The types and numbers of kinesins and dyneins transporting endocytic cargoes modulate their motility and response to tau.

Organelles and vesicular cargoes are transported by teams of kinesin and dynein motors along microtubules. We isolated endocytic organelles from cells at different stages of maturation and reconstituted their motility along microtubules in vitro. We asked how the sets of motors transporting a cargo determine its motility and response to the microtubule-associated protein tau. Here, we find that phagosomes move in both directions along microtubules, but the directional bias changes during maturation. Early phagosomes exhibit retrograde-biased transport while late phagosomes are directionally unbiased. Correspondingly, early and late phagosomes are bound by different numbers and combinations of kinesins-1, -2, -3, and dynein. Tau stabilizes microtubules and directs transport within neurons. While single-molecule studies show that tau differentially regulates the motility of kinesins and dynein in vitro, less is known about its role in modulating the trafficking of endogenous cargoes transported by their native teams of motors. Previous studies showed that tau preferentially inhibits kinesin motors, which biases late phagosome transport towards the microtubule minus-end. Here, we show that tau strongly inhibits long-range, dynein-mediated motility of early phagosomes. Tau reduces forces generated by teams of dynein motors on early phagosomes and accelerates dynein unbinding under load. Thus, cargoes differentially respond to tau, where dynein complexes on early phagosomes are more sensitive to tau inhibition than those on late phagosomes. Mathematical modeling further explains how small changes in the number of kinesins and dynein on cargoes impact the net directionality but also that cargoes with different sets of motors respond differently to tau.

Tau, microtubule dynamics, and axonal transport: New paradigms for neurodegenerative disease.

The etiology of Tauopathies, a diverse class of neurodegenerative diseases associated with the Microtubule Associated Protein (MAP) Tau, is usually described by a common mechanism in which Tau dysfunction results in the loss of axonal microtubule stability. Here, we reexamine and build upon the canonical disease model to encompass other Tau functions. In addition to regulating microtubule dynamics, Tau acts as a modulator of motor proteins, a signaling hub, and a scaffolding protein. This diverse array of functions is related to the dynamic nature of Tau isoform expression, post-translational modification (PTM), and conformational flexibility. Thus, there is no single mechanism that can describe Tau dysfunction. The effects of specific pathogenic mutations or aberrant PTMs need to be examined on all of the various functions of Tau in order to understand the unique etiology of each disease state.

The pathogenic R5L mutation disrupts formation of Tau complexes on the microtubule by altering local N-terminal structure.

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 arginine to leucine mutation at position 5 in Tau (R5L), associated with PSP, on Tau-microtubule interactions using an in vitro reconstituted system. Contrary to the canonical disease-state theory, we determine that the R5L mutation does not reduce Tau affinity for the microtubule using total internal reflection fluorescence microscopy. Rather, the R5L mutation decreases the ability of Tau to form larger-order complexes, or Tau patches, at high concentrations of Tau. Using 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.

The N-terminal disease-associated R5L Tau mutation increases microtubule shrinkage rate due to disruption of microtubule-bound Tau patches.

Regulation of the neuronal microtubule cytoskeleton is achieved through the coordination of microtubule-associated proteins (MAPs). MAP-Tau, the most abundant MAP in the axon, functions to modulate motor motility, participate in signaling cascades, as well as directly mediate microtubule dynamics. Tau misregulation is associated with a class of neurodegenerative diseases, known as tauopathies, including progressive supranuclear palsy, Pick's disease, and Alzheimer's disease. Many disease-associated mutations in Tau are found in the C-terminal microtubule-binding domain. These mutations decrease microtubule-binding affinity and are proposed to reduce microtubule stability, leading to disease. N-terminal disease-associated mutations also exist, but the mechanistic details of their downstream effects are not as clear. Here, we investigate the effect of the progressive supranuclear palsy-associated N-terminal R5L mutation on Tau-mediated microtubule dynamics using an in vitro reconstituted system. We show that the R5L mutation does not alter Tau interactions with tubulin by fluorescence correlation spectroscopy. Using total internal reflection fluorescence microscopy, we determined that the R5L mutation has no effect on microtubule growth rate, catastrophe frequency, or rescue frequency. Rather, the R5L mutation increases microtubule shrinkage rate. We determine this is due to disruption of Tau patches, larger order Tau complexes known to form on the GDP-microtubule lattice. Altogether, these results provide insight into the role of Tau patches in mediating microtubule dynamics and suggesting a novel mechanism by which mutations in the N-terminal projection domain reduce microtubule stability.

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.

Polyglutamylation of tubulin's C-terminal tail controls pausing and motility of kinesin-3 family member KIF1A.

The kinesin-3 family member KIF1A plays a critical role in site-specific neuronal cargo delivery during axonal transport. KIF1A cargo is mislocalized in many neurodegenerative diseases, indicating that KIF1A's highly efficient, superprocessive motility along axonal microtubules needs to be tightly regulated. One potential regulatory mechanism may be through posttranslational modifications (PTMs) of axonal microtubules. These PTMs often occur on the C-terminal tails of the microtubule tracks, act as molecular "traffic signals" helping to direct kinesin motor cargo delivery, and include C-terminal tail polyglutamylation important for KIF1A cargo transport. KIF1A initially interacts with microtubule C-terminal tails through its K-loop, a positively charged surface loop of the KIF1A motor domain. However, the role of the K-loop in KIF1A motility and response to perturbations in C-terminal tail polyglutamylation is underexplored. Using single-molecule imaging, we present evidence that KIF1A pauses on different microtubule lattice structures, linking multiple processive segments together and contributing to KIF1A's characteristic superprocessive run length. Furthermore, modifications of the KIF1A K-loop or tubulin C-terminal tail polyglutamylation reduced KIF1A pausing and overall run length. These results suggest a new mechanism to regulate KIF1A motility via pauses mediated by K-loop/polyglutamylated C-terminal tail interactions, providing further insight into KIF1A's role in axonal transport.

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.

Acetylated Microtubules Are Preferentially Bundled Leading to Enhanced Kinesin-1 Motility.

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.

Kinesin's neck-linker determines its ability to navigate obstacles on the microtubule surface.

The neck-linker is a structurally conserved region among most members of the kinesin superfamily of molecular motor proteins that is critical for kinesin's processive transport of intracellular cargo along the microtubule surface. Variation in the neck-linker length has been shown to directly modulate processivity in different kinesin families; for example, kinesin-1, with a shorter neck-linker, is more processive than kinesin-2. Although small differences in processivity are likely obscured in vivo by the coupling of most cargo to multiple motors, longer and more flexible neck-linkers may allow different kinesins to navigate more efficiently around the many obstacles, including microtubule-associated proteins (MAPs), that are found on the microtubule surface within cells. We hypothesize that, due to its longer neck-linker, kinesin-2 can more easily navigate obstacles (e.g., MAPs) on the microtubule surface than kinesin-1. We used total internal reflection fluorescence microscopy to observe single-molecule motility from different kinesin-1 and kinesin-2 neck-linker chimeras stepping along microtubules in the absence or presence of two Tau isoforms, 3RS-Tau and 4RL-Tau, both of which are MAPs that are known to differentially affect kinesin-1 motility. Our results demonstrate that unlike kinesin-1, kinesin-2 is insensitive to the presence of either Tau isoform, and appears to have the ability to switch protofilaments while stepping along the microtubule when challenged by an obstacle, such as Tau. Thus, although kinesin-1 may be more processive, the longer neck-linker length of kinesin-2 allows it to be better optimized to navigate the complex microtubule landscape. These results provide new insight, to our knowledge, into how kinesin-1 and kinesin-2 may work together for the efficient delivery of cargo in cells.

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.

Modification of the neck-linker of KIF18A alters Microtubule subpopulation preference.

Kinesins support many diverse cellular processes, including facilitating cell division through mechanical regulation of the mitotic spindle. However, how kinesin activity is controlled to facilitate this process is not well understood. Interestingly, posttranslational modifications have been identified within the enzymatic region of all 45 mammalian kinesins, but the significance of these modifications has gone largely unexplored. Given the critical role of the enzymatic region in facilitating nucleotide and microtubule binding, it may serve as a primary site for kinesin regulation. Consistent with this idea, a phosphomimetic mutation at S357 in the neck-linker of KIF18A alters the localization of KIF18A within the spindle from kinetochore microtubules to nonkinetochore microtubules at the periphery of the spindle. Changes in localization of KIF18A-S357D are accompanied by defects in mitotic spindle positioning and the ability to promote mitotic progression. This altered localization pattern is mimicked by a shortened neck-linker mutant, suggesting that KIF18A-S357D may cause the motor to adopt a shortened neck-linker-like state that decreases KIF18A accumulation at the plus-ends of kinetochore microtubules. These findings demonstrate that posttranslational modifications in the enzymatic region of kinesins could be important for biasing their localization to particular microtubule subpopulations.

Optogenetic control of kinesin-1, -2, -3 and dynein reveals their specific roles in vesicular transport.

Each cargo in a cell employs a unique set of motor proteins for its transport. To dissect the roles of each type of motor, we developed optogenetic inhibitors of endogenous kinesin-1, -2, -3 and dynein motors and examined their effect on the transport of early endosomes, late endosomes, and lysosomes. While kinesin-1, -3, and dynein transport vesicles at all stages of endocytosis, kinesin-2 primarily drives late endosomes and lysosomes. Transient optogenetic inhibition of kinesin-1 or dynein causes both early and late endosomes to move more processively by relieving competition with opposing motors. Kinesin-2 and -3 support long-range transport, and optogenetic inhibition reduces the distances that their cargoes move. These results suggest that the directionality of transport is controlled through regulating kinesin-1 and dynein activity. On vesicles transported by several kinesin and dynein motors, modulating the activity of a single type of motor on the cargo is sufficient to direct motility.

Single-molecule motility: statistical analysis and the effects of track length on quantification of processive motion.

In vitro, single-molecule motility assays allow for the direct characterization of molecular motor properties including stepping velocity and characteristic run length. Although application of these techniques in vivo is feasible, the challenges involved in sample preparation, as well as the added complexity of the cell and its systems, result in a reduced ability to collect large datasets, as well as difficulty in simultaneous observation of the components of the motility system, namely motor and track. To address these challenges, we have developed simulations to characterize motility datasets as a function of sample size, processive run length of the motor, and distribution of track lengths. We introduce the use of a simple bootstrapping technique that allows for the quantification of measurement uncertainty and a Monte Carlo permutation resampling scheme for the measurement of statistical significance and the estimation of required sample size. In addition, we have found that, despite conventional wisdom, the measured characteristic run length is directly coupled to the characteristic track length that describes the microtubule length distribution. To be able to make comparisons between motility experiments performed on different track populations as well as make measurements of motility when motors and tracks cannot be simultaneously resolved, we have developed a theoretical framework for the determination of the effect that track length has on observed characteristic run lengths. This shows good agreement with in vitro motility experiments on two kinesin constructs walking on microtubule populations of different characteristic track lengths.

Kinetics of presynaptic filament assembly in the presence of single-stranded DNA binding protein and recombination mediator protein.

Enzymes of the RecA/Rad51 family catalyze DNA strand exchange reactions that are important for homologous recombination and for the accurate repair of DNA double-strand breaks. RecA/Rad51 recombinases are activated by their assembly into presynaptic filaments on single-stranded DNA (ssDNA), a process that is regulated by ssDNA binding protein (SSB) and mediator proteins. Mediator proteins stimulate strand exchange by accelerating the rate-limiting displacement of SSB from ssDNA by the incoming recombinase. The use of mediators is a highly conserved strategy in recombination, but the precise mechanism of mediator activity is unknown. In this study, the well-defined bacteriophage T4 recombination system (UvsX recombinase, Gp32 SSB, and UvsY mediator) is used to examine the kinetics of presynaptic filament assembly on native ssDNA in vitro. Results indicate that the ATP-dependent assembly of UvsX presynaptic filaments on Gp32-covered ssDNA is limited by a salt-sensitive nucleation step in the absence of mediator. Filament nucleation is selectively enhanced and rendered salt-resistant by mediator protein UvsY, which appears to stabilize a prenucleation complex. This mechanism potentially explains how UvsY promotes presynaptic filament assembly at physiologically relevant ionic strengths and Gp32 concentrations. Other data suggest that presynaptic filament assembly involves multiple nucleation events, resulting in many short UvsX-ssDNA filaments or clusters, which may be the relevant form for recombination in vivo. Together, these findings provide the first detailed kinetic model for presynaptic filament assembly involving all three major protein components (recombinase, mediator, and SSB) on native ssDNA.

Modification of the Neck Linker of KIF18A Alters Microtubule Subpopulation Preference.

Kinesins support many diverse cellular processes, including facilitating cell division through mechanical regulation of the mitotic spindle. However, how kinesin activity is controlled to facilitate this process is not well understood. Interestingly, post-translational modifications have been identified within the enzymatic region of all 45 mammalian kinesins, but the significance of these modifications has gone largely unexplored. Given the critical role of the enzymatic region in facilitating nucleotide and microtubule binding, it may serve as a primary site for kinesin regulation. Consistent with this idea, a phosphomimetic mutation at S357 in the neck-linker of KIF18A alters the localization of KIF18A within the spindle from kinetochore microtubules to peripheral microtubules. Changes in localization of KIF18A-S357D are accompanied by defects in mitotic spindle positioning and the ability to promote mitotic progression. This altered localization pattern is mimicked by a shortened neck-linker mutant, suggesting that KIF18A-S357D may cause the motor to adopt a shortened neck-linker like state that prevents KIF18A from accumulating at the plus-ends of kinetochore microtubules. These findings demonstrate that post-translational modifications in the enzymatic region of kinesins could be important for biasing their localization to particular microtubule subpopulations.

The nucleotide-binding state of microtubules modulates kinesin processivity and the ability of Tau to inhibit kinesin-mediated transport.

The ability of Tau to act as a potent inhibitor of kinesin's processive run length in vitro suggests that it may actively participate in the regulation of axonal transport in vivo. However, it remains unclear how kinesin-based transport could then proceed effectively in neurons, where Tau is expressed at high levels. One potential explanation is that Tau, a conformationally dynamic protein, has multiple modes of interaction with the microtubule, not all of which inhibit kinesin's processive run length. Previous studies support the hypothesis that Tau has at least two modes of interaction with microtubules, but the mechanisms by which Tau adopts these different conformations and their functional consequences have not been investigated previously. In the present study, we have used single molecule imaging techniques to demonstrate that Tau inhibits kinesin's processive run length in an isoform-dependent manner on GDP-microtubules stabilized with either paclitaxel or glycerol/DMSO but not guanosine-5'-((α,ß)-methyleno)triphosphate (GMPCPP)-stabilized microtubules. Furthermore, the order of Tau addition to microtubules before or after polymerization has no effect on the ability of Tau to modulate kinesin motility regardless of the stabilizing agent used. Finally, the processive run length of kinesin is reduced on GMPCPP-microtubules relative to GDP-microtubules, and kinesin's velocity is enhanced in the presence of 4-repeat long Tau but not the 3-repeat short isoform. These results shed new light on the potential role of Tau in the regulation of axonal transport, which is more complex than previously recognized.

Switch I closure simultaneously promotes strong binding to actin and ADP in smooth muscle myosin.

The motor protein myosin uses energy derived from ATP hydrolysis to produce force and motion. Important conserved components (P-loop, switch I, and switch II) help propagate small conformational changes at the active site into large scale conformational changes in distal regions of the protein. Structural and biochemical studies have indicated that switch I may be directly responsible for the reciprocal opening and closing of the actin and nucleotide-binding pockets during the ATPase cycle, thereby aiding in the coordination of these important substrate-binding sites. Smooth muscle myosin has displayed the ability to simultaneously bind tightly to both actin and ADP, although it is unclear how both substrate-binding clefts could be closed if they are rigidly coupled to switch I. Here we use single tryptophan mutants of smooth muscle myosin to determine how conformational changes in switch I are correlated with structural changes in the nucleotide and actin-binding clefts in the presence of actin and ADP. Our results suggest that a closed switch I conformation in the strongly bound actomyosin-ADP complex is responsible for maintaining tight nucleotide binding despite an open nucleotide-binding pocket. This unique state is likely to be crucial for prolonged tension maintenance in smooth muscle.

Tau differentially regulates the transport of early endosomes and lysosomes.

Microtubule-associated proteins (MAPs) modulate the motility of kinesin and dynein along microtubules to control the transport of vesicles and organelles. The neuronal MAP tau inhibits kinesin-dependent transport. Phosphorylation of tau at Tyr-18 by fyn kinase results in weakened inhibition of kinesin-1. We examined the motility of early endosomes and lysosomes in cells expressing wild-type (WT) tau and phosphomimetic Y18E tau. We quantified the effects on motility as a function of the tau expression level. Lysosome motility is strongly inhibited by tau. Y18E tau preferentially inhibits lysosomes in the cell periphery, while centrally located lysosomes are less affected. Early endosomes are more sensitive to tau than lysosomes and are inhibited by both WT and Y18E tau. Our results show that different cargoes have disparate responses to tau, likely governed by the types of kinesin motors driving their transport. In support of this model, kinesin-1 and -3 are strongly inhibited by tau while kinesin-2 and dynein are less affected. In contrast to kinesin-1, we find that kinesin-3 is strongly inhibited by phosphorylated tau.

Loop 1 dynamics in smooth muscle myosin: isoform specific differences modulate ADP release.

Isoforms of the smooth muscle (SM) myosin motor domain differ in the presence or absence of a seven amino acid insert in a flexible surface loop spanning the nucleotide-binding pocket known as Loop 1. The presence of this insert leads to a two-fold increase in actin sliding velocity and ADP release rate between these isoforms, although the effect of Loop 1 on the kinetics of ADP release remains unclear. To further investigate the role of the Loop 1 insert in modulating ADP release in SM myosin we have inserted a single tryptophan residue into Loop 1 of both isoforms as a probe of local structural dynamics. By monitoring the dynamics of Loop 1 in relation to the release of ADP we have observed a unique movement of Loop 1 in the inserted isoform, preceding nucleotide release, which is absent in the non-inserted isoform. This movement is sequence dependent as alanine replacement of the insert residues abolishes the transition and slows ADP release. Thus movement of Loop 1 is a critical factor in increasing the ADP release rate in the inserted faster isoform of SM myosin.

KIF18A's neck linker permits navigation of microtubule-bound obstacles within the mitotic spindle.

KIF18A (kinesin-8) is required for mammalian mitotic chromosome alignment. KIF18A confines chromosome movement to the mitotic spindle equator by accumulating at the plus-ends of kinetochore microtubule bundles (K-fibers), where it functions to suppress K-fiber dynamics. It is not understood how the motor accumulates at K-fiber plus-ends, a difficult feat requiring the motor to navigate protein dense microtubule tracks. Our data indicate that KIF18A's relatively long neck linker is required for the motor's accumulation at K-fiber plus-ends. Shorter neck linker (sNL) variants of KIF18A display a deficiency in accumulation at the ends of K-fibers at the center of the spindle. Depletion of K-fiber-binding proteins reduces the KIF18A sNL localization defect, whereas their overexpression reduces wild-type KIF18A's ability to accumulate on this same K-fiber subset. Furthermore, single-molecule assays indicate that KIF18A sNL motors are less proficient in navigating microtubules coated with microtubule-associated proteins. Taken together, these results support a model in which KIF18A's neck linker length permits efficient navigation of obstacles to reach K-fiber ends during mitosis.