Dynactin is an essential cofactor for the microtubule motor cytoplasmic dynein-1. We report the structure of the 23 subunit dynactin complex by cryo-electron microscopy to 4.0Å. Our reconstruction reveals how dynactin is built around a filament containing eight copies of the actin related protein Arp1 and one of β-actin. Capped at each end by distinct protein complexes, the length of the filament is defined by elongated peptides that emerge from the α-helical shoulder domain. A further 8.2Å structure of the complex between dynein, dynactin and the motility inducing cargo adaptor Bicaudal-D2 shows how the translational symmetry of the dynein tail matches that of the dynactin filament. The Bicaudal-D2 coiled coil runs between dynein and dynactin to stabilize the mutually dependent interactions between all three components.Dynactin works with the cytoplasmic dynein-1 motor (dynein) to transport cargos along the microtubule cytoskeleton (1-3). They maintain the cell's spatial organization, return components from the cell's periphery and assist with cellular division (4). Mutations in either complex cause neurodegeneration (5) and both can be co-opted by viruses that travel to the nucleus (6). Dynein and dynactin are similar in size and complexity. Dynein contains two copies of 6 different proteins and has a mass of 1.4 MDa. Dynactin, at about 1.0 MDa, contains more than 20 subunits, corresponding to 12 different proteins. Dynactin is built around a filament of actin related protein 1 (Arp1). In analogy to actin, the filament has a barbed and a pointed end; each capped by a different protein complex. On top sits the shoulder domain (7) from which emerges a long projection, corresponding to dynactin's largest subunit p150 Glued (DCTN1) (8).Despite the presence of a dynein binding site in p150 Glued (9-11), purified dynein and dynactin only form a stable complex in the presence of the cargo adaptor Bicaudal D2 † To whom correspondence should be addressed: cartera@mrc-lmb.cam.ac.uk. Author contributions L.U. prepared dynactin and determined the TDB structure. K.Z. determined the structure of dynactin. A.G.D. and M.Y. determined the DHC N-terminus crystal structure. C.M. and M.A.S. prepared the dynein tail complex. N.A.P. and C.V.R performed mass spectrometry. A.P.C. initiated the project and designed the experiments.

Cytoplasmic dynein is an approximately 1.4 MDa multi‐protein complex that transports many cellular cargoes towards the minus ends of microtubules. Several in vitro studies of mammalian dynein have suggested that individual motors are not robustly processive, raising questions about how dynein‐associated cargoes can move over long distances in cells. Here, we report the production of a fully recombinant human dynein complex from a single baculovirus in insect cells. Individual complexes very rarely show directional movement in vitro. However, addition of dynactin together with the N‐terminal region of the cargo adaptor BICD2 (BICD2N) gives rise to unidirectional dynein movement over remarkably long distances. Single‐molecule fluorescence microscopy provides evidence that BICD2N and dynactin stimulate processivity by regulating individual dynein complexes, rather than by promoting oligomerisation of the motor complex. Negative stain electron microscopy reveals the dynein–dynactin–BICD2N complex to be well ordered, with dynactin positioned approximately along the length of the dynein tail. Collectively, our results provide insight into a novel mechanism for coordinating cargo binding with long‐distance motor movement.

The postsynaptic density (PSD) of central excitatory synapses is essential for postsynaptic signaling, and its components are heterogeneous among different neuronal subtypes and brain structures. Here we report large scale relative and absolute quantification of proteins in PSDs purified from adult rat forebrain and cerebellum. PSD protein profiles were determined using the cleavable ICAT strategy and LC-MS/MS. A total of 296 proteins were identified and quantified with 43 proteins exhibiting statistically significant abundance change between forebrain and cerebellum, indicating marked molecular heterogeneity of PSDs between different brain regions. Moreover we utilized absolute quantification strategy, in which synthetic isotope-labeled peptides were used as internal standards, to measure the molar abundance of 32 key PSD proteins in forebrain and cerebellum. These data confirm the abundance of calcium/calmodulin-dependent protein kinase II and PSD-95 and reveal unexpected stoichiometric ratios between glutamate receptors, scaffold proteins, and signaling molecules in the PSD. Our data also demonstrate that the absolute quantification method is well suited for targeted quantitative proteomic analysis. Overall this study delineates a crucial molecular difference between forebrain and cerebellar PSDs and provides a quantitative framework for measuring the molecular stoichiometry of the PSD. Molecular & Cellular Proteomics 5:1158 -1170, 2006.In excitatory synapses of the mammalian brain, the postsynaptic density (PSD) 1 is a specialized membrane-associated structure containing a high concentration of glutamate receptors, cell adhesion molecules, and associated scaffold proteins and signaling enzymes (1-3). Glutamate receptors in the PSD are assembled into large protein complexes by binding to PDZ domain-containing scaffold proteins. In a well characterized example, the cytoplasmic C termini of NR2 subunits of the NMDA-type glutamate receptor interact with the PDZ domains of the PSD-95 family of scaffold proteins, which are highly enriched in the PSD (2). PSD-95 in turn binds to cytoplasmic signaling proteins such as SynGAP, the synaptic GTPase-activating protein (GAP) for Ras/Rap small GTPases (4, 5). Assembly of such protein complexes facilitates specific coupling of postsynaptic receptors to trafficking mechanisms and downstream signaling pathways that control synaptic strength, cytoskeletal rearrangements, and nuclear responses (1). Many if not most of the protein constituents of the PSD are dynamically influenced by synaptic activity via mechanisms such as protein phosphorylation, local translation, ubiquitination, degradation (6, 7), and protein translocation into and out of synapses (8). Altered composition and structural remodeling of the PSD are believed to play critical roles in the formation/elimination and plasticity of synapses. Because it is amenable to biochemical purification and because of its compact size (a few hundred nanometers in diameter and 20 -40 nm thick), the PSD is a highly suitable "o...

In neurons, the distinct molecular composition of axons and dendrites is established through polarized targeting mechanisms, but it is currently unclear how nonpolarized cargoes, such as mitochondria, become uniformly distributed over these specialized neuronal compartments. Here, we show that TRAK family adaptor proteins, TRAK1 and TRAK2, which link mitochondria to microtubule-based motors, are required for axonal and dendritic mitochondrial motility and utilize different transport machineries to steer mitochondria into axons and dendrites. TRAK1 binds to both kinesin-1 and dynein/dynactin, is prominently localized in axons, and is needed for normal axon outgrowth, whereas TRAK2 predominantly interacts with dynein/dynactin, is more abundantly present in dendrites, and is required for dendritic development. These functional differences follow from their distinct conformations: TRAK2 preferentially adopts a head-to-tail interaction, which interferes with kinesin-1 binding and axonal transport. Our study demonstrates how the molecular interplay between bidirectional adaptor proteins and distinct microtubule-based motors drives polarized mitochondrial transport.