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PubMed1983

Activity patterns of aminoacyl-tRNA synthetases, tRNA methylases, arginyltransferase and tubulin: tyrosine ligase during development and ageing of Caenorhabditis elegans.

As a step in the characterization of development and ageing in the nematode Caenorhabditis elegans, the activities of different groups of enzymes that supposedly exert modulating functions in and after protein synthesis have been determined. From embryonic (E), the four juvenile larval stages (L1-L4) and the gravid adult (A,A+), the selection of defined developmental stages extends to two different preparations of aged nematodes (S10, S12). Some aminoacyl-tRNA synthetase activities remain nearly unchanged in all stages up to the adult, some increase continuously during the larval stages and the remaining activities show stage-specific alterations. Upon ageing all activities except the one for tryptophan decrease sharply, tRNA methylase activities increase from E to L4, decrease from L4 to adult and to aged nematodes with only qualitative alterations in substrate specificity. The activity of tubulin: tyrosine ligase exhibits a parallel pattern, while arginyltransferase activity has a plateau between L2 and L4. The results are consistent with the idea of a modulation of protein synthesis and other cellular processes by quantitative activity changes during development and ageing.

Acyltransferases↗
PubMed1989

Characteristics of microtubules at the different stages of neuronal differentiation and maturation.

The developing nervous system has proved to be a very powerful tool to analyze how MT are involved in basic biological processes such as cell proliferation, cell migration, cell shaping, and transport. A better knowledge of the basic events occurring during neurogenesis also affords us the possibility of establishing the basis of experiments and trying to solve unanswered and important questions. Despite the considerable value of cell culture, we need to use more discrete regions of the developing brain in situ in order to analyze the MT and their modifications into cells developing their "natural" environment. One major problem remains the question of the mode of assembly and disassembly, that is, the behavior of MT in living cells. Dynamic instability and/or treadmilling are accurate interpretations of the dynamics of MT at least in vitro or in cell culture, but we do need more information on what happens in situ and in vitro. One of the main tasks of cell biologists is to devise satisfactory tests to approach this fundamental question. In this view, pharmacological manipulation of embryos treated in whole-embryo culture systems might be a possible way. Microtubules are ubiquitous cell components. However, the extensive heterogeneity of MAP and tubulin in the CNS confers on the neurons a wide range of capabilities of assembly of these proteins and suggests that the neuron has a unique potential of a relation between MT composition and cell function. We have seen that each major event during neurogenesis is related to a specific series of modifications of the MT components. It remains to be determined if there is a causal or just a correlative relationship between the appearance of specific isotypes and the occurrence of specific events and/or functions. We have also to determine the exact spatial and temporal relations among the different isotypes of MT proteins, tubulin, and MAP. Is there a close correspondence between a tubulin and a MAP isotype? Can the appearance of one isotype of tubulin influence the appearance and the assembly of a specific MAP, or vice versa? Recent results obtained with the Tyr- and Glu-MT shed light on these questions and suggest a whole series of possibilities for cells to modulate the structure, behavior, and function of MT in specific domains of the neuron or in specific regions of the brain, by only a minute modification of the molecule of tubulin. Microtubule protein heterogeneity raises also a number of questions.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2003

Proteomics-based identification of differentially expressed genes in human gliomas: down-regulation of SIRT2 gene.

A number of chromosomal abnormalities including 19q deletions have been associated with the formation of human gliomas. In this study, we employed a proteomics-based approach to identify possible genes involved in glioma tumorigenesis which may serve as potential diagnostic molecular markers for this type of cancer. By comparing protein spots from gliomas and non-tumor tissues using two-dimensional (2D) gel electrophoresis, we identified 11 up-regulated proteins and four down-regulated proteins in gliomas. Interestingly, we also discovered that a group of cytoskeleton-related proteins are differentially regulated in gliomas, suggesting the involvement of cytoskeleton modulation in glioma pathogenesis. We then focused on the cytoskeleton-related protein, SIRT2 (sirtuin homologue 2) tubulin deacetylase, which was down-regulated in gliomas. SIRT2 is located at 19q13.2, a region known to be frequently deleted in human gliomas. Subsequent Northern blot analysis revealed that RNA expression of SIRT2 was dramatically diminished in 12 out of 17 gliomas and glioma cell lines, in agreement with proteomic data. Furthermore, ectopic expression of SIRT2 in glioma cell lines led to the perturbation of the microtubule network and caused a remarkable reduction in the number of stable clones expressing SIRT2 as compared to that of a control vector in colony formation assays. These results suggest that SIRT2 may act as a tumor suppressor gene in human gliomas possibly through the regulation of microtubule network and may serve as a novel molecular marker for gliomas. Additional proteins were also identified, whose function in gliomas was previously unsuspected.

Blotting, Northern↗
PubMed2000

[Conquering drug resistance in lung cancer].

The emergence of drug resistance of the tumors is the critical problem in lung cancer. The multifactorial mechanisms of cisplatin resistance are major problems. The reversal agents against MDR have been evaluated clinically. Increased adverse effects and modulation of pharmacokinetics in combined with cytotoxic drugs are the problems in clinical setting. The new ABC transporters related with MDR were cloned and characterized. Modulators for these transporters could be evaluated their clinical efficacy. High-frequent mutations of beta-tubulin in clinical samples are reported as a predictive marker for paclitaxel. Target-based drugs might conquer the resistance to conventional drugs. Prediction of the drug sensitivity and resistance in each patient will be expected using high-throghput gene screening system.

ATP-Binding Cassette Transporters↗
PubMed1997

Colon cancer. Molecular biology of the APC protein.

The discovery of a tumor suppressor gene opens a new pathway to discovery of the fundamental mechanisms that underlie tumor initiation and progression. An inherited tumor suppressor gene is of special interest in that it defines a step in the tumorigenesis pathway that can be rate limiting in development of that tumor type. In the case of colon cancer, we were fortunate in identifying an inherited tumor suppressor gene, the APC gene, that plays a major etiologic role in both the inherited disease, familial adenomatous polyposis (FAP), and in sporadic colon polyps. Characterization of the molecular biology of that gene, and the underlying mechanisms that result in the development of colon tumors, could provide new approaches to both colon cancer diagnostics, therapeutics and chemopreventives. We have embarked, therefore, on a series of exploratory studies designed to provides clues to possible functional roles for the APC protein. We have found through immunocytochemistry that APC protein is distributed throughout the cell, in both the cytoplasm and nucleus. Furthermore, within the nucleus much of the APC protein seems associated with the nucleoli. The cytoplasmic label is distributed in a punctate pattern, with concentrations at the leading edge of migrating cells at the ends of microtubules. Furthermore, following an extraction of the cells that leaves behind primarily cytoskeletal and nuclear scaffold structures, we see strong APC staining of these structures. The yeast two-hybrid system has offered a number of potentially interacting partners for APC, including a new binding site for alpha-tubulin. These results, and others recent discoveries concerning APC, suggest a rather global role for APC protein, modulating cellular activity and signal transduction pathway from the cell periphery to the nucleus.

Adenomatous Polyposis Coli Protein↗
PubMed2001

Conformational properties of alpha-tubulin tail peptide: implications for tail-body interaction.

The carboxy-terminal segments of the alpha/beta-tubulins are flexible regions rich in acidic amino acid residues. It is generally believed that these regions play crucial roles in tubulin polymerization and interaction with many ligands, including colchicine. Exactly how these effects are exerted are not known at present. One such interesting aspect is the pH dependence of colchicine-tubulin interaction and the influence of the alpha-tail on the binding interaction. We have investigated the location of the colchicine-binding site on tubulin by docking. It has been located on the alpha/beta interface on the N-terminal side, which is also supported by much of the solution data. This location is too far from the tail regions, suggesting that influence of the tail region is transmitted by a pH-dependent conformational change. Two-dimensional NMR studies indicate that at pH 7 a 13-residue peptide corresponding to alpha-tubulin tail shows little NOE constraints, suggesting extended conformation. On the contrary, at pH 5, a relatively compact structure was deduced from the interproton NOE constraints. Pulsed field gradient measurement of diffusion constant indicates that the peptide at pH 5 is substantially faster diffusing than at pH 7. The Perrin factors calculated from diffusion data indicates that the peptide structure at pH is significantly more compact than at pH 7. Temperature coefficients of several amide protons at pH 5 fall below 5 ppb/(o)K, indicating a degree of protection. A difference is also seen in the CD spectra obtained at different pHs, consistent with the NMR data. We have investigated the probable spatial organization of the tail of the alpha-subunit of tubulin, in the high pH extended form and the low pH compact form. On the basis of correlation of pH dependence of many properties of tubulin and the conformation of the alpha-tail peptide, we propose that the intrinsic conformational preference of the tail-region modulate the tail-body interaction, which in turn has important bearing on colchicine binding properties.

Binding Sites↗
PubMed2004

3-O-methylfunicone, a secondary metabolite produced by Penicillium pinophilum, induces growth arrest and apoptosis in HeLa cells.

3-O-Methylfunicone (OMF) is a secondary metabolite produced by the soil fungus Penicillium pinophilum which has cytostatic properties. The aim of this study was to investigate the mechanisms by which such properties are exerted, with special reference to any anti-proliferative and apoptotic potential, on HeLa cells. OMF treatment caused about 44% inhibition of cell growth after 24 h, and modifications in the tubulin fibre organization. In addition, a significant increase in p21 mRNA expression and a decrease in cyclin D1 and Cdk4 mRNA expression resulted at the same time. Apoptosis induction was demonstrated by the annexin V assay, cytofluorimetric analysis of the DNA content of the sub-G1 fraction and DNA laddering. Taken together, our data showed that the compound inhibits proliferation of HeLa cells by several mechanisms, such as disruption of tubulin fibres, cell cycle arrest and apoptosis induction. The capacity of the compound to affect the cell cycle and to modulate apoptosis is indicative of a potential for the development of a new agent for cancer chemotherapy.

Annexin A5↗
PubMed2004

Complex formation with kinesin motor domains affects the structure of microtubules.

Microtubules are highly dynamic components of the cytoskeleton. They are important for cell movement and they are involved in a variety of transport processes together with motor proteins, such as kinesin. The exact mechanism of these transport processes is not known and so far the focus has been on structural changes within the motor domains, but not within the underlying microtubule structure. Here we investigated the interaction between kinesin and tubulin and our experimental data show that microtubules themselves are changing structure during that process. We studied unstained, vitrified samples of microtubules composed of 15 protofilaments using cryo electron microscopy and helical image analysis. 3D maps of plain microtubules and microtubules decorated with kinesin have been reconstructed to approximately 17A resolution. The alphabeta-tubulin dimer could be identified and, according to our data, alpha- and beta-tubulin adopt different conformations in plain microtubules. Significant differences were detected between maps of plain microtubules and microtubule-kinesin complexes. Most pronounced is the continuous axial inter-dimer contact in the microtubule-kinesin complex, suggesting stabilized protofilaments along the microtubule axis. It seems, that mainly structural changes within alpha-tubulin are responsible for this observation. Lateral effects are less pronounced. Following our data, we believe, that microtubules play an active role in intracellular transport processes through modulations of their core structure.

Cryoelectron Microscopy↗
PubMed2006

The carboxy-terminus of Alp4 alters microtubule dynamics to induce oscillatory nuclear movement led by the spindle pole body in Schizosaccharomyces pombe.

Alp4 is an essential component of the S. pombe gamma-tubulin complex. Overproduction of the carboxy-terminus of Alp4 induces oscillatory nuclear movement led by the spindle pole body (SPB). The movement is not dependent on cytoplasmic dynein dhc1, or kinesin-related proteins pkl1 and klp2. Rates of SPB movement correlate with elongation rates of microtubules (MTs) extending backwards from the moving SPB (backward-extending MTs), showing that pushing forces exerted by backward-extending MTs move the nucleus via the SPB. These backward-extending MTs are more stable than those of control cells and, thus, are able to push the SPB further towards the cell end, inducing nuclear oscillation with larger amplitudes than in control cells. SPB movement is biased towards the new end of the cell where levels of the CLIP170 homolog Tip1 increase, suggesting that the movement is related to MT-mediated cell polarity control. These results demonstrate that the carboxy-terminus of Alp4 alters MT dynamics and induces nuclear oscillation by modulating a nuclear positioning mechanism based on the balance of MT pushing forces, and suggest that regulation of gamma-tubulin complex activity is important for controlling MT dynamics and nuclear positioning.

Biological Clocks↗
PubMed1996

Related enhancers in the intron of the beta1 tubulin gene of Drosophila melanogaster are essential for maternal and CNS-specific expression during embryogenesis.

Expression of the beta1 tubulin gene of Drosophila melanogaster is under complex developmental control. For high levels of transcription in the embryonic central nervous system (CNS) different modules dispersed over 3 kb have to co-operate. Combination of a core promoter with either far upstream localized enhancer elements or, alternatively, with an enhancer from the intron results in expression limited to only a few neuronal cells. Cooperation of all three modules, however, leads to high level expression in most neuronal cells of the CNS. In the intron, we identified a 6 bp core element which is essential for transcription in the CNS, as well as an 8 bp element required for maternal expression. Interestingly, both motifs are quite similar, with CAAAAT as the CNS core and CAAAAAT as the maternal enhancer core. Specific binding of proteins from nuclear extracts to the CNS-specific element could be demonstrated. We suggest that the beta1 tubulin gene represents an ideal marker gene to elucidate connections between pro-neural or neurogenic genes and downstream target genes throughout the CNS.

Animals↗
PubMed1995

Differential effects of paclitaxel (Taxol) analogs modified at positions C-2, C-7, and C-3' on tubulin polymerization and polymer stabilization: identification of a hyperactive paclitaxel derivative.

Our finding that an analog of paclitaxel (Taxol) modified at position C-2 (2-debenzoyl-2-(m-azidobenzoyl)paclitaxel) was substantially more active than paclitaxel in promoting tubulin assembly [Chaudhary et al. (1994) J. Am. Chem. Soc. 116, 4097-4098] led us to perform an analysis of the modulating effects of microtubule-associated proteins, GTP, and temperature on assembly and polymer stability. The analog always showed superior activity to paclitaxel in inducing polymerization where it fails to occur without drug, probably indicating a greater ability than paclitaxel to "hypernucleate" assembly. In contrast, much smaller differences in effects on polymer stability were observed. The analysis was extended to a large series of derivatives modified at positions C-2, C-7, C-10, and C-3', including docetaxel, a clinically important analog of paclitaxel. While analog stabilization of polymer was frequently observed, neither qualitative nor quantitative analysis of this property reliable predicted whether a compound would have enhanced hypernucleation activity relative to that of paclitaxel. Stabilization was often observed at substoichiometric analog concentrations, while even superstoichiometric concentrations of most compounds failed to induce extensive tubulin polymerization at low temperatures or in the absence of microtubule-associated proteins or GTP. Docetaxel was intermediate in activity between paclitaxel and 2-debenzoyl-2-(m-azidobenzoyl)paclitaxel in promoting assembly reactions. We conclude that the hypernucleation of tubulin assembly and polymer stabilization observed with paclitaxel represent two distinct properties of the drug. Our findings suggest that paclitaxel, docetaxel, and 2-debenzoyl-2-(m-azidobenzoyl)paclitaxel are able to interact with progressively smaller assemblages of tubulin at low temperatures or in the absence of microtubule-associated proteins or GTP.

Drug Stability↗
PubMed2006

Cortical thinning and hydrocephalus in mice lacking the immunoglobulin superfamily member CDO.

CDO is a cell surface immunoglobulin superfamily member that positively regulates myogenic differentiation in vitro and in vivo and signals to posttranslationally activate myogenic basic helix-loop-helix (bHLH) transcription factors. The Cdo gene is also expressed in the dorsal aspect and midline structures of the developing central nervous system, and mice lacking CDO on the C57BL/6 background display holoprosencephaly with approximately 80% penetrance, resulting in perinatal lethality. We report here that a fraction of Cdo-/- mice from this background have additional defects in brain development, including hydrocephalus and cortical thinning. Primary neural progenitor cultures from E14.5 Cdo-/- mutants display reduced proliferation, which may underlie the thinning. The cortical preplate and cortices of mutant animals also show reduced staining for beta-tubulin III, indicating defective neuronal differentiation. CDO levels are strongly increased in cultured C17.2 neuronal precursor cells stimulated to differentiate; modulation of CDO levels in these cells by overexpression or interfering RNA approaches enhances or diminishes differentiation, respectively. Cotransfection of CDO enhances the activity of the neurogenic bHLH factor, neurogenin1, in reporter assays and enhances heterodimerization of neurogenin1 and E47. These results indicate that CDO promotes neuronal differentiation and support the hypothesis that CDO coordinates differentiation of multiple cell lineages by regulating the activity of tissue-specific bHLH factors.

Animals↗
PubMed1986

Tau microheterogeneity: an immunological approach with monoclonal antibodies.

The family of tau polypeptides purified from mammalian brain exhibit both extensive heterogeneity and large similarities in their chemical, physical, and functional properties. All the tau isoforms generated at a transcriptional or posttranscriptional level share the property of interacting with tubulin dimers in a specific manner. They strengthen longitudinal interactions between tubulin dimers and thus may stabilize microtubules once they are formed. Mild proteolysis or phosphorylation does not remove but only modulates the tau specific function that is probably related to the conserved sequences of the molecules. Monoclonal antibodies raised against tau were found to recognize epitopes conserved not only between species but also in different tissues. Using indirect immunofluorescence, a specific staining pattern was observed on rat neuronal cells and also on human skin fibroblasts. The same antibodies did not recognize glial cells, suggesting that these cells either do not contain detectable levels of tau or contain tau molecules different from the neuronal ones. These data suggest that tau protein is widely distributed, highly conserved, and may be preferentially associated with special subclasses of microtubules.

Aging↗
PubMed1995

Beta-tubulin and P-glycoprotein: major determinants of vincristine accumulation in B-CLL cells.

Vincristine (VCR) accumulation in chronic lymphatic leukemia of B-cell origin (B-CLL) has recently been shown not to be inversely correlated to P-glycoprotein (PGP) levels. Therefore, we studied, in addition to PGP expression and accumulation of VCR, the cellular beta-tubulin content in quiescent and rhIL-2 activated B-CLL cells. VCR mediates cytotoxicity by binding to tubulin. Constitutive beta-tubulin levels in B-CLL cells varied considerably. Upon activation with rhIL-2, beta-tubulin expression increased significantly. Therefore, tubulin levels could be correlated over a wide range to VCR accumulation. When the PGP-mediated drug efflux was blocked by verapamil (VRP), tubulin levels correlated linearly to VCR accumulation. All B-CLL cases expressed PGP at different levels. There was no linear correlation between PGP expression and VCR accumulation. A modulation factor m was defined as a quotient of VCR accumulation in the presence and absence of VRP to define the extent by which VRP inhibited a steady-state accumulation of VCR. The factor allowed discrimination between B-CLLs expressing low versus high PGP, irrespective of the levels of tubulin. However, PGP and beta-tubulin levels together were predictive for VCR accumulation in steady state. There was no uniform-accumulation defect for VCR in B-cell CLL because beta-tubulin and PGP were expressed independently. Non PGP-mediated VCR transport seems to play a minor role in B-cell CLL. Leukemia-associated varying of cytoskeletal organization in B-cell CLL might be one reason for the diverse cellular responses to receptor-mediated signals.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1989

Phospholipids differently modulate the activity of cytosolic protein-tyrosine kinase from porcine spleen.

Effect of membrane phospholipids on the activity of cytosolic protein-tyrosine kinase from porcine spleen (CPTK-40) has been studied. Using poly(Glu Na, Tyr)4:1 as a substrate, phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine had stimulatory effects on that phosphorylation activity, however phosphatidic acid had inhibitory and phosphatidylinositol had no effects. Similar results were obtained using[Val5]angiotensin II as a substrate. On the other hand using basic protein (H2B histone and myelin basic protein) as substrates, phosphatidic acid stimulated the activity of CPTK-40, while phosphatidylinositol inhibited the activity. Phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine caused different effect on the activity of CPTK-40 depending on the substrate employed. However using acidic protein (tubulin and casein) as substrates, the activity of CPTK-40 was neither stimulated nor inhibited by any phospholipids. These results suggest that phospholipids may modulate the activity of CPTK-40.

Animals↗
PubMed2006

Survivin modulates microtubule dynamics and nucleation throughout the cell cycle.

Survivin is a member of the chromosomal passenger complex implicated in kinetochore attachment, bipolar spindle formation, and cytokinesis. However, the mechanism by which survivin modulates these processes is unknown. Here, we show by time-lapse imaging of cells expressing either green fluorescent protein (GFP)-alpha-tubulin or the microtubule plus-end binding protein GFP-EB1 that depletion of survivin by small interfering RNAs (siRNAs) increased both the number of microtubules nucleated by centrosomes and the incidence of microtubule catastrophe, the transition from microtubule growth to shrinking. In contrast, survivin overexpression reduced centrosomal microtubule nucleation and suppressed both microtubule dynamics in mitotic spindles and bidirectional growth of microtubules in midbodies during cytokinesis. siRNA depletion or pharmacologic inhibition of another chromosomal passenger protein Aurora B, had no effect on microtubule dynamics or nucleation in interphase or mitotic cells even though mitosis was impaired. We propose a model in which survivin modulates several mitotic events, including spindle and interphase microtubule organization, the spindle assembly checkpoint and cytokinesis through its ability to modulate microtubule nucleation and dynamics. This pathway may affect the microtubule-dependent generation of aneuploidy and defects in cell polarity in cancer cells, where survivin is commonly up-regulated.

Animals↗
PubMed2004

The mammalian exocyst, a complex required for exocytosis, inhibits tubulin polymerization.

The exocyst is a 734-kDa complex essential for development. Perturbation of its function results in early embryonic lethality. Extensive investigation has revealed that this complex participates in multiple biological processes, including protein synthesis and vesicle/protein targeting to the plasma membrane. In this article we report that the exocyst may also play a role in modulating microtubule dynamics. Using monoclonal antibodies, we observed that endogenous exocyst subunits co-localized with microtubules and mitotic spindles in normal rat kidney cells. To test for a functional relationship between the exocyst complex and microtubules, we established an in vitro exocyst reconstitution assay and studied exocyst effect on microtubule dynamics. We found that the exocyst complex reconstituted from eight recombinant exocyst subunits inhibited tubulin polymerization in vitro. Deletion of exocyst subunit sec5, sec6, sec15, or exo70 diminished its tubulin polymerization inhibition activity. Surprisingly, exocyst subunit exo70 itself was also capable of inhibiting tubulin polymerization, although exocyst complex with exo70 deletion did not lose its activity completely. Overexpression of exo70 in NRK cells resulted in microtubule network disruption and the formation of filopodia-like plasma membrane protrusions. The formation of these membrane protrusions was greatly hampered by stabilizing microtubules with taxol. Overexpression of exo84, an exocyst subunit that did not show tubulin polymerization inhibition activity, did not cause this phenotype. Results shown in this article, along with a previous report that localized microtubule instability induces plasma membrane addition, implicates a novel role for the exocyst in modulating microtubule dynamics underlying exocytosis.

Animals↗
PubMed1988

Spindle microtubule dynamics: modulation by metabolic inhibitors.

Recent experiments have shown that spindle microtubules are exceedingly dynamic. Measurements of fluorescence recovery after photobleaching (FRAP), in cells previously microinjected with fluorescent tubulin, provide quantitative information concerning the rate of turnover, or exchange, of tubulin subunits with the population of microtubules in living cells at steady state. In an effort to elucidate the pathways and factors that regulate tubulin exchange with microtubules in living cells, we have investigated the energy requirements for tubulin turnover as measured by FRAP. Spindle morphology was not detectably altered in cells incubated with 5 mM sodium azide and 1 mM 2-deoxyglucose (Az/DOG) for 5 minutes, as assayed by polarized light microscopy and antitubulin immunofluorescence. In FRAP experiments on these ATP-depleted cells, the average rate of recovery and the average percent of bleached fluorescence recovered were reduced to 37% and 30% of controls, respectively. When the inhibitors were removed, cells continued through mitosis, and rapid FRAP was restored. In the presence of azide and glucose, the rate of recovery and percent of fluorescence recovered were only slightly reduced, demonstrating that energy production via glycolysis can support microtubule turnover. Longer incubations with Az/DOG altered the microtubule organization in mitotic cells: astral microtubules lengthened and spindle fibers shortened. Furthermore, both astral and spindle microtubules became resistant to nocodazole-induced disassembly under these conditions. Together these observations indicate that microtubule dynamics require ATP and suggest a relationship between microtubule organization and turnover.

Antimetabolites↗