RESUMEN
Interactions between microtubule and actin networks are thought to be crucial for mechanical and signalling events at the cell cortex. Cytoplasmic dynein has been proposed to mediate many of these interactions. Here, we report that dynein is localized to the cortex at adherens junctions in cultured epithelial cells and that this localization is sensitive to drugs that disrupt the actin cytoskeleton. Dynein is recruited to developing contacts between cells, where it localizes with the junctional proteins beta-catenin and E-cadherin. Microtubules project towards these early contacts and we hypothesize that dynein captures and tethers microtubules at these sites. Dynein immunoprecipitates with beta-catenin, and biochemical analysis shows that dynein binds directly to beta-catenin. Overexpression of beta-catenin disrupts the cellular localization of dynein and also dramatically perturbs the organization of the cellular microtubule array. In cells overexpressing beta-catenin, the centrosome becomes disorganized and microtubules no longer appear to be anchored at the cortex. These results identify a novel role for cytoplasmic dynein in capturing and tethering microtubules at adherens junctions, thus mediating cross-talk between actin and microtubule networks at the cell cortex.
Asunto(s)
Uniones Adherentes/metabolismo , Proteínas del Citoesqueleto/metabolismo , Dineínas/metabolismo , Microtúbulos/metabolismo , Transactivadores , Uniones Adherentes/química , Antineoplásicos/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Células Cultivadas , Cromatografía de Afinidad , Proteínas del Citoesqueleto/genética , Citoesqueleto/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Microscopía Fluorescente , Nocodazol/farmacología , Pruebas de Precipitina , Unión Proteica , Tiazoles/farmacología , Tiazolidinas , beta CateninaRESUMEN
Cytoplasmic dynein is an intracellular motor responsible for endoplasmic reticulum-to-Golgi vesicle trafficking and retrograde axonal transport. The accessory protein dynactin has been proposed to mediate the association of dynein with vesicular cargo. Dynactin contains a 37-nm filament made up of the actin-related protein, Arp1, which may interact with a vesicle-associated spectrin network. Here, we demonstrate that Arp1 binds directly to the Golgi-associated betaIII spectrin isoform. We identify two Arp1-binding sites in betaIII spectrin, one of which overlaps with the actin-binding site conserved among spectrins. Although conventional actin binds weakly to betaIII spectrin, Arp1 binds robustly in the presence of excess F-actin. Dynein, dynactin, and betaIII spectrin co-purify on vesicles isolated from rat brain, and betaIII spectrin co-immunoprecipitates with dynactin from rat brain cytosol. In interphase cells, betaIII spectrin and dynactin both localize to cytoplasmic vesicles, co-localizing most significantly in the perinuclear region of the cell. In dividing cells, betaIII spectrin and dynactin co-localize to the developing cleavage furrow and mitotic spindle, a novel localization for betaIII spectrin. We hypothesize that the interaction between betaIII spectrin and Arp1 recruits dynein and dynactin to intracellular membranes and provides a direct link between the microtubule motor complex and its membrane-bounded cargo.
Asunto(s)
Actinas/química , Proteínas de Microfilamentos , Proteínas Asociadas a Microtúbulos/química , Espectrina/química , Espectrina/metabolismo , Dominios Homologos src , Actinas/metabolismo , Animales , Sitios de Unión , Encéfalo/metabolismo , Células COS , Membrana Celular/metabolismo , Citoplasma/metabolismo , Citosol/metabolismo , Complejo Dinactina , Electroforesis en Gel de Poliacrilamida , Glutatión Transferasa/metabolismo , Immunoblotting , Inmunohistoquímica , Pruebas de Precipitina , Unión Proteica , Isoformas de Proteínas , Estructura Terciaria de Proteína , Ratas , Técnicas del Sistema de Dos HíbridosRESUMEN
We reconstituted dynein-driven, dynactin-dependent vesicle transport using protein-free liposomes and soluble components from squid axoplasm. Dynein and dynactin, while necessary, are not the only essential cytosolic factors; axonal spectrin is also required. Spectrin is resident on axonal vesicles, and rebinds from cytosol to liposomes or proteolysed vesicles, concomitant with their dynein-dynactin-dependent motility. Binding of purified axonal spectrin to liposomes requires acidic phospholipids, as does motility. Using dominant negative spectrin polypeptides and a drug that releases PH domains from membranes, we show that spectrin is required for linking dynactin, and thereby dynein, to acidic phospholipids in the membrane. We verify this model in the context of liposomes, isolated axonal vesicles, and whole axoplasm. We conclude that spectrin has an essential role in retrograde axonal transport.
Asunto(s)
Transporte Axonal/fisiología , Dineínas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Fosfolípidos/metabolismo , Espectrina/metabolismo , Ácidos/metabolismo , Animales , Axones/metabolismo , Vesículas Citoplasmáticas/metabolismo , Decapodiformes , Complejo Dinactina , Concentración de Iones de Hidrógeno , Liposomas/metabolismo , Microscopía por Video , Microtúbulos/metabolismo , Transporte de Proteínas/fisiologíaRESUMEN
Dynactin is a multisubunit complex and a required cofactor for most, or all, of the cellular processes powered by the microtubule-based motor cytoplasmic dynein. Using a dynein affinity column, the previously uncharacterized p62 subunit of dynactin was isolated and microsequenced. Two peptide sequences were used to clone human cDNAs encoding p62. Sequence analysis of the predicted human polypeptide of 53 kDa revealed a highly conserved pattern of eleven cysteine residues, eight of which fit the consensus sequence for a Zn(2+)-binding RING domain. We have characterized p62 as an integral component of 20 S dynactin by biochemical and immunocytochemical methods. Affinity chromatography experiments demonstrate that p62 binds directly to the Arp1 subunit of dynactin. Immunocytochemistry with antibodies to p62 demonstrates that this polypeptide has a punctate cytoplasmic distribution as well as centrosomal distribution typical of dynactin. In transfected cells, overexpression of p62 did not disrupt microtubule organization or the integrity of the Golgi but did cause both cytosolic and nuclear distribution of the protein, suggesting that this polypeptide may be targeted to the nucleus at very high expression levels.
Asunto(s)
Secuencia Conservada , Cisteína/análisis , Proteínas de Unión al ADN/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Receptores de Esteroides , Factores de Transcripción/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Factor de Transcripción COUP II , Factores de Transcripción COUP , Línea Celular , Clonación Molecular , ADN Complementario , Complejo Dinactina , Humanos , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/genética , Unión Proteica , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
Since the initial discovery of cytoplasmic dynein, it has become apparent that this microtubule-based motor is involved in several cellular functions including cell division and intracellular transport. Another multisubunit complex, dynactin, may be required for most, if not all, cytoplasmic dynein-driven activities and may provide clues to dynein's functional diversity. Recent genetic and biochemical findings have illuminated the cellular roles of dynein and dynactin and provided insight into the functional mechanism of this complex motor.
Asunto(s)
División Celular/fisiología , Citoplasma/metabolismo , Dineínas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Anafase , Animales , Aspergillus nidulans/metabolismo , Transporte Biológico , Núcleo Celular/metabolismo , Complejo Dinactina , Aparato de Golgi/metabolismo , Humanos , Cinetocoros/metabolismo , Saccharomyces cerevisiae/metabolismo , Huso Acromático/metabolismoRESUMEN
Dynactin is a required activator for the molecular motor cytoplasmic dynein, and is likely to be essential for normal neuronal development. Previously we mapped the human gene encoding the p150Glued subunit of dynactin to 2p13, in the vicinity of the locus linked to limb-girdle muscular dystrophy (LGMB2B). We now report the genomic organization of DCTN1. We have identified 32 exons in the gene which spans approximately 25 kb. Alternative splicing of several of the exons generates functionally distinct isoforms of the p150Glued polypeptide.
Asunto(s)
Cromosomas Humanos Par 2 , Proteínas Asociadas a Microtúbulos/genética , Distrofias Musculares/genética , Empalme Alternativo , Secuencia de Aminoácidos , Secuencia de Bases , Encéfalo/metabolismo , Línea Celular , Mapeo Cromosómico , Clonación Molecular , Cartilla de ADN , Complejo Dinactina , Dineínas/metabolismo , Exones , Biblioteca de Genes , Humanos , Intrones , Proteínas Asociadas a Microtúbulos/biosíntesis , Proteínas Asociadas a Microtúbulos/química , Datos de Secuencia Molecular , Neuronas/metabolismo , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/químicaRESUMEN
Dynactin, a multisubunit complex that binds to the microtubule motor cytoplasmic dynein, may provide a link between dynein and its cargo. Many subunits of dynactin have been characterized, elucidating the multifunctional nature of this complex. Using a dynein affinity column, p22, the smallest dynactin subunit, was isolated and microsequenced. The peptide sequences were used to clone a full-length human cDNA. Database searches with the predicted amino acid sequence of p22 indicate that this polypeptide is novel. We have characterized p22 as an integral component of dynactin by biochemical and immunocytochemical methods. Affinity chromatography experiments indicate that p22 binds directly to the p150(Glued) subunit of dynactin. Immunocytochemistry with antibodies to p22 demonstrates that this polypeptide localizes to punctate cytoplasmic structures and to the centrosome during interphase, and to kinetochores and to spindle poles throughout mitosis. Antibodies to p22, as well as to other dynactin subunits, also revealed a novel localization for dynactin to the cleavage furrow and to the midbodies of dividing cells; cytoplasmic dynein was also localized to these structures. We therefore propose that dynein/dynactin complexes may have a novel function during cytokinesis.
Asunto(s)
Proteínas Asociadas a Microtúbulos/química , Mitosis/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , División Celular/fisiología , Línea Celular , Clonación Molecular , Dipodomys , Complejo Dinactina , Dineínas/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Proteínas Asociadas a Microtúbulos/metabolismo , Datos de Secuencia Molecular , Proteínas Recombinantes/metabolismo , Análisis de Secuencia , Transfección/genéticaRESUMEN
Amid the continual flux of membranes and proteins through the Golgi, the distinctive structure and compartmentalization of the cisternal stacks are preserved. Microtubules and associated motors are required to maintain Golgi structure and for transport to and from the organelle. There is also evidence for Golgi-associated myosins. Recent research has identified a novel Golgi-associated spectrin-based network. In this review, we discuss evidence for this network and the possible roles for spectrin in maintaining Golgi structure and in vesicular transport to and from the Golgi. Overall the link between the cytoskeleton and the Golgi appears to be dynamic in nature, in keeping with the continuous flux of proteins and lipids through this organelle.
Asunto(s)
Citoesqueleto/fisiología , Aparato de Golgi/fisiología , Espectrina/fisiología , Animales , Línea Celular , Citoesqueleto/metabolismo , Aparato de Golgi/metabolismo , Humanos , Espectrina/metabolismoRESUMEN
Dynactin is a multisubunit complex that binds to the minus-end-directed microtubule motor cytoplasmic dynein and may provide a link between the motor and its cargo. Results from genetic studies in Saccharomyces cerevisiae, Neurospora crassa, Aspergillus nidulans, and Drosophila have suggested that cytoplasmic dynein and dynactin function in the same cellular pathways. p150Glued, a vertebrate homologue of the Drosophila gene Glued, is the largest polypeptide in the dynactin complex with multiple protein interactions. Centractin, the most abundant dynactin subunit polypeptide, forms an actin-like filament at the base of the complex. Studies on dynamitin, the 50-kDa dynactin subunit, predict a role for dynactin in mitotic spindle assembly. Other subunits of dynactin have also been cloned and characterized; these studies have provided insight into the role of the complex in essential cellular processes.
Asunto(s)
Transporte Axonal/fisiología , Núcleo Celular/metabolismo , Dineínas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Transporte Biológico/fisiología , Complejo DinactinaRESUMEN
Cytoplasmic dynein is one of the major motor proteins involved in intracellular transport. It is a protein complex consisting of four subunit classes: heavy chains, intermediate chains (ICs), light intermediate chains, and light chains. In a previous study, we had generated new monoclonal antibodies to the ICs and mapped the ICs to the base of the motor. Because the ICs have been implicated in targeting the motor to cargo, we tested whether these new antibodies to the intermediate chain could block the function of cytoplasmic dynein. When cytoplasmic extracts of Xenopus oocytes were incubated with either one of the monoclonal antibodies (m74-1, m74-2), neither organelle movement nor network formation was observed. Network formation and membrane transport was blocked at an antibody concentration as low as 15 micrograms/ml. In contrast to these observations, no effect was observed on organelle movement and tubular network formation in the presence of a control antibody at concentrations as high as 0.5 mg/ml. After incubating cytoplasmic extracts or isolated membranes with the monoclonal antibodies m74-1 and m74-2, the dynein IC polypeptide was no longer detectable in the membrane fraction by SDS-PAGE immunoblot, indicating a loss of cytoplasmic dynein from the membrane. We used a panel of dynein IC truncation mutants and mapped the epitopes of both antibodies to the N-terminal coiled-coil domain, in close proximity to the p150Glued binding domain. In an IC affinity column binding assay, both antibodies inhibited the IC-p150Glued interaction. Thus these findings demonstrate that direct IC-p150Glued interaction is required for the proper attachment of cytoplasmic dynein to membranes.
Asunto(s)
Citoplasma/metabolismo , Dineínas/metabolismo , Corteza Motora/metabolismo , Oocitos/metabolismo , Orgánulos/metabolismo , Animales , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Citoplasma/química , Complejo Dinactina , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Epítopos/análisis , Proteínas Asociadas a Microtúbulos/inmunología , Proteínas Asociadas a Microtúbulos/metabolismo , Oocitos/citología , Unión Proteica , XenopusRESUMEN
Fast axonal transport is characterized by the bidirectional, microtubule-based movement of membranous organelles. Cytoplasmic dynein is necessary but not sufficient for retrograde transport directed from the synapse to the cell body. Dynactin is a heteromultimeric protein complex, enriched in neurons, that binds to both microtubules and cytoplasmic dynein. To determine whether dynactin is required for retrograde axonal transport, we examined the effects of anti-dynactin antibodies on organelle transport in extruded axoplasm. Treatment of axoplasm with antibodies to the p150(Glued) subunit of dynactin resulted in a significant decrease in the velocity of microtubule-based organelle transport, with many organelles bound along microtubules. We examined the molecular mechanism of the observed inhibition of motility, and we demonstrated that antibodies to p150(Glued) disrupted the binding of cytoplasmic dynein to dynactin and also inhibited the association of cytoplasmic dynein with organelles. In contrast, the anti-p150(Glued) antibodies had no effect on the binding of dynactin to microtubules nor on cytoplasmic dynein-driven microtubule gliding. These results indicate that the interaction between cytoplasmic dynein and the dynactin complex is required for the axonal transport of membrane-bound vesicles and support the hypothesis that dynactin may function as a link between the organelle, the microtubule, and cytoplasmic dynein during vesicle transport.
Asunto(s)
Transporte Axonal/fisiología , Axones/metabolismo , Citoplasma/metabolismo , Dineínas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Animales , Anticuerpos/farmacología , Transporte Biológico/efectos de los fármacos , Sistema Libre de Células , Decapodiformes , Complejo Dinactina , Proteínas Asociadas a Microtúbulos/inmunología , Microtúbulos/efectos de los fármacos , Microtúbulos/ultraestructura , Orgánulos/fisiología , Unión ProteicaRESUMEN
Reversible protein phosphorylation has been implicated in the regulation of organelle transport by cytoplasmic dynein. Motor function may be modulated directly by the phosphorylation of dynein or through the phosphorylation of an accessory factor. Dynactin binds to cytoplasmic dynein and is a required activator for dynein-driven vesicular motility. In metabolic labeling studies we have determined that the p150Glued subunit of dynactin is a phosphoprotein. Treatment of Rat2 cells with okadaic acid or with activators of protein kinase A or protein kinase C caused a marked increase in the incorporation of 32P into p150Glued; the increased phosphorylation correlated with activated vesicular transport. Phosphoamino-acid analysis of p150Glued isolated from cells treated with okadaic acid or with activators of either protein kinase A or protein kinase C indicated exclusive labeling of phosphoserine. These results suggest that the phosphorylation of dynactin may serve to regulate intracellular transport catalyzed by cytoplasmic dynein.
Asunto(s)
Proteínas Asociadas a Microtúbulos/metabolismo , Animales , Transporte Biológico Activo/efectos de los fármacos , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Citoplasma/metabolismo , Complejo Dinactina , Dineínas/química , Dineínas/metabolismo , Activación Enzimática/efectos de los fármacos , Proteínas Asociadas a Microtúbulos/química , Microtúbulos/metabolismo , Modelos Biológicos , Ácido Ocadaico/farmacología , Orgánulos/metabolismo , Fosforilación , Conformación Proteica , Proteína Quinasa C/metabolismo , Ratas , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
p150Glued (Dynactin 1) is a component of the dynactin complex that is essential for retrograde axonal transport in neurons. The mouse p150Glued cDNA was isolated from mouse brain by RT-PCR. The complete sequence of the full length mouse cDNA was determined, including 19 bp of 5' UTR, 3843 bp of coding sequence, and 162 bp of 3' UTR. The predicted protein has a molecular mass of 142 kDa and exhibits 95% amino acid sequence identity to the predicted amino acid sequence of human p150Glued (DCTN1). The mouse Dctn1 gene was previously mapped in the central region of mouse chromosome 6, close to the neuromuscular disease gene mnd2. Northern blot analysis, complete sequencing of the cDNA, Western blot, and functional tests of the protein did not detect any abnormalities of p150Glued in mnd2 mice.
Asunto(s)
Dineínas/genética , Proteínas de Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/genética , Mutación , Enfermedades Neuromusculares/genética , Secuencia de Aminoácidos , Animales , ADN Complementario , Complejo Dinactina , Estudios de Evaluación como Asunto , Humanos , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
We have isolated a 27-kDa protein that binds to cytoplasmic dynein. Microsequencing of a 17-amino acid peptide of this polypeptide yielded a sequence which completely matched the predicted sequence of the beta subunit of casein kinase II, a highly conserved serine/threonine kinase. Affinity chromatography using a dynein column indicates that both the alpha and beta subunits of casein kinase II are retained by the column from rat brain cytosol. Although dynactin is also bound to the column, casein kinase II is not a dynactin subunit. Casein kinase II does not co-immunoprecipitate with dynactin, and it binds to a dynein intermediate chain column which has been preblocked with excess p150(Glued), a treatment that inhibits the binding of dynactin from cytosol. Bacterially expressed and purified rat dynein intermediate chain can be phosphorylated by casein kinase II in vitro. Further, native cytoplasmic dynein purified from rat brain can also be phosphorylated by casein kinase II in vitro. We propose that CKII may be involved in the regulation of dynein function possibly by altering its cargo specificity or its ability to interact with dynactin.
Asunto(s)
Citoplasma/metabolismo , Proteínas de Unión al ADN/metabolismo , Dineínas/metabolismo , Proteínas Asociadas a Microtúbulos , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Quinasa de la Caseína II , Complejo Dinactina , Técnicas In Vitro , Proteínas de Microtúbulos/metabolismo , Peso Molecular , Fosforilación , Conformación Proteica , RatasRESUMEN
Huntington's disease (HD) is an inherited neurodegenerative disease caused by expansion of a polyglutamine repeat in the HD protein huntingtin. Huntingtin's localization within the cell includes an association with cytoskeletal elements and vesicles. We previously identified a protein (HAP1) which binds to huntingtin in a glutamine repeat length-dependent manner. We now report that HAP1 interacts with cytoskeletal proteins, namely the p150 Glued subunit of dynactin and the pericentriolar protein PCM-1. Structural predictions indicate that both HAP1 and the interacting proteins have a high probability of forming coiled coils. We examined the interaction of HAP1 with p150 Glued . Binding of HAP1 to p150 Glued (amino acids 879-1150) was confirmed in vitro by binding of p150 Glued to a HAP1-GST fusion protein immobilized on glutathione-Sepharose beads. Also, HAP1 co-immunoprecipitated with p150 Glued from brain extracts, indicating that the interaction occurs in vivo . Like HAP1, p150 Glued is highly expressed in neurons in brain and both proteins are enriched in a nerve terminal vesicle-rich fraction. Double label immunofluorescence experiments in NGF-treated PC12 cells using confocal microscopy revealed that HAP1 and p150 Glued partially co-localize. These results suggest that HAP1 might function as an adaptor protein using coiled coils to mediate interactions among cytoskeletal, vesicular and motor proteins. Thus, HAP1 and huntingtin may play a role in vesicle trafficking within the cell and disruption of this function could contribute to the neuronal dysfunction and death seen in HD.
Asunto(s)
Liasas de Carbono-Oxígeno , Proteínas de Ciclo Celular , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Animales , Autoantígenos/metabolismo , Secuencia de Bases , Química Encefálica , Línea Celular , Cromatografía de Afinidad , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , ADN Complementario/genética , Complejo Dinactina , Humanos , Cinesinas/genética , Sustancias Macromoleculares , Microscopía Confocal , Proteínas Asociadas a Microtúbulos/química , Datos de Secuencia Molecular , Proteínas Nucleares/química , Células PC12 , Unión Proteica , Conformación Proteica , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiaeRESUMEN
Centractin (Arp1), an actin-related protein, is a component of the dynactin complex. To investigate potential functions of the protein, we used transient transfections to overexpress centractin in mammalian cells. We observed that the overexpressed polypeptide formed filamentous structures that were significantly longer and more variable in length than those observed in the native dynactin complex. The centractin filaments were distinct from conventional actin in subunit composition and pharmacology as demonstrated by the absence of immunoreactivity of these filaments with an actin-specific antibody, by resistance to treatment with the drug cytochalasin D, and by the inability to bind phalloidin. We examined the transfected cells for evidence of specific associations of the novel centractin filaments with cellular organelles or cytoskeletal proteins. Using immunocytochemistry we observed the colocalization of Golgi marker proteins with the centractin polymers. Additional immunocytochemical analysis using antibodies to non-erythroid spectrin (fodrin) and Golgi-spectrin (beta I sigma *) revealed that spectrin colocalized with the centractin filaments in transfected cells. Biochemical assays demonstrated that spectrin was present in dynactin-enriched cellular fractions, was coimmunoprecipitated from rat brain cytosol using antibodies to dynactin subunits, and was coeluted with dynactin using affinity chromatography. Immunoprecipitations and affinity chromatography also revealed that actin is not a bona fide component of dynactin. Our results indicate that spectrin is associated with the dynactin complex. We suggest a model in which dynactin associates with the Golgi through an interaction between the centractin filament of the dynactin complex and a spectrin-linked cytoskeletal network.
Asunto(s)
Actinas/metabolismo , Aparato de Golgi/química , Proteínas de Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos , Espectrina/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/análisis , Actinas/química , Actinas/genética , Animales , Transporte Biológico/fisiología , Biomarcadores , Línea Celular/química , Línea Celular/metabolismo , Cromatografía de Afinidad , Citoesqueleto/química , Citoesqueleto/metabolismo , Complejo Dinactina , Expresión Génica/fisiología , Aparato de Golgi/metabolismo , Macropodidae , Proteínas de Microtúbulos/análisis , Proteínas de Microtúbulos/aislamiento & purificación , Microtúbulos/metabolismo , Datos de Secuencia Molecular , Polímeros , Pruebas de Precipitina , Conejos , Homología de Secuencia de Aminoácido , Espectrina/análisis , Espectrina/aislamiento & purificación , Fracciones Subcelulares/química , TransfecciónRESUMEN
The neuron moves protein and membrane from the cell body to the synapse and back via fast and slow axonal transport. Little is known about the mechanism of microtubule movement in slow axonal transport, although cytoplasmic dynein, the motor for retrograde fast axonal transport of membranous organelles, has been proposed to also slide microtubules down the axon. We previously showed that most of the cytoplasmic dynein moving in the anterograde direction in the axon is associated with the microfilaments and other proteins of the slow component b (SCb) transport complex. The dynactin complex binds dynein, and it has been suggested that dynactin also associates with microfilaments. We therefore examined the role of dynein and dynactin in slow axonal transport. We find that most of the dynactin is also transported in SCb, including dynactin, which contains the neuron-specific splice variant p135(Glued), which binds dynein but not microtubules. Furthermore, SCb dynein binds dynactin in vitro. SCb dynein, like dynein from brain, binds microtubules in an ATP-sensitive manner, whereas brain dynactin binds microtubules in a salt-dependent manner. Dynactin from SCb does not bind microtubules, indicating that the binding of dynactin to microtubules is regulated and suggesting that the role of SCb dynactin is to bind dynein, not microtubules. These data support a model in which dynactin links the cytoplasmic dynein to the SCb transport complex. Dynein then may interact transiently with microtubules to slide them down the axon at the slower rate of SCa.
Asunto(s)
Transporte Axonal/fisiología , Dineínas/metabolismo , Proteínas de Microtúbulos/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Axones/química , Axones/fisiología , Reactivos de Enlaces Cruzados , Citoplasma/química , Complejo Dinactina , Dineínas/análisis , Dineínas/química , Isomerismo , Masculino , Proteínas de Microtúbulos/análisis , Proteínas de Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Dyneins are multisubunit mechanochemical enzymes capable of interacting with microtubules to generate force. Axonemal dyneins produce the motive force for ciliary and flagellar beating by inducing sliding between adjacent microtubules within the axoneme. Cytoplasmic dyneins translocate membranous organelles and chromosomes toward the minus ends of cytoplasmic microtubules. Dynactin is an accessory complex implicated in tethering cytoplasmic dynein to membranous organelles and mitotic kinetochores. In the studies described here, we have identified a number of new dynein genes and determined their mouse chromosomal locations by interspecific backcross analysis. We have also mapped several dynein and dynactin genes cloned previously. Our studies provide the first comprehensive attempt to map dynein and dynactin genes in mammals and provide a basis for the further analysis of dynein function in development and disease.
Asunto(s)
Mapeo Cromosómico , Dineínas/genética , Proteínas Asociadas a Microtúbulos , Secuencia de Aminoácidos , Animales , Clonación Molecular , Cruzamientos Genéticos , Complejo Dinactina , Genes/genética , Ratones , Ratones Endogámicos C57BL , Proteínas de Microtúbulos/genética , Datos de Secuencia Molecular , Muridae , Especificidad de Órganos , Polimorfismo de Longitud del Fragmento de Restricción , ARN Mensajero/análisis , RatasRESUMEN
P150Glued is the largest subunit of dynactin, which binds to cytoplasmic dynein and activates vesicle transport along microtubules. We have isolated human cDNAs encoding p150Glued as well as a 135-kDa isoform; these isoforms are expressed in human brain by alternative mRNA splicing of the human DCTN1 gene. The p135 isoform lacks the consensus microtubule-binding motif shared by members of the p150Glued/Glued/CLIP-170/BIK1 family of microtubule-associated proteins and, therefore, is predicted not to bind directly to microtubules. We used transient transfection assays and in vitro microtubule-binding assays to demonstrate that the p150 isoform binds to microtubules, but the p135 isoform does not. However, both isoforms bind to cytoplasmic dynein, and both partition similarly into cytosolic and membrane cellular fractions. Sequential immunoprecipitations with an isoform-specific antibody for p150 followed by a pan-isoform antibody revealed that, in brain, these polypeptides assemble to form distinct complexes, each of which sediments at approximately 20 S. On the basis of these observations, we hypothesize that there is a conserved neuronal function for a distinct form of the dynactin complex that cannot bind directly to cellular microtubules.