RESUMEN

PURPOSE: Although lorlatinib, the third generation of echinoderm microtubule protein 4-anaplastic lymphoma kinase (EML4-ALK) tyrosine kinase inhibitor (TKI), overcame the previous generation ALK-TKIs' drug resistance problems, but the mechanism of lorlatinib resistance remained unclear. Furthermore, optimal chemotherapy for lorlatinib-resistant non-small cell lung cancer (NSCLC) patients was still unknown. METHODS: A lorlatinib-resistant NSCLC cell line SNU-2535LR was generated by gradually increasing dose of lorlatinib to crizotinib-resistant cell line SNU-2535 in vitro. To study the resistance mechanism of SNU-2535LR cells, we applied CCK-8 assay to detect the sensitivity of crizotinib and the reverse effect of APR-246, a p53 activator, on lorlatinib-induced resistance and different chemotherapy drugs to SNU-2535LR cells. We also detected the expressions of EML4-ALK-related proteins of SNU-2535LR cells via western blot.Please confirm that author names have been identified correctly and are presented in the right order.Dear Editor:     I have carefully confirmed that the author names have been identified correctly and are presented in right order.Thank you very much!                                                                     Your sincerely BoXie RESULTS: The sensitivity of SNU-2535LR cells to lorlatinib was decreased significantly than that of SNU-2535 cells. EML4-ALK fusion was decreased both at protein level and DNA level in SNU-2535LR cells. More interesting, the crizotinib-resistant mutation ALK p.G1269A disappeared, while new TP53 mutation emerged in SNU-2535LR cells. APR-246 can reverse the lorlatinib resistance in SNU-2535LR cells, with a reversal index of 4.768. Compared with SNU-2535 cells, the sensitivity of SNU-2535LR cells to gemcitabine, docetaxel and paclitaxel was significantly increased (P < 0.05), but decreased to cisplatin (P < 0.05). CONCLUSION: This study demonstrated that the combination of p53 protein agonist and lorlatinib may provide a new therapeutic strategy for NSCLC patients with lorlatinib resistance and TP53 mutation. Furthermore, the results also provide guidance for selecting optimal chemo-regimens for NSCLC patients after ALK-TKIs failure.

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Aminopiridinas , Quinasa de Linfoma Anaplásico/genética , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Línea Celular , Cisplatino/uso terapéutico , Crizotinib/uso terapéutico , Docetaxel/uso terapéutico , Resistencia a Antineoplásicos/genética , Humanos , Lactamas , Lactamas Macrocíclicas/farmacología , Lactamas Macrocíclicas/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Proteínas de Microtúbulos/genética , Mutación , Paclitaxel/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirazoles , Proteína p53 Supresora de Tumor/genética

RESUMEN

The cilia and flagella of eukaryotic cells serve many functions, exhibiting remarkable conservation of both structure and molecular composition in widely divergent eukaryotic organisms. SPAG6 and SPAG16 are the homologous in the mice to Chlamydomonas reinhardtii PF16 and PF20. Both proteins are associated with the axonemal central apparatus and are essential for ciliary and flagellar motility in mammals. Recent data derived from high-throughput studies revealed expression of these genes in tissues that do not contain motile cilia. However, the distribution of SPAG6 and SPAG16 in ciliated and non-ciliated tissues is not completely understood. In this work, we performed a quantitative analysis of the expression of Spag6 and Spag16 genes in parallel with the immune-localization of the proteins in several tissues of adult mice. Expression of mRNA was higher in the testis and tissues bearing motile cilia than in the other analyzed tissues. Both proteins were present in ciliated and non-ciliated tissues. In the testis, SPAG6 was detected in spermatogonia, spermatocytes, and in the sperm flagella whereas SPAG16 was found in spermatocytes and in the sperm flagella. In addition, both proteins were detected in the cytoplasm of cells from the brain, spinal cord, and ovary. A small isoform of SPAG16 was localized in the nucleus of germ cells and some neurons. In a parallel set of experiments, we overexpressed EGFP-SPAG6 in cultured cells and observed that the protein co-localized with a subset of acetylated cytoplasmic microtubules. A role of these proteins stabilizing the cytoplasmic microtubules of eukaryotic cells is discussed.

Asunto(s)

Cilios/genética , Proteínas de Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/genética , Neuronas/metabolismo , Animales , Chlamydomonas reinhardtii/genética , Cilios/metabolismo , Epéndimo/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Masculino , Ratones , Proteínas de Microtúbulos/aislamiento & purificación , Proteínas Asociadas a Microtúbulos/aislamiento & purificación , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Espermatocitos/metabolismo , Espermatogonias/metabolismo

RESUMEN

Neurons are highly differentiated cells responsible for the conduction and transmission of information in the nervous system. The proper function of a neuron relies on the compartmentalization of their intracellular domains. Differentiated neuroblastoma cells have been extensively used to study and understand the physiology and cell biology of neuronal cells. Here, we show that differentiation of N1E-115 neuroblastoma cells is more pronounced upon exposure of a chemical analog of cyclic AMP (cAMP), db-cAMP. We next analysed the expression of key microtubule-regulating proteins in differentiated cells and the expression and activation of key cAMP players such as EPAC, PKA and AKAP79/150. Most of the microtubule-promoting factors were up regulated during differentiation of N1E-115 cells, while microtubule-destabilizing proteins were down regulated. We observed an increase in tubulin post-translational modifications related to microtubule stability. As expected, db-cAMP increased PKA- and EPAC-dependent signalling. Consistently, pharmacological modulation of EPAC activity instructed cell differentiation, number of neurites, and neurite length in N1E-115 cells. Moreover, disruption of the PKA-AKAP interaction reduced these morphometric parameters. Interestingly, PKA and EPAC act synergistically to induce neuronal differentiation in N1E-115. Altogether these results show that the changes observed in the differentiation of N1E-115 cells proceed by regulating several microtubule-stabilizing factors, and the acquisition of a neuronal phenotype is a process involving concerted although independent functions of EPAC and PKA.

Asunto(s)

Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de Microtúbulos/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , Diferenciación Celular , Humanos , Transducción de Señal

RESUMEN

The brahma gene encodes the catalytic subunit of the Drosophila melanogaster BRM chromatin-remodeling complexes. Screening for mutations that interact with brahma, we isolated the dominant-negative Pearl-2 allele of gammaTub23C. gammaTub23C encodes one of the two gamma-tubulin isoforms in Drosophila and is essential for zygotic viability and normal adult patterning. gamma-Tubulin is a subunit of microtubule organizer complexes. We show that mutations in lethal (1) discs degenerate 4, which encodes the Grip91 subunit of microtubule organizer complexes, suppress the recessive lethality and the imaginal phenotypes caused by gammaTub23C mutations. The genetic interactions between gammaTub23C and chromatin-remodeling mutations suggest that gamma-tubulin might have a role in regulating gene expression.

Asunto(s)

Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Transactivadores/genética , Tubulina (Proteína)/genética , Alelos , Animales , Proteínas de Ciclo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Microtúbulos/genética , Proteínas de Microtúbulos/metabolismo , Mutación , Transactivadores/metabolismo , Tubulina (Proteína)/metabolismo

RESUMEN

Butterflies in the nymphalid subfamily Ithomiinae are brightly colored and involved in mimicry. Here we present a phylogenetic hypothesis for 23 of the 24 species in the genus Ithomia, based on seven different gene regions, representing 5 linkage groups and 4469 bp. We sequenced varying length regions of the following genes: (1) elongation factor 1alpha (Ef1alpha; 1028 bp); (2) tektin (tektin; 715 bp); (3) wingless (wg; 405 bp); (4) ribosomal protein L5 (RpL5; 722 bp, exons 1, 2, 3, and introns 1 and 2); and (5) mitochondrial cytochrome oxidase I, II (Co1 and Co2 and intervening leucine tRNA; 1599 bp). The results show incongruence between some genetic loci, although when alternate topologies are compared statistically it was generally true that one topology was supported by a majority of loci sampled. This highlights the need to sample widely across the genome in order to obtain a well-supported phylogenetic hypothesis. A combined evidence topology is presented based on a Bayesian analysis of all the gene regions, except the fast-evolving RpL5. The resulting hypothesis is concordant with the most probable relationships determined from our topological comparisons, although in some parts of the tree relationships remain weakly supported. The tree suggests diversification has largely occurred within biogeographic regions such as Central America, the Amazon, the southern and northern Andes, with only occasional dispersal (or vicariance) between such regions. This phylogenetic hypothesis can now be used to investigate patterns of diversification across the genus, such as the potential role of color pattern changes in speciation.

Asunto(s)

Mariposas Diurnas/genética , Filogenia , Animales , Mariposas Diurnas/clasificación , ADN Mitocondrial , Proteínas de Microtúbulos/genética , Mitocondrias/genética

RESUMEN

A chimeric synthetic peptide incorporating immunodominant epitope of the p19 gag protein (116-134) and the gp46 env protein (178-200) of HTLV-II virus, separated by two glycine residues, was synthesized by conventional solid-phase peptide synthesis. The antigenic activity of this peptide was evaluated by Ultramicro Enzyme-linked immunosorbent assay (UMELISA) by using panels of anti-HTLV-II positive sera (n = 9), anti-HTLV-I/II positive sera (n = 2), HTLV-positive (untypeable) serum samples (n = 1),and anti-HTLV-I positive sera (n = 14), while specificity was evaluated with samples from healthy blood donors (n = 20). The efficacy of the chimeric peptide in solid-phase immunoassays was compared with the monomeric peptides. Data demonstrated that the chimeric peptide was the most reactive because it detected antibodies to virus efficiently. This may be related to peptide adsorption to the solid surface and epitope accessibility to the antibodies. The results indicate that chimeric peptide as coating antigen is very useful for the immunodiagnosis of HTLV-II infection.

Asunto(s)

Productos del Gen env/química , Antígenos HTLV-II/química , Antígenos HTLV-II/inmunología , Leucemia de Células T/sangre , Leucemia de Células T/inmunología , Proteínas de Microtúbulos , Fosfoproteínas/química , Proteínas Oncogénicas de Retroviridae/química , Productos del Gen env/inmunología , Humanos , Leucemia de Células T/diagnóstico , Péptidos/síntesis química , Péptidos/inmunología , Fosfoproteínas/inmunología , Proteínas Recombinantes de Fusión/síntesis química , Proteínas Recombinantes de Fusión/inmunología , Proteínas Oncogénicas de Retroviridae/inmunología , Estatmina , Productos del Gen env del Virus de la Inmunodeficiencia Humana

RESUMEN

A mutation in the ilvC gene of Sinorhizobium meliloti 1021 determines a symbiotically defective phenotype. ilvC mutants obtained from different S. meliloti wild-type strains are able to induce root hair deformation on alfalfa roots and show variable activation of the common nodulation genes nodABC. All of these mutants are noninfective. The presence of extra copies of nodD3-syrM in an IlvC- background does not promote nod expression but allows the detection of low levels of Nod factor production. The sulphation of the Nod factor metabolites, however, is not affected. Furthermore, IlvC- strains induce a specific pattern of starch accumulation on alfalfa roots as well as of early nodulin expression. Hence, the pleiotropic action of the ilvC gene in S. meliloti may reveal novel complexities involved in the symbiotic interaction.

Asunto(s)

Medicago sativa/microbiología , Sinorhizobium meliloti/genética , Oxidorreductasas de Alcohol/genética , Genes Bacterianos , Cetoácido Reductoisomerasa , Medicago sativa/citología , Medicago sativa/metabolismo , Proteínas de Microtúbulos/genética , Datos de Secuencia Molecular , Mutación , Regiones Promotoras Genéticas , Sinorhizobium meliloti/enzimología , Simbiosis

RESUMEN

The ultrasensitive response of biological systems is a more sensitive one than that expected from the classical hyperbola of Michaelis-Menten kinetics, and whose physiological relevance depends upon the range of variation of substrate or effector for which ultrasensitivity is observed. Triggering and modulation of the ultrasensitive response in enzymatic and cellular systems are reviewed. Several demonstrations of ultrasensitive behavior in cellular systems and its impact on the amplification properties in signalling cascades and metabolic pathways are also highlighted. It is shown that ubiquitous cytoskeletal proteins may up- or downmodulate ultrasensitivity under physico-chemical conditions resembling those predominant in cells.

Asunto(s)

Enzimas/fisiología , Sustancias Macromoleculares , Proteínas de Microtúbulos/fisiología , Transducción de Señal/fisiología , Animales , Humanos , Cinética , Modelos Teóricos , Especificidad por Sustrato/fisiología

RESUMEN

We have previously reported the posttranslational addition of [14C]-arginine in the N-terminus of several soluble rat brain proteins. One of these proteins was identified as the microtubule-associated protein, the stable tubule only polypeptide (STOP). However, despite the fact that the biological significance of arginylation is not completely understood, some evidence associates it with proteolysis via the ubiquitin pathway. Since this degradative via is exacerbated as a response to stress, we studied in vitro the posttranslational [14C]-arginylation of cytosolic brain proteins of rats subjected to hyperthermia in vivo. Immediately after subjecting the animals to hyperthermia, a minor reduction (16%) in the acceptor capacity of [14C]-arginine into proteins was observed in comparison with animals maintained at 28 degrees C. However, in the animals allowed to recover for 3 h, an increase (46%) in the arginylation was observed concomitantly with a significant accumulation of the heat shock protein (70 kDa; hsp 70) when compared to the control animals. These findings suggest that the posttranslational arginylation of proteins participate in the heat shock response. The STOP protein of the soluble brain fraction of control animals, which in Western blot appears as a doublet band (125 and 130 kDa, respectively), is seen, after the hyperthermic treatment, as a single band of 125 kDa. The amount of 125 kDa protein, as well as the in vitro incorporation of [14C]-arginine, increases after hyperthermia in comparison with control animals. Following hyperthermic treatment, we observed a decrease in the amount of in vivo [35S]-methionine-labeled brain proteins. We speculate that, as observed for STOP protein, the increase in the degradation of protein that occurs in hyperthermia, would produce an increase in the amount of arginine acceptor proteins.

Asunto(s)

Arginina/metabolismo , Encéfalo/metabolismo , Hipertermia Inducida , Proteínas de Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Radioisótopos de Carbono , Citosol/metabolismo , Proteínas HSP70 de Choque Térmico/biosíntesis , Proteínas de Choque Térmico/biosíntesis , Masculino , Metionina/metabolismo , Proteínas de Microtúbulos/biosíntesis , Proteínas de Microtúbulos/aislamiento & purificación , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/aislamiento & purificación , Ratas , Ratas Wistar , Radioisótopos de Azufre

RESUMEN

We investigated the effects of the drug 14-keto-stypodiol diacetate (SDA) extracted from the seaweed product Stypopodium flabelliforme, in inhibiting the cell growth and tumor invasive behavior of DU-145 human prostate cells. In addition, the molecular action of the drug on microtubule assembly was analyzed. The effects of this diterpenoid drug in cell proliferation of DU-145 tumor cells in culture revealed that SDA at concentrations of 5 microM decreased cell growth by 14%, while at 45 microM a 61% decrease was found, as compared with control cells incubated with the solvent but in the absence of the drug. To study their effects on the cell cycle, DU-145 cells were incubated with increasing concentrations of SDA and the distribution of cell-cycle stages was analyzed by flow cytometry. Interestingly, the data showed that 14-keto-stypodiol diacetate dramatically increased the proportion of cells in the G2/M phases, and decreased the number of cells at the S phase of mitosis, as compared with appropriate controls. Studies on their action on the in vitro assembly of microtubules using purified brain tubulin, showed that SDA delayed the lag period associated to nucleation events during assembly, and decreased significantly the extent of polymerization. The studies suggest that this novel derivative from a marine natural product induces mitotic arrest of tumor cells, an effect that could be associated to alterations in the normal microtubule assembly process. On the other hand, a salient feature of this compound is that it affected protease secretion and the in vitro invasive capacity, both properties of cells from metastases. The secretion of plasminogen activator (u-PA) and the capacity of DU-145 cells to migrate through a Matrigel-coated membrane were significantly inhibited in the presence of micromolar concentrations of SDA. These results provide new keys to analyze the functional relationships between protease secretion, invasive behavior of tumor cells and the microtubule network.

Asunto(s)

División Celular/efectos de los fármacos , Microtúbulos/efectos de los fármacos , Neoplasias de la Próstata/patología , Quinonas/farmacología , Citometría de Flujo , Humanos , L-Lactato Deshidrogenasa/metabolismo , Masculino , Proteínas de Microtúbulos/aislamiento & purificación , Microtúbulos/metabolismo , Invasividad Neoplásica , Activadores Plasminogénicos/antagonistas & inhibidores , Activadores Plasminogénicos/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Algas Marinas , Células Tumorales Cultivadas

RESUMEN

On the basis of experimental data obtained in vitro, we propose that differential segregation of actin and tubulin in the cytoplasm may be a regulatory mechanism of metabolic fluxes. The results presented point out that the same enzymes may be differentially modulated at different locations in the cytoplasm, depending on the cytoskeletal protein present at that location, its concentration, polymeric status, or geometric arrangement. Essentially, actin or microtubular protein would exert their effect on enzymatic catalysis through displacement of the equilibrium of enzyme oligomers either to active or less active species. The latter was corroborated by mathematical modeling of the dynamic coupling between microtubular protein assembly-disassembly and pyruvate kinase activity. From these results, a precise biochemical meaning can be given to the putative linkage existing between the mechanisms by which cells rearrange their cytoplasmic architecture and the dynamics of biochemical reactions taking place concomitantly.

Asunto(s)

Actinas/fisiología , Proteínas del Citoesqueleto/análisis , Enzimas/metabolismo , Proteínas de Microtúbulos/fisiología , Regulación Alostérica , Animales , Biopolímeros , Proteínas del Citoesqueleto/ultraestructura , Proteínas de Microtúbulos/química , Modelos Estadísticos , Ratas , Tubulina (Proteína)/fisiología

RESUMEN

Tau proteins play major regulatory roles in the organization and integrity of the cytoskeletal network. In neurons, a specific axonal compartmentalization of tau has been shown. However, recent studies demonstrate that tau displays a widespread distribution in a variety of non-neuronal cell types. These proteins have been found in human fibroblasts and in several transformed cell lines. The heterogeneous family of tau is formed by a set of molecular species that share common peptide sequences. There is a single gene that contains several exons encoding for the six different tau isoforms in mammalian brain. Alternative splicing of a common RNA transcript as well as post-translational modifications contribute to its heterogeneity. Tau isoforms generated by splicing differ from one another by having either three or four repeats in their C-terminal half, and a variable number of inserts in their N-terminal moiety. These repeats have been shown to constitute microtubule-binding motifs. In this review some relevant aspects of tau function and its regulation are analyzed. Three major topics are discussed. The first one focuses on the tau roles in regulating the interactions between microtubules with actin filaments and with intermediate filament systems. Another problem deals with the question of whether tau isoforms segregate into functionally different subsets of microtubules in axonal processes, or tau associates with these polymers in a random fashion. The third question that emerges is the involvement of tau and tau-like proteins in morphogenetic events. The regulation of the interactions of DMAP-85, a recently discovered tau-like protein, with the cytoskeleton during development of Drosophila melanogaster is analyzed.

Asunto(s)

Proteínas de Microtúbulos/fisiología , Neuronas/fisiología , Proteínas tau/fisiología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Sitios de Unión , Diferenciación Celular/fisiología , Drosophila melanogaster/crecimiento & desarrollo , Datos de Secuencia Molecular , Proteínas tau/inmunología

RESUMEN

Tau proteins play major regulatory roles in the organization and integrity of the cystoskeletal networks. In neurons, a specific axonal compartmentalization of tau has been shown. However, recent studies demonstrate that tau displays a widespread distribution in a variety of non-neuronal cell types. These proteins have been found in human fibroblasts and in several transformed cell lines. The heterogenous family of tau is formed by a set of molecular species that share common peptide sequences. There is a single gene that contains several exons enconding for the six different tau isoforms in mammalian brain. Alternative splicing of a common RNA transcript as well as post-translational modifications contribute to its heterogeneity. Tau isoforms generated by splicing differ from one another by having either three or four repeats in their C-terminal half, and a variable number of inserts in their N-terminal moiety. These repeats have been shown to constitute microtubule-binding motifs. In this review some relevant aspects of tau function and its regulation are analysed. Three major topics are discussed. The first one focuses on the tau roles in regulating the interactions between microtubules with actin filaments and with intermediate filment systems. Another problem deals with the question of whether tau isoforms segregate into functionally different subsets of microtubules in axonal processes, or tau associates with these polymers in a random fashion. The third question that emerges is the involvement of tau and tau-like proteins in morphogenetic events. The regulation of the interactions of DMAP-85, a recently discovered tau-like protein, with the cytoskeleton during development of Drosophila melanogaster is analyzed.

Asunto(s)

Animales , Anticuerpos Monoclonales/inmunología , Drosophila melanogaster/crecimiento & desarrollo , Neuronas/fisiología , Proteínas de Microtúbulos/fisiología , Proteínas tau/fisiología , Sitios de Unión , Diferenciación Celular/fisiología , Proteínas tau/inmunología , Química Encefálica

RESUMEN

The fluxes through HK/G6PDH and PK/LDH coupled-enzymatic reactions were quantified in the presence of physiological concentrations (1-15 microM) of polymerized or non-polymerized microtubular protein (MTP) from rat brain and in a permeabilized yeast cell system. In vitro enzymatic fluxes were increased by either polymerized or nonpolymerized brain MTP mainly in the lower range of MTP concentration. At fixed MTP concentrations in the flux stimulatory range of HK/G6PDH (1 mg/ml MTP) or PK/LDH (0.4 mg/ml MTP), a hyperbolic and sigmoidal response to NADP and PEP, respectively, was detected. That dependence varied according to the polymeric status of MTP. The specificity of the phenomenon observed in vitro, was tested for the PK/LDH and HK/G6PDH enzymatic couples in the presence of neutral polymers such as glycogen (< or = 10 mg/ml), poly(ethylene glycol) (up to 10% w/w) or G-actin (< or = 1 mg/ml). In permeabilized Saccharomyces cerevisiae cells, the PK-catalyzed flux was sensitive to microtubule disruption by nocodazole (15 micrograms/ml). The HK/G6PDH system was not affected by nocodazole showing values of kinetic parameters close to those obtained in vitro in the presence of polymerized brain MTP. Indirect immunofluorescence with specific antibodies against tubulin allowed to confirm the microtubules disruption in the presence of nocodazole in permeabilized yeast cells under the same conditions in which enzymes were assayed intracellularly. The experimental evidence is in agreement with the observed phenomenon of increase in fluxes in the enzymatic reactions assayed to be specifically induced by MTP either in vitro or in situ. The results presented are discussed in terms of the assembly of large supramolecular structures as a supraregulatory mechanism of synchronization of systemic cellular processes such as metabolic fluxes.

Asunto(s)

Carbono/metabolismo , Proteínas de Microtúbulos/fisiología , Actinas/farmacología , Animales , Biopolímeros , Química Encefálica/fisiología , Catálisis , Proteínas Fúngicas/fisiología , Glucosafosfato Deshidrogenasa/metabolismo , Hexoquinasa/metabolismo , Técnicas In Vitro , Cinética , L-Lactato Deshidrogenasa/metabolismo , Proteínas del Tejido Nervioso/fisiología , Piruvato Quinasa/metabolismo , Ratas , Saccharomyces cerevisiae

RESUMEN

We show here that antisense MAP2 oligonucleotides inhibit neurite outgrowth in cultured cerebellar macroneurons. Unlike control neurons, which first extend a lamellipodial veil followed by a consolidation phase during which the cells extend minor neurites, MAP2-suppressed cells persist with lamellipodia and later become rounded. The induction of microtubules containing tyrosinated tubulin, which parallels neurite outgrowth in control neurons, was blocked under antisense conditions. The small but significant increase in acetylated microtubules was not affected. In contrast, the suppression of tau, which selectively blocks axonal elongation, completely prevented the increase of acetylated microtubules, but did not modify the induction of labile microtubules. These results suggest that MAP2 and tau have different functions: the initial establishment of neurites depends upon MAP2, whereas further neurite elongation depends upon tau and microtubule stabilization.

Asunto(s)

Cerebelo/fisiología , Proteínas Asociadas a Microtúbulos/genética , Neuritas/fisiología , Neuronas/fisiología , Oligonucleótidos Antisentido/farmacología , Análisis de Varianza , Animales , Secuencia de Bases , Células Cultivadas , Cerebelo/citología , Embrión de Mamíferos , Cinética , Proteínas de Microtúbulos/análisis , Proteínas de Microtúbulos/metabolismo , Datos de Secuencia Molecular , Neuritas/efectos de los fármacos , Neuritas/ultraestructura , Neuronas/efectos de los fármacos , Neuronas/ultraestructura , Ratas , Factores de Tiempo

RESUMEN

Microtubule protein preparations purified from rat brain were used to study the effect of polycations and polyanions on the release of the COOH-terminal tyrosine of the alpha-chain of tubulin catalyzed by tubulin carboxypeptidase. (1) Most of the polycations and polyanions tested, independently of the ionogenic group, inhibited the reaction in a concentration-dependent fashion. Under steady-state conditions, detyrosination of the microtubule pool was inhibited to the same degree as occurred with the non-assembled tubulin pool, except in the case of chondroitin sulphate. This compound inhibited detyrosination of the non-assembled tubulin pool, but not that of microtubules. (2) Heparin, the most potent inhibitor tested, produced the dissociation of the carboxypeptidase from microtubules. Many, but not all, of the other microtubule-associated polypeptides were also dissociated by heparin. (3) Polylysine counteracted the inhibitory and dissociating effects of heparin. (4) Heparin protected tubulin carboxypeptidase against inactivation. Our results and previous reports describing, in nervous tissue, the presence of proteoglycans, RNA and basic proteins that inhibit detyrosination, suggest that tubulin carboxypeptidase might be physiologically modulated by electrically charged macromolecules.

Asunto(s)

Carboxipeptidasas/metabolismo , Proteínas de Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Animales , Aniones , Encéfalo/enzimología , Cationes , Sulfatos de Condroitina/farmacología , Heparina/farmacología , Cinética , Poli A/farmacología , Ácido Poliglutámico/farmacología , Polilisina/farmacología , Protaminas/farmacología , Ratas