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The microtubule-associated protein (MAP) Tau is an intrinsically disordered protein (IDP) primarily expressed in axons, where it functions to regulate microtubule dynamics, modulate motor protein motility, and participate in signaling cascades. Tau misregulation and point mutations are linked to neurodegenerative diseases, including Progressive Supranuclear Palsy (PSP), Pick’s Disease and Alzheimer’s disease. Many disease-associated mutations in Tau occur in the C-terminal microtubule-binding domain of the protein. Effects of C-terminal mutations in Tau have led to the widely accepted disease-state theory that missense mutations in Tau reduce microtubule-binding affinity or increase Tau propensity to aggregate. Here, we investigate the effect of an N-terminal disease-associated mutation in Tau, R5L, on Tau-microtubule interactions using an in vitro reconstituted system. Contrary to the canonical disease-state theory, we determine the R5L mutation does not reduce Tau affinity for the microtubule using Total Internal Reflection Fluorescence (TIRF) Microscopy. Rather, the R5L mutation decreases the ability of Tau to form larger order complexes, or Tau patches, at high concentrations of Tau. Using Nuclear Magnetic Resonance (NMR), we show that the R5L mutation results in a local structural change that reduces interactions of the projection domain in the presence of microtubules. Altogether, these results challenge both the current paradigm of how mutations in Tau lead to disease and the role of the projection domain in modulating Tau behavior on the microtubule surface.Significance StatementThe microtubule-associated protein Tau is strongly linked to a number of neurological diseases. Disease onset is typically associated with weakened interaction with the microtubule, but this widely accepted model is based on hyperphosphorylation or mutations within the C-terminal microtubule-binding domain of Tau. Here, we find an N-terminal disease-associated mutation in Tau, R5L, does not reduce Tau affinity for microtubules, but instead modifies the N-terminal structure, altering Tau’s behavior and ability to condense on the microtubule surface. Our findings challenge the current paradigms of both how mutations in Tau lead to disease and the functional role of the N-terminal region of Tau.

The conserved kinesin-5 bipolar tetrameric motors slide apart microtubules during mitotic spindle assembly and elongation. Kinesin-5 bipolar organization originates from its conserved tetrameric helical minifilament, which position the C-terminal tail domains of two subunits near the N-terminal motor domains of two anti-parallel subunits (Scholey et al, 2014). This unique tetrameric structure enables kinesin-5 to simultaneously engage two microtubules and transmit forces between them, and for multiple kinesin-5 motors to organize via tail to motor interactions during microtubule sliding (Bodrug et al, 2020). Here, we show how these two structural adaptations, the kinesin-5 tail-motor domain interactions and the length of the tetrameric minifilament, determine critical aspects of kinesin-5 motility and sliding mechanisms. An x-ray structure of the 34-nm kinesin-5 minifilament reveals how the dual dimeric N-terminal coiled-coils emerge from the tetrameric central bundle. Using this structure, we generated active bipolar mini-tetrameric motors from Drosophila and human orthologs, which are half the length of native kinesin-5. Using single-molecule motility assays, we show that kinesin-5 tail domains promote mini-tetramers static pauses that punctuate processive motility. During such pauses, kinesin-5 mini-tetramers form multi-motor clusters mediated via tail to motor domain cross-interactions. These clusters undergo slow and highly processive motility and accumulate at microtubule plus-ends. In contrast to native kinesin-5, mini-tetramers require tail domains to initiate microtubule crosslinking. Although mini-tetramers are highly strained in initially aligning microtubules, they slide microtubules more efficiently than native kinesin-5, due to their decreased minifilament flexibility. Our studies reveal that the conserved kinesin-5 motor-tail mediated clustering and the length of the tetrameric minifilament are key features for sliding motility and are critical in organizing microtubules during mitotic spindle assembly and elongation.

The cell relies on an intricate system of molecular highways and motors to transport proteins, organelles and other vesicular cargoes to their proper locations. Microtubules, long filaments that form a network throughout the cell, act as highways. The motor proteins kinesin and dynein associate with cargoes and transport them along microtubules. Rather than simply acting as passive tracks, microtubules contain signals that regulate kinesin and dynein to target cargoes to specific locations in the cell. These signals include the organization of the microtubule network, chemical modifications that alter the microtubule surface properties and mechanics, and microtubuleassociated proteins that modulate the motility of motor proteins and microtubule polymerization.

Microtubule-associated proteins (MAPs) regulate microtubule polymerization, dynamics, and organization. In addition, MAPs alter the motility of kinesin and dynein to control trafficking along microtubules. MAP7 (ensconsin, E-MAP-115) is a ubiquitous MAP that organizes the microtubule cytoskeleton in mitosis and neuronal branching. MAP7 also recruits kinesin-1 to microtubules. To understand how the activation of kinesin-1 by MAP7 regulates the motility of organelles transported by ensembles of kinesin and dynein, we isolated organelles and reconstituted their motility in vitro. In the absence of MAP7, isolated phagosomes exhibit approximately equal fractions of plus- and minus-end–directed motility along microtubules. MAP7 causes a pronounced shift in motility; phagosomes move toward the plus-end ∼80% of the time, and kinesin teams generate more force. To dissect MAP7-mediated regulation of kinesin-driven transport, we examined its effects on the motility and force generation of single and teams of full-length kinesin-1 motors. We find that MAP7 does not alter the force exerted by a single kinesin-1 motor, but instead increases its binding rate to the microtubule. For ensembles of kinesin, a greater number of kinesin motors are simultaneously engaged and generating force to preferentially target organelles toward the microtubule plus-end.

Microtubule-associated proteins (MAPs) play well-characterized roles in regulating microtubule polymerization, dynamics, and organization. In addition, MAPs control trans-port along microtubules by regulating the motility of kinesin and dynein. MAP7 (ensconsin, E-MAP-115) is a ubiquitous MAP that organizes the microtubule cytoskeleton in mitosis and neuronal branching. MAP7 also promotes the interaction of kinesin-1 with microtubules. We expressed and purified full-length kinesin-1 and MAP7 in Sf9 cells. In single-molecule motiity assays, MAP7 recruits kinesin-1 to microtubules, increasing the frequency of both diffusive and processive runs. Optical trapping assays on beads transported by single and teams of kinesin-1 motors indicate that MAP7 increases the relative binding rate of kinesin-1 and the number of motors simultaneously engaged in ensembles. To examine the role of MAP7 in regulating bidirectional transport, we isolated late phagosomes along with their native set of kinesin-1, kinesin-2, and dynein motors. Bidirectional cargoes exhibit a clear shift towards plus-end directed motility on MAP7-decorated microtubules due to increased forces exerted by kinesin teams. Collectively, our results indicate that MAP7 enhances kinesin-1 recruitment to microtubules and targets organelle transport to the plus end.

The motor proteins kinesin and dynein transport organelles, mRNA, proteins, and signaling molecules along the microtubule cytoskeleton. In addition to serving as tracks for transport, the microtubule cytoskeleton directs intracellular trafficking by regulating the activity of motor proteins through the organization of the filament network, microtubule-associated proteins, and tubulin posttranslational modifications. However, it is not well understood how these factors influence motor motility, and in vitro assays and live cell observations often produce disparate results. To systematically examine the factors that contribute to cytoskeleton-based regulation of motor protein motility, we extracted intact microtubule networks from cells and tracked the motility of single fluorescently labeled motor proteins on these cytoskeletons. We find that tubulin acetylation alone does not directly affect kinesin-1 motility. However, acetylated microtubules are predominantly bundled, and bundling enhances kinesin run lengths and provides a greater number of available kinesin binding sites. The neuronal MAP tau is also not sensitive to tubulin acetylation, but enriches preferentially on highly curved regions of microtubules where it strongly inhibits kinesin motility. Taken together, these results suggest that the organization of the microtubule network is a key contributor to the regulation of motor-based transport.

Abnormal axonal transport is associated with neuronal disease. We identified a role for DHC-1, the C. elegans dynein heavy chain, in maintaining neuronal cargo distribution. Surprisingly, this does not involve dynein's role as a retrograde motor in cargo transport, hinging instead on its ability to inhibit microtubule (MT) dynamics. Neuronal MTs are highly static, yet the mechanisms and functional significance of this property are not well understood. In disease-mimicking dhc-1 alleles, excessive MT growth and collapse occur at the dendrite tip, resulting in the formation of aberrant MT loops. These unstable MTs act as cargo traps, leading to ectopic accumulations of cargo and reduced availability of cargo at normal locations. Our data suggest that an anchored dynein pool interacts with plus-end-out MTs to stabilize MTs and allow efficient retrograde transport. These results identify functional significance for neuronal MT stability and suggest a mechanism for cellular dysfunction in dynein-linked disease., Microtubule dynamics are essential for axonal transport. In C. elegans, the authors show that dynein heavy chain regulates the spatial distribution of dendritic microtubules which ensures correct transport progression.

Optical tweezers have been instrumental in uncovering the mechanisms motor proteins use to generate and react to force. While optical traps have primarily been applied to purified, in vitro systems, emerging methods enable measurements in living cells where the actively fluctuating, viscoelastic environment and varying refractive index complicate calibration of the instrument. Here, we describe techniques to calibrate optical traps in living cells using the forced response to sinusoidal oscillations and spontaneous fluctuations, and to measure the forces exerted by endogenous ensembles of kinesin and dynein motor proteins as they transport cargoes in the cell.

Intracellular trafficking of organelles often involves cytoskeletal track switching. Organelles such as melanosomes are transported by multiple motors including kinesin-2, dynein, and myosin-V, which drive switching between microtubules and actin filaments during dispersion and aggregation. Here, we used optical trapping to determine the unitary and ensemble forces of kinesin-2, and to reconstitute cargo switching at cytoskeletal intersections in a minimal system with kinesin-2 and myosin-V motors bound to beads. Single kinesin-2 motors exerted forces up to ∼5 pN, similar to kinesin-1. However, kinesin-2 motors were more likely to detach at submaximal forces, and the duration of force maintenance was short as compared to kinesin-1. In multimotor assays, force increased with kinesin-2 density but was not affected by the presence of myosin-V. In crossed filament assays, switching frequencies of motor-bound beads were dependent on the starting track. At equal average forces, beads tended to switch from microtubules onto overlying actin filaments consistent with the relatively faster detachment of kinesin-2 at near-maximal forces. Thus, in addition to relative force, switching probability at filament intersections is determined by the dynamics of motor-filament interaction, such as the quick detachment of kinesin-2 under load. This may enable fine-tuning of filament switching in the cell.

Recent experiments using single-molecule techniques have characterized the mechanical properties of single kinesin molecules in vitro at a range of loads and ATP concentrations. These experiments have shown that kinesin moves processively along microtubules by alternately advancing each of its motor domains in a hand-over-hand fashion, using Brownian motion and the energy from ATP hydrolysis. We have extended the theoretical analysis of kinesin through a mechanistic model that is capable of describing transient and steady-state behavior. Transient dynamics are needed to describe the effect of external perturbations (e.g. interactions with other kinesin molecules). Quantitative metrics are tailored to characterize the synchronization of nonlinear, nonsmooth systems such as kinesin. These metrics are employed to analyze the simulation results and to quantify the effect of the cargo linker stiffness, the load, and the difference in intrinsic velocity on the synchronization of two coupled motor proteins. Herein, the mechanistic model and the new analysis techniques are demonstrated for the case of two coupled kinesin motors.

The motor proteins kinesin and dynein transport organelles, mRNA, proteins, and signaling molecules along the microtubule cytoskeleton. In addition to serving as tracks for transport, the microtubule cytoskeleton directs intracellular trafficking by regulating the activity of motor proteins through the organization of the filament network, microtubule-associated proteins (MAPs), and tubulin post-translational modifications (PTMs). However, it is not well understood how these factors influence motor motility, as in vitro assays and live cell observations often produce disparate results, likely due in part to the interdependencies between MAPs, PTMs, and cytoskeletal organization. For example, the MAP tau strongly inhibits kinesin-1 motility in purified systems, yet kinesin-1 drives long-range transport in neuronal axons where microtubules are heavily decorated with tau. While tau directly affects motility by acting as an obstacle to transport, tau also influences motor activity by promoting tubulin acetylation and acting to stabilize and bundle microtubules. To systematically examine the factors that contribute to cytoskeleton-based regulation of motor protein motility in a simple in vitro system, we extracted intact, native microtubule networks from cells and tracked the motility of single fluorescently-labeled motor proteins on these cytoskeletons using Total Internal Reflection Fluorescence (TIRF) microscopy. Using this system, we analyzed the effect of tubulin post-translational modifications such as acetylation on kinesin-1 track preference. Further, we examined the binding of the MAP tau on extracted microtubule networks and its influence on motor behavior. This work is supported by NSERC.

Kinesin-1 is a processive molecular motor that converts the energy from ATP hydrolysis and Brownian motion into directed movement. Single-molecule techniques have allowed the experimental characterization of single kinesins in vitro at a range of loads and ATP concentrations, and shown that each kinesin molecule moves processively along microtubules by alternately advancing each of its motor domains in a hand-over-hand fashion. Existing models of kinesin movement focus on time and space invariant loads, and hence are not well suited to describing transient dynamics. However, kinesin must undergo transient dynamics when external perturbations (e.g., interactions with other kinesin molecules) cause the load on each motor to change in time. We have developed a mechanistic model that describes, deterministically, the average motion of kinesin under time and space varying loads. The diffusion is modeled using a novel approach inspired by the classical closed form solution for the mean first-passage time. In the new approach, the potential in which the free motor domain diffuses is time varying and updated at each instant during the motion. The mechanistic model is able to predict experimental force-velocity data over a wide range of ATP concentrations (1 μM–10 mM). This mechanistic approach to modeling the mechanical behavior of the motor domains of kinesin allows rational and efficient characterization of the mechanochemical coupling, and provides predictions of kinesin with time-varying loads, which is critical for modeling coordinated transport involving several kinesin molecules.

Kinesin, dynein, and myosin transport intracellular cargoes including organelles, membrane-bound vesicles, and mRNA along the cytoskeleton. These motor proteins work collectively in teams to transport cargoes over long distances and navigate around obstacles in the cell. In addition, several types of motors often interact on the same cargo to allow bidirectional transport and switching between the actin and microtubule networks. To examine transport of native cargoes in a simplified in vitro system, techniques have been developed to isolate endogenous cargoes and reconstitute their motility. Isolated cargoes can be tracked and manipulated with high precision using total internal reflection fluorescence microscopy and optical trapping. Through use of native cargoes, we can examine vesicular transport in a minimal system while retaining endogenous motor stoichiometry and the biochemical and mechanical characteristics of both motor and cargo.

Many cellular cargoes move bidirectionally along microtubules, driven by teams of plus- and minus-end–directed motor proteins. To probe the forces exerted on cargoes during intracellular transport, we examined latex beads phagocytosed into living mammalian macrophages. These latex bead compartments (LBCs) are encased in membrane and transported along the cytoskeleton by a complement of endogenous kinesin-1, kinesin-2, and dynein motors. The size and refractive index of LBCs makes them well-suited for manipulation with an optical trap. We developed methods that provide in situ calibration of the optical trap in the complex cellular environment, taking into account any variations among cargoes and local viscoelastic properties of the cytoplasm. We found that centrally and peripherally directed forces exerted on LBCs are of similar magnitude, with maximum forces of ∼20 pN. During force events greater than 10 pN, we often observe 8-nm steps in both directions, indicating that the stepping of multiple motors is correlated. These observations suggest bidirectional transport of LBCs is driven by opposing teams of stably bound motors that operate near force balance.

SummaryMicrotubules undergo alternating periods of growth and shortening, known as dynamic instability. These dynamics allow microtubule plus ends to explore cellular space. The “search and capture” model posits that selective anchoring of microtubule plus ends at the cell cortex may contribute to cell polarization, spindle orientation, or targeted trafficking to specific cellular domains [1–3]. Whereas cytoplasmic dynein is primarily known as a minus-end-directed microtubule motor for organelle transport, cortically localized dynein has been shown to capture and tether microtubules at the cell periphery in both dividing and interphase cells [3–7]. To explore the mechanism involved, we developed a minimal in vitro system, with dynein-bound beads positioned near microtubule plus ends using an optical trap. Dynein induced a significant reduction in the lateral diffusion of microtubule ends, distinct from the effects of other microtubule-associated proteins such as kinesin-1 and EB1. In assays with dynamic microtubules, dynein delayed barrier-induced catastrophe of microtubules. This effect was ATP dependent, indicating that dynein motor activity was required. Computational modeling suggests that dynein delays catastrophe by exerting tension on individual protofilaments, leading to microtubule stabilization. Thus, dynein-mediated capture and tethering of microtubules at the cortex can lead to enhanced stability of dynamic plus ends.

SummaryThe microtubule motors kinesin and dynein function collectively to drive vesicular transport. High-resolution tracking of vesicle motility in the cell indicates that transport is often bidirectional, characterized by frequent directional changes. However, the mechanisms coordinating the collective activities of oppositely oriented motors bound to the same cargo are not well understood. To examine motor coordination, we purified neuronal transport vesicles and analyzed their motility via automated particle tracking with nanometer resolution. The motility of purified vesicles reconstituted in vitro closely models the movement of LysoTracker-positive vesicles in primary neurons, where processive bidirectional motility is interrupted with frequent directional switches, diffusional movement, and pauses. Quantitative analysis indicates that vesicles copurify with a low number of stably bound motors: one to five dynein and one to four kinesin motors. These observations compare well to predictions from a stochastic tug-of-war model, where transport is driven by the force-dependent kinetics of teams of opposing motors in the absence of external regulation. Together, these observations indicate that vesicles move robustly with a small complement of tightly bound motors and suggest an efficient regulatory scheme for bidirectional motility where small changes in the number of engaged motors manifest in large changes in the motility of cargo.

Motor proteins are biological enzymes that convert chemical energy to mechanical work in cells. Kinesin-1 is a motor protein that transports vesicles along microtubules and is widely believed to be responsible for anterograde transport of synaptic vesicles in neurons. Advances in single-molecule techniques have shown that single kinesin motors are capable of processive movement along the microtubule at a maximum velocity of approximately 1 microm/s . The velocity decreases roughly linearly in response to load until reaching stall at a load of approximately 6 pN. Several theoretical models have been proposed that describe the steady-state motion of single kinesin motors. Growing evidence suggests that kinesin functions collectively in cells, whereby several motors work in a coordinated manner to transport a vesicle. A transient description is required to describe collective dynamics, as the interactions among coupled motors induce time-varying forces on each motor. Herein a mechanistic model of kinesin is proposed that is capable of accurately describing transient and steady-state dynamics. Each domain of the protein is modeled via a mechanical potential. The mechanical potentials are related explicitly to the chemical kinetics of each motor domain. The mechanistic model was used to simulate the collective behavior of coupled kinesin motors under varying loads, cargo linker stiffnesses, and numbers of motors. To analyze the simulations of coordinated transport, several metrics were developed that are specifically tailored to characterizing the synchronization of nonlinear nonsmooth oscillators such as kinesin. The model results suggest that, in the cell, the dynamics of coupled motors under low loads are loosely correlated. When the load is increased, such as when the cargo encounters an obstacle such as another vesicle or the cytoskeleton, motors become more correlated in response to increased loads, allowing them to produce greater forces. Increasing the number of motors involved in the transport does not appreciably increase the dimension of the trajectory, implying large numbers of motors are able to function in a highly correlated manner without becoming fully synchronized.

Kinesin-1 is a motor protein essential to intracellular transport that converts the energy from ATP hydrolysis to directed movement along microtubules. Experimental and theoretical characterization of kinesin-1 has focused on single-molecule experiments. These experiments show that one motor is capable of transporting a cargo at speeds of about 1 μm/sec and maintaining contact with the microtubule for about 100 steps. In the cell, it is widely thought that several kinesin-1 motors cooperate to transport a cargo. Through a mechanistic model, we have extended the theoretical analysis of kinesin to describing transient and steady state behavior. A transient description is essential when studying collective behavior, as interaction between motors introduces time-varying loads. Herein, we interpret the kinesin motors as nonlinear, non-smooth oscillators and we employ metrics to characterize their cooperativity and to quantify their synchronization. These metrics are used to investigate the effect of the cargo linker stiffness, the load, and the difference in intrinsic velocity on the synchronization of two mechanically coupled motors.

While the myosin and kinesin superfamilies achieve diverse functions through a large number of motors, each specialized to its role, cytoplasmic dynein accomplishes varied roles in the cell through diverse interacting partners. We hypothesized that in addition to its role in bidirectional organelle transport, mammalian dynein might also play role in tethering microtubules at the cortex, analogous to the well-characterized function of dynein in S. cerevisiae. We identified a direct interaction between dynein and the neuronal cell adhesion molecule NCAM180 that recruits dynein to the cortex. To test the ability of dynein to tether microtubule plus ends, microtubules containing short biotinylated segments at the minus end were attached to the coverslip, allowing the non-biotinylated plus-ends to exhibit diffusive movements. When dynein-coated beads were brought near to the plus-end using an optical trap, the microtubule became tethered, while beads coated with BSA or kinesin-1 did not effectively restrict lateral fluctuations. In dynamic microtubule assays, plus-ends tended to associate with dynein-coated beads and continue to grow, remaining in contact with a bead for 94+/-18 s (mean+/-SEM). In contrast, encounters between growing plus-ends and BSA-coated beads often led to catastrophe, decreasing the contact time to 45+/-16 s. In live cell assays we also measured a significant increase in microtubule dwell time in the vicinity of NCAM patches (189+/-12 s, compared to 100+/-7 s in controls). Together, these data support a mechanism in which dynein is anchored to the cell cortex by NCAM180 and transiently tethers the plus-ends of microtubules. Tethered microtubules likely provide a direct connection between the cell center and sites of cell-cell interaction, either mechanically or as preferred routes for transport, creating a powerful mechanism for cells to respond to or produce stimuli in the extracellular environment.

The motor proteins dynein and kinesin function collectively to achieve long-range, bidirectional transport along microtubules. Transport in live cells, imaged using phase microscopy, exhibits distinct modes of motility, with fast, unidirectional movement in both anterograde and retrograde directions as well as saltatory, bidirectional movement. To examine transport in a simplified environment, we isolated axonal transport vesicles from transgenic mice expressing GFP-dynamitin. The fluorescent vesicles were imaged with high resolution using total internal reflection fluorescence microscopy. Using automated tracking software, we tracked all vesicles associated with polarity-marked microtubules. The purified vesicles move bidirectionally with 40% of motility in the anterograde direction and 60% in the retrograde direction, similar to the bidirectional population of vesicles observed in live cells. Inhibitory antibodies to dynein modulate the direction of transport. We compared the predictions of a simple tug-of-war model, proposed by Muller et al., [PNAS, 2008] to the observed motility in vitro, and found good agreement when 6-7 dynein motors and 1 kinesin motor are active. This prediction is in striking agreement to quantitation of motor numbers through photobleaching and quantitative western blotting, which estimate approximately 6 dynein motors per vesicle and a ratio of 6.3±0.7 dynein motors to each kinesin-1 motor. Together, the analysis of vesicle transport in live cells, purified vesicles in vitro, and mathematical modeling indicate that vesicles move robustly with a small complement of motors. The results suggest an efficient regulatory scheme where small changes in the number of active motors manifest in large changes in the motility of the cargo.

Many cellular cargos move bidirectionally along microtubules and actin, driven by teams of plus- and minus-end directed motor proteins. To probe the forces exerted on cargos in intracellular transport, we examined latex beads phagocytosed into living mammalian macrophages. These latex bead compartments (LBCs) are encased in membrane and transported along the cytoskeleton by a complement of endogenous kinesin, dynein, and myosin motors. The size and refractive index of LBCs makes them well-suited to manipulation with an optical trap. We developed novel methods to calibrate the optical trap in the complex, viscoelastic environment of the cytoplasm using spectra of thermal motions and forced sinusoidal positioning of the trap laser and the microscope stage. This method provides an accurate, in situ characterization of the fluctuating motions and forces on LBCs inside living cells without reliance on calibrations performed outside of the cell or on different beads, as in previous studies. Correlation analysis of quadrant photodiode signals is used to minimize spurious signals from scattering particles out of the LBC’s plane. For both LBCs in live cells and purified LBCs in vitro, we find that centrally and peripherally directed forces exerted on LBCs in live cells, or plus- and minus-end directed forces on microtubules in vitro, are of similar magnitude. In vitro and in live cells, the force traces are indicative of transport by multiple motors, with maximum forces of 15-20 pN and detachment occurring in multiple steps. These observations suggest bidirectional transport of LBCs is driven by opposing teams of stably-bound motors that operate near force balance. Supported by NIH grant GM087253 to ELFH and YEG and GM089077 to AGH.

The motor protein kinesin converts the energy from ATP hydrolysis and Brownian motion into directed movement. There is increasing evidence suggesting that several kinesin motors cooperate to transport cargoes. Recent experiments also suggest that the collective behavior of kinesin differs significantly from single-molecule behavior. We study the collective behavior of Neurospora kinesin (Nkin) in vitro. By laser trapping latex beads attached to microtubules through biotin-streptavidin linkages, we are able to apply forces to microtubules being transported by several kinesins attached to the coverslip. The density of motors on the coverslip is related to the average number of motors involved in the transport. We experimentally characterize the transport for a range of loads and motor densities.