The structure of the dynactin complex and its interaction with dynein.

The dynactin complex contains proteins including p150 that interacts with cytoplasmic dynein and an actin-related protein Arp1 that forms a minifilament. Proteins including Arp11 and p62 locate at the pointed end of the Arp1 filament, but their biochemical functions are unclear (Schroer TA. Dynactin. Annu Rev Cell Dev Biol 2004;20:759-779). In Aspergillus nidulans, loss of Arp11 or p62 causes the same nuclear distribution (nud) defect displayed by dynein mutants, indicating that these pointed-end proteins are essential for dynein function. We constructed a strain with S-tagged p150 of dynactin that allows us to pull down components of the dynactin and dynein complexes. Surprisingly, while the ratio of pulled-down Arp1 to S-p150 in Arp11-depleted cells is clearly lower than that in wild-type cells, the ratio of pulled-down dynein to S-p150 is significantly higher. We further show that the enhanced dynein-dynactin interaction in Arp11-depleted cells is also present in the soluble fraction and therefore is not dependent upon the affinity of these proteins to the membrane. We suggest that loss of the pointed-end proteins alters the Arp1 filament in a way that affects the conformation of p150 required for its proper interaction with the dynein motor.

SUMMARYKinesin-1 activity is regulated by autoinhibition. Intramolecular interactions within the kinesin heavy chain (KHC) are proposed to be one facet of motor regulation. The KHC also binds to the kinesin light chain (KLC), which has been implicated in both autoinhibition and activation of the motor. We show that the KLC inhibits the kinesin-microtubule interaction independently from the proposed intramolecular interaction within KHC. Cargo-adaptor proteins that bind the KLC stimulated processive movement, but the landing rate of activated kinesin complexes remained low. Microtubule-associated protein 7 (MAP7) enhanced motility by increasing the landing rate and run length of the activated kinesin motors. Our results support a model whereby the motor activity of the kinesin is regulated by synergistic inhibition mechanisms and that cargo-adaptor binding to the KLC releases both mechanisms. However, a non-motor MAP is required for robust microtubule association of the activated motor. Thus, human kinesin is regulated by synergistic autoinhibition and activation mechanisms.

Motor Protein Receptors: Moonlighting on Other Jobs

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

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.

Human complex II is a key protein complex that links two essential energy-producing processes: the tricarboxylic acid cycle and oxidative phosphorylation. Deficiencies due to mutagenesis have been shown to cause mitochondrial disease and some types of cancers. However, the structure of this complex is yet to be resolved, hindering a comprehensive understanding of the functional aspects of this molecular machine. Here, we have determined the structure of human complex II in the presence of ubiquinone at 2.86Å resolution by cryoelectron microscopy, showing it comprises two water-soluble subunits, SDHA and SDHB, and two membrane-spanning subunits, SDHC and SDHD. This structure allows us to propose a route for electron transfer. In addition, clinically relevant mutations are mapped onto the structure. This mapping provides a molecular understanding to explain why these variants have the potential to produce disease.

Cytoplasmic dynein-1 is a minus-end-directed microtubule motor that transports a variety of cargoes including early endosomes, late endosomes and other organelles. In many cell types, dynein accumulates at the microtubule plus end, where it interacts with its cargo to be moved toward the minus end. Dynein binds to its various cargoes via the dynactin complex and specific cargo adapters. Dynactin and some of the coiled-coil-domain-containing cargo adapters not only link dynein to cargo but also activate dynein motility, which implies that dynein is activated by its cellular cargo. Structural studies indicate that a dynein dimer switches between the autoinhibited phi state and an open state; and the binding of dynactin and a cargo adapter to the dynein tails causes the dynein motor domains to have a parallel configuration, allowing dynein to walk processively along a microtubule. Recently, the dynein regulator LIS1 has been shown to be required for dynein activation in vivo, and its mechanism of action involves preventing dynein from switching back to the autoinhibited state. In this review, we will discuss our current understanding of dynein activation and point out the gaps of knowledge on the spatial regulation of dynein in live cells. In addition, we will emphasize the importance of studying a complete set of dynein regulators for a better understanding of dynein regulation in vivo.

The minus-end-directed microtubule motor cytoplasmic dynein requires the dynactin complex for in vivo functions. The backbone of the vertebrate dynactin complex is the Arp1 (actin-related protein 1) mini-filament whose barbed end binds to the heterodimeric actin capping protein. However, it is unclear whether the capping protein is a dynactin component in lower eukaryotic organisms, especially because it does not appear to be a component of the budding yeast dynactin complex. Here our biochemical data show that the capping protein is a component of the dynactin complex in the filamentous fungus Aspergillus nidulans. Moreover, deletion of the gene encoding capping protein alpha (capA) results in a defect in both nuclear distribution and early-endosome transport, two dynein-mediated processes. However, the defect in either process is less severe than that exhibited by a dynein heavy chain mutant or the ∆p25 mutant of dynactin. In addition, loss of capping protein does not significantly affect the assembly of the dynactin Arp1 filament or the formation of the dynein-dynactin-∆C-HookA (Hook in A. nidulans) complex. These results suggest that fungal capping protein is not important for Arp1 filament assembly but its presence is required for enhancing dynein function in vivo.

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.

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.

The p150Glued CAP-Gly Domain Regulates Initiation of Retrograde Transport at Synaptic Termini

A major function of the acinar cells of the lacrimal gland is the production and stimulated release of tear proteins into ocular surface fluid. We investigate the participation of cytoplasmic dynein in carbachol-stimulated traffic to the apical plasma membrane in primary rabbit lacrimal acinar epithelial cells. Confocal fluorescence microscopy revealed a major carbachol-induced, microtubule-dependent recruitment of cytoplasmic dynein and the dynactin complex into the subapical region. Colocalization studies, sorbitol density gradient/phase partitioning analysis and microtubule-affinity purification of membranes showed that some dynein and dynactin complex were associated with VAMP2-enriched membranes. Adenovirus-mediated overexpression of p50/dynamitin inhibited the recruitment and colocalization of dynein, the dynactin complex and VAMP2 in the subapical region. Nocodazole treatment and p50/dynamitin overexpression also depleted subapical stores of rab3D in resting acini, suggesting that dynein activity was also involved in maintenance of rab3D-enriched secretory vesicles. These data implicate cytoplasmic dynein in stimulated traffic to the apical plasma membrane in these secretory epithelial cells.

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

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.

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