Molecular motors: strategies to get along

The majority of active transport in the cell is driven by three classes of molecular motors: the kinesin and dynein families that move toward the plus-end and minus-end of microtubules, respectively, and the unconventional myosin motors that move along actin filaments. Each class of motor has different properties, but in the cell they often function together. In this review we summarize what is known about their single-molecule properties and the possibilities for regulation of such properties. In view of new results on cytoplasmic dynein, we attempt to rationalize how these different classes of motors might work together as part of the intracellular transport machinery. We propose that kinesin and myosin are robust and highly efficient transporters, but with somewhat limited room for regulation of function. Because cytoplasmic dynein is less efficient and robust, to achieve function comparable to the other motors it requires a number of accessory proteins as well as multiple dyneins functioning together. This necessity for additional factors, as well as dynein's inherent complexity, in principle allows for greatly increased control of function by taking the factors away either singly or in combination. Thus, dynein's contribution relative to the other motors can be dynamically tuned, allowing the motors to function together differently in a variety of situations.     

Microtubule‐binding protein doublecortin‐like kinase 1 (DCLK1) guides kinesin‐3‐mediated cargo transport to dendrites

In neurons, the polarized distribution of vesicles and other cellular materials is established through molecular motors that steer selective transport between axons and dendrites. It is currently unclear whether interactions between kinesin motors and microtubule‐binding proteins can steer polarized transport. By screening all 45 kinesin family members, we systematically addressed which kinesin motors can translocate cargo in living cells and drive polarized transport in hippocampal neurons. While the majority of kinesin motors transport cargo selectively into axons, we identified five members of the kinesin‐3 (KIF1) and kinesin‐4 (KIF21) subfamily that can also target dendrites. We found that microtubule‐binding protein doublecortin‐like kinase 1 (DCLK1) labels a subset of dendritic microtubules and is required for KIF1‐dependent dense‐core vesicles (DCVs) trafficking into dendrites and dendrite development. Our study demonstrates that microtubule‐binding proteins can provide local signals for specific kinesin motors to drive polarized cargo transport.     

The kinases male germ cell‐associated kinase and cell cycle‐related kinase regulate kinesin‐2 motility in Caenorhabditis elegans neuronal cilia

Kinesin‐2 motors power anterograde intraflagellar transport (IFT), a highly ordered process that assembles and maintains cilia. However, it remains elusive how kinesin‐2 motors are regulated in vivo. Here, we performed forward genetic screens to isolate suppressors that rescue the ciliary defects of OSM‐3‐kinesin (homolog of mammalian homodimeric kinesin‐2 KIF17) mutants in Caenorhabditis elegans. We identified the C. elegans dyf‐5 and dyf‐18, which encode the homologs of mammalian male germ cell‐associated kinase and cell cycle‐related kinase, respectively. Using time‐lapse fluorescence microscopy, we show that DYF‐5 and DYF‐18 are IFT cargo molecules and are enriched at the distal segments of sensory cilia. Mutations of dyf‐5 and dyf‐18 generate elongated cilia and ectopic localization of the heterotrimeric kinesin‐2 (kinesin‐II) at the ciliary distal segments. Genetic analyses reveal that dyf‐5 and dyf‐18 are important for stabilizing the interaction between IFT particles and OSM‐3‐kinesin. Our data suggest that DYF‐5 and DYF‐18 act in the same pathway to promote handover between kinesin‐II and OSM‐3 in sensory cilia.      

How kinesin motor proteins drive mitotic spindle function: Lessons from molecular assays

Kinesins are enzymes that use the energy of ATP to perform mechanical work. There are approximately 14 families of kinesins within the kinesin superfamily. Family classification is derived primarily from alignments of the sequences of the core motor domain. For this reason, the enzymatic behavior and motility of each motor generally reflects its family. At the cellular level, kinesin motors perform a variety of functions during cell division and within the mitotic spindle to ensure that chromosomes are segregated with the highest fidelity possible. The cellular functions of these motors are intimately related to their mechanical and enzymatic properties at the single molecule level. For this reason, motility studies designed to evaluate the activity of purified molecular motors are a requirement in order to understand, mechanistically, how these motors make the mitotic spindle work and what can cause the spindle to fail. This review will focus on a selection of illustrative kinesins, which have been studied at the molecular level in order to inform our understanding of their function in cells. In addition, the review will endeavor to point out some kinesins that have been studied extensively but which still lack sufficient molecular underpinnings to fully predict their contribution to spindle function.     

Functional asymmetry in kinesin and dynein dimers

Active transport along the microtubule lattice is a complex process that involves both the Kinesin and Dynein superfamily of motors. Transportation requires sophisticated regulation much of which occurs through the motor's tail domain. However, a significant portion of this regulation also occurs through structural changes that arise in the motor and the microtubule upon binding. The most obvious structural change being the manifestation of asymmetry. To a first approximation in solution, kinesin dimers exhibit twofold symmetry, and microtubules exhibit helical symmetry. The higher symmetries of both the kinesin dimers and microtubule lattice are lost on formation of the kinesin–microtubule complex. Loss of symmetry has functional consequences such as an asymmetric hand‐over‐hand mechanism in plus‐end‐directed kinesins, asymmetric microtubule binding in the Kinesin‐14 family, spatially biased stepping in dynein and cooperative binding of additional motors to the microtubule. This review focusses on how the consequences of asymmetry affect regulation of motor heads within a dimer, dimers within an ensemble of motors, and suggests how these asymmetries may affect regulation of active transport within the cell.     

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.     

Crystal structure of kinesin regulated by Ca(2+)-calmodulin

Kinesins orchestrate cell division by controlling placement of chromosomes. Kinesins must be precisely regulated or else cell division fails. Calcium, a universal second messenger in eukaryotes, and calmodulin regulate some kinesins by causing the motor to dissociate from its biological track, the microtubule. Our focus was the mechanism of calcium regulation of kinesin at atomic resolution. Here we report the crystal structure of kinesin-like calmodulin-binding protein (KCBP) from potato, which was resolved to 2.3 A. The structure reveals three subdomains of the regulatory machinery located at the C terminus extension of the kinesin motor. Calmodulin that is activated by Ca2+ ions binds to an alpha-helix positioned on the microtubule-binding face of kinesin. A negatively charged segment following this helix competes with microtubules. A mimic of the conventional kinesin neck, connecting the calmodulin-binding helix to the KCBP motor core, links the regulatory machine to the kinesin catalytic cycle. Together with biochemical data, the crystal structure suggests that Ca(2+)-calmodulin inhibits the binding of KCBP to microtubules by blocking the microtubule-binding sites on KCBP.     

Clonal tests of conventional kinesin function during cell proliferation and differentiation

Null mutations in the Drosophila Kinesin heavy chain gene (Khc), which are lethal during the second larval instar, have shown that conventional kinesin is critical for fast axonal transport in neurons, but its functions elsewhere are uncertain. To test other tissues, single imaginal cells in young larvae were rendered null for Khc by mitotic recombination. Surprisingly, the null cells produced large clones of adult tissue. The rates of cell proliferation were not reduced, indicating that conventional kinesin is not essential for cell growth or division. This suggests that in undifferentiated cells vesicle transport from the Golgi to either the endoplasmic reticulum or the plasma membrane can proceed at normal rates without conventional kinesin. In adult eye clones produced by null founder cells, there were some defects in differentiation that caused mild ultrastructural changes, but they were not consistent with serious problems in the positioning or transport of endoplasmic reticulum, mitochondria, or vesicles. In contrast, defective cuticle deposition by highly elongated Khc null bristle shafts suggests that conventional kinesin is critical for proper secretory vesicle transport in some cell types, particularly ones that must build and maintain long cytoplasmic extensions. The ubiquity and evolutionary conservation of kinesin heavy chain argue for functions in all cells. We suggest interphase organelle movements away from the cell center are driven by multilayered transport mechanisms; that is, individual organelles can use kinesin-related proteins and myosins, as well as conventional kinesin, to move toward the cell periphery. In this case, other motors can compensate for the loss of conventional kinesin except in cells that have extremely long transport tracks.     

Kinesin‐2 motors adapt their stepping behavior for processive transport on axonemes and microtubules

Two structurally distinct filamentous tracks, namely singlet microtubules in the cytoplasm and axonemes in the cilium, serve as railroads for long‐range transport processes in vivo. In all organisms studied so far, the kinesin‐2 family is essential for long‐range transport on axonemes. Intriguingly, in higher eukaryotes, kinesin‐2 has been adapted to work on microtubules in the cytoplasm as well. Here, we show that heterodimeric kinesin‐2 motors distinguish between axonemes and microtubules. Unlike canonical kinesin‐1, kinesin‐2 takes directional, off‐axis steps on microtubules, but it resumes a straight path when walking on the axonemes. The inherent ability of kinesin‐2 to side‐track on the microtubule lattice restricts the motor to one side of the doublet microtubule in axonemes. The mechanistic features revealed here provide a molecular explanation for the previously observed partitioning of oppositely moving intraflagellar transport trains to the A‐ and B‐tubules of the same doublet microtubule. Our results offer first mechanistic insights into why nature may have co‐evolved the heterodimeric kinesin‐2 with the ciliary machinery to work on the specialized axonemal surface for two‐way traffic.     

Processive cytoskeletal motors studied with single-molecule fluorescence techniques

Processive cytoskeletal motors from the myosin, kinesin, and dynein families walk on actin filaments and microtubules to drive cellular transport and organization in eukaryotic cells. These remarkable molecular machines are able to take hundreds of successive steps at speeds of up to several microns per second, allowing them to effectively move vesicles and organelles throughout the cytoplasm. Here, we focus on single-molecule fluorescence techniques and discuss their wide-ranging applications to the field of cytoskeletal motor research. We cover both traditional fluorescence and sub-diffraction imaging of motors, providing examples of how fluorescence data can be used to measure biophysical parameters of motors such as coordination, stepping mechanism, gating, and processivity. We also outline some remaining challenges in the field and suggest future directions.     

Functional analysis of mouse C-terminal kinesin motor KifC2

Proteins of the kinesin superfamily define a class of microtubule-dependent motors that play crucial roles in cell division and intracellular transport. In the mouse, several kinesin motors have been characterized and are suggested to play roles in axonal and/or dendritic transport. One such kinesin is KifC2. Sequence and secondary structure analysis revealed that KifC2 is a member of the C-terminal motor family. Northern and Western blot analyses indicated that KifC2 is specifically expressed in both the central and peripheral nervous systems. The cellular locations of the KifC2 proteins were found to be mainly in neural cell bodies and dendrites but also in axons. To understand the in vivo function of the KifC2 gene, we used homologous recombination in embryonic stem cells to construct knockout mouse strains for the KifC2 gene. Homozygous KifC2 mutants were viable and reproduced normally, and their development was apparently normal. These results suggest that KifC2 is dispensable for normal neural development and behavior in the mouse.     

Carboxypeptidase E cytoplasmic tail-driven vesicle transport is key for activity-dependent secretion of peptide hormones

Vesicular transport of peptide hormones from the cell body to the plasma membrane for activity-dependent secretion is important for endocrine function, but how it is achieved is unclear. Here we uncover a mechanism in which the cytoplasmic tail of transmembrane carboxypeptidase E (CPE) found in proopiomelanocotin (POMC)/ACTH vesicles interacts with microtubule-based motors to control transport of these vesicles to the release site in pituitary cells. Overexpression of the CPE tail in live cells significantly reduced the velocity and distance of POMC/ACTH- and CPE-containing vesicle movement into the cell processes. Biochemical studies showed that the CPE tail interacted with dynactin, which, in turn, recruited microtubule plus-end motors kinesin 2 and kinesin 3. Overexpression of the CPE tail inhibited the stimulated secretion of ACTH from AtT20 cells. Thus, the CPE cytoplasmic tail interaction with dynactin-kinesin 2/kinesin 3 plays an important role in the transport of POMC vesicles for activity-dependent secretion.     

Symposium 4: Dynamics of the Neuronal Cytoskeleton. Actin‐based vesicle transport in nerve cell extracts: implications for neurodegenerative diseases

Neurodegenerative diseases may result in part from defects in motor‐driven vesicle transport in neuronal cells. Myosin‐V, an actin‐based motor that is highly enriched in the brain, mediates the movement of vesicles on cortical actin filaments. Recent evidence suggests that the globular tail of myosin‐V interacts with the microtubule‐based motor, kinesin, to form a ‘hetero‐motor’ complex on vesicles. The complex of these two motors, one microtubule‐based and the other actin‐based, facilitates the movement of vesicles from microtubules to actin filaments. Based on our studies of vesicle transport by these two motors in extracts of squid neurons, we hypothesize that one of the functions of the tail–tail interaction is to provide feedback between the two proteins to allow seamless transition of vesicles from microtubules to actin filaments. To study the interactions of the globular tail domain of myosin‐V to kinesin and to neuronal vesicles, we used a GST‐tagged globular tail fragment in motility assays. The MyoV tail fragment inhibited vesicle transport by 81–91% and thereby exhibited a dominant negative effect. These data show that the recombinant protein blocked the activity of native myosin‐V presumably by binding to vesicles and competing away the native myosin‐V motors. The GST‐MyoV‐tail fragment pulled down kinesin by immunoprecipitation from squid brain homogenates and therefore it exhibited binding properties of native myosin‐V. These data show that the headless myosin‐V fragment is an effective inhibitor of vesicle transport in cell extracts. These studies support the hypothesis that tail–tail interactions may be a mechanism for feedback between myosin‐V and kinesin to allow transition of vesicles from microtubules to actin filaments. Acknowledgements: Supported by NSF grant MCB9974709.     

The axonal transport motor kinesin‐2 navigates microtubule obstacles via protofilament switching

Axonal transport involves kinesin motors trafficking cargo along microtubules that are rich in microtubule‐associated proteins (MAPs). Much attention has focused on the behavior of kinesin‐1 in the presence of MAPs, which has overshadowed understanding the contribution of other kinesins such as kinesin‐2 in axonal transport. We have previously shown that, unlike kinesin‐1, kinesin‐2 in vitro motility is insensitive to the neuronal MAP Tau. However, the mechanism by which kinesin‐2 efficiently navigates Tau on the microtubule surface is unknown. We hypothesized that mammalian kinesin‐2 side‐steps to adjacent protofilaments to maneuver around MAPs. To test this, we used single‐molecule imaging to track the characteristic run length and protofilament switching behavior of kinesin‐1 and kinesin‐2 motors in the absence and presence of 2 different microtubule obstacles. Under all conditions tested, kinesin‐2 switched protofilaments more frequently than kinesin‐1. Using computational modeling that recapitulates run length and switching frequencies in the presence of varying roadblock densities, we conclude that kinesin‐2 switches protofilaments to navigate around microtubule obstacles. Elucidating the kinesin‐2 mechanism of navigation on the crowded microtubule surface provides a refined view of its contribution in facilitating axonal transport.      

Balancing self-renewal and differentiation by asymmetric division: insights from brain tumor suppressors in Drosophila neural stem cells

Balancing self-renewal and differentiation of stem cells is an important issue in stem cell and cancer biology. Recently, the Drosophila neuroblast (NB), neural stem cell has emerged as an excellent model for stem cell self-renewal and tumorigenesis. It is of great interest to understand how defects in the asymmetric division of neural stem cells lead to tumor formation. Here, we review recent advances in asymmetric division and the self-renewal control of Drosophila NBs. We summarize molecular mechanisms of asymmetric cell division and discuss how the defects in asymmetric division lead to tumor formation. Gain-of-function or loss-of-function of various proteins in the asymmetric machinery can drive NB overgrowth and tumor formation. These proteins control either the asymmetric protein localization or mitotic spindle orientation of NBs. We also discuss other mechanisms of brain tumor suppression that are beyond the control of asymmetric division.     

MAPping out distribution routes for kinesin couriers

In the crowded environment of eukaryotic cells, diffusion is an inefficient distribution mechanism for cellular components. Long‐distance active transport is required and is performed by molecular motors including kinesins. Furthermore, in highly polarised, compartmentalised and plastic cells such as neurons, regulatory mechanisms are required to ensure appropriate spatio‐temporal delivery of neuronal components. The kinesin machinery has diversified into a large number of kinesin motor proteins as well as adaptor proteins that are associated with subsets of cargo. However, many mechanisms contribute to the correct delivery of these cargos to their target domains. One mechanism is through motor recognition of sub‐domain‐specific microtubule (MT) tracks, sign‐posted by different tubulin isoforms, tubulin post‐translational modifications, tubulin GTPase activity and MT‐associated proteins (MAPs). With neurons as a model system, a critical review of these regulatory mechanisms is presented here, with a particular focus on the emerging contribution of compartmentalised MAPs. Overall, we conclude that – especially for axonal cargo – alterations to the MT track can influence transport, although in vivo, it is likely that multiple track‐based effects act synergistically to ensure accurate cargo distribution.     

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.      

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 .     

Kinesin velocity increases with the number of motors pulling against viscoelastic drag

Although the properties of single kinesin molecular motors are well understood, it is not clear whether multiple motors pulling a single vesicle in a cell cooperate or interfere with one another. To learn how small numbers of motors interact, microtubule gliding assays were carried out with full-length Drosophila kinesin in a novel motility medium containing xanthan, a stiff, water-soluble polysaccharide. At 2 mg/ml xanthan, the zero-shear viscosity of this medium is 1,000 times the viscosity of water, similar to cellular viscosity. To mimic the rheological drag force on the motors when attached to a vesicle in a cell, we attached a 2 μm bead to one end of the microtubule (MT). During gliding assays in our novel medium, the moving bead exerted a drag force of 4–15 pN on the kinesins pulling the MT. The velocity of MTs with an attached bead increased with MT length and with kinesin concentration. The increase with MT length arose because the number of motors is directly proportional to MT length. Our results show that small numbers of kinesins cooperate constructively when pulling against a viscoelastic drag. In the absence of a bead but still in the viscous medium, MT velocity was independent of MT length and kinesin concentration because the thin MT, like a snake moving through grass, was able to move between xanthan molecules with little resistance. A minimal shared-load model in which the number of motors is proportional to MT length fits the observed dependence of gliding velocity on MT length and kinesin concentration.