DYNC1H1 mutations associated with neurological diseases compromise processivity of dynein-dynactin-cargo adaptor complexes.

Mutations in the human DYNC1H1 gene are associated with neurological diseases. DYNC1H1 encodes the heavy chain of cytoplasmic dynein-1, a 1.4-MDa motor complex that traffics organelles, vesicles, and macromolecules toward microtubule minus ends. The effects of the DYNC1H1 mutations on dynein motility, and consequently their links to neuropathology, are not understood. Here, we address this issue using a recombinant expression system for human dynein coupled to single-molecule resolution in vitro motility assays. We functionally characterize 14 DYNC1H1 mutations identified in humans diagnosed with malformations in cortical development (MCD) or spinal muscular atrophy with lower extremity predominance (SMALED), as well as three mutations that cause motor and sensory defects in mice. Two of the human mutations, R1962C and H3822P, strongly interfere with dynein's core mechanochemical properties. The remaining mutations selectively compromise the processive mode of dynein movement that is activated by binding to the accessory complex dynactin and the cargo adaptor Bicaudal-D2 (BICD2). Mutations with the strongest effects on dynein motility in vitro are associated with MCD. The vast majority of mutations do not affect binding of dynein to dynactin and BICD2 and are therefore expected to result in linkage of cargos to dynein-dynactin complexes that have defective long-range motility. This observation offers an explanation for the dominant effects of DYNC1H1 mutations in vivo. Collectively, our results suggest that compromised processivity of cargo-motor assemblies contributes to human neurological disease and provide insight into the influence of different regions of the heavy chain on dynein motility.

In vitro reconstitution of a highly processive recombinant human dynein complex.

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.

Pericentrosomal targeting of Rab6 secretory vesicles by Bicaudal-D-related protein 1 (BICDR-1) regulates neuritogenesis.

Membrane and secretory trafficking are essential for proper neuronal development. However, the molecular mechanisms that organize secretory trafficking are poorly understood. Here, we identify Bicaudal-D-related protein 1 (BICDR-1) as an effector of the small GTPase Rab6 and key component of the molecular machinery that controls secretory vesicle transport in developing neurons. BICDR-1 interacts with kinesin motor Kif1C, the dynein/dynactin retrograde motor complex, regulates the pericentrosomal localization of Rab6-positive secretory vesicles and is required for neural development in zebrafish. BICDR-1 expression is high during early neuronal development and strongly declines during neurite outgrowth. In young neurons, BICDR-1 accumulates Rab6 secretory vesicles around the centrosome, restricts anterograde secretory transport and inhibits neuritogenesis. Later during development, BICDR-1 expression is strongly reduced, which permits anterograde secretory transport required for neurite outgrowth. These results indicate an important role for BICDR-1 as temporal regulator of secretory trafficking during the early phase of neuronal differentiation.

Probing intracellular motor protein activity using an inducible cargo trafficking assay.

Although purified cytoskeletal motor proteins have been studied extensively with the use of in vitro approaches, a generic approach to selectively probe actin and microtubule-based motor protein activity inside living cells is lacking. To examine specific motor activity inside living cells, we utilized the FKBP-rapalog-FRB heterodimerization system to develop an in vivo peroxisomal trafficking assay that allows inducible recruitment of exogenous and endogenous kinesin, dynein, and myosin motors to drive specific cargo transport. We demonstrate that cargo rapidly redistributes with distinct dynamics for each respective motor, and that combined (antagonistic) actions of more complex motor combinations can also be probed. Of importance, robust cargo redistribution is readily achieved by one type of motor protein and does not require the presence of opposite-polarity motors. Simultaneous live-cell imaging of microtubules and kinesin or dynein-propelled peroxisomes, combined with high-resolution particle tracking, revealed that peroxisomes frequently pause at microtubule intersections. Titration and washout experiments furthermore revealed that motor recruitment by rapalog-induced heterodimerization is dose-dependent but irreversible. Our assay directly demonstrates that robust cargo motility does not require the presence of opposite-polarity motors, and can therefore be used to characterize the motile properties of specific types of motor proteins.

The mammalian dynein-dynactin complex is a strong opponent to kinesin in a tug-of-war competition.

Kinesin and dynein motors transport intracellular cargos bidirectionally by pulling them in opposite directions along microtubules, through a process frequently described as a 'tug of war'. While kinesin produces 6 pN of force, mammalian dynein was found to be a surprisingly weak motor (0.5-1.5 pN) in vitro, suggesting that many dyneins are required to counteract the pull of a single kinesin. Mammalian dynein's association with dynactin and Bicaudal-D2 (BICD2) activates its processive motility, but it was unknown how this affects dynein's force output. Here, we show that formation of the dynein-dynactin-BICD2 (DDB) complex increases human dynein's force production to 4.3 pN. An in vitro tug-of-war assay revealed that a single DDB successfully resists a single kinesin. Contrary to previous reports, the clustering of many dyneins is not required to win the tug of war. Our work reveals the key role of dynactin and a cargo adaptor protein in shifting the balance of forces between dynein and kinesin motors during intracellular transport.

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

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 4.0 angstroms. Our reconstruction reveals how dynactin is built around a filament containing eight copies of the actin-related protein Arp1 and one of ß-actin. The filament is capped at each end by distinct protein complexes, and its length is defined by elongated peptides that emerge from the α-helical shoulder domain. A further 8.2 angstrom 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.

The GRIP1/14-3-3 pathway coordinates cargo trafficking and dendrite development.

Regulation of cargo transport via adaptor molecules is essential for neuronal development. However, the role of PDZ scaffolding proteins as adaptors in neuronal cargo trafficking is still poorly understood. Here, we show by genetic deletion in mice that the multi-PDZ domain scaffolding protein glutamate receptor interacting protein 1 (GRIP1) is required for dendrite development. We identify an interaction between GRIP1 and 14-3-3 proteins that is essential for the function of GRIP1 as an adaptor protein in dendritic cargo transport. Mechanistically, 14-3-3 binds to the kinesin-1 binding region in GRIP1 in a phospho-dependent manner and detaches GRIP1 from the kinesin-1 motor protein complex thereby regulating cargo transport. A single point mutation in the Thr956 of GRIP1 in transgenic mice impairs dendritic development. Together, our results show a regulatory role for GRIP1 during microtubule-based transport and suggest a crucial function for 14-3-3 proteins in controlling kinesin-1 motor attachment during neuronal development.

Bicaudal d family adaptor proteins control the velocity of Dynein-based movements.

Cargo transport along microtubules is driven by the collective function of microtubule plus- and minus-end-directed motors (kinesins and dyneins). How the velocity of cargo transport is driven by opposing teams of motors is still poorly understood. Here, we combined inducible recruitment of motors and adaptors to Rab6 secretory vesicles with detailed tracking of vesicle movements to investigate how changes in the transport machinery affect vesicle motility. We find that the velocities of kinesin-based vesicle movements are slower and more homogeneous than those of dynein-based movements. We also find that Bicaudal D (BICD) adaptor proteins can regulate dynein-based vesicle motility. BICD-related protein 1 (BICDR-1) accelerates minus-end-directed vesicle movements and affects Rab6 vesicle distribution. These changes are accompanied by reduced axonal outgrowth in neurons, supporting their physiological importance. Our study suggests that adaptor proteins can modulate the velocity of dynein-based motility and thereby control the distribution of transport carriers.

A role for Bicaudal-D2 in radial cerebellar granule cell migration.

Bicaudal-D (BICD) belongs to an evolutionary conserved family of dynein adaptor proteins. It was first described in Drosophila as an essential factor in fly oogenesis and embryogenesis. Missense mutations in a human BICD homologue, BICD2, have been linked to a dominant mild early onset form of spinal muscular atrophy. Here we further examine the in vivo function of BICD2 in Bicd2 knockout mice. BICD2-deficient mice develop disrupted laminar organization of cerebral cortex and the cerebellum, pointing to impaired radial neuronal migration. Using astrocyte and granule cell specific inactivation of BICD2, we show that the cerebellar migration defect is entirely dependent upon BICD2 expression in Bergmann glia cells. Proteomics analysis reveals that Bicd2 mutant mice have an altered composition of extracellular matrix proteins produced by glia cells. These findings demonstrate an essential non-cell-autonomous role of BICD2 in neuronal cell migration, which might be connected to cargo trafficking pathways in glia cells.

TRAK/Milton motor-adaptor proteins steer mitochondrial trafficking to axons and dendrites.

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.

BICD2, dynactin, and LIS1 cooperate in regulating dynein recruitment to cellular structures.

Cytoplasmic dynein is the major microtubule minus-end-directed cellular motor. Most dynein activities require dynactin, but the mechanisms regulating cargo-dependent dynein-dynactin interaction are poorly understood. In this study, we focus on dynein-dynactin recruitment to cargo by the conserved motor adaptor Bicaudal D2 (BICD2). We show that dynein and dynactin depend on each other for BICD2-mediated targeting to cargo and that BICD2 N-terminus (BICD2-N) strongly promotes stable interaction between dynein and dynactin both in vitro and in vivo. Direct visualization of dynein in live cells indicates that by itself the triple BICD2-N-dynein-dynactin complex is unable to interact with either cargo or microtubules. However, tethering of BICD2-N to different membranes promotes their microtubule minus-end-directed motility. We further show that LIS1 is required for dynein-mediated transport induced by membrane tethering of BICD2-N and that LIS1 contributes to dynein accumulation at microtubule plus ends and BICD2-positive cellular structures. Our results demonstrate that dynein recruitment to cargo requires concerted action of multiple dynein cofactors.

Mixed microtubules steer dynein-driven cargo transport into dendrites.

BACKGROUND: To establish and maintain their polarized morphology, neurons employ active transport driven by molecular motors to sort cargo between axons and dendrites. However, the basic traffic rules governing polarized transport on neuronal microtubule arrays are unclear. RESULTS: Here we show that the microtubule minus-end-directed motor dynein is required for the polarized targeting of dendrite-specific cargo, such as AMPA receptors. To directly examine how dynein motors contribute to polarized dendritic transport, we established a trafficking assay in hippocampal neurons to selectively probe specific motor protein activity. This revealed that, unlike kinesins, dynein motors drive cargo selectively into dendrites, governed by their mixed microtubule array. Moreover, axon-specific cargos, such as presynaptic vesicle protein synaptophysin, are redirected to dendrites by coupling to dynein motors. Quantitative modeling demonstrated that bidirectional dynein-driven transport on mixed microtubules provides an efficient mechanism to establish a stable density of continuously renewing vesicles in dendrites. CONCLUSIONS: These results demonstrate a powerful approach to study specific motor protein activity inside living cells and imply a key role for dynein in dendritic transport. We propose that dynein establishes the initial sorting of dendritic cargo and additional motor proteins assist in subsequent delivery.

Basic mechanisms for recognition and transport of synaptic cargos.

Synaptic cargo trafficking is essential for synapse formation, function and plasticity. In order to transport synaptic cargo, such as synaptic vesicle precursors, mitochondria, neurotransmitter receptors and signaling proteins to their site of action, neurons make use of molecular motor proteins. These motors operate on the microtubule and actin cytoskeleton and are highly regulated so that different cargos can be transported to distinct synaptic specializations at both pre- and post-synaptic sites. How synaptic cargos achieve specificity, directionality and timing of transport is a developing area of investigation. Recent studies demonstrate that the docking of motors to their cargos is a key control point. Moreover, precise spatial and temporal regulation of motor-cargo interactions is important for transport specificity and cargo recruitment. Local signaling pathways Ca2+ influx, CaMKII signaling and Rab GTPase activity regulate motor activity and cargo release at synaptic locations. We discuss here how different motors recognize their synaptic cargo and how motor-cargo interactions are regulated by neuronal activity.

A role for the Rab6B Bicaudal-D1 interaction in retrograde transport in neuronal cells.

The Rab6 subfamily of small GTPases consists of three different isoforms: Rab6A, Rab6A' and Rab6B. Both Rab6A and Rab6A' are ubiquitously expressed whereas Rab6B is predominantly expressed in brain. Recent studies have shown that Rab6A' is the isoform regulating the retrograde transport from late endosomes via the Golgi to the ER and in the transition from anaphase to metaphase during mitosis. Since the role of Rab6B is still ill defined, we set out to characterize its intracellular environment and dynamic behavior. In a Y-2H search for novel Rab6 interacting proteins, we identified Bicaudal-D1, a large coiled-coil protein known to bind to the dynein/dynactin complex and previously shown to be a binding partner for Rab6A/Rab6A'. Co-immunoprecipitation studies and pull down assays confirmed that Bicaudal-D1 also interacts with Rab6B in its active form. Using confocal laser scanning microscopy it was established that Rab6B and Bicaudal-D1 co-localize at the Golgi and vesicles that align along microtubules. Furthermore, both proteins co-localized with dynein in neurites of SK-N-SH cells. Live cell imaging revealed bi-directional movement of EGFP-Rab6B structures in SK-N-SH neurites. We conclude from our data that the brain-specific Rab6B via Bicaudal-D1 is linked to the dynein/dynactin complex, suggesting a regulatory role for Rab6B in the retrograde transport of cargo in neuronal cells.

Relative and absolute quantification of postsynaptic density proteome isolated from rat forebrain and cerebellum.

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.