Kinesin superfamily proteins and their various functions and dynamics

Kinesin superfamily proteins (KIFs) are motor proteins that transport membranous organelles and macromolecules fundamental for cellular functions along microtubules. Their roles in transport in axons and dendrites have been studied extensively, but KIFs are also used in intracellular transport in general. Recent findings have revealed that in many cases, the specific interaction of cargoes and motors is mediated via adaptor/scaffolding proteins. Cargoes are sorted to precise destinations, such as axons or dendrites. KIFs also participate in polarized transport in epithelial cells as shown in the apical transport of annexin XIIIb-containing vesicles by KIFC3. KIFs play important roles in higher order neuronal activity; transgenic mice overexpressing KIF17, which transports N-methyl-d-asp (NMDA) receptors to dendrites, show enhanced memory and learning. KIFs also play significant roles in neuronal development and brain wiring: KIF2A suppresses elongation of axon collaterals by its unique microtubule-depolymerizing activity. X-ray crystallography has revealed the structural uniqueness of KIF2 underlying the microtubule-depolymerizing activity. In addition, single molecule biophysics and optical trapping have shown that the motility of monomeric KIF1A is caused by biased Brownian movement, and X-ray crystallography has shown how the conformational changes occur for KIF1A to move during ATP hydrolysis. These multiple approaches in analyzing KIF functions will illuminate many basic mechanisms underlying intracellular events and will be a very promising and fruitful area for future studies.     

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.     

β‐Tubulin mutations that cause severe neuropathies disrupt axonal transport

Microtubules are fundamental to neuronal morphogenesis and function. Mutations in tubulin, the major constituent of microtubules, result in neuronal diseases. Here, we have analysed β‐tubulin mutations that cause neuronal diseases and we have identified mutations that strongly inhibit axonal transport of vesicles and mitochondria. These mutations are in the H12 helix of β‐tubulin and change the negative charge on the surface of the microtubule. This surface is the interface between microtubules and kinesin superfamily motor proteins (KIF). The binding of axonal transport KIFs to microtubules is dominant negatively disrupted by these mutations, which alters the localization of KIFs in neurons and inhibits axon elongation in vivo. In humans, these mutations induce broad neurological symptoms, such as loss of axons in the central nervous system and peripheral neuropathy. Thus, our data identified the critical region of β‐tubulin required for axonal transport and suggest a molecular mechanism for human neuronal diseases caused by tubulin mutations.     

CRMP-2 directly binds to cytoplasmic dynein and interferes with its activity

The active transport of proteins and organelles is critical for cellular organization and function in eukaryotic cells. A substantial portion of long-distance transport depends on the opposite polarity of the kinesin and dynein family molecular motors to move cargo along microtubules. It is increasingly clear that many cargo molecules are moved bi-directionally by both sets of motors; however, the regulatory mechanism that determines the directionality of transport remains unclear. We previously reported that collapsin response mediator protein-2 (CRMP-2) played key roles in axon elongation and neuronal polarization. CRMP-2 was also found to associate with the anterograde motor protein Kinesin-1 and was transported with other cargoes toward the axon terminal. In this study, we investigated the association of CRMP-2 with a retrograde motor protein, cytoplasmic dynein. Immunoprecipitation assays showed that CRMP-2 interacted with cytoplasmic dynein heavy chain. Dynein heavy chain directly bound to the N-terminus of CRMP-2, which is the distinct side of CRMP-2's kinesin light chain-binding region. Furthermore, over-expression of the dynein-binding fragments of CRMP-2 prevented dynein-driven microtubule transport in COS-7 cells. Given that CRMP-2 is a key regulator of axon elongation, this interference with cytoplasmic dynein function by CRMP-2 might have an important role in axon formation, and neuronal development.     

Action in the axon: generation and transport of signaling endosomes

Neurons extend axonal processes over long distances, necessitating efficient transport mechanisms to convey target-derived neurotrophic survival signals from remote distal axons to cell bodies. Retrograde transport, powered by dynein motors, supplies cell bodies with survival signals in the form of 'signaling endosomes'. In this review, we will discuss new advances in our understanding of the motor proteins that bind to and move signaling components in a retrograde direction and discuss mechanisms that might specify distinct neuronal responses to spatially restricted neurotrophin signals. Disruption of retrograde transport leads to a variety of neurodegenerative diseases, highlighting the role of retrograde transport of signaling endosomes for axonal maintenance and the importance of efficient transport for neuronal survival and function.     

Effects of dynein inhibitor on the number of motor proteins transporting synaptic cargos

Synaptic cargo transport by kinesin and dynein in hippocampal neurons was investigated by noninvasively measuring the transport force based on nonequilibrium statistical mechanics. Although direct physical measurements such as force measurement using optical tweezers are difficult in an intracellular environment, the noninvasive estimations enabled enumerating force-producing units (FPUs) carrying a cargo comprising the motor proteins generating force. The number of FPUs served as a barometer for stable and long-distance transport by multiple motors, which was then used to quantify the extent of damage to axonal transport by dynarrestin, a dynein inhibitor. We found that dynarrestin decreased the FPU for retrograde transport more than for anterograde transport. This result indicates the applicability of the noninvasive force measurements. In the future, these measurements may be used to quantify damage to axonal transport resulting from neuronal diseases, including Alzheimer’s, Parkinson’s, and Huntington’s diseases.     

Biochemical and molecular characterization of diseases linked to motor proteins

Recent studies have revealed that kinesin, dynein and myosin each form large superfamilies and participate in many different intracellular transport systems. Importantly, these motor proteins play significant roles in the pathogenesis of a variety of diseases. Studies using knockout mice for kinesin KIF1B have led to the identification of the cause of a human hereditary neuropathy, Charcot-Marie-Tooth disease type 2A. The function of members of the dynein superfamily whose existence has previously only been confirmed through genome databases, has been revealed by studies of immotile cilia syndrome. Unconventional myosins have been shown to function in the inner-ear cells by examination of hereditary human hearing impairment and studies using mouse models. In addition, some diseases are caused by mutations, not in the motor itself, but in the proteins associated with the motor proteins. Here, we discuss the relationship of these motor proteins and how they contribute to disease in molecular terms.     

Organelle transport along microtubules - the role of KIFs

Organelle transporters are very important for cellular morphogenesis and other cellular functions, conveying and targeting important materials to the correct destination, often at considerable velocities. One of the first proteins to be identified as a motor was kinesin, and recently at least 10 new kinesin superfamily proteins (KIFs) have been described. Characterization of some of them reveals that each member can convey a specific organelle or cargo, although there is some redundancy. It has also become clear that there are distinct subclasses of KIFs that form monomeric, heterodimeric and homodimeric motors. Here, Nobutaka Hirokawa reviews what is known about the kinesin superfamily and discusses how a study of the different types of motors is helping to elucidate the mechanism of mechanical force generation.     

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.      

Hither and yon: a review of bi-directional microtubule-based transport

Active transport is critical for cellular organization and function, and impaired transport has been linked to diseases such as neuronal degeneration. Much long distance transport in cells uses opposite polarity molecular motors of the kinesin and dynein families to move cargos along microtubules. It is increasingly clear that many cargos are moved by both sets of motors, and frequently reverse course. This review compares this bi-directional transport to the more well studied uni-directional transport. It discusses some bi-directionally moving cargos, and critically evaluates three different physical models for how such transport might occur. It then considers the evidence for the number of active motors per cargo, and how the net or average direction of transport might be controlled. The likelihood of a complex linking the activities of kinesin and dynein is also discussed. The paper concludes by reviewing elements of apparent universality between different bi-directionally moving cargos and by briefly considering possible reasons for the existence of bi-directional transport.     

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 .     

Effect of fuel concentration and force on collective transport by a team of dynein motors

Motor proteins are essential components of intracellular transport inside eukaryotic cells. These protein molecules use chemical energy obtained from hydrolysis of ATP to produce mechanical forces required for transporting cargos inside cells, from one location to another, in a directed manner. Of these motors, cytoplasmic dynein is structurally more complex than other motor proteins involved in intracellular transport, as it shows force and fuel (ATP) concentration dependent step‐size. Cytoplasmic dynein motors are known to work in a team during cargo transport and force generation. Here, we use a complete Monte‐Carlo model of single dynein constrained by in vitro experiments, which includes the effect of both force and ATP on stepping as well as detachment of motors under force. We then use our complete Monte‐Carlo model of single dynein motor to understand collective cargo transport by a team of dynein motors, such as dependence of cargo travel distance and velocity on applied force and fuel concentration. In our model, cargos pulled by a team of dynein motors do not detach rapidly under higher forces, confirming the experimental observation of longer persistence time of dynein team on microtubule under higher forces.     

Cargo-carrying motor vehicles on the neuronal highway: transport pathways and neurodegenerative disease

Within axons vital cargoes must be transported over great distances along microtubule tracks to maintain neuronal viability. Essential to this system are the molecular motors, kinesin and dynein, which transport a variety of neuronal cargoes. Elucidating the transport pathways, the identity of the cargoes transported, and the regulation of motor-cargo complexes are areas of intense investigation. Evidence suggests that essential components, including signaling proteins, neuroprotective and repair molecules, and vesicular and cytoskeletal components are all transported. In addition newly emerging data indicate that defects in axonal transport pathways may contribute to the initiation or progression of chronic neuronal dysfunction. In this review we concentrate on microtubule-based motor proteins, their linkers, and cargoes and discuss how factors in the axonal transport pathway contribute to disease states. As additional cargo complexes and transport pathways are identified, an understanding of the role these pathways play in the development of human disease will hopefully lead to new diagnostic and treatment strategies.     

Motor-cargo interactions: the key to transport specificity

Eukaryotic cells organize their cytoplasm by moving different organelles and macromolecular complexes along microtubules and actin filaments. These movements are powered by numerous motor proteins that must recognize their respective cargoes in order to function. Recently, several proteins that interact with motors have been identified by yeast two-hybrid and biochemical analyses, and their roles in transport are now being elucidated. In several cases, analysis of the binding partners helped to identify new transport pathways, new types of cargo, and transport regulated at the level of motor-cargo binding. We discuss here how different motors of the kinesin, dynein and myosin families recognize their cargo and how motor-cargo interactions are regulated.     

Effects of ALS-related SOD1 mutants on dynein- and KIF5-mediated retrograde and anterograde axonal transport

Transport of material and signals between extensive neuronal processes and the cell body is essential to neuronal physiology and survival. Slowing of axonal transport has been shown to occur before the onset of symptoms in amyotrophic lateral sclerosis (ALS). We have previously shown that several familial ALS-linked copper–zinc superoxide dismutase (SOD1) mutants (A4V, G85R, and G93A) interacted and colocalized with the retrograde dynein–dynactin motor complex in cultured cells and affected tissues of ALS mice. We also found that the interaction between mutant SOD1 and the dynein motor played a critical role in the formation of large inclusions containing mutant SOD1. In this study, we showed that, in contrast to the dynein situation, mutant SOD1 did not interact with anterograde transport motors of the kinesin-1 family (KIF5A, B and C). Using dynein and kinesin accumulation at the sciatic nerve ligation sites as a surrogate measurement of axonal transport, we also showed that dynein mediated retrograde transport was slower in G93A than in WT mice at an early presymptomatic stage. While no decrease in KIF5A-mediated anterograde transport was detected, the slowing of anterograde transport of dynein heavy chain as a cargo was observed in the presymptomatic G93A mice. The results from this study along with other recently published work support that mutant SOD1 might only interact with and interfere with some kinesin members, which, in turn, could result in the impairment of a selective subset of cargos. Although it remains to be further investigated how mutant SOD1 affects different axonal transport motor proteins and various cargos, it is evident that mutant SOD1 can induce defects in axonal transport, which, subsequently, contribute to the propagation of toxic effects and ultimately motor neuron death in ALS.     

Kinesin superfamily protein 2A (KIF2A) functions in suppression of collateral branch extension

Through interactions with microtubules, the kinesin superfamily of proteins (KIFs) could have multiple roles in neuronal function and development. During neuronal development, postmitotic neurons develop primary axons extending toward targets, while other collateral branches remain short. Although the process of collateral branching is important for correct wiring of the brain, the mechanisms involved are not well understood. In this study, we analyzed kif2a(-/-) mice, whose brains showed multiple phenotypes, including aberrant axonal branching due to overextension of collateral branches. In kif2a(-/-) growth cones, microtubule-depolymerizing activity decreased. Moreover, many individual microtubules showed abnormal behavior at the kif2a(-/-) cell edge. Based on these results, we propose that KIF2A regulates microtubule dynamics at the growth cone edge by depolymerizing microtubules and that it plays an important role in the suppression of collateral branch extension.     

A novel motor, KIF13A, transports mannose-6-phosphate receptor to plasma membrane through direct interaction with AP-1 complex

Intracellular transport mediated by kinesin superfamily proteins (KIFs) is a highly regulated process. The molecular mechanism of KIFs binding to their respective cargoes remains unclear. We report that KIF13A is a novel plus end-directed microtubule-dependent motor protein and associates with beta 1-adaptin, a subunit of the AP-1 adaptor complex. The cargo vesicles of KIF13A contained AP-1 and mannnose-6-phosphate receptor (M6PR). Overexpression of KIF13A resulted in mislocalization of the AP-1 and the M6PR. Functional blockade of KIF13A reduced cell surface expression of the M6PR. Thus, KIF13A transports M6PR-containing vesicles and targets the M6PR from TGN to the plasma membrane via direct interaction with the AP-1 adaptor complex.     

A kinesin medley: biochemical and functional heterogeneity

Over the past decade, a remarkable number and diversity of molecular motors have been described in eukaryotic cells. In addition to the identification of novel forms of myosin and dynein, the kinesins have been defined as an entirely new family of molecular motors. There may be as many as 30 different genes in a single organism encoding members of the kinesin superfamily. Why is such diversity in molecular motors needed? The biochemical and functional diversity of the originally defined form of kinesin provides some insights into the roles of molecular motors in cellular dynamics.     

Cytoplasmic dynein subunit heterogeneity: implications for axonal transport

The formation and maintenance of neuronal synapses is dependent on the active transport of material between the cell body and the axon terminal. Cytoplasmic dynein is one motor for microtubule-based axonal transport. Two pools of cytoplasmic dynein have been identified in the axon. They are distinguished by their intermediate and light intermediate chain subunits. Each pool is transported at different rates down the axon in association with different proteins or organelles. This review presents several models to discuss the potential functional roles of these different pools of cytoplasmic dynein during axonal transport.     

CRMP/UNC-33 organizes microtubule bundles for KIF5-mediated mitochondrial distribution to axon

Neurons are highly specialized cells with polarized cellular processes and subcellular domains. As vital organelles for neuronal functions, mitochondria are distributed by microtubule-based transport systems. Although the essential components of mitochondrial transport including motors and cargo adaptors are identified, it is less clear how mitochondrial distribution among somato-dendritic and axonal compartment is regulated. Here, we systematically study mitochondrial motors, including four kinesins, KIF5, KIF17, KIF1, KLP-6, and dynein, and transport regulators in C. elegans PVD neurons. Among all these motors, we found that mitochondrial export from soma to neurites is mainly mediated by KIF5/UNC-116. Interestingly, UNC-116 is especially important for axonal mitochondria, while dynein removes mitochondria from all plus-end dendrites and the axon. We surprisingly found one mitochondrial transport regulator for minus-end dendritic compartment, TRAK-1, and two mitochondrial transport regulators for axonal compartment, CRMP/UNC-33 and JIP3/UNC-16. While JIP3/UNC-16 suppresses axonal mitochondria, CRMP/UNC-33 is critical for axonal mitochondria; nearly no axonal mitochondria present in unc-33 mutants. We showed that UNC-33 is essential for organizing the population of UNC-116-associated microtubule bundles, which are tracks for mitochondrial trafficking. Disarrangement of these tracks impedes mitochondrial transport to the axon. In summary, we identified a compartment-specific transport regulation of mitochondria by UNC-33 through organizing microtubule tracks for different kinesin motors other than microtubule polarity.