presence of specific amino acid residues in the target molecules. To discover new and effective anthelmintic drugs, we used a medicinal chemistry approach to synthesize a series of BZM derivatives that exploited the BZM moiety as a template. We have previously found that one compound, 2-(trifluoromethyl)-1H-benzimidazole (RCB20), has better in vitro and in vivo activity than albendazole sulfoxide (ABZSO). In the present study, the effect of RCB20 and ABZSO treatment on expression of Taenia crassiceps cysticerci cytoskeletal proteins such as actin, myosin II, and tubulin isoforms was examined. The effects of RCB20 and ABZSO after 11days treatment of the parasites was evaluated by light, confocal, and electron microscopy, and by immunochemistry and immunohistochemistry. The RCB20-induced effects were more rapid than the ABZSO-induced effects on the parasites. In the RCB20-treated parasites, we observed gross-structural damage at the whole parasite level, particularly in the inner tissues and flame cells. Changes in the expression patterns of the cytoskeletal proteins, as assessed by immunohistochemistry and immunoblotting, revealed that the most important drug-induced effect on the parasites was a reduction in the expression level of tyrosinated α-tubulins. Our research findings suggest that RCB20 treatment affected posttranslational modification of parasite α-tubulin molecules, which involved removal of the α-tubulin carboxy-terminal tyrosine.

Direct binding of TUBB3 with DCC couples netrin-1 signaling to intracellular microtubule dynamics in axon outgrowth and guidance

The coupling of axon guidance cues, such as netrin-1, to microtubule (MT) dynamics is essential for growth cone navigation in the developing nervous system. However, whether axon guidance signaling regulates MT dynamics directly or indirectly is unclear. Here, we report that TUBB3, the most dynamic β-tubulin isoform in neurons, directly interacts with the netrin receptor DCC, and that netrin-1 induces this interaction in primary neurons. TUBB3 colocalizes with DCC in the growth cones of primary neurons and MT dynamics is required for netrin-1-promoted association of TUBB3 with DCC. Netrin-1 not only increases co-sedimentation of DCC with polymerized MT, but also promotes MT dynamics in the growth cone. Knocking down TUBB3 inhibits netrin-1-induced MT dynamics, axon outgrowth and attraction in vitro and causes defects in commissural axon projection in the embryo. These results indicate that TUBB3 directly links netrin signaling pathways to MT dynamics and plays an important role in guiding commissural axons in vivo.

Unpolymerized βII Tubulin in Regulation of Mitocondrial Function in Muscle Cells

The importance of microtubular system in shaping the organization of intracellular energy metabolism and regulating mitochondrial functioning is becoming increasingly more evident. Our previous studies with cardiac cells have shown that regulation of mitochondrial outer membrane (MOM) permeability for ADP by dimeric αβ-tubulin is important for efficient cross-talk between mitochondria and contraction apparatus through phoshocreatine energy transfer pathway. This regulation was specifically related with tubulin isoform βII after showing its mitochondrial localization in cardiac cells and verifying its concomitant expression with mitochondrial creatine kinase (MtCK). However the exact mechanism of this regulation is still rather elusive and studied mainly in cardiac cells. To determine if βII tubulin expression is specific only to oxidative muscle cells with high MtCK activity and to gain further insight to the role of βII tubulin in energy metabolism, we have analyzed the relationship between βII tubulin expression, mitochondrial respiration regulation and their intracellular positioning in striated muscles with different metabolic phenotype. In this study we provide further proof for the functional importance of βII tubulin in regulation of mitochondrial respiration in striated muscles. We show that both oxidative and glycolytic muscles express βII tubulin, but the presence of unpolymerized βII tubulin is significantly lower in glycolytic muscle cells concomitant with higher MOM permeability for ADP. Analysis of mitochondria and βII tubulin localization reveals that in oxidative muscle cells mitochondria are positioned in close vicinity to βII tubulin with high degree of colocalization which is much less prevalent in glycolytic muscles. Together our results show that βII tubulin displays both structural and regulatory role in striated muscle cells and its distribution and polymerization level has direct impact on regulation of mitochondrial ADP sensitivity and efficiency of mitochondria coupling with contraction apparatus.

Tubulin isoforms identified in the brain by MALDI in-source decay

Identification of biomarkers is a major issue for enhancement of chemotherapies. The molecular characterization of tissues necessitates the identification of thousands of biomolecules each participating in physiopathological processes. MALDI in-source decay (ISD) fragmentation has already been proven to be effective for protein characterization. However, the difficulty to identify proteins from complex mixtures such as tissue sections can limit the applications of this technique. In this study, we evidenced that tubulin has an unusual fragmentation pathway in the MALDI source. This striking property allowed the detecting of several mouse brain tubulin isotypes simultaneously by simply using laser fragmentation. Tubulin isoforms are consistent markers of a bad prognosis of solid tumors and could be the target of targeted chemotherapies. Such a direct molecular printout of tubulin in tissues is a milestone that should be useful either at preclinical or clinical stage.

P1-013 - High Expression of Acetyl-Tubulin as a Chemoresistant Biomarker in Human Docetaxel-Resistance Prostate Cancer Cells

ABSTRACT Background Docetaxel-based chemotherapy has generally been considered as one of the effective treatments for prostate cancer. Unfortunately, clinical treatment with docetaxel often encounters a number of undesirable side-effects, including drug resistance. Therefore, it has become essential to identify molecular events that may be associated with the development of docetaxel resistance. Tubulin isoforms have been previously examined for their resistant ability to docetaxel in many cancers, but their real mechanisms remained unclear. In this study, we evaluated the feasibility of employing docetaxel as a cytotoxic agent for PC3 cells and to examine the role of acetyl-tubulin in docetaxel-resistant prostate cancer. Methods A series of docetaxel-resistant PC3/DX cell subclones was established by chronically exposing PC3 to progressively increased concentrations of docetaxel. Herein, we characterized the docetaxel-mediated cytoxicity and molecular events in PC3 and PC3/DX cells by the MTT assay, western blotting, RT–PCR and real-time PCR. Results Our results showed that levels of acetyl-tubulin, a-tubulin, b-tubulin, γ-tubulin and bIII-tubulin were significantly higher expression in PC3/DX than in parental PC3 cells by western blotting analysis. PC/DX with greater resistance to docetaxel had higher levels of acetyl-tubulin than in PC3 cells. The expression of acetyl-tubulin was gradually increased by docetaxel in a dose- and time-dependent manner in PC3 cells. Histone deacetylase 6, a deacetyl enzyme of tubulin, mRNA and protein levels were significantly decreased in PC3/DX than in PC3 cells. Interestingly, we also found an evident up-regulation of acetyl-tubulin protein expression after recombinant epidermal growth factor treatment in a time-dependent manner in PC3 cells. Conclusions Up-regulation of acetyl-tubulin may play an important chemoresistant role in docetaxel-resistant prostate cancer.

Hematologic β-Tubulin VI Isoform Exhibits Genetic Variability That Influences Paclitaxel Toxicity

Cellular microtubules composed of α-β-tubulin heterodimers that are essential for cell shape, division, and intracellular transport are valid targets for anticancer therapy. However, not all the conserved but differentially expressed members of the β-tubulin gene superfamily have been investigated for their role in these settings. In this study, we examined roles for the hematologic isoform β-tubulin VI and functional genetic variants in the gene. β-tubulin VI was highly expressed in blood cells with a substantial interindividual variability (seven-fold variation in mRNA). We characterized DNA missense variations leading to Q43P, T274M, and R307H, and a rare nonsense variant, Y55X. Because variations in the hematologic target of microtubule-binding drugs might alter their myelosuppressive action, we tested their effect in cell lines stably expressing the different β-tubulin VI full-length variants, finding that the T274M change significantly decreased sensitivity to paclitaxel-induced tubulin polymerization. Furthermore, patients treated with paclitaxel and carrying β-tubulin VI T274M exhibited a significantly lower thrombocytopenia than wild-type homozygous patients (P = 0.031). Together, our findings define β-tubulin VI as a hematologic isotype with significant genetic variation in humans that may affect the myelosuppresive action of microtubule-binding drugs. A polymorphism found in a tubulin isoform expressed only in hemapoietic cells may contribute to the patient variation in myelosuppression that occurs after treatment with microtubule-binding drugs.

Microtubule Dynamics of S. Pombe Tubulin

We have developed a fission yeast system for expression and purification of single isoform tubulin. Replacing the non-essential S. pombe α2 tubulin gene with a second copy of the α1 encoding gene creates a strain expressing single α1 and β tubulin isoforms. We obtain yields of about 10 mg of highly purified (>99%) single isoform tubulin from an 80 l culture. Mass spectroscopy of the purified tubulin detects no post-translational modifications. Dynamic microtubules were nucleated from axonemes, recorded by video enhanced DIC microscopy and microtubule lengths measured. Microtubule growth rates increase linearly with tubulin concentration at both the fast and slow growing microtubule ends. Rate constants were determined using a simple bimolecular model. α1β tubulin has fast end kon 5.5 uM−1 s−1 and koff 6.7 s−1, and slow end kon 3.1 uM−1 s−1 and koff 2.8 s−1. Mixed isoform α1α2β has fast end kon 7.6 uM s−1 and koff 15.1 s−1 and slow end kon 2.6 uM−1 s−1 and koff 5.0 s−1. The Kd for both microtubule ends was ∼2uM for α1α2β and ∼1.1 uM for α1β suggesting similar binding affinities for tubulin heterodimers at fast and slow ends, but different kinetics. Following catastrophe the rate of rapid shrinkage was independent of the free tubulin concentration and about 2x faster at the fast compared to the slow ends: 218 ± 16 s−1 compared to 124 ± 23 s−1 for α1α2β and 263 ± 26 s−1 compared to 111 ± 29 s−1 for α1β. We conclude that S. pombe microtubule dynamics are qualitatively similar to those of brain tubulin microtubules but the kinetic rates are different, consistent with S. pombe tubulin assembling at lower concentrations than brain tubulin. We are now comparing S. pombe microtubule growth with that of brain tubulin at higher resolution.

Quantitative Study of Tubulin Distrubution and Mitochondrial Dynamics in Skeletal Muscle Cells

The comparative studies of regulation of oxidative phosphorylation as a function of mitochondrial organization in healthy and cancerous cardiac muscle cells have revealed that the mechanisms of regulation of the energy metabolism depend on the cellular organization (Biochim Biophys Acta, 2010, 1797: 678-97). The aim of this study is to evaluate mitochondrial dynamics and organization of mitochondria into intracellular energy units in adult skeletal muscle cells as a function of the energy metabolic pattern and mitochondrial interactions with cytoskeletal proteins. Among the latter, intracellular distribution of tubulin isoform was evaluated in rat oxidative and glycolytic skeletal muscle cells by Western blot analysis, immunofluorescence and functional methods. Investigation of mitochondrial dynamics in living skeletal muscle fibers was conducted using fluorescent probe labelling analyzed by fluorescent confocal microscopy. Fast scanning of cells was carried out using a line scanning LSM7 LIVE and LSM710 confocal microscope. The fast and simultaneous line scanning in two fluorescent channels synchronized with the piezo stage axial displacements allow rapid 3D imaging of the mitochondrial dynamics referenced to the structural elements of sarcomers. The analysis of position of mitochondrial fluorescence centres, or virtual mass centres, is studied by applying the gradient clustering algorithm. Among the results obtained, we report specific tubulin isoforms distribution profiles in oxidative and glycolytic muscles that could rely to their energy meatbolic pattern. This study was performed in the framework of the ANR project SYBECAR, France.

MAP1B 1–126 interacts with tubulin isoforms and induces neurite outgrowth and neuronal death of cultured cortical neurons

Previously we reported that overexpression of MAP1B containing N-terminal 126 amino acids promoted neuronal death. Here, we identified α-, β-, and βIII-tubulins as proteins interacting with MAP1B 1–126 by two-hybrid and pull-down assays. Transfection experiments indicated that MAP1B 1–126 interacts with microtubules, but to a much lesser extent than two previously reported microtubule-binding domains. Overexpression of MAP1B 1–126 induced both neurite extension and neuronal death, suggesting that MAP1B 1–126 could be involved in neuronal degeneration and aberrant sprouting.