Cytoplasmic dynein: a key player in neurodegenerative and neurodevelopmental diseases

Cytoplasmic dynein is the most important molecular motor driving the movement of a wide range of cargoes towards the minus ends of microtubules.As a molecular motor protein,dynein performs a variety of basic cellular functions including organelle transport and centrosome assembly.In the nervous system,dynein has been demonstrated to be responsible for axonal retrograde transport.Many studies have revealed direct or indirect evidence of dynein in neurodegenerative diseases such as amyotrophic lateral sclerosis,Charcot-Marie-Tooth disease,Alzheimer’s disease,Parkinson’s disease and Huntington’s disease.Among them,a number of mutant proteins involved in various neurodegenerative diseases interact with dynein.Axonal transport disruption is presented as a common feature occurring in neurodegenerative diseases.Dynein heavy chain mutant mice also show features of neurodegenerative diseases.Moreover,defects of dynein-dependent processes such as autophagy or clearance of aggregation-prone proteins are found in most of these diseases.Lines of evidence have also shown that dynein is associated with neurodevelopmental diseases.In this review,we focus on dynein involvement in different neurological diseases and discuss potential underlying mechanisms.     

Coupling viruses to dynein and kinesin-1

It is now clear that transport on microtubules by dynein and kinesin family motors has an important if not critical role in the replication and spread of many different viruses. Understanding how viruses hijack dynein and kinesin motors using a limited repertoire of proteins offers a great opportunity to determine the molecular basis of motor recruitment. In this review, we discuss the interactions of dynein and kinesin-1 with adenovirus, the α herpes viruses: herpes simplex virus (HSV1) and pseudorabies virus (PrV), human immunodeficiency virus type 1 (HIV-1) and vaccinia virus. We highlight where the molecular links to these opposite polarity motors have been defined and discuss the difficulties associated with identifying viral binding partners where the basis of motor recruitment remains to be established. Ultimately, studying microtubule-based motility of viruses promises to answer fundamental questions as to how the activity and recruitment of the dynein and kinesin-1 motors are coordinated and regulated during bi-directional transport.     

A single protofilament is sufficient to support unidirectional walking of Dynein and Kinesin

Cytoplasmic dynein and kinesin are two-headed microtubule motor proteins that move in opposite directions on microtubules. It is known that kinesin steps by a 'hand-over-hand' mechanism, but it is unclear by which mechanism dynein steps. Because dynein has a completely different structure from that of kinesin and its head is massive, it is suspected that dynein uses multiple protofilaments of microtubules for walking. One way to test this is to ask whether dynein can step along a single protofilament. Here, we examined dynein and kinesin motility on zinc-induced tubulin sheets (zinc-sheets) which have only one protofilament available as a track for motor proteins. Single molecules of both dynein and kinesin moved at similar velocities on zinc-sheets compared to microtubules, clearly demonstrating that dynein and kinesin can walk on a single protofilament and multiple rows of parallel protofilaments are not essential for their motility. Considering the size and the motile properties of dynein, we suggest that dynein may step by an inchworm mechanism rather than a hand-over-hand mechanism.     

A direct interaction between cytoplasmic dynein and kinesin I may coordinate motor activity

Cytoplasmic dynein and kinesin I are both unidirectional intracellular motors. Dynein moves cargo toward the cell center, and kinesin moves cargo toward the cell periphery. There is growing evidence that bi-directional motility is regulated in the cell, potentially through direct interactions between oppositely oriented motors. We have identified a direct interaction between cytoplasmic dynein and kinesin I. Using the yeast two-hybrid assay and affinity chromatography, we demonstrate that the intermediate chain of dynein binds to kinesin light chains 1 and 2. The interaction is both direct and specific. Co-immunoprecipitation experiments demonstrate an interaction between endogenous proteins in rat brain cytosol. Double-label immunocytochemistry reveals a partial co-localization of vesicle-associated motor proteins. Together these observations suggest that soluble motors can interact, potentially allowing kinesin I to actively localize dynein to cellular sites of function. There is also a vesicle population with both dynein and kinesin I bound that may be capable of bi-directional motility along cellular microtubules.     

Tau directs intracellular trafficking by regulating the forces exerted by kinesin and dynein teams

Organelles, proteins, and mRNA are transported bidirectionally along microtubules by plus‐end directed kinesin and minus‐end directed dynein motors. Microtubules are decorated by microtubule‐associated proteins (MAPs) that organize the cytoskeleton, regulate microtubule dynamics and modulate the interaction between motor proteins and microtubules to direct intracellular transport. Tau is a neuronal MAP that stabilizes axonal microtubules and crosslinks them into bundles. Dysregulation of tau leads to a range of neurodegenerative diseases known as tauopathies including Alzheimer's disease (AD). Tau reduces the processivity of kinesin and dynein by acting as an obstacle on the microtubule. Single‐molecule assays indicate that kinesin‐1 is more strongly inhibited than kinesin‐2 or dynein, suggesting tau might act to spatially modulate the activity of specific motors. To investigate the role of tau in regulating bidirectional transport, we isolated phagosomes driven by kinesin‐1, kinesin‐2, and dynein and reconstituted their motility along microtubules. We find that tau biases bidirectional motility towards the microtubule minus‐end in a dose‐dependent manner. Optical trapping measurements show that tau increases the magnitude and frequency of forces exerted by dynein through inhibiting opposing kinesin motors. Mathematical modeling indicates that tau controls the directional bias of intracellular cargoes through differentially tuning the processivity of kinesin‐1, kinesin‐2, and dynein. Taken together, these results demonstrate that tau modulates motility in a motor‐specific manner to direct intracellular transport, and suggests that dysregulation of tau might contribute to neurodegeneration by disrupting the balance of plus‐ and minus‐end directed transport.      

Molecular motors in neuronal development, intracellular transport and diseases

Molecular motors such as kinesin superfamily proteins (KIFs), dynein superfamily proteins and myosin superfamily proteins have diverse and fundamental roles in many cellular processes, including neuronal development and the pathogenesis of neuronal diseases. During neuronal development, KIFs take significant roles in the regulation of axon-collateral branch extension, which is essential for brain wiring. Cytoplasmic dynein together with LIS1 takes pivotal roles in neocortical layer formation. In axons, anterograde transport is mediated by KIFs, whereas retrograde transport is mediated mainly by cytoplasmic dynein, and dysfunction of motors results in neurodegenerative diseases. In dendrites, the transport of NMDA and AMPA receptors is mediated by KIFs, and the motor has been shown to play a significant part in establishing learning and memory.     

The cytoplasmic dynein and kinesin motors have interdependent roles in patterning the Drosophila oocyte

BACKGROUND: Motor proteins of the minus end-directed cytoplasmic dynein and plus end-directed kinesin families provide the principal means for microtubule-based transport in eukaryotic cells. Despite their opposing polarity, these two classes of motors may cooperate in vivo. In Drosophila circumstantial evidence suggests that dynein acts in the localization of determinants and signaling factors during oogenesis. However, the pleiotropic requirement for dynein throughout development has made it difficult to establish its specific role. RESULTS: We analyzed dynein function in the oocyte by disrupting motor activity through temporally restricted expression of the dynactin subunit, dynamitin. Our results indicate that dynein is required for several processes that impact patterning; such processes include localization of bicoid (bcd) and gurken (grk) mRNAs and anchoring of the oocyte nucleus to the cell cortex. Surprisingly, dynein function is sensitive to reduction in kinesin levels, and germ line clones lacking kinesin show defects in dorsal follicle cell fate, grk mRNA localization, and nuclear attachment that are similar to those resulting from the loss of dynein. Significantly, dynein and dynactin localization is perturbed in these animals. Conversely, kinesin localization also depends on dynein activity. CONCLUSIONS: We demonstrate that dynein is required for nuclear anchoring and localization of cellular determinants during oogenesis. Strikingly, mutations in the kinesin motor also disrupt these processes and perturb dynein and dynactin localization. These results indicate that the activity of the two motors is interdependent and suggest a model in which kinesin affects patterning indirectly through its role in the localization and recycling of dynein.     

Dynein and kinesin share an overlapping microtubule-binding site

Dyneins and kinesins move in opposite directions on microtubules. The question of how the same-track microtubules are able to support movement in two directions remains unanswered due to the absence of details on dynein-microtubule interactions. To address this issue, we studied dynein-microtubule interactions using the tip of the microtubule-binding stalk, the dynein stalk head (DSH), which directly interacts with microtubules upon receiving conformational change from the ATPase domain. Biochemical and cryo-electron microscopic studies revealed that DSH bound to tubulin dimers with a periodicity of 80 A, corresponding to the step size of dyneins. The DSH molecule was observed as a globular corn grain-like shape that bound the same region as kinesin. Biochemical crosslinking experiments and image analyses of the DSH-kinesin head-microtubule complex revealed competition between DSH and the kinesin head for microtubule binding. Our results demonstrate that dynein and kinesin share an overlapping microtubule-binding site, and imply that binding at this site has an essential role for these motor proteins.     

Microtubule motors in spindle and chromosome motility.

Many of the kinesin microtubule motor proteins discovered during the past 8-9 years have roles in spindle assembly and function or chromosome movement during meiosis or mitosis. The discovery of kinesin motor proteins with a clear involvement in spindle and chromosome motility, together with recent evidence that cytoplasmic dynein plays a role in chromosome distribution, has attracted great interest. The identification of microtubule motors that function in chromosome distribution represents a major advance in understanding the forces that underlie chromosome and spindle movements during cell division.     

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.     

The p150(Glued) CAP-Gly domain regulates initiation of retrograde transport at synaptic termini

p150(Glued) is the major subunit of dynactin, a complex that functions with dynein in minus-end-directed microtubule transport. Mutations within the p150(Glued) CAP-Gly microtubule-binding domain cause neurodegenerative diseases through an unclear mechanism. A p150(Glued) motor neuron degenerative disease-associated mutation introduced into the Drosophila Glued locus generates a partial loss-of-function allele (Gl(G38S)) with impaired neurotransmitter release and adult-onset locomotor dysfunction. Disruption of the p150(Glued) CAP-Gly domain in neurons causes a specific disruption of vesicle trafficking at?terminal boutons (TBs), the distal-most ends of synapses. Gl(G38S) larvae accumulate endosomes along with dynein and kinesin motor proteins within swollen TBs, and genetic analyses show that kinesin and p150(Glued) function cooperatively at TBs to coordinate transport. Therefore, the p150(Glued) CAP-Gly domain regulates dynein-mediated retrograde transport at synaptic termini, and this function of dynactin is disrupted by a mutation that causes motor?neuron disease.     

Axonopathy-Inducing 1,2-Diacetylbenzene Forms Adducts with Motor and Cytoskeletal Proteins Required for Axonal Transport

The aromatic hydrocarbon 1,2-diacetylbenzene (1,2-DAB) is a protein-reactive γ-diketone metabolite of the neurotoxic solvent 1,2-diethylbenzene (1,2-DEB). The effect of neurotoxic 1,2-DAB and its non-neurotoxic isomer 1,3-DAB has been studied on motor proteins and cytoskeletal proteins of rat spinal cord (SC). For in vitro studies, SC slices were incubated with 1, 2, 5, 10 mM of DAB isomers for 30 min at 37°C. For in vivo studies, rats received (i.p.) 20 mg/kg/day of 1,2-DAB or 1,3-DAB, or vehicle (2% acetone in saline), 5 days a week for 2 weeks. Spinal cord and sciatic nerve proteins were subjected to Western blotting using monoclonal mouse antibodies to NF-M, kinesin, dynein, and tau. Proteins were quantified and paired mean comparisons performed to assess concentration-dependent changes in native protein bands. In vitro, 1,2-DAB produced a concentration-dependent decrease of motor and cytoskeletal proteins. While dynein and tau appeared similarly affected by 1,2-DAB, kinesin was most affected by the toxicant. In vivo, 1,2-DAB affected motor and cytoskeletal proteins of sciatic nerves and spinal cord differentially. In general, sciatic nerve proteins were much more affected than spinal cord proteins. The results show that motor proteins that drive axonal transport anterogradely (kinesin) and retrogradely (dynein), cytoskeletal protein NF-M, which is slowly transported in the anterograde direction, and microtubule-associated protein, tau, which is involved in axonal transport, are differentially impacted by 1,2-DAB. By contrast, non-neurotoxic isomer 1,3-diacetylbenzene (1,3-DAB), had no adverse effect on neural proteins either in vitro or in vivo. 2D-Differential in gel electrophoresis (2D-DIGE) of sciatic nerves from neurotoxic 1,2-DAB and non-neurotoxic 1,3-DAB treated rats revealed 197 and 304 protein spots, respectively. This paper is dedicated to my long-time friend Naren L. Banik, Ph.D.     

Kinesin and dynein-dynactin at intersecting microtubules: motor density affects dynein function

Kinesin and cytoplasmic dynein are microtubule-based motor proteins that actively transport material throughout the cell. Microtubules can intersect at a variety of angles both near the nucleus and at the cell periphery, and the behavior of molecular motors at these intersections has implications for long-range transport efficiency and accuracy. To test motor function at microtubule intersections, crossovers were arranged in vitro using flow to orient successive layers of filaments. Single kinesin and cytoplasmic dynein-dynactin molecules fused with green-fluorescent protein, and artificial bead cargos decorated with multiple motors, were observed while they encountered intersections. Single kinesins tend to cross intersecting microtubules, whereas single dynein-dynactins have a more varied response. For bead cargos, kinesin motion is independent of motor number. Dynein beads with high motor numbers pause, but their actions become more varied as the motor number decreases. These results suggest that regulating the number of active dynein molecules could change a motile cargo into one that is anchored at an intersection, consistent with dynein's proposed transport and tethering functions in the cell.     

Motor proteins for cytoplasmic microtubules.

It has been thought that motile structures within the cell are driven toward the plus and minus ends of microtubules by the ATPases, kinesin and dynein, respectively. Recently obtained data indicate that this model is far too simplistic. Kinesin is now understood to be one representative of a family of proteins. Another member of the kinesin family has been found to generate force toward the microtubule minus end. Evidence for either a bidirectional dynein, or closely related retrograde and anterograde forms of dynein has also received potent new support. The discovery of a third potential microtubule motor, the GTPase, 'dynamin', complicates matters further.     

Is the dynein motor a winch?

Dyneins are the largest and most complex of the three classes of linear motor proteins in eukaryotic cells. The mass of the dynein motor domain is about ten times that of the other microtubule motor, kinesin. Dynein's homology with the AAA+ superfamily of mechanoenzymes distinguishes it from both kinesin and myosin, which share a common fold and ancestry as members of the G-protein superfamily. In contrast to the other motor proteins, little is known about the mechanism of dynein; its three-dimensional structure is unknown even at low resolution. Recent two-dimensional images from electron microscopy have revealed new details of its structure and how this changes to produce movement. These and the recently solved crystal structure of another AAA+ protein, ClpB, offer tantalising hints about dynein's mechanism, suggesting it may act like a molecular winch.     

Motor neurons rely on motor proteins

The importance of active axonal transport to the neuron has been highlighted by the recent discoveries that mutations in microtubule motor proteins result in neurodegenerative diseases. Mutations affecting microtubule motor function have been shown to cause hereditary forms of Charcot-Marie-Tooth disease (type 2A), hereditary spastic paraplegia and motor neuron disease. Although motor neurons appear to be uniquely susceptible to defects in axonal transport, recent work has identified links between perturbations in axonal transport and the pathogenesis of other neurodegenerative diseases such as Huntington's disease and Alzheimer's disease. More broadly, cytoskeletal abnormalities might also be at the root of related disorders such as spinal muscular atrophy, supporting a key role for axonal transport in the pathogenesis of many neurodegenerative diseases.     

Molecular motors in neurons: transport mechanisms and roles in brain function, development, and disease

The kinesin, dynein, and myosin superfamily molecular motors have fundamental roles in neuronal function, plasticity, morphogenesis, and survival by transporting cargos such as synaptic vesicle precursors, neurotransmitter and neurotrophic factor receptors, and mRNAs within axons, dendrites, and synapses. Recent studies have begun to clarify the mechanisms of cargo selection and directional transport in subcellular compartments. Furthermore, molecular genetics has revealed unexpected roles for molecular motors in brain wiring, neuronal survival, neuronal plasticity, higher brain function, and control of central nervous system and peripheral nervous system development. Finally, it is also evident that molecular motors are critically involved in neuronal disease pathogenesis. Thus, molecular motor research is becoming an exciting frontier of neuroscience.     

Kinesin family in murine central nervous system

In neuronal axons, various kinds of membranous components are transported along microtubules bidirectionally. However, only two kinds of mechanochemical motor proteins, kinesin and brain dynein, had been identified as transporters of membranous organelles in mammalian neurons. Recently, a series of genes that encode proteins closely related to kinesin heavy chain were identified in several organisms including Schizosaccharomyces pombe, Aspergillus niddulans, Saccharomyces cerevisiae, Caenorhabditus elegans, and Drosophila. Most of these members of the kinesin family are implicated in mechanisms of mitosis or meiosis. To address the mechanism of intracellular organelle transport at a molecular level, we have cloned and characterized five different members (KIF1-5), that encode the microtubule-associated motor domain homologous to kinesin heavy chain, in murine brain tissue. Homology analysis of amino acid sequence indicated that KIF1 and KIF5 are murine counterparts of unc104 and kinesin heavy chain, respectively, while KIF2, KIF3, and KIF4 are as yet unidentified new species. Complete amino acid sequence of KIF3 revealed that KIF3 consists of NH2-terminal motor domain, central alpha-helical rod domain, and COOH-terminal globular domain. Complete amino acid sequence of KIF2 revealed that KIF2 consists of NH2-terminal globular domain, central motor domain, and COOH-terminal alpha-helical rod domain. This is the first identification of the kinesin-related protein which has its motor domain at the central part in its primary structure. Northern blot analysis revealed that KIF1, KIF3, and KIF5 are expressed almost exclusively in murine brain, whereas KIF2 and KIF4 are expressed in brain as well as in other tissues. All these members of the kinesin family are expressed in the same type of neurons, and thus each one of them may transport its specific organelle in the murine central nervous system.     

Endogenous GSK‐3/Shaggy Regulates Bidirectional Axonal Transport of the Amyloid Precursor Protein

Neurons rely on microtubule (MT) motor proteins such as kinesin‐1 and dynein to transport essential cargos between the cell body and axon terminus. Defective axonal transport causes abnormal axonal cargo accumulations and is connected to neurodegenerative diseases, including Alzheimer's disease (AD). Glycogen synthase kinase 3 (GSK‐3) has been proposed to be a central player in AD and to regulate axonal transport by the MT motor protein kinesin‐1. Using genetic, biochemical and biophysical approaches in Drosophila melanogaster, we find that endogenous GSK‐3 is a required negative regulator of both kinesin‐1‐mediated and dynein‐mediated axonal transport of the amyloid precursor protein (APP), a key contributor to AD pathology. GSK‐3 also regulates transport of an unrelated cargo, embryonic lipid droplets. By measuring the forces motors generate in vivo, we find that GSK‐3 regulates transport by altering the activity of kinesin‐1 motors but not their binding to the cargo. These findings reveal a new relationship between GSK‐3 and APP, and demonstrate that endogenous GSK‐3 is an essential in vivo regulator of bidirectional APP transport in axons and lipid droplets in embryos. Furthermore, they point to a new regulatory mechanism in which GSK‐3 controls the number of active motors that are moving a cargo .     

Connecting vesicle transport to the cytoskeleton

The cytoskeleton is crucial for the efficient and polarized transport of vesicles in intracellular membrane-sorting pathways. Recent studies have identified specific kinesin, dynein, and myosin motor proteins that mediate defined membrane transport steps. Important clues have also been uncovered about the nature of motor-protein receptors on vesicular cargoes and the molecular mechanisms of motor-protein regulation.