Importance of anisotropy in detachment rates for force production and cargo transport by a team of motor proteins

Many cellular processes are driven by collective forces generated by a team consisting of multiple molecular motor proteins. One aspect that has received less attention is the detachment rate of molecular motors under mechanical force/load. While detachment rate of kinesin motors measured under backward force increases rapidly for forces beyond stall‐force; this scenario is just reversed for non‐yeast dynein motors where detachment rate from microtubule decreases, exhibiting a catch‐bond type behavior. It has been shown recently that yeast dynein responds anisotropically to applied load, i.e. detachment rates are different under forward and backward pulling. Here, we use computational modeling to show that these anisotropic detachment rates might help yeast dynein motors to improve their collective force generation in the absence of catch‐bond behavior. We further show that the travel distance of cargos would be longer if detachment rates are anisotropic. Our results suggest that anisotropic detachment rates could be an alternative strategy for motors to improve the transport properties and force production by the team.     

Bidirectional Transport by Molecular Motors: Enhanced Processivity and Response to External Forces

Intracellular transport along cytoskeletal filaments is often mediated by two teams of molecular motors that pull on the same cargo and move in opposite directions along the filaments. We have recently shown theoretically that this bidirectional transport can be understood as a stochastic tug-of-war between the two motor teams. Here, we further develop our theory to investigate the experimentally accessible dynamic behavior of cargos transported by strong motors such as kinesin-1 or cytoplasmic dynein. By studying the run and binding times of such a cargo, we show that the properties of biological motors, such as the large ratio of stall/detachment force and the small ratio of superstall backward/forward velocity, are favorable for bidirectional cargo transport, leading to fast motion and enhanced diffusion. In addition, cargo processivity is shown to be strongly enhanced by transport via several molecular motors even if these motors are engaged in a tug-of-war. Finally, we study the motility of a bidirectional cargo under force. Frictional forces arising, e.g., from the viscous cytoplasm, lead to peaks in the velocity distribution, while external forces as exerted, e.g., by an optical trap, lead to hysteresis effects. Our results, in particular our explicit expressions for the cargo binding time and the distance of the peaks in the velocity relation under friction, are directly accessible to in vitro as well as in vivo experiments.     

Measuring Molecular Motor Forces In Vivo: Implications for Tug-of-War Models of Bidirectional Transport

Molecular motor proteins use the energy released from ATP hydrolysis to generate force and haul cargoes along cytoskeletal filaments. Thus, measuring the force motors generate amounts to directly probing their function. We report on optical trapping methodology capable of making precise in vivo stall-force measurements of individual cargoes hauled by molecular motors in their native environment. Despite routine measurement of motor forces in vitro, performing and calibrating such measurements in vivo has been challenging. We describe the methodology recently developed to overcome these difficulties, and used to measure stall forces of both kinesin-1 and cytoplasmic dynein-driven lipid droplets in Drosophila embryos. Critically, by measuring the cargo dynamics in the optical trap, we find that there is memory: it is more likely for a cargo to resume motion in the same direction—rather than reverse direction—after the motors transporting it detach from the microtubule under the force of the optical trap. This suggests that only motors of one polarity are active on the cargo at any instant in time and is not consistent with the tug-of-war models of bidirectional transport where both polarity motors can bind the microtubules at all times. We further use the optical trap to measure in vivo the detachment rates from microtubules of kinesin-1 and dynein-driven lipid droplets. Unlike what is commonly assumed, we find that dynein’s but not kinesin’s detachment time in vivo increases with opposing load. This suggests that dynein’s interaction with microtubules behaves like a catch bond.     

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 .     

Engineered Tug‐of‐War Between Kinesin and Dynein Controls Direction of Microtubule Based Transport In Vivo

Bidirectional transport of membrane organelles along microtubules (MTs) is driven by plus‐end directed kinesins and minus‐end directed dynein bound to the same cargo. Activities of opposing MT motors produce bidirectional movement of membrane organelles and cytoplasmic particles along MT transport tracks. Directionality of MT‐based transport might be controlled by a protein complex that determines which motor type is active at any given moment of time, or determined by the outcome of a tug‐of‐war between MT motors dragging cargo organelles in opposite directions. However, evidence in support of each mechanisms of regulation is based mostly on the results of theoretical analyses or indirect experimental data. Here, we test whether the direction of movement of membrane organelles in vivo can be controlled by the tug‐of‐war between opposing MT motors alone, by attaching a large number of kinesin‐1 motors to organelles transported by dynein to minus‐ends of MTs. We find that recruitment of kinesin significantly reduces the length and velocity of minus‐end‐directed dynein‐dependent MT runs, leading to a reversal of the overall direction of dynein‐driven organelles in vivo. Therefore, in the absence of external regulators tug‐of‐war between opposing MT motors alone is sufficient to determine the directionality of MT transport in vivo.      

Motor Domain Phosphorylation Modulates Kinesin-1 Transport

Disruptions in microtubule motor transport are associated with a variety of neurodegenerative diseases. Post-translational modification of the cargo-binding domain of the light and heavy chains of kinesin has been shown to regulate transport, but less is known about how modifications of the motor domain affect transport. Here we report on the effects of phosphorylation of a mammalian kinesin motor domain by the kinase JNK3 at a conserved serine residue (Ser-175 in the B isoform and Ser-176 in the A and C isoforms). Phosphorylation of this residue has been implicated in Huntington disease, but the mechanism by which Ser-175 phosphorylation affects transport is unclear. The ATPase, microtubule-binding affinity, and processivity are unchanged between a phosphomimetic S175D and a nonphosphorylatable S175A construct. However, we find that application of force differentiates between the two. Placement of negative charge at Ser-175, through phosphorylation or mutation, leads to a lower stall force and decreased velocity under a load of 1 piconewton or greater. Sedimentation velocity experiments also show that addition of a negative charge at Ser-175 favors the autoinhibited conformation of kinesin. These observations imply that when cargo is transported by both dynein and phosphorylated kinesin, a common occurrence in the cell, there may be a bias that favors motion toward the minus-end of microtubules. Such bias could be used to tune transport in healthy cells when properly regulated but contribute to a disease state when misregulated.     

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.     

Motor Dynamics Underlying Cargo Transport by Pairs of Kinesin-1 and Kinesin-3 Motors

Intracellular cargo transport by kinesin family motor proteins is crucial for many cellular processes, particularly vesicle transport in axons and dendrites. In a number of cases, the transport of specific cargo is carried out by two classes of kinesins that move at different speeds and thus compete during transport. Despite advances in single-molecule characterization and modeling approaches, many questions remain regarding the effect of intermotor tension on motor attachment/reattachment rates during cooperative multimotor transport. To understand the motor dynamics underlying multimotor transport, we analyzed the complexes of kinesin-1 and kinesin-3 motors attached through protein scaffolds moving on immobilized microtubules in vitro. To interpret the observed behavior, simulations were carried out using a model that incorporated motor stepping, attachment/detachment rates, and intermotor force generation. In single-molecule experiments, isolated kinesin-3 motors moved twofold faster and had threefold higher landing rates than kinesin-1. When the positively charged loop 12 of kinesin-3 was swapped with that of kinesin-1, the landing rates reversed, indicating that this “K-loop” is a key determinant of the motor reattachment rate. In contrast, swapping loop 12 had negligible effects on motor velocities. Two-motor complexes containing one kinesin-1 and one kinesin-3 moved at different speeds depending on the identity of their loop 12, indicating the importance of the motor reattachment rate on the cotransport speed. Simulations of these loop-swapped motors using experimentally derived motor parameters were able to reproduce the experimental results and identify best fit parameters for the motor reattachment rates for this geometry. Simulation results also supported previous work, suggesting that kinesin-3 microtubule detachment is very sensitive to load. Overall, the simulations demonstrate that the transport behavior of cargo carried by pairs of kinesin-1 and -3 motors are determined by three properties that differ between these two families: the unloaded velocity, the load dependence of detachment, and the motor reattachment rate.     

Microtubule C‐Terminal Tails Can Change Characteristics of Motor Force Production

Control of intracellular transport is poorly understood, and functional ramifications of tubulin isoform differences between cell types are mostly unexplored. Motors' force production and detachment kinetics are critical for their group function, but how microtubule (MT) details affect these properties – if at all – is unknown. We investigated these questions using both a vesicular transport human kinesin, kinesin‐1, and also a mitotic kinesin likely optimized for group function, kinesin‐5, moving along either bovine brain or MCF7(breast cancer) MTs. We found that kinesin‐1 functioned similarly on the two sets of MTs – in particular, its mean force production was approximately the same, though due to its previously reported decreased processivity, the mean duration of kinesin‐1 force production was slightly decreased on MCF7 MTs. In contrast, kinesin‐5's function changed dramatically on MCF7 MTs: its average detachment force was reduced and its force–velocity curve was different. In spite of the reduced detachment force, the force–velocity alteration surprisingly improved high‐load group function for kinesin‐5 on the cancer‐cell MTs, potentially contributing to functions such as spindle‐mediated chromosome separation. Significant differences were previously reported for C‐terminal tubulin tails in MCF7 versus bovine brain tubulin. Consistent with this difference being functionally important, elimination of the tails made transport along the two sets of MTs similar.     

An RNA‐binding atypical tropomyosin recruits kinesin‐1 dynamically to oskar mRNPs

Localization and local translation of oskar mRNA at the posterior pole of the Drosophila oocyte directs abdominal patterning and germline formation in the embryo. The process requires recruitment and precise regulation of motor proteins to form transport‐competent mRNPs. We show that the posterior‐targeting kinesin‐1 is loaded upon nuclear export of oskar mRNPs, prior to their dynein‐dependent transport from the nurse cells into the oocyte. We demonstrate that kinesin‐1 recruitment requires the DmTropomyosin1‐I/C isoform, an atypical RNA‐binding tropomyosin that binds directly to dimerizing oskar 3′UTRs. Finally, we show that a small but dynamically changing subset of oskar mRNPs gets loaded with inactive kinesin‐1 and that the motor is activated during mid‐oogenesis by the functionalized spliced oskar RNA localization element. This inefficient, dynamic recruitment of Khc decoupled from cargo‐dependent motor activation constitutes an optimized, coordinated mechanism of mRNP transport, by minimizing interference with other cargo‐transport processes and between the cargo‐associated dynein and kinesin‐1.     

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.      

Lipase-catalyzed synthesis of fatty acid sugar ester using extremely supersaturated sugar solution in ionic liquids

Artificial nanotransport systems inspired by intracellular transport processes have been investigated for over a decade using the motor protein kinesin and microtubules. However, only unidirectional cargo transport has been achieved for the purpose of nanotransport in a microfluidic system. Here, we demonstrate bidirectional nanotransport by integrating kinesin and dynein motor proteins. Our molecular system allows microtubule orientation of either polarity in a microfluidic channel to construct a transport track. Each motor protein acts as a nanoactuators that transports microspheres in opposite directions determined by the polarity of the oriented microtubules: kinesin-coated microspheres move toward the plus end of microtubules, whereas dynein-coated microspheres move toward the minus end. We demonstrate both unidirectional and bidirectional transport using kinesin- and dynein-coated microspheres on microtubules oriented and glutaraldehyde-immobilized in a microfluidic channel. Tracking and statistical analysis of microsphere movement demonstrate that 87-98% of microspheres move in the designated direction at a mean velocity of 0.22-0.28 microm/s for kinesin-coated microspheres and 0.34-0.39 microm/s for dynein-coated microspheres. This bidirectional nanotransport goes beyond conventional unidirectional transport to achieve more complex artificial nanotransport in vitro.     

Mechanical coupling of microtubule-dependent motor teams during peroxisome transport in Drosophila S2 cells

BackgroundIntracellular transport requires molecular motors that step along cytoskeletal filaments actively dragging cargoes through the crowded cytoplasm. Here, we explore the interplay of the opposed polarity motors kinesin-1 and cytoplasmic dynein during peroxisome transport along microtubules in Drosophila S2 cells.MethodsWe used single particle tracking with nanometer accuracy and millisecond time resolution to extract quantitative information on the bidirectional motion of organelles. The transport performance was studied in cells expressing a slow chimeric plus-end directed motor or the kinesin heavy chain. We also analyzed the influence of peroxisomes membrane fluidity in methyl-β-ciclodextrin treated cells. The experimental data was also confronted with numerical simulations of two well-established tug of war scenarios.Results and conclusionsThe velocity distributions of retrograde and anterograde peroxisomes showed a multimodal pattern suggesting that multiple motor teams drive transport in either direction. The chimeric motors interfered with the performance of anterograde transport and also reduced the speed of the slowest retrograde team. In addition, increasing the fluidity of peroxisomes membrane decreased the speed of the slowest anterograde and retrograde teams.General significanceOur results support the existence of a crosstalk between opposed-polarity motor teams. Moreover, the slowest teams seem to mechanically communicate with each other through the membrane to trigger transport.     

Productive Cooperation among Processive Motors Depends Inversely on Their Mechanochemical Efficiency

Subcellular cargos are often transported by teams of processive molecular motors, which raises questions regarding the role of motor cooperation in intracellular transport. Although our ability to characterize the transport behaviors of multiple-motor systems has improved substantially, many aspects of multiple-motor dynamics are poorly understood. This work describes a transition rate model that predicts the load-dependent transport behaviors of multiple-motor complexes from detailed measurements of a single motor's elastic and mechanochemical properties. Transition rates are parameterized via analyses of single-motor stepping behaviors, load-rate-dependent motor-filament detachment kinetics, and strain-induced stiffening of motor-cargo linkages. The model reproduces key signatures found in optical trapping studies of structurally defined complexes composed of two kinesin motors, and predicts that multiple kinesins generally have difficulties in cooperating together. Although such behavior is influenced by the spatiotemporal dependence of the applied load, it appears to be directly linked to the efficiency of kinesin's stepping mechanism, and other types of less efficient and weaker processive motors are predicted to cooperate more productively. Thus, the mechanochemical efficiencies of different motor types may determine how effectively they cooperate together, and hence how motor copy number contributes to the regulation of cargo motion.     

Regulation of ER–Golgi Intermediate Compartment Tubulation and Mobility by COPI Coats,Motor Proteins and Microtubules

Little is known about the formation and regulation of endoplasmic reticulum (ER)–Golgi transport intermediates, although previous studies suggest that cargo is the main regulator of their morphology. In this study, we analyze the role of coat protein I (COPI) and cytoskeleton in the formation of tubular ER–Golgi intermediate compartment (ERGIC) and also show that partial COPI detachment by means of low temperature (15°C) or brefeldin A induces the formation of transient tubular ERGIC elements. Most of them moved from the cell periphery to the perinuclear area and were 2.5× slower than vesicles. Time‐lapse analysis of living cells demonstrates that the ERGIC elements are able to shift very fast from tubular to vesicular forms and vice versa, suggesting that the amount of cargo is not the determining factor for ERGIC morphology. Both the partial microtubule depolymerization and the inhibition of uncoating of the membranes result in the formation of long tubules that grow from round ERGICs and form at complex network. Interestingly, both COPI detachment and microtubule depolymerization induce a redistribution of kinesin from peripheral ERGIC elements to the Golgi area, while dynein distribution is not affected. However, both kinesin and dynein downregulation by RNA interference induced ERGIC tubulation. The tubules induced by kinesin depletion were static, whereas those resulting from dynein depletion were highly mobile. Our results strongly suggest that the interaction of motor proteins with COPI‐coated membranes and microtubules is a key regulator of ERGIC morphology and mobility.     

Models of motor-assisted transport of intracellular particles

One-dimensional models are presented for the macroscopic intracellular transport of vesicles and organelles by molecular motors on a network of aligned intracellular filaments. A motor-coated vesicle or organelle is described as a diffusing particle binding intermittently to filaments, when it is transported at the motor velocity. Two models are treated in detail: 1) a unidirectional model, where only one kind of motor is operative and all filaments have the same polarity; and 2) a bidirectional model, in which filaments of both polarities exist (for example, a randomly polarized actin network for myosin motors) and/or particles have plus-end and minus-end motors operating on unipolar filaments (kinesin and dynein on microtubules). The unidirectional model provides net particle transport in the absence of a concentration gradient. A symmetric bidirectional model, with equal mixtures of filament polarities or plus-end and minus-end motors of the same characteristics, provides rapid transport down a concentration gradient and enhanced dispersion of particles from a point source by motor-assisted diffusion. Both models are studied in detail as a function of the diffusion constant and motor velocity of bound particles, and their rates of binding to and detachment from filaments. These models can form the basis of more realistic models for particle transport in axons, melanophores, and the dendritic arms of melanocytes, in which networks of actin filaments and microtubules coexist and motors for both types of filament are implicated.     

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.     

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.     

Assessing the Impact of Electrostatic Drag on Processive Molecular Motor Transport

The bidirectional movement of intracellular cargo is usually described as a tug-of-war among opposite-directed families of molecular motors. While tug-of-war models have enjoyed some success, recent evidence suggests underlying motor interactions are more complex than previously understood. For example, these tug-of-war models fail to predict the counterintuitive phenomenon that inhibiting one family of motors can decrease the functionality of opposite-directed transport. In this paper, we use a stochastic differential equations modeling framework to explore one proposed physical mechanism, called microtubule tethering, that could play a role in this “co-dependence” among antagonistic motors. This hypothesis includes the possibility of a trade-off: weakly bound trailing molecular motors can serve as tethers for cargoes and processing motors, thereby enhancing motor–cargo run lengths along microtubules; however, this introduces a cost of processing at a lower mean velocity. By computing the small- and large-time mean-squared displacement of our theoretical model and comparing our results to experimental observations of dynein and its “helper protein” dynactin, we find some supporting evidence for microtubule tethering interactions. We extrapolate these findings to predict how dynein–dynactin might interact with the opposite-directed kinesin motors and introduce a criterion for when the trade-off is beneficial in simple systems.     

Dynactin polices two-way organelle traffic

How is the bidirectional motion of organelles controlled? In this issue, Deacon et al. (2003) reveal the unexpected finding that dynactin (previously known to control dynein-based motility) binds to kinesin II and regulates anterograde movement of Xenopus melanosomes. This result suggests that dynactin may be a key player in coordinating vesicle traffic in this system.The movement of intracellular cargo is essential for cell survival. In animal cells, membranous organelles are propelled through the cytoplasm by microtubule-based motor proteins. Anterograde movement toward microtubule plus ends at the cell periphery is driven by motor proteins of the kinesin superfamily, whereas retrograde movement toward minus ends at the cell center is largely accomplished by cytoplasmic dynein. In most cells, organelles do not travel smoothly in one direction but frequently switch between plus and minus end–directed travel. The net time spent traveling in the plus versus the minus end direction determines the steady-state distribution of an organelle population within a cell. A long-standing question for those studying organelle transport is how this bidirectional trafficking is coordinated. Is the binding of kinesin and dynein to vesicles mutually exclusive, or are these motors bound at the same time but with their activities coordinately regulated? What molecule(s) might be responsible for linking kinesin and dynein activities? In this issue, Vladimir Gelfand''s group (Deacon et al., 2003) addresses these questions by studying the motor proteins kinesin II and cytoplasmic dynein that move pigment granules in Xenopus melanophore cells. Their results are surprising; the dynactin complex, previously known to bind to cytoplasmic dynein and anchor it to organelles, also interacts with kinesin II and is necessary for plus end–directed motion. The ability of dynactin to physically interact with these two opposite polarity motors suggests that it may be the long sought-after molecular switch that coordinates bidirectional movement in this system.Previous studies hinted that the actions of dynein and kinesin may be controlled via dynactin. Dynactin is a large, multimeric protein complex. Its p150^Glued subunit has binding sites for both microtubules and the intermediate chain of dynein and is thought to be responsible for the association of dynein with many of its cargo organelles (Karki and Holzbaur, 1995; Vaughan and Vallee, 1995; Waterman-Storer et al., 1995). Curiously, the treatment of extruded squid axoplasm with antibodies against p150^Glued inhibited both the anterograde and retrograde movement of organelles along microtubules (Waterman-Storer et al., 1997). These antibodies were known to inhibit the interaction of dynactin with dynein, but their effect on anterograde movement was more difficult to explain. However, genetic studies yielded similar results. Martin et al. (1999) found that mutations in either p150^Glued, the cytoplasmic dynein heavy chain, or kinesin I inhibited both retrograde and anterograde fast axonal transport in Drosophila larvae. This phenotype potentially could be explained by stalled retrograde vesicles sterically blocking the movement of anterograde cargo, but the authors also suggested the possibility of a physical linkage between kinesin, dynein, and dynactin. This theory was further tested by tracking the movement of lipid droplets in Drosophila embryos (Gross et al., 2002b). A mild defect in the dynein heavy chain impaired several aspects of minus end–directed transport of lipid droplets: run lengths, velocities, and the opposing optical trap force required to halt droplet movement were all decreased. Surprisingly, this mutation produced similar effects on droplets moving toward the microtubule plus ends. Embryos expressing a mutant p150^Glued protein that partially impaired dynactin function also exhibited impaired movement in both the plus and minus end directions. Collectively, these results suggested that dynactin might be involved in coordinating the bidirectional movement of organelles. However, these studies did not provide a molecular explanation of how this mechanism might work.To study the mechanism of coordination of bidirectional vesicle movement, Deacon et al. (2003) used Xenopus melanophores due to the unique ability to experimentally control the directional movement of their pigmented melanosomes (Daniolos et al., 1990). Upon treatment of melanophores with melatonin, the cAMP concentration in the cytoplasm drops and the melanosomes move with a net minus end–directed bias and aggregate toward the cell center. Treatment with melanocyte-stimulating hormone (MSH)^* restores cAMP levels, and the melanosomes exhibit a plus end–directed bias and disperse throughout the cell. Aggregation is accomplished by cytoplasmic dynein (Nilsson and Wallin, 1997), whereas dispersion requires the combined actions of kinesin II and the actin-based motor myosin V (Rogers and Gelfand, 1998; Tuma et al., 1998; Gross et al., 2002a). Kinesin II is a heterotrimeric protein consisting of two motor subunits and a third nonmotor subunit known as kinesin-associated protein (KAP) (Cole et al., 1992). KAP is thought to be involved in binding kinesin II to its cargo, although the mechanism for this interaction is not known.The role of dynactin in melanosome transport was investigated by disrupting dynactin function via the overexpression of dynamitin (Echeverri et al., 1996), a crucial subunit that holds the dynactin complex together. To ensure that all observed melanosome movement occurred on the microtubule cytoskeleton, actin filaments were depolymerized with latrunculin B. Here, the authors report that melanosome movement to both the plus and minus ends of microtubules was inhibited by dynamitin overexpression, suggesting a role for dynactin in coordinating bidirectional movement. They considered whether this result might be explained if both kinesin II and dynein bound to dynactin and thereby docked onto membranes. To test this idea, kinesin II was immunoprecipitated with a series of antibodies, and the authors found that dynactin was pulled down along with this kinesin motor in all cases. The reverse experiment of immunoprecipitating with p150^Glued antibodies also brought down kinesin II. Blot overlays of purified melanosomes with p150^Glued detected an interaction with a 115-kD protein, the expected size of Xenopus KAP. Subsequent overlay and affinity pull-down experiments with purified proteins confirmed the direct binding of p150^Glued to KAP. By constructing a series of GST fusion proteins, Deacon et al. (2003) were able to map the site of this interaction to residues 600–811 of p150^Glued and the COOH-terminal domain of KAP. Interestingly, this region of p150^Glued also interacts with the dynein intermediate chain, which raised the question of whether kinesin II and dynein might compete for binding to dynactin. Using a blot overlay competition assay, the authors found that the COOH-terminal KAP domain blocked the binding of p150^Glued to the dynein intermediate chain, whereas the NH₂-terminal KAP domain, used as a control, did not. This result confirms that the two motors cannot bind dynactin simultaneously.If these biochemical results are relevant to melanosome movement, then overexpression of KAP should inhibit both anterograde and retrograde traffic. Indeed, overexpession of Xenopus KAP or just its COOH-terminal fragment inhibited bidirectional melanosome movement. As a control, NH₂-terminal KAP had no effect on retrograde movement and only a small effect on anterograde movement, perhaps due to interactions with the kinesin II motor subunits. Together, the results of Deacon et al. (2003) demonstrate that kinesin II, via its KAP subunit, binds to the p150^Glued subunit of dynactin and that this interaction is important for kinesin II–mediated movement of melanosomes.Although the authors identify the p150^Glued subunit of dynactin as a key player in coordinating the bidirectional movement of melanosomes, the mechanism is still unclear. Their biochemical results showing competitive binding to dynactin suggest that binding of kinesin II and dynein to melanosomes may be mutually exclusive events; however, previous work has shown that this is not the case. In a recent paper from the same authors (Gross et al., 2002a), as well as an earlier study from Reese and Haimo (Reese and Haimo, 2000), the relative amounts of kinesin II and dynein bound to purified melanosomes did not change when cells were treated with melatonin to stimulate aggregation or with MSH to stimulate dispersion. Thus, it is possible that proteins other than dynactin might bind kinesin II and dynein to melanosomes. This question also could be addressed by determining if motor binding to melanophores is diminished in cells overexpressing KAP or dynamitin. Unfortunately, Deacon et al. (2003) were not able to answer this question by biochemical isolation of melanosomes and motor quantitation because transfected cells were only a small percentage of the total population. Another possible model is that dynactin is not needed for recruiting kinesin II and dynein to melanosomes but is somehow involved in regulating the activation or organization of motors already bound to the membrane.Future studies will no doubt explore whether dynactin is involved in bidirectional transport in systems other than melanophores. In intraflagellar transport, kinesin II and cytoplasmic dynein 2 are involved in moving nonmembranous particles between the cell body and the tip of the flagella or cilia (Rosenbaum and Witman, 2002). It will be interesting to determine whether dynactin plays a role in this type of cargo transport. In neurons, kinesin I is responsible for moving organelles from the cell body to the axon terminal. As discussed above, Martin et al. (1999) found that mutations in either kinesin I heavy chain, dynein, or p150^Glued all produced the same phenotype in Drosophila larvae neurons, suggesting that dynactin may play a role in coordinating bidirectional movement in this system as well. Immunoprecipitation of neuronal p150^Glued, however, brought down only dynein but not kinesin I. This finding may result from the fact that kinesin I, which possesses a light chain unrelated to the KAP subunit, could be linked indirectly to dynactin by another protein. Thus, this study by Deacon et al. (2003) has opened up a new area of exploration and dynactin will undoubtedly receive closer scrutiny from kinesin researchers in the future.