Dynein Processivity Research Articles

Traffic control: adaptor proteins guide dynein-cargo takeoff.

The precise movement of intracellular components requires active transport by molecular motors along the filamentous tracks of the cytoskeleton. While yeast cytoplasmic dynein can walk for some distance along microtubules, mammalian dynein is non‐processive. This has raised the question of how this motor can transport cargo. In two recent papers by the Carter, Bullock and Vale labs, mammalian dynein processivity has now been successfully reconstituted in vitro in the presence of adaptor proteins.

Activation of cytoplasmic dynein motility by dynactin-cargo adapter complexes.

Cytoplasmic dynein is a molecular motor that transports a large variety of cargoes (e.g., organelles, messenger RNAs, and viruses) along microtubules over long intracellular distances. The dynactin protein complex is important for dynein activity in vivo, but its precise role has been unclear. Here, we found that purified mammalian dynein did not move processively on microtubules in vitro. However, when dynein formed a complex with dynactin and one of four different cargo-specific adapter proteins, the motor became ultraprocessive, moving for distances similar to those of native cargoes in living cells. Thus, we propose that dynein is largely inactive in the cytoplasm and that a variety of adapter proteins activate processive motility by linking dynactin to dynein only when the motor is bound to its proper cargo.

The influence of dynein processivity control, MAPs, and microtubule ends on directional movement of a localising mRNA.

Many cellular constituents travel along microtubules in association with multiple copies of motor proteins. How the activity of these motors is regulated during cargo sorting is poorly understood. In this study, we address this issue using a novel in vitro assay for the motility of localising Drosophila mRNAs bound to native dynein-dynactin complexes. High precision tracking reveals that individual RNPs within a population undergo either diffusive, or highly processive, minus end-directed movements along microtubules. RNA localisation signals stimulate the processive movements, with regulation of dynein-dynactin's activity rather than its total copy number per RNP, responsible for this effect. Our data support a novel mechanism for multi-motor translocation based on the regulation of dynein processivity by discrete cargo-associated features. Studying the in vitro responses of RNPs to microtubule-associated proteins (MAPs) and microtubule ends provides insights into how an RNA population could navigate the cytoskeletal network and become anchored at its destination in cells. DOI: http://dx.doi.org/10.7554/eLife.01596.001.

BicD2 and Dynactin Convert a Non-Processive Cytoplasmic Dynein to an Ultra-Processive Directional Motor

Cytoplasmic dynein is the predominant minus-end directed microtubule motor in metazoan cells. Dynein transports diverse cargoes over long distances in neurons, and the motor is thought to be adapted for a myriad of cellular functions through the use of several accessory protein factors that impinge on its basic biophysical characteristics. One of these accessory factors is the multisubunit dynactin complex, which has been implicated in dynein-based cargo transport and the modulation of dynein processivity and directionality. While isolated dynein from Saccharomyces has been shown to be a strongly processive motor, dynein from other organisms displays weakly processive, bidirectional or diffusive motility. Here we show that, on its own, cytoplasmic dynein from humans and other metazoans is not a processive motor. Previous attempts to study dynein-dynactin co-complexes have found relatively modest effects on dynein processivity and directionality by dynactin. We utilize the evolutionarily conserved coiled-coil adapter protein BicD2 to strongly induce the formation of a stable dynein-dynactin-BicD2 (DDB) supercomplex that is over 2MDa in size. Using multicolor single-molecule microscopy, we have found that, remarkably, the purified DDB supercomplex is unidirectional and ultra-processive, displaying run-lengths that greatly exceed the previously observed enhancement of single dynein run-lengths by dynactin. The DDB supercomplex accumulates at microtubule minus-ends and displays characteristics expected of a processive cargo transport motor. Our data suggest that the dynein motor is more plastic that previously thought, able to transition from a non-processive motor to an ultra-processive mode of motility upon association with external regulatory factors.

Microtubule Motors: Doin’ It without Dynactin

The minus-end directed microtubule motor protein cytoplasmic dynein contributes to many aspects of cell division and it is generally believed that these mitotic functions require the dynein activator and processivity factor, dynactin. New research now shows that dynein accomplishes many of its mitotic functions without dynactin.

The Mechanism of Cytoplasmic Dynein Motility

Cytoplasmic dynein is a homodimeric AAA+ motor that transports a multitude of cargos towards the microtubule minus end. The mechanism of dynein processivity and interhead coordination remain unclear due to its large size (2.6 MDa) and the complexity of its structure. In contrast to kinesins and myosins, we directly observed that dynein heads move independently along the microtubule. Stepping behavior of the heads varies as a function of interhead separation and establishing the basis of high variability in dynein step size. By engineering the mechanical and catalytic properties of the dynein motor domain, we show that a rigid linkage between monomers and dimerization between N-terminal tail domains are not essential for processive movement. Instead, dynein processivity minimally requires the linker domain of one active monomer to be attached to an inert MT tether retaining only the MT-binding domain. The release of a dynein monomer from the MT can be mediated either by nucleotide binding or external load. However, nucleotide dependent release is inhibited when force was applied to the linker domain. Force dependent release is significantly asymmetric, with faster release towards the minus-end. On the basis of these measurements, we developed a model that describes the basis of dynein processivity, directionality and force generation.

From the Cell Membrane to the Nucleus: Unearthing Transport Mechanisms for Dynein

Mutations in the motor protein cytoplasmic dynein have been found to cause Charcot-Marie-Tooth disease, spinal muscular atrophy, and severe intellectual disabilities in humans. In mouse models, neurodegeneration is observed. We sought to develop a novel model which could incorporate the effects of mutations on distance travelled and velocity. A mechanical model for the dynein mediated transport of endosomes is derived from first principles and solved numerically. The effects of variations in model parameter values are analysed to find those that have a significant impact on velocity and distance travelled. The model successfully describes the processivity of dynein and matches qualitatively the velocity profiles observed in experiments.

Engineered Dynein Mutants Reveal Minimal Structural and Catalytic Requirements for Processive Motility

Cytoplasmic dynein is a molecular motor responsible for minus-end directed transport along microtubules. In contrast to kinesins and myosins, the detailed mechanism of dynein processivity and force generation remains unclear. Dynein’s structure and evolutionary origin are different from these motors, suggesting unique mechanistic features. In this work, we engineered novel dynein constructs with altered mechanical, chemical and geometric properties to test the roles of rigid linkage between monomers, interaction between the ATPase rings and the proposed linker swing mechanism in maintaining dynein processivity. We found that a rigid linkage between monomers and dimerization through the N-terminal tail domains are not essential for dynein processivity. Instead, processivity minimally requires the linker domain of one monomer with an active ATPase ring to be attached to a partner monomer, which can be replaced by an inert protein retaining the microtubule-binding domain. To understand how one active head could provide motility to an inactive partner, we quantified the force-dependent microtubule release rates of dynein monomers. A clear directional asymmetry for detachment was observed, with significantly faster release towards the minus-end, providing insight into dynein’s directionality. These results led us to a detailed mechanistic model of dynein processivity, directionality and force generation.

Evolution of the eukaryotic dynactin complex, the activator of cytoplasmic dynein

BackgroundDynactin is a large multisubunit protein complex that enhances the processivity of cytoplasmic dynein and acts as an adapter between dynein and the cargo. It is composed of eleven different polypeptides of which eight are unique to this complex, namely dynactin1 (p150Glued), dynactin2 (p50 or dynamitin), dynactin3 (p24), dynactin4 (p62), dynactin5 (p25), dynactin6 (p27), and the actin-related proteins Arp1 and Arp10 (Arp11).ResultsTo reveal the evolution of dynactin across the eukaryotic tree the presence or absence of all dynactin subunits was determined in most of the available eukaryotic genome assemblies. Altogether, 3061 dynactin sequences from 478 organisms have been annotated. Phylogenetic trees of the various subunit sequences were used to reveal sub-family relationships and to reconstruct gene duplication events. Especially in the metazoan lineage, several of the dynactin subunits were duplicated independently in different branches. The largest subunit repertoire is found in vertebrates. Dynactin diversity in vertebrates is further increased by alternative splicing of several subunits. The most prominent example is the dynactin1 gene, which may code for up to 36 different isoforms due to three different transcription start sites and four exons that are spliced as differentially included exons.ConclusionsThe dynactin complex is a very ancient complex that most likely included all subunits in the last common ancestor of extant eukaryotes. The absence of dynactin in certain species coincides with that of the cytoplasmic dynein heavy chain: Organisms that do not encode cytoplasmic dynein like plants and diplomonads also do not encode the unique dynactin subunits. The conserved core of dynactin consists of dynactin1, dynactin2, dynactin4, dynactin5, Arp1, and the heterodimeric actin capping protein. The evolution of the remaining subunits dynactin3, dynactin6, and Arp10 is characterized by many branch- and species-specific gene loss events.

The Mechanism of Cytoplasmic Dynein Processivity

Cytoplasmic dynein is a homodimeric AAA+ motor that transports a multitude of cargos towards the microtubule minus end. It is currently unknown how the two catalytic head domains interact and move relative to each other during processive movement. We have tracked the relative positions of both heads with nanometer precision and directly observed that the heads move independently along the microtubule. The heads remain widely separated and the stepping behavior of the heads varies as a function of interhead separation. Consistent with a lack of tight coordination, only one active head is sufficient for processive movement and the active head drags its inactive partner head forward. Only a single active ATPase ring is sufficient for processivity, and the linker swing provides required force to drive minus end directed motion. These results show that dynein is the first dimeric motor that moves processively without interhead coordination, a mechanism fundamentally distinct from hand-over-hand motion of kinesin and myosin.

Cytoplasmic Dynein Moves Through Uncoordinated Stepping of the AAA+ Ring Domains

Cytoplasmic dynein is a homodimeric AAA+ motor that transports a multitude of cargos toward the microtubule minus end. How the two catalytic head domains interact and move relative to each other during processive movement is unclear. Here, we tracked the relative positions of both heads with nanometer precision and directly observed the heads moving independently along the microtubule. The heads remained widely separated, and their stepping behavior varied as a function of interhead separation. One active head was sufficient for processive movement, and an active head could drag an inactive partner head forward. Thus, dynein moves processively without interhead coordination, a mechanism fundamentally distinct from the hand-over-hand stepping of kinesin and myosin.

Mutually Exclusive Cytoplasmic Dynein Regulation by NudE-Lis1 and Dynactin

Cytoplasmic dynein is responsible for a wide range of cellular roles. How this single motor protein performs so many functions has remained a major outstanding question for many years. Part of the answer is thought to lie in the diversity of dynein regulators, but how the effects of these factors are coordinated in vivo remains unexplored. We previously found NudE to bind dynein through its light chain 8 (LC8) and intermediate chain (IC) subunits (1), the latter of which also mediates the dynein-dynactin interaction (2). We report here that NudE and dynactin bind to a common region within the IC, and compete for this site. We find LC8 to bind to a novel sequence within NudE, without detectably affecting the dynein-NudE interaction. We further find that commonly used dynein inhibitory reagents have broad effects on the interaction of dynein with its regulatory factors. Together these results reveal an unanticipated mechanism for preventing dual regulation of individual dynein molecules, and identify the IC as a nexus for regulatory interactions within the dynein complex.

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.

Two-Dimensional Analysis of the Dynein Stepping Mechanism

Cytoplasmic dynein powers the transport of diverse macromolecules within eukaryotic cells, enabling them to effectively organize their contents, move, divide and respond to signals. One of our goals is to understand how dynein moves processively along microtubules to perform these functions. Our initial analysis of recombinant S. cerevisiae cytoplasmic dynein stepping was a one-dimensional analysis, in which all movements were projected along the axis of the microtubule (Reck-Peterson et al., 2006). However, as we also observed dynein side-stepping in this earlier study, we have now performed a complete analysis of dynein stepping in two-dimensions. Our analysis uses a new two-dimensional step finding algorithm to determine dynein's true step size, stepping angle and frequency of off-axis stepping. In these experiments we have used high-precision single molecule fluorescence microscopy to analyze the stepping behavior of dynein labeled with a single quantum dot on its tail domain. When we analyze dynein's step size using on-axis projection we detect a predominant step size of 8nm, consistent with our previous studies. However, our two-dimensional analysis methods reveal a predominant step size of ∼12nm. By modeling the theoretical steps available to dynein taking into account the microtubule lattice and curvature, as well as the possible distance of the fluorescent probe from the microtubule surface, we hypothesize that dynein does not typically step on the same protofilament. Furthermore, we find that dynein's side steps occur with equal frequency to the left and right, suggesting symmetry in dynein's stepping mechanism. Our results suggest that dynein move on the same face of the microtubule, but with considerably more flexibility than the opposite polarity motor conventional kinesin.Reck-Peterson, S.L., Yildiz, Y., Carter, A.P., Gennerich, A., Zhang, N., and Vale, R.D. (2006). Single molecule analysis of dynein processivity and stepping behavior. Cell 126, 335-348.

Neuronal migration defects in the Loa dynein mutant mouse

BackgroundCytoplasmic dynein and its regulatory proteins have been implicated in neuronal and non-neuronal cell migration. A genetic model for analyzing the role of cytoplasmic dynein specifically in these processes has, however, been lacking. The Loa (Legs at odd angles) mouse with a mutation in the dynein heavy chain has been the focus of an increasing number of studies for its role in neuron degeneration. Despite the location of this mutation in the tail domain of the dynein heavy chain, we previously found a striking effect on coordination between the two dynein motor domains, resulting in a defect in dynein run length in vitro and in vivo.ResultsWe have now tested for effects of the Loa mutation on neuronal migration in the developing neocortex. Loa homozygotes showed clear defects in neocortical lamination and neuronal migration resulting from a reduction in the rate of radial migration of bipolar neurons.ConclusionsThese results present a new genetic model for understanding the dynein pathway and its functions during neuronal migration. They also provide the first evidence for a link between dynein processivity and somal movement, which is essential for proper development of the brain.

Cytoplasmic Dynein is not a Classical Duty Ratio Motor

Cytoplasmic dynein is not a classical duty ratio motorThe mechanical cross bridge cycle of cytoplasmic dynein has often been compared with that of myosin. Cytoplasmic dynein and myosin 5 are both organelle bound motors responsible for transport of their cargo over a long distance. It has also been demonstrated that myosin 5 is a processive motor and that its processivity is regulated by the duty ratio; i.e., the ratio between bound and free state during the cross bridge cycle. As the binding of ATP leads to the dissociation of the motor filament complex a decrease of the ATP concentration results in an increased duty ratio.It was the aim of our study to investigate whether the processivity of cytoplasmic dynein is also governed by the duty ratio. With the optical trap single molecule measurements were carried out in a two bead dumbbell approach. At 100 μM ATP consecutive 8 nm steps up to stall force were observed often resulting in repeated 8nm forward and backward steps at stall force. To our surprise however, at low ATP concentrations dynein underwent only single binding events with an apparent working stroke of 8nm.These results can not be explained by a simple one site model for ATP binding where processivity is governed by the duty ratio. In contrast to myosin, dynein possesses two essential ATP binding sites. At low ATP concentrations we hypothesis that only one of the ATP binding sites is occupied, thereby resulting in a loss of processivity.

Cytoplasmic Dynein Travel Cut Short by a Neurodegenerative Mutation in its Tail

Cytoplasmic dynein is a minus-end directed microtubule motor responsible for cellular functions including fast retrograde axonal transport. The Loa mouse strain carries a mutation within the tail domain of the dynein heavy chain gene, and develops progressive motor neuron degeneration similar to amyotrophic lateral sclerosis, associated with a decreased rate of retrograde axonal transport. To understand the molecular basis for transport impairment and its role in motor neuron degeneration, we have conducted a detailed biochemical and biophysical study of purified mutant and wild-type mouse dynein. The mutant dynein was identical in subunit composition to the wild type protein but showed mild, but reproducible dissociation during sucrose gradient centrifugation (Ori-McKenney et al., 2009, MBC abstr., in press). ATPase activity for the mutant dynein exhibited a higher Km for microtubules, and the mutant's microtubule binding was reduced in the presence of ATP. Using optical trap and quantum dot assays, we found that Loa mutation drastically reduced the single motor processivity of dynein without affecting its velocity or force production. Surprisingly, small increase in buffer ionic strength further exaggerated the difference between the mutant and wild-type travel. Our analysis of motor motion under load showed no difference in the stepping behavior between mutant and wild-type dynein. We propose that the Loa mutation introduces defects in the interhead communication leading to decreased processivity, possibly via weakened heavy chain-intermediate chain interaction. Our study provides the first indication that a tail mutation can affect dynein function. Furthermore, our results provide the first link between altered processivity and disease. Supported by RO1GM070676, AHA0825278F, GM47434, and the CUMC MNC.Authors Jing Xu and Kassandra M. Ori-McKenney contributed equally to this work. Steven P. Gross and Richard B. Vallee are co-senior authors.

Regulation of the processivity and intracellular localization of Saccharomyces cerevisiae dynein by dynactin

Dynactin, a large multisubunit complex, is required for intracellular transport by dynein; however, its cellular functions and mechanism of action are not clear. Prior studies suggested that dynactin increases dynein processivity by tethering the motor to the microtubule through its own microtubule binding domains. However, this hypothesis could not be tested without a recombinant source of dynactin. Here, we have produced recombinant dynactin and dynein in Saccharomyces cerevisiae, and examined the effect of dynactin on dynein in single-molecule motility assays. We show that dynactin increases the run length of single dynein motors, but does not alter the directionality of dynein movement. Enhancement of dynein processivity by dynactin does not require the microtubule (MT) binding domains of Nip100 (the yeast p150(Glued) homolog). Dynactin lacking these MT binding domains also supports the proper localization and function of dynein during nuclear segregation in vivo. Instead, a segment of the coiled-coil of Nip100 is required for these activities. Our results directly demonstrate that dynactin increases the processivity of dynein through a mechanism independent of microtubule tethering.

Measurement Of The Protein Friction Between The Yeast Kinesin-8 Kip3p And Microtubules

Several proteins have been shown to undergo 'one-dimensional' diffusion along the surface of microtubules. Diffusion is thought to enhance the rate of targeting of proteins to the microtubule end for the depolymerizing kinesin-13 and the polymerase XMAP215, or to increase the processivity of kinesin-1 and dynein. According to the Einstein-Smolukowski relation, the diffusion coefficient, D, is related to the friction coefficient, gamma, according to D = kT/gamma. This relation, however, has not been experimentally tested for individual bio-molecules. We measured both the diffusional and frictional properties of single yeast kinesin-8 motor proteins, Kip3p, interacting with microtubule filaments in the ADP state. Using single molecule fluorescence we found that that the diffusion coefficient was 5400±1400 nm2/s with an average lifetime on the microtubule lattice of 8 s. Using an optical trap to drag a microsphere coated with Kip3p along microtubules, we measured a single molecule drag coefficient of 790±230 nNs/m at low speeds. Thus we verified the Einstein-Smolukowski relation. For larger speeds and drag forces, we measured a non-linear force-velocity relation which was well fit by a model in which Kip3p is diffusing in a periodic potential with an 8-nm periodicity and a barrier height between binding sites of 14±2 kT. This finding of an 8-nm periodicity is supported by an analysis of the positional fluctuations. Our measurements are a step towards resolving the molecular mechanism underlying protein friction an important parameter for active protein locomotion limiting the efficiency.

Regulation of Dynactin through the Differential Expression of p150Glued Isoforms

Cytoplasmic dynein and dynactin interact to drive microtubule-based transport in the cell. The p150Glued subunit of dynactin binds to dynein, and directly to microtubules. We have identified alternatively spliced isoforms of p150Glued that are expressed in a tissue-specific manner and which differ significantly in their affinity for microtubules. Live cell assays indicate that these alternatively spliced isoforms also differ significantly in their microtubule plus end-tracking activity, suggesting a mechanism by which the cell may regulate the dynamic localization of dynactin. To test the function of the microtubule-binding domain of p150Glued, we used RNAi to deplete the endogenous polypeptide from HeLa cells, followed by rescue with constructs encoding either the full-length polypeptide or an isoform lacking the microtubule-binding domain. Both constructs fully rescued defects in Golgi morphology induced by depletion of p150Glued, indicating that an independent microtubule-binding site in dynactin may not be required for dynactin-mediated trafficking in some mammalian cell types. In neurons, however, a mutation within the microtubule-binding domain of p150Glued results in motor neuron disease; here we investigate the effects of four other mutations in highly conserved domains of the polypeptide (M571T, R785W, R1101K, and T1249I) associated in genetic studies with Amyotrophic Lateral Sclerosis. Both biochemical and cellular assays reveal that these amino acid substitutions do not result in functional differences, suggesting that these sequence changes are either allelic variants or contributory risk factors rather than causative for motor neuron disease. Together, these studies provide further insight into the regulation of dynein-dynactin function in the cell.