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1.
J Biol Chem ; 285(23): 17507-13, 2010 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-20371874

RESUMO

Microtubule growth proceeds through the endwise addition of nucleotide-bound tubulin dimers. The microtubule wall is composed of GDP-tubulin subunits, which are thought to come exclusively from the incorporation of GTP-tubulin complexes at microtubule ends followed by GTP hydrolysis within the polymer. The possibility of a direct GDP-tubulin incorporation into growing polymers is regarded as hardly compatible with recent structural data. Here, we have examined GTP-tubulin and GDP-tubulin incorporation into polymerizing microtubules using a minimal assembly system comprised of nucleotide-bound tubulin dimers, in the absence of free nucleotide. We find that GDP-tubulin complexes can efficiently co-polymerize with GTP-tubulin complexes during microtubule assembly. GDP-tubulin incorporation into microtubules occurs with similar efficiency during bulk microtubule assembly as during microtubule growth from seeds or centrosomes. Microtubules formed from GTP-tubulin/GDP-tubulin mixtures display altered microtubule dynamics, in particular a decreased shrinkage rate, apparently due to intrinsic modifications of the polymer disassembly properties. Thus, although microtubules polymerized from GTP-tubulin/GDP-tubulin mixtures or from homogeneous GTP-tubulin solutions are both composed of GDP-tubulin subunits, they have different dynamic properties, and this may reveal a novel form of microtubule "structural plasticity."


Assuntos
Guanosina Difosfato/química , Microtúbulos/metabolismo , Polímeros/química , Tubulina (Proteína)/química , Animais , Bioquímica/métodos , Centrossomo/metabolismo , Microscopia Crioeletrônica/métodos , Dimerização , Filtração , Guanosina Trifosfato/química , Humanos , Hidrólise , Microtúbulos/química , Nucleotídeos/química , Moduladores de Tubulina/química
2.
Curr Opin Cell Biol ; 15(1): 111-7, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12517712

RESUMO

Microtubule nucleation is the process in which several tubulin molecules interact to form a microtubule seed. Microtubule nucleation occurs spontaneously in purified tubulin solutions, and molecular intermediates between tubulin dimers and microtubules have been identified. Microtubule nucleation is enhanced in tubulin solutions by the addition of gamma-tubulin or various gamma-tubulin complexes. In vivo, microtubule assembly is usually seeded by gamma-tubulin ring complexes. Recent studies suggest, however, that microtubule nucleation can occur in the absence of gamma-tubulin, and that gamma-tubulin may have other cell functions apart from being a major component of the gamma-tubulin ring complex.


Assuntos
Células Eucarióticas/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)/biossíntese , Animais , Células Eucarióticas/ultraestrutura , Humanos , Microtúbulos/ultraestrutura , Modelos Biológicos , Polímeros/metabolismo
3.
J Cell Biol ; 174(6): 839-49, 2006 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-16954346

RESUMO

Tubulin-tyrosine ligase (TTL), the enzyme that catalyzes the addition of a C-terminal tyrosine residue to alpha-tubulin in the tubulin tyrosination cycle, is involved in tumor progression and has a vital role in neuronal organization. We show that in mammalian fibroblasts, cytoplasmic linker protein (CLIP) 170 and other microtubule plus-end tracking proteins comprising a cytoskeleton-associated protein glycine-rich (CAP-Gly) microtubule binding domain such as CLIP-115 and p150 Glued, localize to the ends of tyrosinated microtubules but not to the ends of detyrosinated microtubules. In vitro, the head domains of CLIP-170 and of p150 Glued bind more efficiently to tyrosinated microtubules than to detyrosinated polymers. In TTL-null fibroblasts, tubulin detyrosination and CAP-Gly protein mislocalization correlate with defects in both spindle positioning during mitosis and cell morphology during interphase. These results indicate that tubulin tyrosination regulates microtubule interactions with CAP-Gly microtubule plus-end tracking proteins and provide explanations for the involvement of TTL in tumor progression and in neuronal organization.


Assuntos
Fibroblastos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Tubulina (Proteína)/metabolismo , Tirosina/metabolismo , Animais , Células Cultivadas , Complexo Dinactina , Fibroblastos/ultraestrutura , Interfase/fisiologia , Camundongos , Microtúbulos/ultraestrutura , Proteínas do Tecido Nervoso/metabolismo , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Polímeros/metabolismo , Estrutura Terciária de Proteína/fisiologia , Fuso Acromático/metabolismo , Fuso Acromático/ultraestrutura
4.
J Cell Biol ; 169(3): 391-7, 2005 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-15883193

RESUMO

MCAK is a member of the kinesin-13 family of microtubule (MT)-depolymerizing kinesins. We show that the potent MT depolymerizer MCAK tracks (treadmills) with the tips of polymerizing MTs in living cells. Tip tracking of MCAK is inhibited by phosphorylation and is dependent on the extreme COOH-terminal tail of MCAK. Tip tracking is not essential for MCAK's MT-depolymerizing activity. We propose that tip tracking is a mechanism by which MCAK is preferentially localized to regions of the cell that modulate the plus ends of MTs.


Assuntos
Compartimento Celular/fisiologia , Polaridade Celular/fisiologia , Cinesinas/metabolismo , Microtúbulos/metabolismo , Animais , Células CHO , Cricetinae , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Microscopia de Vídeo , Microtúbulos/ultraestrutura , Fosforilação , Polímeros/metabolismo , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico/fisiologia , Proteínas Recombinantes de Fusão/metabolismo
5.
J Cell Biol ; 157(5): 807-17, 2002 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-12034773

RESUMO

The p160-Rho-associated coiled-coil-containing protein kinase (ROCK) is identified as a new centrosomal component. Using immunofluorescence with a variety of p160ROCK antibodies, immuno EM, and depletion with RNA interference, p160ROCK is principally bound to the mother centriole (MC) and an intercentriolar linker. Inhibition of p160ROCK provoked centrosome splitting in G1 with the MC, which is normally positioned at the cell center and shows little motion during G1, displaying wide excursions around the cell periphery, similar to its migration toward the midbody during cytokinesis. p160ROCK inhibition late after anaphase in mitosis triggered MC migration to the midbody followed by completion of cell division. Thus, p160ROCK is required for centrosome positioning and centrosome-dependent exit from mitosis.


Assuntos
Centríolos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Anáfase/fisiologia , Animais , Anticorpos , Bovinos , Centríolos/ultraestrutura , Clonagem Molecular , Fase G1/fisiologia , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Microscopia Imunoeletrônica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Coelhos , Quinases Associadas a rho
6.
Mol Biol Cell ; 17(3): 1041-50, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16371510

RESUMO

The activation of the cyclin-dependent kinase Cdk1 at the transition from interphase to mitosis induces important changes in microtubule dynamics. Cdk1 phosphorylates a number of microtubule- or tubulin-binding proteins but, hitherto, tubulin itself has not been detected as a Cdk1 substrate. Here we show that Cdk1 phosphorylates beta-tubulin both in vitro and in vivo. Phosphorylation occurs on Ser172 of beta-tubulin, a site that is well conserved in evolution. Using a phosphopeptide antibody, we find that a fraction of the cell tubulin is phosphorylated during mitosis, and this tubulin phosphorylation is inhibited by the Cdk1 inhibitor roscovitine. In mitotic cells, phosphorylated tubulin is excluded from microtubules, being present in the soluble tubulin fraction. Consistent with this distribution in cells, the incorporation of Cdk1-phosphorylated tubulin into growing microtubules is impaired in vitro. Additionally, EGFP-beta3-tubulin(S172D/E) mutants that mimic phosphorylated tubulin are unable to incorporate into microtubules when expressed in cells. Modeling shows that the presence of a phosphoserine at position 172 may impair both GTP binding to beta-tubulin and interactions between tubulin dimers. These data indicate that phosphorylation of tubulin by Cdk1 could be involved in the regulation of microtubule dynamics during mitosis.


Assuntos
Proteína Quinase CDC2/metabolismo , Microtúbulos/metabolismo , Mitose/fisiologia , Tubulina (Proteína)/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Fosfo-Específicos/metabolismo , Bovinos , Células HCT116 , Células HeLa , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Fosfopeptídeos/metabolismo , Fosforilação , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de Proteína , Serina/metabolismo , Tubulina (Proteína)/química , Células Tumorais Cultivadas
7.
J Neurochem ; 104(3): 745-56, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18199119

RESUMO

The microtubule-associated stable tubule only polypeptide (STOP) protein plays a key-role in neuron architecture and synaptic plasticity. Recent studies suggest that schizophrenia is associated with alterations in the synaptic connectivity. Mice invalidated for the STOP gene display phenotype reminiscent of some schizophrenic-like symptoms, such as behavioral disturbances, dopamine (DA) hyper-reactivity, and possible hypoglutamatergia, partly improved by antipsychotic treatment. In the present work, we examined potential alterations in some DAergic key proteins and behaviors in STOP knockout mice. Whereas the densities of the DA transporter, the vesicular monoamine transporter and the D(1) receptor were not modified, the densities of the D(2) and D(3) receptors were decreased in some DAergic regions in mutant versus wild-type mice. Endogenous DA levels were selectively decreased in DAergic terminals areas, although the in vivo DA synthesis was diminished both in cell bodies and terminal areas. The DA uptake was decreased in accumbic synaptosomes, but not significantly altered in striatal synaptosomes. Finally, STOP knockout mice were hypersensitive to acute and subchronic locomotor effects of cocaine, although the drug equally inhibited DA uptake in mutant and wild-type mice. Altogether, these data showed that deletion of the ubiquitous STOP protein elicited restricted alterations in DAergic neurotransmission, preferentially in the meso-limbic pathway.


Assuntos
Dopamina/metabolismo , Proteínas Associadas aos Microtúbulos/deficiência , Receptores Dopaminérgicos/metabolismo , Transmissão Sináptica/fisiologia , Análise de Variância , Animais , Gânglios da Base/metabolismo , Cocaína/farmacologia , Inibidores da Captação de Dopamina/farmacologia , Relação Dose-Resposta a Droga , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Atividade Motora/genética , Radiografia , Transmissão Sináptica/efeitos dos fármacos , Sinaptossomos/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Proteínas Vesiculares de Transporte de Monoamina/metabolismo
8.
Neuropharmacology ; 52(8): 1691-700, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17512560

RESUMO

Mice deficient in the microtubule stabilizing protein STOP (stable tubule only polypeptide) show synaptic plasticity anomalies in hippocampus, dopamine hyper-reactivity in the limbic system and severe behavioral deficits. Some of these disturbances are alleviated by long-term antipsychotic treatment. Therefore, this mouse line represents a pertinent model for some aspects of schizophrenia symptomatology. Numerous data support dysfunction of nicotinic neurotransmission in schizophrenia and epidemiological studies show increased tobacco use in schizophrenic patients, in whom nicotine has been reported to improve cognitive deficits and impairment in sensory gating. In this study, we examined potential alterations in cholinergic (ACh) and nicotinic components and functions in STOP mutant mice. STOP KO mice displayed no variation of the density of ACh esterase and beta2* nicotinic receptors (nAChRs), large reductions in the density of vesicular ACh transporter and alpha6* nAChRs and marked increases in the density of alpha7 nAChRs, in some brain areas. STOP KO mice were hypersensitive to the stimulating locomotor effect of nicotine and, interestingly, their impaired performance in learning the cued version of the water maze were improved by administration of the preferential alpha7 nAChR agonist choline. Altogether, our data show that the deletion of the ubiquitous STOP protein elicited restricted alterations in ACh components. They also suggest that nicotinic neurotransmission can be deficient in STOP KO mice and that mutant mice can represent a meaningful model to study some nicotinic dysfunctions and therapeutic treatments.


Assuntos
Colina/uso terapêutico , Regulação da Expressão Gênica/genética , Deficiências da Aprendizagem , Proteínas Associadas aos Microtúbulos/deficiência , Nootrópicos/uso terapêutico , Receptores Nicotínicos/metabolismo , Acetilcolinesterase/metabolismo , Análise de Variância , Animais , Autorradiografia , Comportamento Animal , Relação Dose-Resposta a Droga , Deficiências da Aprendizagem/tratamento farmacológico , Deficiências da Aprendizagem/genética , Deficiências da Aprendizagem/metabolismo , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Atividade Motora/genética , Tempo de Reação/efeitos dos fármacos , Receptores Nicotínicos/genética , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismo , Receptor Nicotínico de Acetilcolina alfa7
9.
J Psychopharmacol ; 21(6): 635-44, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17050659

RESUMO

Stable tubule-only polypeptide (STOP) proteins are a family of microtubule associated proteins (MAPs) important in microtubule stabilization. Data indicating a role for microtubules in synaptic function has come from studies of the STOP null mouse, which exhibits synaptic deficits, in association with behavioural changes that are alleviated by antipsychotic treatment. These findings suggested that STOP mutant mice may be useful in studies of synaptic function, and could be especially relevant to schizophrenia, postulated to be a disorder of the synapse. Moreover, a genetic association between STOP and schizophrenia has been reported. This study aimed to further characterize synaptic alterations in STOP null and heterozygous mice. Using in situ hybridization histochemistry, the mRNA expression of three pre-synaptic (synaptophysin; growth associated protein-43 (GAP-43); vesicular glutamate transporter-1 (VGlut1)) and two post-synaptic (spinophilin; MAP2) proteins, was quantified in female STOP null (n = 7), heterozygous (n = 5) and wild type (n = 6) mice. For STOP null and heterozygous mice, synaptophysin, VGlut1, GAP-43 and spinophilin mRNAs were decreased in the hippocampus, whilst in addition in the null mice, synaptophysin, VGlut1 and spinophilin mRNAs were decreased in the cerebellum. Alterations in synaptic protein mRNA expression were also detected in the frontal and occipital cortex. MAP2 mRNA expression was unchanged in all brain regions. The profile of mRNA changes is broadly similar to that observed in schizophrenia. Together the data provide supporting evidence for a role for microtubules in synaptic function, and suggest that STOP, or other microtubule proteins, may contribute to the synaptic pathology of schizophrenia.


Assuntos
Encéfalo/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , RNA Mensageiro/metabolismo , Sinapses/metabolismo , Animais , Feminino , Proteína GAP-43/metabolismo , Hibridização In Situ , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Associadas aos Microtúbulos/genética , Proteínas do Tecido Nervoso/genética , Esquizofrenia/metabolismo , Sinaptofisina/metabolismo , Proteína Vesicular 1 de Transporte de Glutamato/metabolismo
10.
Mol Biol Cell ; 14(11): 4605-17, 2003 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12960437

RESUMO

A number of key cellular functions, such as morphological differentiation and cell motility, are closely associated with changes in cytoskeletal dynamics. Many of the principal signaling components involved in actin cytoskeletal dynamics have been identified, and these have been shown to be critically involved in cell motility. In contrast, signaling to microtubules remains relatively uncharacterized, and the importance of signaling pathways in modulation of microtubule dynamics has so far not been established clearly. We report here that the Rho-effector ROCK and the multiadaptor proto-oncoprotein Cbl can profoundly affect the microtubule cytoskeleton. Simultaneous inhibition of these two signaling molecules induces a dramatic rearrangement of the microtubule cytoskeleton into microtubule bundles. The formation of these microtubule bundles, which does not involve signaling by Rac, Cdc42, Crk, phosphatidylinositol 3-kinase, and Abl, is sufficient to induce distinct neurite-like extensions in NIH 3T3 fibroblasts, even in the absence of microfilaments. This novel microtubule-dependent function that promotes neurite-like extensions is not dependent on net changes in microtubule polymerization or stabilization, but rather involves selective elongation and reorganization of microtubules into long bundles.


Assuntos
Extensões da Superfície Celular/metabolismo , Citoesqueleto/metabolismo , Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Oncogênicas de Retroviridae/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/fisiologia , Amidas/farmacologia , Animais , Extensões da Superfície Celular/fisiologia , Clonagem Molecular , Citoesqueleto/fisiologia , Inibidores Enzimáticos/farmacologia , Imunofluorescência , Genes abl/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Microtúbulos/fisiologia , Células NIH 3T3 , Proteína Oncogênica v-cbl , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas c-akt , Piridinas/farmacologia , Proteínas Oncogênicas de Retroviridae/fisiologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Quinases Associadas a rho
11.
Biol Psychiatry ; 60(11): 1224-30, 2006 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-16806091

RESUMO

BACKGROUND: Recent data suggest that cytoskeletal defects may play a role in schizophrenia. We previously imitated features of schizophrenia in an animal model by disrupting gene coding for a microtubule-associated protein called STOP. STOP-null mice display synaptic defects in glutamatergic neurons, hyper-dopaminergy, and severe behavioral disorders. Synaptic and behavioral deficits are amended by neuroleptic treatment in STOP-null mice, providing an attractive model to test new antipsychotic agents. We examined the effects of a taxol-related microtubule stabilizer, epothilone D. METHODS: Mice were treated either with vehicle alone or with epothilone D. Treatment effects on synaptic function were assessed using electron-microscopy quantification of synaptic vesicle pools and electrophysiology in the CA1 region of the hippocampus. Dopamine transmission was investigated using electrochemical assays. Behavior was principally assessed using tests of maternal skills. RESULTS: In STOP-null mice, treatment with epothilone D increased synaptic vesicle pools, ameliorated both short- and long-term forms of synaptic plasticity in glutamatergic neurons, and had a dramatic beneficial effect on mouse behavior. CONCLUSIONS: A microtubule stabilizer can have a beneficial effect on synaptic function and behavior, suggesting new possibilities for treatment of schizophrenia.


Assuntos
Comportamento Animal/efeitos dos fármacos , Epotilonas/administração & dosagem , Neurônios/efeitos dos fármacos , Esquizofrenia , Transmissão Sináptica/efeitos dos fármacos , Moduladores de Tubulina/administração & dosagem , Animais , Comportamento Animal/fisiologia , Células Cultivadas , Modelos Animais de Doenças , Estimulação Elétrica/métodos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Comportamento Exploratório/efeitos dos fármacos , Feminino , Hipocampo/patologia , Masculino , Comportamento Materno/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/deficiência , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/fisiologia , Plasticidade Neuronal/efeitos da radiação , Esquizofrenia/tratamento farmacológico , Esquizofrenia/patologia , Esquizofrenia/fisiopatologia , Transmissão Sináptica/fisiologia
12.
Behav Brain Res ; 163(2): 257-64, 2005 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-16046005

RESUMO

Schizophrenia is a chronic and debilitating disease which is thought to arise from a neuro-developmental disorder. Both the stable tubule-only polypeptide (STOP) protein and the N-methyl-D-aspartate (NMDA) NR1 subunit are involved in neuronal development and physiology. It has therefore been postulated that transgenic mice lacking either the STOP or the NMDAR1 gene would show a 'schizophrenic-like' phenotype. Here, STOP knockout and NMDA NR1 hypomorphic mice were assessed in a behavioural measure that can be used to detect schizophrenic-like phenotypes: a change in sensorimotor gating, measured through prepulse inhibition (PPI). STOP knockout mice were further assessed in another measure of 'schizophrenic-like behaviour': hyperlocomotion. The PPI deficit exhibited by both the STOP knockout and NMDA knockdown mice could not be reversed by acute treatment with the atyptical antipsychotic, clozapine (1 mg/kg, i.p.) but the hyperlocomotion shown by the STOP knockout mice was reversed with the same acute dose of clozapine.


Assuntos
Transtornos Neurológicos da Marcha/genética , Transtornos Neurológicos da Marcha/fisiopatologia , Proteínas Associadas aos Microtúbulos/deficiência , Receptores de N-Metil-D-Aspartato/deficiência , Córtex Somatossensorial/fisiopatologia , Estimulação Acústica/métodos , Animais , Antipsicóticos/administração & dosagem , Temperatura Corporal/efeitos dos fármacos , Temperatura Corporal/genética , Peso Corporal/efeitos dos fármacos , Peso Corporal/genética , Clozapina/administração & dosagem , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Transtornos Neurológicos da Marcha/tratamento farmacológico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Atividade Motora/genética , Inibição Neural/efeitos dos fármacos , Inibição Neural/genética , Fenciclidina/farmacologia , Reflexo Acústico/efeitos dos fármacos , Reflexo Acústico/genética , Teste de Desempenho do Rota-Rod/métodos , Córtex Somatossensorial/efeitos dos fármacos , Natação , Fatores de Tempo
13.
Dis Model Mech ; 6(1): 72-83, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22773755

RESUMO

Mutations in SPG4, encoding the microtubule-severing protein spastin, are responsible for the most frequent form of hereditary spastic paraplegia (HSP), a heterogeneous group of genetic diseases characterized by degeneration of the corticospinal tracts. We previously reported that mice harboring a deletion in Spg4, generating a premature stop codon, develop progressive axonal degeneration characterized by focal axonal swellings associated with impaired axonal transport. To further characterize the molecular and cellular mechanisms underlying this mutant phenotype, we have assessed microtubule dynamics and axonal transport in primary cultures of cortical neurons from spastin-mutant mice. We show an early and marked impairment of microtubule dynamics all along the axons of spastin-deficient cortical neurons, which is likely to be responsible for the occurrence of axonal swellings and cargo stalling. Our analysis also reveals that a modulation of microtubule dynamics by microtubule-targeting drugs rescues the mutant phenotype of cortical neurons. Together, these results contribute to a better understanding of the pathogenesis of SPG4-linked HSP and ascertain the influence of microtubule-targeted drugs on the early axonal phenotype in a mouse model of the disease.


Assuntos
Adenosina Trifosfatases/deficiência , Adenosina Trifosfatases/genética , Animais , Transporte Axonal , Axônios/efeitos dos fármacos , Axônios/patologia , Células Cultivadas , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Modelos Animais de Doenças , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Camundongos , Camundongos Knockout , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Modelos Neurológicos , Mutação , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Nocodazol/farmacologia , Paclitaxel/farmacologia , Paraplegia Espástica Hereditária/tratamento farmacológico , Paraplegia Espástica Hereditária/genética , Paraplegia Espástica Hereditária/metabolismo , Paraplegia Espástica Hereditária/patologia , Espastina , Vimblastina/farmacologia
14.
PLoS One ; 7(3): e33490, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22432029

RESUMO

Localization of CAP-Gly proteins such as CLIP170 at microtubule+ends results from their dual interaction with α-tubulin and EB1 through their C-terminal amino acids -EEY. Detyrosination (cleavage of the terminal tyrosine) of α-tubulin by tubulin-carboxypeptidase abolishes CLIP170 binding. Can detyrosination affect EB1 and thus regulate the presence of CLIP170 at microtubule+ends as well? We developed specific antibodies to discriminate tyrosinated vs detyrosinated forms of EB1 and detected only tyrosinated EB1 in fibroblasts, astrocytes, and total brain tissue. Over-expressed EB1 was not detyrosinated in cells and chimeric EB1 with the eight C-terminal amino acids of α-tubulin was only barely detyrosinated. Our results indicate that detyrosination regulates CLIPs interaction with α-tubulin, but not with EB1. They highlight the specificity of carboxypeptidase toward tubulin.


Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Tirosina/metabolismo , Animais , Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Encéfalo/metabolismo , Bovinos , Fibroblastos/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/química , Microtúbulos/química , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tubulina (Proteína)/química , Tubulina (Proteína)/imunologia , Tubulina (Proteína)/metabolismo
15.
Methods Mol Biol ; 777: 71-86, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21773921

RESUMO

Alpha tubulin comprises a C-terminal tyrosine residue, which is subject to cyclic removal from the peptide chain by a still uncharacterized carboxypeptidase and re-addition to the chain by a tubulin tyrosine ligase. We have shown in different animal or human models that the presence or absence of the tyrosine residue had dramatic consequences for both tumor progression and neuronal organization. In cells, tubulin detyrosination impairs the proper localization of CAP-Gly proteins at microtubule + end, compromises the activity of microtubule-depolymerizing motors of the Kinesin 13 family, and favors both spastin microtubule-severing activity and kinesin 1 processivity. The biochemical basis for these cellular effects of tubulin detyrosination can now be investigated in reconstituted systems in vitro using homogeneous solutions of polymerizable tyrosinated or detyrosinated tubulin.


Assuntos
Isoformas de Proteínas/isolamento & purificação , Tubulina (Proteína)/isolamento & purificação , Tirosina/química , Animais , Humanos , Isoformas de Proteínas/química , Tubulina (Proteína)/química
16.
PLoS One ; 5(9): e12753, 2010 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-20856814

RESUMO

BACKGROUND: STOP (Stable Tubulin-Only Polypeptide) null mice show behavioral deficits, impaired synaptic plasticity, decrease in synaptic vesicular pools and disturbances in dopaminergic transmission, and are considered a neurodevelopmental model of schizophrenia. Olfactory neurons highly express STOP protein and are continually generated throughout life. Experimentally-induced loss of olfactory neurons leads to epithelial regeneration within two months, providing a useful model to evaluate the role played by STOP protein in adult olfactory neurogenesis. METHODOLOGY/PRINCIPAL FINDINGS: Immunocytochemistry and electron microscopy were used to study the structure of the glomerulus in the main olfactory bulb and neurogenesis in the neurosensorial epithelia. In STOP null mice, olfactory neurons showed presynaptic swellings with tubulovesicular profiles and autophagic-like structures. In olfactory and vomeronasal epithelia, there was an increase in neurons turnover, as shown by the increase in number of proliferating, apoptotic and immature cells with no changes in the number of mature neurons. Similar alterations in peripheral olfactory neurogenesis have been previously described in schizophrenia patients. In STOP null mice, regeneration of the olfactory epithelium did not modify these anomalies; moreover, regeneration resulted in abnormal organisation of olfactory terminals within the olfactory glomeruli in STOP null mice. CONCLUSIONS/SIGNIFICANCE: In conclusion, STOP protein seems to be involved in the establishment of synapses in the olfactory glomerulus. Our results indicate that the olfactory system of STOP null mice is a well-suited experimental model (1) for the study of the mechanism of action of STOP protein in synaptic function/plasticity and (2) for pathophysiological studies of the mechanisms of altered neuronal connections in schizophrenia.


Assuntos
Proteínas Associadas aos Microtúbulos/deficiência , Neurogênese , Neurônios Receptores Olfatórios/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Bulbo Olfatório/citologia , Bulbo Olfatório/metabolismo , Mucosa Olfatória/inervação , Mucosa Olfatória/metabolismo , Esquizofrenia/genética , Esquizofrenia/metabolismo , Sinapses/metabolismo
18.
PLoS One ; 4(4): e5405, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19404406

RESUMO

BACKGROUND: During development, neuronal growth cones integrate diffusible and contact guidance cues that are conveyed to both actin and microtubule (MT) cytoskeletons and ensure axon outgrowth and pathfinding. Although several post-translational modifications of tubulin have been identified and despite their strong conservation among species, their physiological roles during development, especially in the nervous sytem, are still poorly understood. METHODOLOGY/FINDINGS: Here, we have dissected the role of a post-translational modification of the last amino acid of the alpha-tubulin on axonal growth by analyzing the phenotype of precerebellar neurons in Tubulin tyrosin ligase knock-out mice (TTL(-/-)) through in vivo, ex vivo and in vitro analyses. TTL(-/-) neurons are devoid of tyrosinated tubulin. Their pathway shows defects in vivo, ex vivo, in hindbrains open-book preparations or in vitro, in a collagen matrix. Their axons still orient toward tropic cues, but they emit supernumerary branches and their growth cones are enlarged and exhibit an emission of mis-oriented filopodia. Further analysis of the TTL(-/-) growth cone intracellular organization also reveals that the respective localization of actin and MT filaments is disturbed, with a decrease in the distal accumulation of Myosin IIB, as well as a concomitant Rac1 over-activation in the hindbrain. Pharmacological inhibition of Rac1 over-activation in TTL(-/-) neurons can rescue Myosin IIB localization. CONCLUSIONS/SIGNIFICANCE: In the growth cone, we propose that tubulin tyrosination takes part in the relative arrangement of actin and MT cytoskeletons, in the regulation of small GTPases activity, and consequently, in the proper morphogenesis, organization and pathfinding of the growth cone during development.


Assuntos
Cones de Crescimento/ultraestrutura , Tubulina (Proteína)/metabolismo , Tirosina/metabolismo , Actinas/metabolismo , Animais , Axônios/ultraestrutura , Citoesqueleto/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Camundongos , Miosina não Muscular Tipo IIB/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas rac1 de Ligação ao GTP/metabolismo
19.
J Cell Biol ; 185(7): 1159-66, 2009 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-19564401

RESUMO

In cells, stable microtubules (MTs) are covalently modified by a carboxypeptidase, which removes the C-terminal Tyr residue of alpha-tubulin. The significance of this selective detyrosination of MTs is not understood. In this study, we report that tubulin detyrosination in fibroblasts inhibits MT disassembly. This inhibition is relieved by overexpression of the depolymerizing motor mitotic centromere-associated kinesin (MCAK). Conversely, suppression of MCAK expression prevents disassembly of normal tyrosinated MTs in fibroblasts. Detyrosination of MTs suppresses the activity of MCAK in vitro, apparently as the result of a decreased affinity of the adenosine diphosphate (ADP)-inorganic phosphate- and ADP-bound forms of MCAK for the MT lattice. Detyrosination also impairs MT disassembly in neurons and inhibits the activity of the neuronal depolymerizing motor KIF2A in vitro. These results indicate that MT depolymerizing motors are directly inhibited by the detyrosination of tubulin, resulting in the stabilization of cellular MTs. Detyrosination of transiently stabilized MTs may give rise to persistent subpopulations of disassembly-resistant polymers to sustain subcellular cytoskeletal differentiation.


Assuntos
Microtúbulos/metabolismo , Proteínas Motores Moleculares/metabolismo , Fuso Acromático/metabolismo , Tubulina (Proteína)/metabolismo , Tirosina/metabolismo , Animais , Forma Celular , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Hipocampo/citologia , Cinesinas/genética , Cinesinas/metabolismo , Camundongos , Camundongos Knockout , Proteínas Motores Moleculares/genética , Neurônios/citologia , Neurônios/metabolismo , Nocodazol/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Moduladores de Tubulina/metabolismo
20.
J Cell Sci ; 121(Pt 9): 1506-13, 2008 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-18411245

RESUMO

Bik1p is the budding yeast counterpart of the CLIP-170 family of microtubule plus-end tracking proteins, which are required for dynein localization at plus ends and dynein-dependent spindle positioning. CLIP-170 proteins make up a CAP-Gly microtubule-binding domain, which sustains their microtubule plus-end tracking behaviour. However, in yeast, Bik1p travels towards plus ends as a cargo of the plus-end-directed kinesin Kip2p. Additionally, Kip2p behaves as a plus-end-tracking protein; hence, it has been proposed that Bik1p might track plus ends principally as a cargo of Kip2p. Here, we examined Bik1p localization in yeast strains expressing mutant tubulin lacking the C-terminal amino acid (Glu tubulin; lacking Phe), the interaction of which with Bik1p is severely impaired compared with wild type. In Glu-tubulin strains, despite the presence of robust Kip2p comets at microtubule plus ends, Bik1p failed to track plus ends. Despite Bik1p depletion at plus ends, dynein positioning at the same plus ends was unperturbed. Video microscopy and genetic evidence indicated that dynein was transported at plus ends in a Kip2p-Bik1p-dependent manner, and was then capable of tracking Bik1p-depleted plus ends. These results indicate that Bik1p interactions with tubulin are important for Bik1p plus-end tracking, and suggest alternative pathways for Bik1p-Kip2p-dependent dynein localization at plus ends.


Assuntos
Cinesinas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/metabolismo , Polaridade Celular , Dineínas/metabolismo , Proteínas dos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitose , Modelos Biológicos , Proteínas Motores Moleculares , Proteínas Mutantes/metabolismo , Transporte Proteico , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Tubulina (Proteína)/metabolismo
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