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PubMed2004

Dynactin is involved in a checkpoint to monitor cell wall synthesis in Saccharomyces cerevisiae.

Checkpoint controls ensure the completion of cell cycle events with high fidelity in the correct order. Here we show the existence of a novel checkpoint that ensures coupling of cell wall synthesis and mitosis. In response to a defect in cell wall synthesis, S. cerevisiae cells arrest the cell-cycle before spindle pole body separation. This arrest results from the regulation of the M-phase cyclin Clb2p at the transcriptional level through the transcription factor Fkh2p. Components of the dynactin complex are required to achieve the G2 arrest whilst keeping cells highly viable. Thus, the dynactin complex has a function in a checkpoint that monitors cell wall synthesis.

Cell Cycle↗

PubMed2005

Molecular dissection of ARP1 regions required for nuclear migration and cell wall integrity checkpoint functions in Saccharomyces cerevisiae.

The dynactin complex is one of the components required for the regulation of the cell wall integrity checkpoint, which ensures the completion of cell wall remodeling before mitosis. The core of the dynactin complex is a backbone filament composed of monomers of an actin-related protein, Arp1, which is also involved in nuclear migration. To examine the molecular basis for the dual functions of the dynactin core subunit Arp1p in yeast, we constructed 32 mutated arp1 alleles. We assessed the effects of the mutations on cell wall integrity checkpoint and nuclear migration functions and identified four categories of mutants: 1) those showing no change from the wild type; 2) those resulting in a defective cell wall integrity checkpoint but normal nuclear migration; 3) those with a normal cell wall integrity checkpoint but defective nuclear migration; and 4) those defective in both the cell wall integrity checkpoint and nuclear migration functions. Our results show a separation of the two functions in the molecular structure of Arp1p and indicate that a local surface region of Arp1p is important in maintaining the cell wall integrity checkpoint function.

Alleles↗

PubMed2005

Sunday Driver links axonal transport to damage signaling.

Neurons transmit long-range biochemical signals between cell bodies and distant axonal sites or termini. To test the hypothesis that signaling molecules are hitchhikers on axonal vesicles, we focused on the c-Jun NH2-terminal kinase (JNK) scaffolding protein Sunday Driver (syd), which has been proposed to link the molecular motor protein kinesin-1 to axonal vesicles. We found that syd and JNK3 are present on vesicular structures in axons, are transported in both the anterograde and retrograde axonal transport pathways, and interact with kinesin-I and the dynactin complex. Nerve injury induces local activation of JNK, primarily within axons, and activated JNK and syd are then transported primarily retrogradely. In axons, syd and activated JNK colocalize with p150Glued, a subunit of the dynactin complex, and with dynein. Finally, we found that injury induces an enhanced interaction between syd and dynactin. Thus, a mobile axonal JNK-syd complex may generate a transport-dependent axonal damage surveillance system.

Adaptor Proteins, Signal Transducing↗

PubMed1998

Identification and molecular characterization of the p24 dynactin light chain.

Intracellular transport along microtubules uses the motor proteins cytoplasmic dynein and kinesin. Cytoplasmic dynein is responsible for movement to the minus ends of microtubules and the evidence indicates that dynein interacts with another protein complex, dynactin. In order to better understand how these proteins function, we have sought to identify and clone the subunit polypeptides of these two complexes, in particular their light chains. Dynactin is made up of eight subunits of approximately 24,000 to 160,000 Da. In order to clone the p24 subunit, the components of purified dynactin were resolved by SDS polyacrylamide gel electrophoresis. The amino acid sequence of a tryptic peptide from the 24,000-Mr region of the gel was obtained and a candidate polypeptide identified by a screen of the databases. This polypeptide has a predicted molecular weight of 20,822 Da. Using an antibody to a different region of this protein, we demonstrate that it copurifies with microtubules and elutes from the microtubule pellet with characteristics similar to those of the dynactin complex and distinct from those of cytoplasmic dynein. This polypeptide co-sediments with dynactin on sucrose density gradients and it also co-immunoprecipitates with dynactin, but not with kinesin or cytoplasmic dynein. Together these results demonstrate that this polypeptide is the p24 subunit of dynactin. Analysis of the predicted amino acid sequence of p24 shows that it is a unique protein that has no significant similarity to known enzymes or other proteins. Structural analysis indicates that most of this protein will form an alpha-helix and that portions of the molecule may participate in the formation of coiled-coils. Since stoichiometric analysis of dynactin indicates that there is one molecule of p24 per dynactin complex, these characteristics suggest that this polypeptide may be involved in protein-protein interactions, perhaps in the assembly of the dynactin complex.

Adenosine Triphosphate↗

PubMed1997

Phosphorylation by p34cdc2 protein kinase regulates binding of the kinesin-related motor HsEg5 to the dynactin subunit p150.

The kinesin-related motor HsEg5 is essential for centrosome separation, and its association with centrosomes appears to be regulated by phosphorylation of tail residue threonine 927 by the p34(cdc2) protein kinase. To identify proteins able to interact with the tail of HsEg5, we performed a yeast two-hybrid screen with a HsEg5 stalk-tail construct as bait. We isolated a cDNA coding for the central, alpha-helical region of human p150(Glued), a prominent component of the dynactin complex. The interaction between HsEg5 and p150(Glued) was enhanced upon activation of p34(CDC28), the budding yeast homolog of p34(cdc2), provided that HsEg5 had a phosphorylatable residue at position 927. Phosphorylation also enhanced the specific binding of p150(Glued) to the tail domain of HsEg5 in vitro, indicating that the two proteins are able to interact directly. Immunofluorescence microscopy revealed co-localization of HsEg5 and p150(Glued) during mitosis but not during interphase, consistent with a cell cycle-dependent association between the two proteins. Taken together, these results suggest that HsEg5 and p150(Glued) may interact in mammalian cells in vivo and that p34(cdc2) may regulate this interaction. Furthermore, they imply that the dynactin complex may functionally interact not only with dynein but also with kinesin-related motors.

Amino Acid Sequence↗

PubMed1999

Cytoplasmic dynein and dynactin as likely candidates for microtubule-dependent apical targeting of pancreatic zymogen granules.

The critical role of microtubules in vectorial delivery of post-Golgi carrier vesicles to the apical cell surface has been established for various polarized epithelial cell types. In the present study we used secretory granules of the rat and chicken pancreas, termed zymogen granules, as model system for apically bound post-Golgi carrier vesicles that underlie the regulated exocytotic pathway. We found that targeting of zymogen granules to the apical cell surface requires an intact microtubule system which contains its colchicine-resistant organizing center and, thus, the microtubular minus ends close to the apical membrane domain. Purified zymogen granules and their membranes were found to be associated with cytoplasmic dynein intermediate and heavy chain and to contain the major components of the dynein activator complex, dynactin, i.e. p150Glued, p62, p50, Arp1, and beta-actin. Kinesin heavy chain and the kinesin receptor, 160 kD kinectin, were not detected as components of zymogen granules. Immunofluorescence staining showed a zymogen granule-like distribution for dynein and dynactin (p150Glued, p62, p50, Arpl) in the apical cytoplasm, whereas kinesin and kinectin were largely concentrated in the basal half of the cells in a pattern similar to the distribution of calreticulin, a component of the endoplasmic reticulum. Secretory granules of non-polarized chromaffin cells of the bovine adrenal medulla, that are assumed to underlie microtubular plus end targeting from the Golgi apparatus to the cell periphery, were not found to be associated with dynein or dynactin. To our knowledge, this is the first demonstration of major components of the dynein-dynactin complex associated with the membrane of a biochemically and functionally well-defined organelle which is considered to underlie a vectorial minus end-driven microtubular transport critically involved in precise delivery of digestive enzymes to the apically located acinar lumen.

Animals↗

PubMed1997

Mutant molecular motors disrupt neural circuits in Drosophila.

A dominant negative mutation, Glued1, that codes for a component of the dynactin complex, disrupted the axonal anatomy of leg sensory neurons in Drosophila. To examine neuron structure in mutant animals, a P[Gal4] enhancer trap targeted expression of lacZ to the sensory neurons and thereby labeled neurons in the femoral chordotonal organ and their axons within the central nervous system. When these sensory axons were examined in the Glued1 mutant specimens, they were observed to arborize abnormally. This anatomical disruption of the sensory axons was associated with a corresponding disruption in a reflex. Normally, the tibial extensor motor neurons were excited when the femoral-tibial joint was flexed, but this resistance reflex was nearly absent in mutant animals. We used the P[Gal4] insertion strains to target expression of tetanus toxin light chain to these sensory neurons in wild-type animals and showed that this blocked the resistance reflex and produced a phenocopy of the Glued result. We conclude that disruption of the dynein-dynactin complex disrupts sensory axon path finding during metamorphosis, and this in turn disrupts synaptic connectivity.

Animals↗

PubMed1999

Dynein-dynactin function and sensory axon growth during Drosophila metamorphosis: A role for retrograde motors.

Mutations in the genes for components of the dynein-dynactin complex disrupt axon path finding and synaptogenesis during metamorphosis in the Drosophila central nervous system. In order to better understand the functions of this retrograde motor in nervous system assembly, we analyzed the path finding and arborization of sensory axons during metamorphosis in wild-type and mutant backgrounds. In wild-type specimens the sensory axons first reach the CNS 6-12 h after puparium formation and elaborate their terminal arborizations over the next 48 h. In Glued1 and Cytoplasmic dynein light chain mutants, proprioceptive and tactile axons arrive at the CNS on time but exhibit defects in terminal arborizations that increase in severity up to 48 h after puparium formation. The results show that axon growth occurs on schedule in these mutants but the final process of terminal branching, synaptogenesis, and stabilization of these sensory axons requires the dynein-dynactin complex. Since this complex functions as a retrograde motor, we suggest that a retrograde signal needs to be transported to the nucleus for the proper termination of some sensory neurons.

Animals↗

PubMed1997

Specific interactions between ATPase subunits of the 26 S protease.

The regulatory complex of the 26 S protease contains at least 15 distinct subunits. Six of these subunits (S4, S6, S6', S7, S8, and S10b) belong to a novel subfamily of presumptive nucleotidases that we call subunit 4 (S4)-like ATPases. Each of these putative ATPases was synthesized in reticulocyte lysate containing [35S]methionine, and the radiolabeled proteins were used in binding studies. S4, S6, S10b, and S6' displayed specific binding to components of the regulatory complex separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) or two-dimensional PAGE. S4 bound to S7, and S6 bound two proteins: S8 and centractin, a component of the dynactin complex. S10b bound to S6' and bound much more weakly to S1 and p50, another component of the dynactin complex. S6' bound to S10b. Two subunits, S7 and S8, did not bind any components present on nitrocellulose membranes, presumably because S7 and S8 are already oligomeric following synthesis. Co-translation and sucrose gradient sedimentation of 35S-labeled ATPases demonstrated the formation of S6'-S10b dimers in solution but revealed more complex associations, namely the formation of trimers and tetramers, among S4, S6, S7, and S8. Progressive COOH-terminal deletions that removed as much as 300 amino acids from S4 had no effect on the binding of S4 to S7. In striking contrast, truncation of 85 NH2-terminal amino acids from S4 abrogated binding, clearly implicating the NH2 terminus of S4 in its specific interaction with S7. Since S4-like ATPases contain putative coiled-coils within the first 150 NH2-terminal amino acids, we propose that coiled-coil interactions are responsible for the specificity of the observed subunit associations and that these associations are important for self-assembly of the regulatory complex.

Adenosine Triphosphatases↗

PubMed1999

Isolation of a new set of Aspergillus nidulans mutants defective in nuclear migration.

In the filamentous fungus Aspergillus nidulans, nuclear migration in the germ tube is mediated by cytoplasmic dynein. We have previously reported the characterization of four nud (nuclear distribution) genes, nudA, nudC, nudF and nudG, involved in this process. The nudA and nudG genes respectively encode for the heavy chain and the 8-kDa light chain of cytoplasmic dynein. In this work, we describe an improved method for the isolation of nud mutants that has led to the identification of at least ten additional nud loci. We have cloned one of the genes, nudK, and determined that it encodes the actin-related protein Arp1, which is a component of the dynactin complex. This provides the first evidence that dynactin is involved in nuclear migration in A. nidulans.

Actins↗

PubMed2000

ARP1 in Golgi organisation and attachment of manchette microtubules to the nucleus during mammalian spermatogenesis.

Actin related protein of vertebrate, Arp1, is a major component of the dynactin complex. To characterise and localise Arp1 during mammalian spermatogenesis, polyclonal antibodies were raised against a human recombinant Arp1. Anti-Arp1 antibodies were used for western-immunoblotting, indirect immunofluorescence and immunoelectron microscopy. In round spermatids, Arp1 was detected at the centrosome and at the Golgi apparatus. In elongated spermatids, Arp1 was predominantly found along microtubules of the manchette and at their site of attachment to the nuclear envelope. In maturing spermatids, Arp1 was still present in the pericentriolar material, but in testicular spermatozoa it was not detectable. These various localisations of Arp1 and their changes during spermatid differentiation suggest that the dynactin complex in association with dynein might contribute to several activities: the functional organisation of the centrosome and of the Golgi apparatus and the shaping of the nucleus by manchette microtubules.

Animals↗

PubMed2006

Dynactin targets Pavarotti-KLP to the central spindle during anaphase and facilitates cytokinesis in Drosophila S2 cells.

The dynactin complex cooperates with the dynein complex in various systems for mitotic completion. Here we analysed the mitotic phenotype of Drosophila S2 cells following the knockdown of the dynactin subunit p150(Glued). We found that p150(Glued)-depleted cells were delayed in metaphase and that the centrosomes were poorly connected to mitotic spindle poles. In addition, anaphase occurred with asynchronous chromosome segregation. Although cyclin B was degraded in these anaphase cells, Aurora B, MEI-S322 and BubR1 were not released from the non-segregating chromosomes. We also found that the density and organisation of the central spindle were compromised, with Aurora B and polo kinases absent from the diminished number of microtubules. Pavarotti-KLP, a component of the centralspindlin complex required for the formation of stable microtubule bundles, was not immediately targeted to the plus ends of the microtubules following anaphase onset as happened in controls. Instead, it accumulated transiently at the cell cortex during early anaphase and its targeting to the central spindle was delayed. These data suggest that the dynactin complex contributes to cytokinesis by promoting stable targeting of the centralspindlin complex to microtubule plus ends at anaphase onset. The contribution of the dynein-dynactin complex to synchronous chromosome segregation and cytokinesis is discussed.

Anaphase↗

PubMed2003

LIS1 at the microtubule plus end and its role in dynein-mediated nuclear migration.

The cytoplasmic dynein complex and its accessory dynactin complex are involved in many cellular activities including nuclear migration in fungi (for review see Karki and Holzbaur, 1999). LIS1, the product of a causal gene for human lissencephaly (smooth brain), has also been implicated in dynein function based on studies in fungi and more recent studies in higher eukaryotic systems (for review see Gupta et al., 2002). Exactly how LIS1 may regulate the behavior of cytoplasmic dynein in various organisms is a fascinating question. In this issue, Lee et al. (2003) describe important new findings in Saccharomyces cerevisiae regarding the role of LIS1 (Pac1) in dynein-mediated nuclear migration.

Cell Movement↗

PubMed2000

A Neurospora crassa Arp1 mutation affecting cytoplasmic dynein and dynactin localization.

Of the actin-related proteins, Arp1 is the most similar to conventional actin, and functions solely as a component of the multisubunit complex dynactin. Dynactin has been identified as an activator of the microtubule-associated motor cytoplasmic dynein. The role of Arp1 within dynactin is two-fold: (1) it serves as a structural scaffold protein for other dynactin subunits; and (2) it has been proposed to link dynactin, and thereby dynein, with membranous cargo via interaction with spectrin. Using the filamentous fungus Neurospora crassa, we have identified genes encoding subunits of cytoplasmic dynein and dynactin. In this study, we describe a genetic screen for N. crassa Arp1 (ro-4) mutants that are defective for dynactin function. We report that the ro-4(E8) mutant is unusual in that it shows alterations in the localization of cytoplasmic dynein and dynactin and in microtubule organization. In the mutant, dynein/dynactin complexes co-localize with bundled microtubules at hyphal tips. Given that dynein transports membranous cargo from hyphal tips to distal regions, the cytoplasmic dynein and dynactin complexes that accumulate along microtubule tracts at hyphal tips in the ro-4(E8) mutant may have either reduced motor activity or be delayed for activation of motor activity following cargo binding.

Actins↗

PubMed2004

Mutations in Caenorhabditis elegans cytoplasmic dynein components reveal specificity of neuronal retrograde cargo.

We describe Caenorhabditis elegans dynein complex mutants, which misaccumulate synaptic proteins at the ends of neuronal processes. Ultrastructural analysis revealed irregularly sized vesicles that likely represent accumulation of cargo. We propose that synaptobrevin, synaptotagmin, and UNC-104 are specific cargoes of the dynein complex. Many cargoes link to dynein via interactions between dynactin and vesicle-associated spectrin. However, loss of spectrin results in only mild and occasional defects in synaptobrevin localization. Thus, the dynein-dynactin complex shows neuronal cargo selectivity without spectrin being a critical component of cargo binding. We observed parallels to progressive motor neuron disease symptoms in these animals. With age, neuronal misaccumulations increase in size and frequency; locomotion becomes progressively slower; and life span is shortened. These mutants provide a model to assess whether defects in transport of specific cargo mediate neuronal dysfunction.

Aging↗

PubMed2001

Apoptotic cleavage of cytoplasmic dynein intermediate chain and p150(Glued) stops dynein-dependent membrane motility.

Cytoplasmic dynein is the major minus end-directed microtubule motor in animal cells, and associates with many of its cargoes in conjunction with the dynactin complex. Interaction between cytoplasmic dynein and dynactin is mediated by the binding of cytoplasmic dynein intermediate chains (CD-IC) to the dynactin subunit, p150(Glued). We have found that both CD-IC and p150(Glued) are cleaved by caspases during apoptosis in cultured mammalian cells and in Xenopus egg extracts. Xenopus CD-IC is rapidly cleaved at a conserved aspartic acid residue adjacent to its NH(2)-terminal p150(Glued) binding domain, resulting in loss of the otherwise intact cytoplasmic dynein complex from membranes. Cleavage of CD-IC and p150(Glued) in apoptotic Xenopus egg extracts causes the cessation of cytoplasmic dynein--driven endoplasmic reticulum movement. Motility of apoptotic membranes is restored by recruitment of intact cytoplasmic dynein and dynactin from control cytosol, or from apoptotic cytosol supplemented with purified cytoplasmic dynein--dynactin, demonstrating the dynamic nature of the association of cytoplasmic dynein and dynactin with their membrane cargo.

Amino Acid Sequence↗

PubMed2002

Disruption of dynein/dynactin inhibits axonal transport in motor neurons causing late-onset progressive degeneration.

To test the hypothesis that inhibition of axonal transport is sufficient to cause motor neuron degeneration such as that observed in amyotrophic lateral sclerosis (ALS), we engineered a targeted disruption of the dynein-dynactin complex in postnatal motor neurons of transgenic mice. Dynamitin overexpression was found to disassemble dynactin, a required activator of cytoplasmic dynein, resulting in an inhibition of retrograde axonal transport. Mice overexpressing dynamitin demonstrate a late-onset progressive motor neuron degenerative disease characterized by decreased strength and endurance, motor neuron degeneration and loss, and denervation of muscle. Previous transgenic mouse models of ALS have shown abnormalities in microtubule-based axonal transport. In this report, we describe a mouse model that confirms the critical role of disrupted axonal transport in the pathogenesis of motor neuron degenerative disease.

Amyotrophic Lateral Sclerosis↗

PubMed2001

Modulation of cytoplasmic dynein ATPase activity by the accessory subunits.

The microtubule-based motor molecule cytoplasmic dynein has been proposed to be regulated by a variety of mechanisms, including phosphorylation and specific interaction with the organelle-associated complex, dynactin. In this study, we examined whether the intermediate chain subunits of cytoplasmic dynein are involved in modulation of ATP hydrolysis, and thereby affect motility. Treatment of testis cytoplasmic dynein under hypertonic salt conditions resulted in separation of the intermediate chains from the remainder of the dynein molecule, and led to a 4-fold enhancement of ATP hydrolysis. This result suggests that the accessory subunits act as negative regulators of dynein heavy chain activity. Comparison of ATPase activities of dyneins with differing intermediate chain isoforms showed significant differences in basal ATP hydrolysis rates, with testis dynein 7-fold more active than dynein from brain. Removal of the intermediate chain subunits led to an equalization of ATPase activity between brain and testis dyneins, suggesting that the accessory subunits are responsible for the observed differences in tissue activity. Finally, our preparative procedures have allowed for the identification and purification of a 1:1 complex of dynein with dynactin. As this interaction is presumed to be mediated by the dynein intermediate chain subunits, we now have defined experimental conditions for further exploration of dynein enzymatic and motility regulation.

Adenosine Triphosphate↗