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At least 19 recordsLinked to original sources

PubMed2000

A molecular genetic analysis of the interaction between the cytoplasmic dynein intermediate chain and the glued (dynactin) complex.

The microtubule motor cytoplasmic dynein performs multiple cellular functions; however, the regulation and targeting of the motor to different cargoes is not well understood. A biochemical interaction between the dynein intermediate chain subunit and the p150-Glued component of the dynein regulatory complex, dynactin, has supported the hypothesis that the intermediate chain is a key modulator of dynein attachment to cellular cargoes. In this report, we identify multiple intermediate chain polypeptides that cosediment with the 19S dynein complex and two differentially expressed transcripts derived from the single cytoplasmic dynein intermediate chain (Cdic) gene that differ in the 3' untranslated region sequence. These results support previous observations of multiple Cdic gene products that may contribute to the specialization of dynein function. Most significantly, we provide genetic evidence that the interaction between the dynein intermediate chain and p150-Glued is functionally relevant. We use a genomic Cdic transgene to show that extra copies of the dynein intermediate chain gene act to suppress the rough eye phenotype of the mutant Glued(1), a mutation in the p150-Glued subunit of dynactin. Furthermore, we show that the interaction between the dynein intermediate chain and p150-Glued is dependent on the dosage of the Cdic gene. This result suggests that the dynein intermediate chain may be a limiting component in the assembly of the dynein complex and that the regulation of the interaction between the dynein intermediate chain and dynactin is critical for dynein function.

3' Untranslated Regions↗

PubMed1998

The yeast dynactin complex is involved in partitioning the mitotic spindle between mother and daughter cells during anaphase B.

Although vertebrate cytoplasmic dynein can move to the minus ends of microtubules in vitro, its ability to translocate purified vesicles on microtubules depends on the presence of an accessory complex known as dynactin. We have cloned and characterized a novel gene, NIP100, which encodes the yeast homologue of the vertebrate dynactin complex protein p150(glued). Like strains lacking the cytoplasmic dynein heavy chain Dyn1p or the centractin homologue Act5p, nip100Delta strains are viable but undergo a significant number of failed mitoses in which the mitotic spindle does not properly partition into the daughter cell. Analysis of spindle dynamics by time-lapse digital microscopy indicates that the precise role of Nip100p during anaphase is to promote the translocation of the partially elongated mitotic spindle through the bud neck. Consistent with the presence of a true dynactin complex in yeast, Nip100p exists in a stable complex with Act5p as well as Jnm1p, another protein required for proper spindle partitioning during anaphase. Moreover, genetic depletion experiments indicate that the binding of Nip100p to Act5p is dependent on the presence of Jnm1p. Finally, we find that a fusion of Nip100p to the green fluorescent protein localizes to the spindle poles throughout the cell cycle. Taken together, these results suggest that the yeast dynactin complex and cytoplasmic dynein together define a physiological pathway that is responsible for spindle translocation late in anaphase.

Amino Acid Sequence↗

PubMed1996

The product of the Drosophila gene, Glued, is the functional homologue of the p150Glued component of the vertebrate dynactin complex.

p150Glued is the largest polypeptide in the dynactin complex, a protein heteromultimer that binds to and may mediate the microtubule-based motor cytoplasmic dynein. Cloning of a cDNA encoding p150Glued from rat revealed 31% amino acid sequence identity with the product of the Drosophila gene, Glued. A dominant Glued mutation results in neuronal disruption; null mutations are lethal. However, the Glued gene product has not been characterized. To determine whether the Glued polypeptide is functionally similar to vertebrate p150Glued, we characterized the Glued protein in the Drosophila S-2 cell line. Antibodies raised against Glued were used to demonstrate that this protein sediments exclusively at 20 S, and associates with microtubules in a salt- and ATP-dependent manner. Immunoprecipitations from S-2 cytosol with the anti-Glued antibody resulted in the co-precipitation of subunits of both cytoplasmic dynein and the dynactin complex. An affinity column with covalently bound Glued protein retained cytoplasmic dynein from S-2 cytosol. Based on these observations, we conclude that Glued is a component of a dynactin complex in Drosophila and binds to cytoplasmic dynein, and therefore the mutant Glued phenotypes can be interpreted as resulting from a disruption in the function of the dynactin complex.

Animals↗

PubMed1998

The role of the dynactin complex in intracellular motility.

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.

Axonal Transport↗

PubMed1999

The APC-associated protein EB1 associates with components of the dynactin complex and cytoplasmic dynein intermediate chain.

Human EB1 is a highly conserved protein that binds to the carboxyl terminus of the human adenomatous polyposis coli (APC) tumor suppressor protein [1], a domain of APC that is commonly deleted in colorectal neoplasia [2]. EB1 belongs to a family of microtubule-associated proteins that includes Schizosaccharomyces pombe Mal3 [3] and Saccharomyces cerevisiae Bim1p [4]. Bim1p appears to regulate the timing of cytokinesis as demonstrated by a genetic interaction with Act5, a component of the yeast dynactin complex [5]. Whereas the predominant function of the dynactin complex in yeast appears to be in positioning the mitotic spindle [6], in animal cells, dynactin has been shown to function in diverse processes, including organelle transport, formation of the mitotic spindle, and perhaps cytokinesis [7] [8] [9] [10]. Here, we demonstrate that human EB1 can be coprecipitated with p150(Glued), a member of the dynactin protein complex. EB1 was also found associated with the intermediate chain of cytoplasmic dynein (CDIC) and with dynamitin (p50), another component of the dynactin complex, but not with dynein heavy chain, in a complex that sedimented at approximately 5S in a sucrose density gradient. The association of EB1 with members of the dynactin complex was independent of APC and was preserved in the absence of an intact microtubule cytoskeleton. The molecular interaction of EB1 with members of the dynactin complex and with CDIC may be important for microtubule-based processes.

Adenomatous Polyposis Coli Protein↗

PubMed1995

Affinity chromatography demonstrates a direct binding between cytoplasmic dynein and the dynactin complex.

We used affinity chromatography to probe for a direct binding interaction between cytoplasmic dynein and dynactin. Purified cytoplasmic dynein was found to bind to an affinity column of p150Glued, the largest polypeptide in the dynactin complex. To test the specificity of the interaction, we loaded rat brain cytosol onto the p150Glued affinity column and observed that cytoplasmic dynein from cytosol was specifically retained on the column. Preincubation of the p150Glued affinity matrix with excess exogenous dynein intermediate chain resulted in a significant reduction of dynein binding, suggesting that p150Glued may be interacting with dynein via this polypeptide. Therefore we constructed an affinity column of recombinant dynein intermediate chain and observed that dynactin was retained from rat brain cytosol. These results demonstrate that the native dynein and dynactin complexes are capable of direct in vitro interaction mediated by a direct binding of the dynein intermediate chain to the p150Glued component of the dynactin complex. We have mapped the site of this interaction to the amino-terminal region of p150Glued, which is predicted to form an alpha-helical coiled-coil. Regulation of the dynein-dynactin interaction may prove to be key in the control mechanism for cytoplasmic dynein-mediated vesicular transport.

Animals↗

PubMed1994

Ultrastructural analysis of the dynactin complex: an actin-related protein is a component of a filament that resembles F-actin.

The dynactin complex visualized by deepetch electron microscopy appears as a short filament 37-nm in length, which resembles F-actin, plus a thinner, laterally oriented filament that terminates in two globular heads. The locations of several of the constituent polypeptides were identified on this structure by applying antibodies to decorate the dynactin complex before processing for electron microscopy. Antibodies to the actin-related protein Arp1 (previously referred to as actin-RPV), bound at various sites along the filament, demonstrating that this protein assembles in a polymer similar to conventional actin. Antibodies to the barbed-end actin-binding protein, capping protein, bound to one end of the filament. Thus, an actin-binding protein that binds conventional actin may also bind to Arp1 to regulate its polymerization. Antibodies to the 62-kD component of the dynactin complex also bound to one end of the filament. An antibody that binds the COOH-terminal region of the 160/150-kD dynactin polypeptides bound to the globular domains at the end of the thin lateral filament, suggesting that the dynactin polypeptide comprises at least part of the sidearm structure.

Actin Cytoskeleton↗

PubMed1999

Cytoplasmic dynein, the dynactin complex, and kinesin are interdependent and essential for fast axonal transport.

In axons, organelles move away from (anterograde) and toward (retrograde) the cell body along microtubules. Previous studies have provided compelling evidence that conventional kinesin is a major motor for anterograde fast axonal transport. It is reasonable to expect that cytoplasmic dynein is a fast retrograde motor, but relatively few tests of dynein function have been reported with neurons of intact organisms. In extruded axoplasm, antibody disruption of kinesin or the dynactin complex (a dynein activator) inhibits both retrograde and anterograde transport. We have tested the functions of the cytoplasmic dynein heavy chain (cDhc64C) and the p150(Glued) (Glued) component of the dynactin complex with the use of genetic techniques in Drosophila. cDhc64C and Glued mutations disrupt fast organelle transport in both directions. The mutant phenotypes, larval posterior paralysis and axonal swellings filled with retrograde and anterograde cargoes, were similar to those caused by kinesin mutations. Why do specific disruptions of unidirectional motor systems cause bidirectional defects? Direct protein interactions of kinesin with dynein heavy chain and p150(Glued) were not detected. However, strong dominant genetic interactions between kinesin, dynein, and dynactin complex mutations in axonal transport were observed. The genetic interactions between kinesin and either Glued or cDhc64C mutations were stronger than those between Glued and cDhc64C mutations themselves. The shared bidirectional disruption phenotypes and the dominant genetic interactions demonstrate that cytoplasmic dynein, the dynactin complex, and conventional kinesin are interdependent in fast axonal transport.

Animals↗

PubMed1994

A yeast actin-related protein homologous to that in vertebrate dynactin complex is important for spindle orientation and nuclear migration.

Spindle orientation controls nuclear migration and segregation during mitosis. In yeast, defects in dynein and astral microtubules lead to abnormal spindle orientation and nuclear migration. Dynactin complex is necessary for dynein-mediated vesicle motility in vitro. The major polypeptide of dynactin complex is an actin-related protein in the family Arp1. We have identified in S. cerevisiae a novel actin-related gene, ACT5, in the Arp1 family. An act5 null mutant has defects in spindle orientation and nuclear migration, as does overexpression of Act5p. The phenotype of a double mutant lacking dynein and Act5p is similar to that of single mutants. Therefore, dynactin complex is in the same pathway as dynein and may be necessary for the action of dynein in vivo.

Actins↗

PubMed1995

The p150Glued component of the dynactin complex binds to both microtubules and the actin-related protein centractin (Arp-1).

p150Glued was first identified as a polypeptide that copurifies with cytoplasmic dynein, the minus-end-directed microtubule-based motor protein, and has more recently been shown to be present as a member of the oligomeric dynactin complex, which includes the actin-related protein centractin (Arp-1). Dynactin is thought to mediate dynein-driven vesicle motility, as well as nuclear transport, in lower eukaryotes. The mechanism by which dynactin may function in these cellular processes is unknown. To examine the role of the dynactin complex in vivo, we overexpressed the rat cDNA encoding p150Glued in Rat-2 fibroblasts. Overexpression of full-length, as well as C-terminal deletion, constructs resulted in the decoration of microtubules with the p150Glued polypeptides. This cellular evidence for microtubule association was corroborated by in vitro microtubule-binding assays. Amino acids 39-150 of p150Glued were determined to be sufficient for microtubule association. We also tested for a direct interaction between p150Glued and centractin. In vitro translated centractin was specifically retained by a p150Glued affinity column, and this interaction was blocked by a synthetic peptide which corresponds to a highly conserved motif from the C terminus of p150Glued. These results demonstrate that p150Glued, a protein implicated in cytoplasmic dynein-based microtubule motility, is capable of direct binding to both microtubules and centractin.

Actins↗

PubMed2000

Dynamics of cytoplasmic dynein in living cells and the effect of a mutation in the dynactin complex actin-related protein Arp1.

Cytoplasmic dynein is a minus-end-directed microtubule motor that participates in multiple cellular activities such as organelle transport and mitotic spindle assembly [1]. To study the dynamic behavior of cytoplasmic dynein in the filamentous fungus Aspergillus nidulans, we replaced the gene for the cytoplasmic dynein heavy chain, nudA, with a gene encoding a green fluorescent protein (GFP)-tagged chimera, GFP-nudA. The GFP-NUDA fusion protein is fully functional in vivo: strains expressing only the GFP-tagged nudA grow as well as wild-type strains. Fluorescence microscopy showed GFP-NUDA to be in comet-like structures that moved in the hyphae toward the growing tip. Retrograde movement of some GFP-NUDA comets after they arrived at the tip was also observed. These dynamics of GFP-NUDA were not observed in cells treated with a microtubule-destabilizing drug, benomyl, suggesting they are microtubule-dependent. The rate of GFP-NUDA tip-ward movement is similar to the rate of cytoplasmic microtubule polymerization toward the hyphal tip, suggesting that GFP-NUDA is associated and moving with the polymerizing ends of microtubules. A mutation in actin-related protein Arp1 of the dynactin complex abolishes the presence of these dynamic GFP-NUDA structures near the hyphal tip, suggesting a targeting role of the dynactin complex.

Aspergillus nidulans↗

PubMed1995

Expression of the p150Glued component of the dynactin complex in developing and adult rat brain.

p150Glued is a component of the dynactin (Glued) complex that has been shown in vitro to be a required activator of cytoplasmic dynein-mediated transport of vesicles along microtubules and, thus, may be an essential component of retrograde axonal transport. In vivo, a dominant mutation in the Drosophila homologue of p150Glued induces aberrant neuronal development when heterozygous and is lethal when homozygous. In order to characterize the role of the dynactin complex in the development and function of vertebrate neurons, the distribution of the p150Glued message was examined via in situ hybridization to serial sections of adult rat brain and to a developmental series of sections. In the adult rat brain, the most intense hybridization observed with the p150Glued probe was in the pyramidal cells of the hippocampus proper, the dentate granule neurons, the cingulate and piriform cortices, the ventromedial hypothalamus, and the granular cell layer of the cerebellum. White-matter fiber tracts and the neuropil were generally devoid of signal. The data indicate that the mRNA encoding p150Glued is highly enriched in the cell bodies of neurons within the central nervous system. In developing rat, p150Glued is expressed at very high levels in neural tissue from the earliest time points assayed. Particularly intense hybridization was observed in the multiple layers of the retina, which is consistent with the phenotype of the Drosophila mutation. Therefore, the distributions observed via in situ hybridization are consistent with an essential role for p150Glued in retrograde axonal transport.

Animals↗

PubMed1997

The presence of the 50-kDa subunit of dynactin complex in the nerve growth cone.

Membrane proteins in growth cone-enriched and growth cone-non-enriched fractions prepared from neonatal mouse brain were separated by lectin-affinity and ion exchange chromatographies, 2D-PAGE, and SDS-PAGE. Partial amino acid sequences of the proteins concentrated in the growth cone-enriched fraction were determined. We found that one such protein, gmp23-48k, corresponds to the 50-kDa subunit (p50) of the dynactin complex. An antibody raised against gmp23-48k strongly reacted with growth cones of differentiated neuronal precursor cells. Immunoblot analyses revealed that gmp23-48k was present both in membrane and in soluble fractions of neonatal brain. However, the amount of gmp23-48k in the membrane fraction greatly decreased in adult brain. These results suggest a special role of membrane-associated gmp23-48k/p50 in synapse formation during brain development.

Amino Acid Sequence↗

PubMed1997

Overexpression of the dynamitin (p50) subunit of the dynactin complex disrupts dynein-dependent maintenance of membrane organelle distribution.

Dynactin is a multisubunit complex that plays an accessory role in cytoplasmic dynein function. Overexpression in mammalian cells of one dynactin subunit, dynamitin, disrupts the complex, resulting in dissociation of cytoplasmic dynein from prometaphase kinetochores, with consequent perturbation of mitosis (Echeverri, C.J., B.M. Paschal, K.T. Vaughan, and R.B. Vallee. 1996. J. Cell Biol. 132:617-634). Based on these results, dynactin was proposed to play a role in linking cytoplasmic dynein to kinetochores and, potentially, to membrane organelles. The current study reports on the dynamitin interphase phenotype. In dynamitin-overexpressing cells, early endosomes (labeled with antitransferrin receptor), as well as late endosomes and lysosomes (labeled with anti-lysosome-associated membrane protein-1 [LAMP-1]), were redistributed to the cell periphery. This redistribution was disrupted by nocodazole, implicating an underlying plus end-directed microtubule motor activity. The Golgi stack, monitored using sialyltransferase, galactosyltransferase, and N-acetylglucosaminyltransferase I, was dramatically disrupted into scattered structures that colocalized with components of the intermediate compartment (ERGIC-53 and ERD-2). The disrupted Golgi elements were revealed by EM to represent short stacks similar to those formed by microtubule-depolymerizing agents. Golgi-to-ER traffic of stack markers induced by brefeldin A was not inhibited by dynamitin overexpression. Time-lapse observations of dynamitin-overexpressing cells recovering from brefeldin A treatment revealed that the scattered Golgi elements do not undergo microtubule-based transport as seen in control cells, but rather, remain stationary at or near their ER exit sites. These results indicate that dynactin is specifically required for ongoing centripetal movement of endocytic organelles and components of the intermediate compartment. Results similar to those of dynamitin overexpression were obtained by microinjection with antidynein intermediate chain antibody, consistent with a role for dynactin in mediating interactions of cytoplasmic dynein with specific membrane organelles. These results suggest that dynamitin plays a pivotal role in regulating organelle movement at the level of motor-cargo binding.

Antigens, CD↗

PubMed1996

p150Glued, the largest subunit of the dynactin complex, is nonessential in Neurospora but required for nuclear distribution.

Dynactin is a multisubunit complex that is required for cytoplasmic dynein, a minus-end-directed, microtubule-associated motor, to efficiently transport vesicles along microtubules in vitro. p150Glued, the largest subunit of dynactin, has been identified in vertebrates and Drosophila and recently has been shown to interact with cytoplasmic dynein intermediate chains in vitro. The mechanism by which dynactin facilitates cytoplasmic dynein-dependent vesicle transport is unknown. We have devised a genetic screen for cytoplasmic dynein/dynactin mutants in the filamentous fungus Neurospora crassa. In this paper, we report that one of these mutants, ro-3, defines a gene encoding an apparent homologue of p150Glued, and we provide genetic evidence that cytoplasmic dynein and dynactin interact in vivo. The major structural features of vertebrate and Drosophila p150Glued, a microtubule-binding site at the N-terminus and two large alpha-helical coiled-coil regions contained within the distal two-thirds of the polypeptide, are conserved in Ro3. Drosophila p150Glued is essential for viability; however, ro-3 null mutants are viable, indicating that dynactin is not an essential complex in N. crassa. We show that N. crassa cytoplasmic dynein and dynactin mutants have abnormal nuclear distribution but retain the ability to organize cytoplasmic microtubules and actin in anucleate hyphae.

Actins↗

PubMed1998

The dynactin complex is required for cleavage plane specification in early Caenorhabditis elegans embryos.

BACKGROUND: During metazoan development, cell diversity arises primarily from asymmetric cell divisions which are executed in two phases: segregation of cytoplasmic factors and positioning of the mitotic spindle - and hence the cleavage plane -relative to the axis of segregation. When polarized cells divide, spindle alignment probably occurs through the capture and subsequent shortening of astral microtubules by a site in the cortex. RESULTS: Here, we report that dynactin, the dynein-activator complex, is localized at cortical microtubule attachment sites and is necessary for mitotic spindle alignment in early Caenorhabditis elegans embryos. Using RNA interference techniques, we eliminated expression in early embryos of dnc-1 (the ortholog of the vertebrate gene for p150(Glued)) and dnc-2 (the ortholog of the vertebrate gene for p50/Dynamitin). In both cases, misalignment of mitotic spindles occurred, demonstrating that two components of the dynactin complex, DNC-1 and DNC-2, are necessary to align the spindle. CONCLUSIONS: Dynactin complexes may serve as a tether for dynein at the cortex and allow dynein to produce forces on the astral microtubules required for mitotic spindle alignment.

Amino Acid Sequence↗

PubMed2002

Aberrant expression of centractin and capping proteins, integral constituents of the dynactin complex, in fetal down syndrome brain.

Down syndrome (DS, trisomy 21) is the most frequent genetic cause of mental retardation. Although known for more than a hundred years the underlying pathomechanisms for the phenotype and impaired brain functions remain elusive. Performing protein hunting in fetal DS brain, we detected a series of cytoskeleton proteins with aberrant expression in fetal DS cortex. Fetal brain cortex samples of controls and DS of the early second trimenon of gestation were used for the experiments. We applied two-dimensional electrophoresis with in-gel digestion of protein spots, subsequent mass spectroscopical (MALDI) identification, and quantification of spots using specific software. Centractin alpha, F-actin capping protein alpha-1, alpha-2 and beta subunits were significantly reduced in fetal DS cortex, whereas dynein intermediate clear 2, dynein intermediate chain 2, and kinesin light chain protein levels were unchanged. Centractins and F-actin capping proteins are major determinants of the cytoskeleton and are involved in pivotal functions including cellular, organelle, and nuclear motility. Deranged centractins and F-actin capping proteins may represent or induce deficient axonal transport and may well contribute to deterioration of the cytoskeleton's mitotic functions in trisomy 21.

Actin Depolymerizing Factors↗

PubMed1999

Neurospora crassa ro-10 and ro-11 genes encode novel proteins required for nuclear distribution.

Movement and distribution of nuclei in fungi have been shown to be dependent on cytoplasmic microtubules and the microtubule-associated motor cytoplasmic dynein. We have isolated hundreds of Neurospora crassa mutants, known as ropy, that are defective in nuclear distribution. Three of the ro genes, ro-1, ro-3 and ro-4, have been shown to encode subunits of either cytoplasmic dynein or the dynein activator complex, dynactin. In this report, we describe the isolation and initial characterization of two additional ro genes, ro-10 and ro-11. ro-10 and ro-11 are non-essential genes that encode novel 24 kDa and 75 kDa proteins respectively. Both ro-10 and ro-11 mutants retain the ability to generate long cytoplasmic microtubule tracks, suggesting that the nuclear distribution defect is not caused by a gross defect in the microtubule cytoskeleton. RO10, as well as RO4 (actin-related protein ARP1, the most abundant subunit of dynactin), appears to be required for the stability of RO3 (p150Glued), the largest subunit of dynactin. We propose that ro-10 mutants lack proper nuclear distribution, because RO10 is either a subunit of dynactin and required for dynactin activity or essential for assembly of the dynactin complex. ro-11 mutations have no effect on RO1 or RO3 levels and have only a very slight effect on the localization pattern of cytoplasmic dynein and dynactin. The role of RO11 in the movement and distribution of nuclei in N. crassa hyphae remains unknown.

Amino Acid Sequence↗