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Results for “Dynactin Complex”

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PubMed1998

The role of microtubule-based motor proteins in maintaining the structure and function of the Golgi complex.

The intimate association between the Golgi complex and the microtubule cytoskeleton plays an important role in Golgi structure and function. Recent evidence indicates that the dynamic flow of material from the ER to the Golgi is crucial to maintaining the integrity of the Golgi complex and its characteristic location within the cell, and it is now clear that this flow is dependent on the ongoing activity of microtubule motor proteins. This review focuses primarily on recent microinjection and expression studies which have explored the role of individual microtubule motor proteins in controlling Golgi dynamics. The collective evidence shows that one or more isoforms of cytoplasmic dynein, together with its cofactor the dynactin complex, are required to maintain a juxtanuclear Golgi complex in fibroblasts. Although questions remain about how dynein and dynactin are linked to the Golgi, there is evidence that the Golgi-spectrin lattice is involved. Kinesin and kinesin-like proteins appear to play a smaller role in Golgi dynamics, though this may be very cell-type specific. Moreover, new evidence about the role of kinesin family members continues to emerge. Thanks in part to recent advances in our understanding of these molecular motors, our current view of the Golgi complex is of an organelle in flux, undergoing constant renewal. Future research will be aimed at elucidating how and to what extent these motor proteins function as regulators of Golgi function.

Animals↗

PubMed1999

Dynamic association of cytoplasmic dynein heavy chain 1a with the Golgi apparatus and intermediate compartment.

Microtubule motors, such as the minus end-directed motor, cytoplasmic dynein, play an important role in maintaining the integrity, intracellular location, and function of the Golgi apparatus, as well as in the translocation of membrane between the endoplasmic reticulum and Golgi apparatus. We have immunolocalised conventional cytoplasmic dynein heavy chain to the Golgi apparatus in cultured vertebrate cells. In addition, we present evidence that cytoplasmic dynein heavy chain cycles constitutively between the endoplasmic reticulum and Golgi apparatus: it colocalises partially with the intermediate compartment, it is found on nocodazole-induced peripheral Golgi elements and, most strikingly, on Brefeldin A-induced tubules that are moving towards microtubule plus ends. The direction of movement of membrane between the endoplasmic reticulum and Golgi apparatus is therefore unlikely to be regulated by controlling motor-membrane interactions: rather, the motors probably remain bound throughout the whole cycle, with their activity being modulated instead. We also report that the overexpression of p50/dynamitin results in the loss of cytoplasmic dynein heavy chain from the membrane of peripheral Golgi elements. These results explain how dynamitin overexpression causes the inhibition of endoplasmic reticulum-to-Golgi transport complex movement towards the centrosomal region, and support the general model that an intact dynactin complex is required for cytoplasmic dynein binding to all cargoes.

Amino Acid Sequence↗

PubMed2005

Rod-Zw10-Zwilch: a key player in the spindle checkpoint.

The spindle checkpoint assures the proper segregation of chromosomes during mitosis. The best-characterized components of the checkpoint were originally identified in budding yeast. But three proteins with no yeast homologs--Rod, Zw10 and Zwilch--also play a crucial, but poorly understood, role in the metazoan spindle checkpoint. Recent work has begun to reveal the function of these proteins. The three form a complex (the RZZ complex), which is required for the recruitment of two better-known components of the kinetochore--the dynein-dynactin complex, and Mad1-Mad2. It has now been established that RZZ is directly or indirectly responsible for both Mad1-Mad2 recruitment to unattached kinetochores and its subsequent shedding from kinetochores following MT attachment, and thus is involved in both the activation and inactivation of the checkpoint. This review (which is part of the Chromosome Segregation and Aneuploidy series) covers recent developments in our understanding of RZZ dynamics and its function in the checkpoint.

Animals↗

PubMed2004

Hsp90-binding immunophilins link p53 to dynein during p53 transport to the nucleus.

The tumor suppressor protein p53 is known to be transported to the nucleus along microtubular tracks by cytoplasmic dynein. However, the connection between p53 and the dynein motor protein complex has not been established. Here, we show that hsp90.binding immunophilins link p53.hsp90 complexes to dynein and that prevention of that linkage in vivo inhibits the nuclear movement of p53. First, we show that p53.hsp90 heterocomplexes from DLD-1 human colon cancer cells contain an immunophilin (FKBP52, CyP-40, or PP5) as well as dynein. p53.hsp90.immunophilin.dynein complexes can be formed by incubating immunopurified p53 with rabbit reticulocyte lysate, and we show by peptide competition that the immunophilins link via their tetratricopeptide repeat domains to p53-bound hsp90 and by means of their PPIase domains to the dynein complex. The linkage of immunophilins to the dynein motor is indirect by means of the dynamitin component of the dynein-associated dynactin complex, and we show that purified FKBP52 binds directly by means of its PPIase domain to purified dynamitin. By using a temperature-sensitive mutant of p53 where cytoplasmic-nuclear movement occurs by shift to permissive temperature, we show that p53 movement is impeded when p53 binding to hsp90 is inhibited by the hsp90 inhibitor radicicol. Also, nuclear movement of p53 is inhibited when immunophilin binding to dynein is competed for by expression of a PPIase domain fragment in the same manner as when dynein linkage to cargo is dissociated by expression of dynamitin. This is the first demonstration of the linkage between an hsp90-chaperoned transcription factor and the system for its retrograde movement to the nucleus both in vitro and in vivo.

Active Transport, Cell Nucleus↗

PubMed2001

Dyneins have run their course in plant lineage.

Flowering plant genomes lack flagellar and cytoplasmic dyneins as well as the proteins that make up the dynactin complex. The mechanisms for organizing the Golgi apparatus, establishing spindle poles, and moving nuclei, vesicles, and chromosomes in flowering plants must be fundamentally different from those in other systems where these processes are dependent upon dynein and dynactin.

Cytoplasm↗

PubMed1992

A vertebrate actin-related protein is a component of a multisubunit complex involved in microtubule-based vesicle motility.

Actin is a cytoskeletal protein which is highly conserved across eukaryotic phyla. Actin filaments, in association with a family of myosin motor proteins, are required for cellular motile processes as diverse as vesicle transport, cell locomotion and cytokinesis. Many organisms have several closely related actin isoforms. In addition to conventional actins, yeasts contain actin-related proteins that are essential for viability. We show here that vertebrates also contain an actin-related protein (actin-RPV). Actin-RPV is a major component of the dynactin complex, an activator of dynein-driven vesicle movement, indicating that unlike conventional actins which work in conjunction with myosin motors, actin-RPV may be involved in cytoplasmic movements via a microtubule-based system.

Actins↗

PubMed2005

Comparative genome analysis reveals a conserved family of actin-like proteins in apicomplexan parasites.

BACKGROUND: The phylum Apicomplexa is an early-branching eukaryotic lineage that contains a number of important human and animal pathogens. Their complex life cycles and unique cytoskeletal features distinguish them from other model eukaryotes. Apicomplexans rely on actin-based motility for cell invasion, yet the regulation of this system remains largely unknown. Consequently, we focused our efforts on identifying actin-related proteins in the recently completed genomes of Toxoplasma gondii, Plasmodium spp., Cryptosporidium spp., and Theileria spp. RESULTS: Comparative genomic and phylogenetic studies of apicomplexan genomes reveals that most contain only a single conventional actin and yet they each have 8-10 additional actin-related proteins. Among these are a highly conserved Arp1 protein (likely part of a conserved dynactin complex), and Arp4 and Arp6 homologues (subunits of the chromatin-remodeling machinery). In contrast, apicomplexans lack canonical Arp2 or Arp3 proteins, suggesting they lost the Arp2/3 actin polymerization complex on their evolutionary path towards intracellular parasitism. Seven of these actin-like proteins (ALPs) are novel to apicomplexans. They show no phylogenetic associations to the known Arp groups and likely serve functions specific to this important group of intracellular parasites. CONCLUSION: The large diversity of actin-like proteins in apicomplexans suggests that the actin protein family has diverged to fulfill various roles in the unique biology of intracellular parasites. Conserved Arps likely participate in vesicular transport and gene expression, while apicomplexan-specific ALPs may control unique biological traits such as actin-based gliding motility.

Actins↗

PubMed1995

A fungal actin-related protein involved in nuclear migration.

The ro-4 mutant of the filamentous fungus Neurospora crassa forms distinctive colonies in which hyphae grow into rope-like aggregates. This unusual morphology coincides with a defect in hyphal nuclear migration. The ro-4 gene was cloned from a cosmid library by complementation of the closely linked pab-2 gene. The deduced 380 amino acid protein is most similar to the vertebrate actin-related protein/centractin. The RO4 protein is not essential for cell viability, and new strains created by inducing point mutations at the ro-4 locus have a phenotype which is very similar to that of the original mutant. This study provides genetic evidence that an actin-related protein plays a role in nuclear motility. Since nuclear motility is believed to be a microtubule-dependent process, the ro-4 gene product may function as a component of the dynactin complex which activates force generation by cytoplasmic dynein.

Actins↗

PubMed1999

Cloning and characterization of a gene (arpA) from Aspergillus oryzae encoding an actin-related protein required for normal nuclear distribution and morphology of conidiophores.

We have isolated the arpA gene from Aspergillus oryzae as a homologue of the Neurospora crassa ro-4 gene. In N. crassa, mutations in the ro-4 gene, which encodes a major component of the dynactin complex Arp1, causes curling of hyphae and abnormalities in nuclear distribution. The arpA gene contains two introns and encodes a polypeptide of 381 amino acids, with a 78% sequence identity to the N. crassa Arp1. Overexpression of the arpA gene causes a defect in nuclear migration into elongating hyphae of germlings in A. oryzae. We constructed arpA disruptant strains of A. oryzae. The arpA null mutants showed poor growth and hyper-branched mycelia, as well as a nuclear distribution defect. Scanning electron microscopy revealed that the arpA null mutant has an aberrant conidiophore morphology with irregular phialides.

Actins↗

PubMed2005

Hrs1p/Mcp6p on the meiotic SPB organizes astral microtubule arrays for oscillatory nuclear movement.

Microtubules and the motor protein dynein play pivotal roles in the movement and positioning of the nucleus and cytoplasmic organelles in a cell. In fission yeast, oscillatory movement of the nucleus termed horsetail nuclear movement (HNM) has been observed during meiotic prophase. HNM is led by an astral microtubule array emanating from the spindle pole body (SPB), a centrosome-equivalent organelle in yeasts, aided by the dynein-dynactin complex, and is proposed to facilitate the alignment of homologous chromosomes necessary for efficient meiotic recombination. Here we show that a meiosis-specific SPB component Hrs1p (also known as Mcp6p) is a key molecule to remodel microtubules into the horsetail-astral array (HAA). Deletion of Hrs1p impaired HAA formation, leading to compromised HNM. Ectopic expression of Hrs1p during the mitotic cell cycle resulted in the formation of a HAA-like astral microtubule array, which drove an oscillatory nuclear movement in interphase cells. Hrs1p interacted with components of the gamma-tubulin ring complex (gamma-TuRC) as well as with a meiotic SPB component. We propose that Hrs1p facilitates formation of the HAA, responsible for the vigorous HNM, by stabilizing connection between the SPB and minus ends of microtubules.

Cell Nucleus↗

PubMed1998

Epithelial and fibroblastoid cells contain numerous cell-type specific putative microtubule-regulating proteins, among which are ezrin and fodrin.

Upon cell junction formation, the microtubules of polarizing epithelial cells become reorganized by unknown signaling mechanisms and regulating proteins. Microtubule-associated (MAPs) and other types of proteins are likely to be involved in this process, but most of these are unknown. Such proteins are called here collectively microtubule-regulating proteins (MRPs). As a first step towards their characterization, we used co-sedimentation of cytosolic proteins of MDCK cells and A72, a dog fibroblastoid line, with an excess of taxol-stabilized MTs, to obtain a cell fraction enriched in putative MRPs ("MRPs"). Additional tests have led to the inventory of around 40 "MRPs" among the 80 proteins present in the microtubule pellet. We also found that "MRPs" are recovered in higher amounts from MDCK cytosol, and that half of these are cell-type specific. These results corroborate data from yeast cells and insect eggs, and show that in mammalian somatic cells too, a large number of proteins seems to be involved in microtubule regulation, and that different cell types use a specific set of MRPs. "MRPs" found in both cell types are the intermediate chain of cytoplasmic dynein, Arp1, the major subunit of the dynactin complex, and CLIP-170. Two MDCK-specific "MRPs" were identified as the actin-binding proteins ezrin and alpha-fodrin. These results are discussed with regard to a possible involvement of ezrin and fodrin in morphogenetic interactions of microtubules with the membrane cytoskeleton in polarizing epithelia upon junction formation.

Actins↗

PubMed2002

Polar transport in the Drosophila oocyte requires Dynein and Kinesin I cooperation.

BACKGROUND: The cytoskeleton and associated motors play an important role in the establishment of intracellular polarity. Microtubule-based transport is required in many cell types for the asymmetric localization of mRNAs and organelles. A striking example is the Drosophila oocyte, where microtubule-dependent processes govern the asymmetric positioning of the nucleus and the localization to distinct cortical domains of mRNAs that function as cytoplasmic determinants. A conserved machinery for mRNA localization and nuclear positioning involving cytoplasmic Dynein has been postulated; however, the precise role of plus- and minus end-directed microtubule-based transport in axis formation is not yet understood. RESULTS: Here, we show that mRNA localization and nuclear positioning at mid-oogenesis depend on two motor proteins, cytoplasmic Dynein and Kinesin I. Both of these microtubule motors cooperate in the polar transport of bicoid and gurken mRNAs to their respective cortical domains. In contrast, Kinesin I-mediated transport of oskar to the posterior pole appears to be independent of Dynein. Beside their roles in RNA transport, both motors are involved in nuclear positioning and in exocytosis of Gurken protein. Dynein-Dynactin complexes accumulate at two sites within the oocyte: around the nucleus in a microtubule-independent manner and at the posterior pole through Kinesin-mediated transport. CONCLUSION: The microtubule motors cytoplasmic Dynein and Kinesin I, by driving transport to opposing microtubule ends, function in concert to establish intracellular polarity within the Drosophila oocyte. Furthermore, Kinesin-dependent localization of Dynein suggests that both motors are components of the same complex and therefore might cooperate in recycling each other to the opposite microtubule pole.

Animals↗

PubMed2002

The human DnaJ protein, hTid-1, enhances binding of a multimer of the herpes simplex virus type 1 UL9 protein to oris, an origin of viral DNA replication.

We have identified cellular proteins that interact with the herpes simplex virus type 1 (HSV-1) origin-binding protein (UL9 protein) by screening a HeLa cell complementary DNA library by using the yeast two-hybrid system. Approximately 7 x 10(5) colonies were screened. Five of the 48 positive clones contained cDNAs that encoded the p150(Glued) component of the dynactin complex, three contained cDNAs for the neural F Box 42-kDa protein (NFB42), which is highly enriched in neural tissue, and three contained hTid-1, a human homologue of the bacterial DnaJ protein. We have focused in this report on the interaction of the viral UL9 protein with the cellular hTid-1. In vitro immunoprecipitation experiments confirmed that hTid-1 interacts with the UL9 protein. Electrophoretic mobility-shift assays indicated that the hTid-1 enhances the binding of UL9 protein to an HSV-1 origin, ori(s), and facilitates formation of the multimer from the dimeric UL9 protein. hTid-1 had no effect on the DNA-dependent ATPase or helicase activities associated with the UL9 protein. These findings implicate hTid-1 in HSV-1 DNA replication, and suggest that this cellular protein may provide a chaperone function analogous to the DnaJ protein in Escherichia coli DNA replication.

Adenosine Triphosphatases↗

PubMed1997

Na,K-ATPase transport from endoplasmic reticulum to Golgi requires the Golgi spectrin-ankyrin G119 skeleton in Madin Darby canine kidney cells.

Spectrin (betaISigma*) and ankyrin (AnkG119) associate with Golgi membranes and the dynactin complex, but their role in vesicle trafficking remains uncertain. We find that the actin-binding domain and membrane-association domain 1 (MAD1) of betaI spectrin together form a constitutive Golgi targeting signal in transfected MDCK cells. Expression of this signal in transfected cells disrupts the endogenous Golgi spectrin skeleton and blocks transport of alpha- and beta-Na,K-ATPase and vesicular stomatitis virus-G protein from the endoplasmic reticulum (ER) but does not disrupt the formation of Golgi stacks, the distribution of beta-COP, or the transport and surface display of E-cadherin. The Golgi spectrin skeleton is thus required for the transport of a subset of membrane proteins from the ER to the Golgi. We postulate that together with polyfunctional adapter proteins such as AnkG119, Golgi spectrin forms a docking complex that acts prior to the cis-Golgi, presumably with vesicular-tubular clusters (VTCs or ERGIC), to sequester specific membrane proteins into vesicles transiting between the ER and Golgi, and subsequently (probably involving other isoforms of spectrin and ankyrin) to mediate cargo transport within the Golgi and to other membrane compartments. We hypothesize that this vesicular spectrin-ankyrin adapter-protein trafficking (or tethering) system (SAATS) mediates the capture and transport of many membrane proteins and acts in conjunction with vesicle-targeting molecules to effect the efficient transport of cargo proteins.

Animals↗

PubMed2006

No association of DYNC1H1 with sporadic ALS in a case-control study of a northern European derived population: a tagging SNP approach.

The cytoplasmic dynein-dynactin complex has been implicated in the aetiology of motor neuron degeneration in both mouse models and human forms of motor neuron disease. We have previously shown that mutations in the cytoplasmic dynein 1 heavy chain 1 gene (Dync1h1) are causal in a mouse model of late-onset motor neuron degeneration but have found no association of the homologous sites in human DYNC1H1 with human motor neuron disease. Here we extend these analyses to investigate the DYNC1H1 genomic locus to determine if it is associated with sporadic amyotrophic lateral sclerosis (ALS) in a northern European-derived population. Among the 16 single nucleotide polymorphisms (SNPs) we examined, just two SNPs (rs2251644 and rs941793) were sufficient to tag the majority of haplotypic variation (r2 > or = 0.85) and these were tested in a case-control association study with 266 North American sporadic ALS patients and 225 matched controls. We found no association between genetic variation at DYNC1H1 and sporadic ALS (rs2251644; p = 0.538, rs941793; p = 0.204, haplotype; p = 0.956). In addition we investigated patterns of diversity at DYNC1H1 in Japanese and Cameroonian populations to establish the evolutionary history for this gene and observed reduced genetic diversity in the northern Europeans suggestive of selection at this locus.

Amyotrophic Lateral Sclerosis↗

PubMed1997

Saccharomyces cerevisiae genes required in the absence of the CIN8-encoded spindle motor act in functionally diverse mitotic pathways.

Kinesin-related Cin8p is the most important spindle-pole-separating motor in Saccharomyces cerevisiae but is not essential for cell viability. We identified 20 genes whose products are specifically required by cell deficient for Cin8p. All are associated with mitotic roles and represent at least four different functional pathways. These include genes whose products act in two spindle motor pathways that overlap in function with Cin8p, the kinesin-related Kip1p pathway and the cytoplasmic dynein pathway. In addition, genes required for mitotic spindle checkpoint function and for normal microtubule stability were recovered. Mutant alleles of eight genes caused phenotypes similar to dyn1 (encodes the dynein heavy chain), including a spindle-positioning defect. We provide evidence that the products of these genes function in concept with dynein. Among the dynein pathway gene products, we found homologues of the cytoplasmic dynein intermediate chain, the p150Glued subunit of the dynactin complex, and human LIS-1, required for normal brain development. These findings illustrate the complex cellular interactions exhibited by Cin8p, a member of a conserved spindle motor family.

Amino Acid Sequence↗

PubMed2006

Chlamydia trachomatis causes centrosomal defects resulting in chromosomal segregation abnormalities.

Chlamydiae traffic along microtubules to the microtubule organizing center (MTOC) to establish an intracellular niche within the host cell. Trafficking to the MTOC is dynein dependent although the activating and cargo-linking function of the dynactin complex is supplanted by unknown chlamydial protein(s). We demonstrate that once localized to the MTOC, the chlamydial inclusion maintains a tight association with cellular centrosomes. This association is sustained through mitosis and leads to a significant increase in supernumerary centrosomes, abnormal spindle poles, and chromosomal segregation defects. Chlamydial infection thus can lead to chromosome instability in cells that recover from infection.

Centrosome↗

PubMed2005

Kv1.5 surface expression is modulated by retrograde trafficking of newly endocytosed channels by the dynein motor.

In this article we have investigated the mechanisms by which retrograde trafficking regulates the surface expression of the voltage-gated potassium channel, Kv1.5. Overexpression of p50/dynamitin, known to disrupt the dynein-dynactin complex responsible for carrying vesicle cargo, substantially increased outward K+ currents in HEK293 cells stably expressing Kv1.5 (0.57+/-0.07 nA/pF, n=12; to 1.18+/-0.2 nA/pF, n=12, P<0.01), as did treatment of the cells with a dynamin inhibitory peptide, which blocks endocytosis. Nocodazole pretreatment, which depolymerizes the microtubule cytoskeleton along which dynein tracks, also doubled Kv1.5 currents in HEK cells and sustained K+ currents in isolated rat atrial myocytes. These increased currents were blocked by 1 mmol/L 4-aminopyridine, and the specific Kv1.5 antagonist, DMM (100 nM). Confocal imaging of both HEK cells and myocytes, as well as experiments testing the sensitivity of the channel in living cells to external Proteinase K, showed that this increase of K+ current density was caused by a redistribution of channels toward the plasma membrane. Coimmunoprecipitation experiments demonstrated a direct interaction between Kv1.5 and the dynein motor complex in both heterologous cells and rat cardiac myocytes, supporting the role of this complex in Kv1.5 trafficking, which required an intact SH3-binding domain in the Kv1.5 N terminus to occur. These experiments highlight a pathway for Kv1.5 internalization from the cell surface involving early endosomes, followed by later trafficking by the dynein motor along microtubules. This work has significant implications for understanding the way Kv channel surface expression is regulated.

Animals↗