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.     

Structural dynamics and multiregion interactions in dynein-dynactin recognition

Cytoplasmic dynein is a 1.2-MDa multisubunit motor protein complex that, together with its activator dynactin, is responsible for the majority of minus end microtubule-based motility. Dynactin targets dynein to specific cellular locations, links dynein to cargo, and increases dynein processivity. These two macromolecular complexes are connected by a direct interaction between dynactin's largest subunit, p150(Glued), and dynein intermediate chain (IC) subunit. Here, we demonstrate using NMR spectroscopy and isothermal titration calorimetry that the binding footprint of p150(Glued) on IC involves two noncontiguous recognition regions, and both are required for full binding affinity. In apo-IC, the helical structure of region 1, the nascent helix of region 2, and the disorder in the rest of the chain are determined from coupling constants, amide-amide sequential NOEs, secondary chemical shifts, and various dynamics measurements. When bound to p150(Glued), different patterns of spectral exchange broadening suggest that region 1 forms a coiled-coil and region 2 a packed stable helix, with the intervening residues remaining disordered. In the 150-kDa complex of p150(Glued), IC, and two light chains, the noninterface segments remain disordered. The multiregion IC binding interface, the partial disorder of region 2 and its potential for post-translational modification, and the modulation of the length of the longer linker by alternative splicing may provide a basis for elegant and multifaceted regulation of binding between IC and p150(Glued). The long disordered linker between the p150(Glued) binding segments and the dynein light chain consensus sequences could also provide an attractive recognition platform for diverse cargoes.     

The gene for the intermediate chain subunit of cytoplasmic dynein is essential in Drosophila

The microtubule motor cytoplasmic dynein powers a variety of intracellular transport events that are essential for cellular and developmental processes. A current hypothesis is that the accessory subunits of the dynein complex are important for the specialization of cytoplasmic dynein function. In a genetic approach to understanding the range of dynein functions and the contribution of the different subunits to dynein motor function and regulation, we have identified mutations in the gene for the cytoplasmic dynein intermediate chain, Dic19C. We used a functional Dic transgene in a genetic screen to recover X-linked lethal mutations that require this transgene for viability. Three Dic mutations were identified and characterized. All three Dic alleles result in larval lethality, demonstrating that the intermediate chain serves an essential function in Drosophila. Like a deficiency that removes Dic19C, the Dic mutations dominantly enhance the rough eye phenotype of Glued(1), a dominant mutation in the gene for the p150 subunit of the dynactin complex, a dynein activator. Additionally, we used complementation analysis to identify an existing mutation, shortwing (sw), as an allele of the dynein intermediate chain gene. Unlike the Dic alleles isolated de novo, shortwing is homozygous viable and exhibits recessive and temperature-sensitive defects in eye and wing development. These phenotypes are rescued by the wild-type Dic transgene, indicating that shortwing is a viable allele of the dynein intermediate chain gene and revealing a novel role for dynein function during wing development.     

Rhodopsin's carboxy-terminal cytoplasmic tail acts as a membrane receptor for cytoplasmic dynein by binding to the dynein light chain Tctex-1.

The interaction of cytoplasmic dynein with its cargoes is thought to be indirectly mediated by dynactin, a complex that binds to the dynein intermediate chain. However, the roles of other dynein subunits in cargo binding have been unknown. Here we demonstrate that dynein translocates rhodopsin-bearing vesicles along microtubules. This interaction occurs directly between the C-terminal cytoplasmic tail of rhodopsin and Tctex-1, a dynein light chain. C-terminal rhodopsin mutations responsible for retinitis pigmentosa inhibit this interaction. Our results point to an alternative docking mechanism for cytoplasmic dynein, provide novel insights into the role of motor proteins in the polarized transport of post-Golgi vesicles, and shed light on the molecular basis of retinitis pigmentosa.     

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.     

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.     

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.     

Characterization of the p22 Subunit of Dynactin Reveals the Localization of Cytoplasmic Dynein and Dynactin to the Midbody of Dividing Cells

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.     

Cytoplasmic dynein binds dynactin through a direct interaction between the intermediate chains and p150Glued

Cytoplasmic dynein is a retrograde microtubule motor thought to participate in organelle transport and some aspects of minus end- directed chromosome movement. The mechanism of binding to organelles and kinetochores is unknown. Based on homology with the Chlamydomonas flagellar outer arm dynein intermediate chains (ICs), we proposed a role for the cytoplasmic dynein ICs in linking the motor protein to organelles and kinetochores. In this study two different IC isoforms were used in blot overlay and immunoprecipitation assays to identify IC- binding partners. In overlays of complex protein samples, the ICs bound specifically to polypeptides of 150 and 135 kD, identified as the p150Glued doublet of the dynactin complex. In reciprocal overlay assays, p150Glued specifically recognized the ICs. Immunoprecipitations from total Rat2 cell extracts, rat brain cytosol, and rat brain membranes further identified the dynactin complex as a specific target for IC binding. using truncation mutants, the sites of interaction were mapped to amino acids 1-123 of IC-1A and amino acids 200-811 of p150Glued. While cytoplasmic dynein and dynactin have been implicated in a common pathway by genetic analysis, our findings identify a direct interaction between two specific component polypeptides and support a role for dynactin as a dynein "receptor". Our data also suggest, however, that this interaction must be highly regulated.     

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.     

Neurodegenerative mutation in cytoplasmic dynein alters its organization and dynein-dynactin and dynein-kinesin interactions

A single amino acid change, F580Y (Legs at odd angles (Loa), Dync1h1(Loa)), in the highly conserved and overlapping homodimerization, intermediate chain, and light intermediate chain binding domain of the cytoplasmic dynein heavy chain can cause severe motor and sensory neuron loss in mice. The mechanism by which the Loa mutation impairs the neuron-specific functions of dynein is not understood. To elucidate the underlying molecular mechanisms of neurodegeneration arising from this mutation, we applied a cohort of biochemical methods combined with in vivo assays to systemically study the effects of the mutation on the assembly of dynein and its interaction with dynactin. We found that the Loa mutation in the heavy chain leads to increased affinity of this subunit of cytoplasmic dynein to light intermediate and a population of intermediate chains and a suppressed association of dynactin to dynein. These data suggest that the Loa mutation drives the assembly of cytoplasmic dynein toward a complex with lower affinity to dynactin and thus impairing transport of cargos that tether to the complex via dynactin. In addition, we detected up-regulation of kinesin light chain 1 (KLC1) and its increased association with dynein but reduced microtubule-associated KLC1 in the Loa samples. We provide a model describing how up-regulation of KLC1 and its interaction with cytoplasmic dynein in Loa could play a regulatory role in restoring the retrograde and anterograde transport in the Loa neurons.     

Cytoplasmic dynein intermediate chain phosphorylation regulates binding to dynactin

Previously, we identified dynactin as a cargo receptor or adaptor for cytoplasmic dynein, mediated by an interaction between the dynein intermediate chain and p150(Glued). To test phosphorylation as a potential regulatory mechanism for this interaction, we analyzed cytoplasmic dynein by two-dimensional gel analysis and detected two intermediate chain variants, one of which was eliminated by phosphatase treatment. Overlay assays demonstrated that p150(Glued) bound dephosphorylated but not phosphorylated intermediate chains. We then subjected the purified cytoplasmic dynein intermediate chain to mass spectrometry and identified a single phosphorylated tryptic fragment corresponding to the p150(Glued)-binding domain. Fragmentation and retention time analysis mapped the phosphorylation site to serine 84. Site-directed mutants designed to mimic the dephosphorylated or phosphorylated intermediate chain disrupted both in vitro phosphorylation and in vivo phosphorylation of transfected proteins. Mutants mimicking the dephosphorylated form bound p150(Glued) in vitro and overexpression perturbed transport of dynein-dependent membranes. Mutants mimicking the phosphorylated form displayed diminished p150(Glued) binding in vitro and did not disrupt dynein-mediated transport when expressed in vivo. These findings represent the first mapping of an intermediate chain phosphorylation site and suggest that this phosphorylation plays an important role in regulating the binding of cytoplasmic dynein to dynactin.     

Actin-related protein 1 and cytoplasmic dynein-based motility - what's the connection?

The actin-related protein Arp1 works in conjunction with the microtubule-based motor cytoplasmic dynein to drive many types of intracellular motility. In vertebrate cells, Arp1 is present exclusively in the form of a 37-nm filament that constitutes the backbone of dynactin, a 1.2-MDa macromolecular complex containing nine other polypeptides. Dynactin has been proposed to function as the link between dynein and its cargo. Recent work indicates that the dynactin subunit p150(Glued) mediates the interaction of the dynactin molecule with dynein and microtubules, leaving the Arp1 filament as a possible cargo-binding domain. Mechanisms for binding of F-actin to membranes are discussed as models of the Arp1-membrane interaction.     

Null Mutants of the Neurospora Actin-related Protein 1 Pointed-End Complex Show Distinct Phenotypes

Dynactin is a multisubunit complex that regulates the activities of cytoplasmic dynein, a microtubule-associated motor. Actin-related protein 1 (Arp1) is the most abundant subunit of dynactin, and it forms a short filament to which additional subunits associate. An Arp1 filament pointed-end--binding subcomplex has been identified that consists of p62, p25, p27, and Arp11 subunits. The functional roles of these subunits have not been determined. Recently, we reported the cloning of an apparent homologue of mammalian Arp11 from the filamentous fungus Neurospora crassa. Here, we report that N. crassa ro-2 and ro-12 genes encode the respective p62 and p25 subunits of the pointed-end complex. Characterization of Delta ro-2, Delta ro-7, and Delta ro-12 mutants reveals that each has a distinct phenotype. All three mutants have reduced in vivo vesicle trafficking and have defects in vacuole distribution. We showed previously that in vivo dynactin function is required for high-level dynein ATPase activity, and we find that all three mutants have low dynein ATPase activity. Surprisingly, Delta ro-12 differs from Delta ro-2 and Delta ro-7 and other previously characterized dynein/dynactin mutants in that it has normal nuclear distribution. Each of the mutants shows a distinct dynein/dynactin localization pattern. All three mutants also show stronger dynein/dynactin-membrane interaction relative to wild type, suggesting that the Arp1 pointed-end complex may regulate interaction of dynactin with membranous cargoes.     

Analysis of the dynein-dynactin interaction in vitro and in vivo

Cytoplasmic dynein and dynactin are megadalton-sized multisubunit molecules that function together as a cytoskeletal motor. In the present study, we explore the mechanism of dynein-dynactin binding in vitro and then extend our findings to an in vivo context. Solution binding assays were used to define binding domains in the dynein intermediate chain (IC) and dynactin p150Glued subunit. Transient overexpression of a series of fragments of the dynein IC was used to determine the importance of this subunit for dynein function in mammalian tissue culture cells. Our results suggest that a functional dynein-dynactin interaction is required for proper microtubule organization and for the transport and localization of centrosomal components and endomembrane compartments. The dynein IC fragments have different effects on endomembrane localization, suggesting that different endomembranes may bind dynein via distinct mechanisms.     

Dynactin is essential for growth cone advance

Dynactin is a multi-subunit complex that serves as a critical cofactor of the microtubule motor cytoplasmic dynein. We previously identified dynactin in the nerve growth cone. However, the function of dynactin in the growth cone is still unclear. Here we show that dynactin in the growth cone is required for constant forward movement of the growth cone. Chromophore-assisted laser inactivation (CALI) of dynamitin, a dynactin subunit, within the growth cone markedly decreases the rate of growth cone advance. CALI of dynamitin in vitro dissociates another dynactin subunit, p150^Glued, from dynamitin. These results indicate that dynactin, especially the interaction between dynamitin and p150^Glued, plays an essential role in growth cone advance.     

Regulation of cytoplasmic dynein function in vivo by the Drosophila Glued complex

The Drosophila Glued gene product shares sequence homology with the p150 component of vertebrate dynactin. Dynactin is a multiprotein complex that stimulates cytoplasmic dynein-mediated vesicle motility in vitro. In this report, we present biochemical, cytological, and genetic evidence that demonstrates a functional similarity between the Drosophila Glued complex and vertebrate dynactin. We show that, similar to the vertebrate homologues in dynactin, the Glued polypeptides are components of a 20S complex. Our biochemical studies further reveal differential expression of the Glued polypeptides, all of which copurify as microtubule-associated proteins. In our analysis of the Glued polypeptides encoded by the dominant mutation, Glued, we identify a truncated polypeptide that fails to assemble into the wild-type 20S complex, but retains the ability to copurify with microtubules. The spatial and temporal distribution of the Glued complex during oogenesis is shown by immunocytochemistry methods to be identical to the pattern previously described for cytoplasmic dynein. Significantly, the pattern of Glued distribution in oogenesis is dependent on dynein function, as well as several other gene products known to be required for proper dynein localization. In genetic complementation studies, we find that certain mutations in the cytoplasmic dynein heavy chain gene Dhc64C act as dominant suppressors or enhancers of the rough eye phenotype of the dominant Glued mutation. Furthermore, we show that a mutation that was previously isolated as a suppressor of the Glued mutation is an allele of Dhc64C. Together with the observed dependency of Glued localization on dynein function, these genetic interactions demonstrate a functional association between the Drosophila dynein motor and Glued complexes.