Kinesin-1-transported liposomes prefer to go straight in 3D microtubule intersections by a mechanism shared by other molecular motors.

Kinesin-1 ensembles maneuver vesicular cargoes through the three-dimensional (3D) intracellular microtubule (MT) network. To define how such cargoes navigate MT intersections, we first determined how many kinesins from an ensemble on a lipid-based cargo simultaneously engage a MT, and then determined the directional outcomes (straight, turn, terminate) for liposome cargoes at perpendicular MT intersections. Run lengths of 350-nm diameter liposomes decorated with up to 20, constitutively active, truncated kinesin-1 KIF5B (K543) were longer than single motor transported cargo, suggesting multiple motor engagement. However, detachment forces of lipid-coated beads with ~20 kinesins, measured using an optical trap, showed no more than three simultaneously engaged motors, with a single engaged kinesin predominating, indicating anticooperative MT binding. At two-dimensional (2D) and 3D in vitro MT intersections, liposomes frequently paused (~2 s), suggesting kinesins simultaneously bind both MTs and engage in a tug-of-war. Liposomes showed no directional outcome bias in 2D (1.1 straight:turn ratio) but preferentially went straight (1.8 straight:turn ratio) in 3D intersections. To explain these data, we developed a mathematical model of liposome transport incorporating the known mechanochemistry of kinesins, which diffuse on the liposome surface, and have stiff tails in both compression and extension that impact how motors engage the intersecting MTs. Our model predicts the ~3 engaged motor limit observed in the optical trap and the bias toward going straight in 3D intersections. The striking similarity of these results to our previous study of liposome transport by myosin Va suggests a "universal" mechanism by which cargoes navigate 3D intersections.

"Spatial Relationships Matter: Kinesin-1 Molecular Motors Transport Liposome Cargo Through 3D Microtubule Intersections In Vitro".

Kinesin-1 ensembles maneuver vesicular cargoes through intersections in the 3-dimensional (3D) intracellular microtubule (MT) network. To characterize directional outcomes (straight, turn, terminate) at MT intersections, we challenge 350 nm fluid-like liposomes transported by ~10 constitutively active, truncated kinesin-1 KIF5B (K543) with perpendicular 2-dimensional (2D) and 3D intersections in vitro. Liposomes frequently pause at 2D and 3D intersections (~2s), suggesting that motor teams can simultaneously engage each MT and undergo a tug-of-war. Once resolved, the directional outcomes at 2D MT intersections have a straight to turn ratio of 1.1; whereas at 3D MT intersections, liposomes more frequently go straight (straight to turn ratio of 1.8), highlighting that spatial relationships at intersections bias directional outcomes. Using 3D super-resolution microscopy (STORM), we define the gap between intersecting MTs and the liposome azimuthal approach angle heading into the intersection. We develop an in silico model in which kinesin-1 motors diffuse on the liposome surface, simultaneously engage the intersecting MTs, generate forces and detach from MTs governed by the motors' mechanochemical cycle, and undergo a tug-of-war with the winning team determining the directional outcome in 3D. The model predicts that 1-3 motors typically engage the MT, consistent with optical trapping measurements. Modeled liposomes also predominantly go straight through 3D intersections over a range of intersection gaps and liposome approach angles, even when obstructed by the crossing MT. Our observations and modeling offer mechanistic insights into how cells might tune the MT cytoskeleton, cargo, and motors to modulate cargo transport.

Double-headed binding of myosin II to F-actin shows the effect of strain on head structure.

Force production in muscle is achieved through the interaction of myosin and actin. Strong binding states in active muscle are associated with Mg·ADP bound to the active site; release of Mg·ADP allows rebinding of ATP and dissociation from actin. Thus, Mg·ADP binding is positioned for adaptation as a force sensor. Mechanical loads on the lever arm can affect the ability of myosin to release Mg·ADP but exactly how this is done is poorly defined. Here we use F-actin decorated with double-headed smooth muscle myosin fragments in the presence of Mg·ADP to visualize the effect of internally supplied tension on the paired lever arms using cryoEM. The interaction of the paired heads with two adjacent actin subunits is predicted to place one lever arm under positive and the other under negative strain. The converter domain is believed to be the most flexible domain within myosin head. Our results, instead, point to the segment of heavy chain between the essential and regulatory light chains as the location of the largest structural change. Moreover, our results suggest no large changes in the myosin coiled coil tail as the locus of strain relief when both heads bind F-actin. The method would be adaptable to double-headed members of the myosin family. We anticipate that the study of actin-myosin interaction using double-headed fragments enables visualization of domains that are typically noisy in decoration with single-headed fragments.

Mechanism of small molecule inhibition of Plasmodium falciparum myosin A informs antimalarial drug design.

Malaria results in more than 500,000 deaths per year and the causative Plasmodium parasites continue to develop resistance to all known agents, including different antimalarial combinations. The class XIV myosin motor PfMyoA is part of a core macromolecular complex called the glideosome, essential for Plasmodium parasite mobility and therefore an attractive drug target. Here, we characterize the interaction of a small molecule (KNX-002) with PfMyoA. KNX-002 inhibits PfMyoA ATPase activity in vitro and blocks asexual blood stage growth of merozoites, one of three motile Plasmodium life-cycle stages. Combining biochemical assays and X-ray crystallography, we demonstrate that KNX-002 inhibits PfMyoA using a previously undescribed binding mode, sequestering it in a post-rigor state detached from actin. KNX-002 binding prevents efficient ATP hydrolysis and priming of the lever arm, thus inhibiting motor activity. This small-molecule inhibitor of PfMyoA paves the way for the development of alternative antimalarial treatments.

Acetylation of fission yeast tropomyosin does not promote differential association with cognate formins.

It was proposed from cellular studies that S. pombe tropomyosin Cdc8 (Tpm) segregates into two populations due to the presence or absence of an amino-terminal acetylation that specifies which formin-mediated F-actin networks it binds, but with no supporting biochemistry. To address this mechanism in vitro, we developed methods for S. pombe actin expression in Sf9 cells. We then employed 3-color TIRF microscopy using all recombinant S. pombe proteins to probe in vitro multicomponent mechanisms involving actin, acetylated and unacetylated Tpm, formins, and myosins. Acetyl-Tpm exhibits tight binding to actin in contrast to weaker binding by unacetylated Tpm. In disagreement with the differential recruitment model, Tpm showed no preferential binding to filaments assembled by the FH1-FH2-domains of two S. pombe formins, nor did Tpm binding have any bias towards the growing formin-bound actin filament barbed end. Although our in vitro findings do not support a direct formin-tropomyosin interaction, it is possible that formins bias differential tropomyosin isoform recruitment through undiscovered mechanisms. Importantly, despite a 12% sequence divergence between skeletal and S. pombe actin, S. pombe myosins Myo2 and Myo51 exhibited similar motile behavior with these two actins, validating key prior findings with these myosins that used skeletal actin.

High-throughput force measurement of individual kinesin-1 motors during multi-motor transport.

Molecular motors often work in teams to move a cellular cargo. Yet measuring the forces exerted by each motor is challenging. Using a sensor made with denatured ssDNA and multi-color fluorescence, we measured picoNewtons of forces and nanometer distances exerted by individual constrained kinesin-1 motors acting together while driving a common microtubule in vitro. We find that kinesins primarily exerted less than 1 pN force, even while the microtubule is bypassing artificial obstacles of 20-100 nanometer size. Occasionally, individual forces increase upon encountering obstacles, although at other times they do not, with the cargo continuing in a directional manner. Our high-throughput technique, which can measure forces by many motors simultaneously, is expected to be useful for many different types of molecular motors.

Coil-to-α-helix transition at the Nup358-BicD2 interface activates BicD2 for dynein recruitment.

Nup358, a protein of the nuclear pore complex, facilitates a nuclear positioning pathway that is essential for many biological processes, including neuromuscular and brain development. Nup358 interacts with the dynein adaptor Bicaudal D2 (BicD2), which in turn recruits the dynein machinery to position the nucleus. However, the molecular mechanisms of the Nup358/BicD2 interaction and the activation of transport remain poorly understood. Here for the first time, we show that a minimal Nup358 domain activates dynein/dynactin/BicD2 for processive motility on microtubules. Using nuclear magnetic resonance titration and chemical exchange saturation transfer, mutagenesis, and circular dichroism spectroscopy, a Nup358 α-helix encompassing residues 2162-2184 was identified, which transitioned from a random coil to an α-helical conformation upon BicD2 binding and formed the core of the Nup358-BicD2 interface. Mutations in this region of Nup358 decreased the Nup358/BicD2 interaction, resulting in decreased dynein recruitment and impaired motility. BicD2 thus recognizes Nup358 through a 'cargo recognition α-helix,' a structural feature that may stabilize BicD2 in its activated state and promote processive dynein motility.

Resistance of Acta2R149C/+ mice to aortic disease is associated with defective release of mutant smooth muscle α-actin from the chaperonin-containing TCP1 folding complex.

Pathogenic variants of the gene for smooth muscle α-actin (ACTA2), which encodes smooth muscle (SM) α-actin, predispose to heritable thoracic aortic disease. The ACTA2 variant p.Arg149Cys (R149C) is the most common alteration; however, only 60% of carriers have a dissection or undergo repair of an aneurysm by 70 years of age. A mouse model of ACTA2 p.Arg149Cys was generated using CRISPR/Cas9 technology to determine the etiology of reduced penetrance. Acta2R149C/+ mice had significantly decreased aortic contraction compared with WT mice but did not form aortic aneurysms or dissections when followed to 24 months, even when hypertension was induced. In vitro motility assays found decreased interaction of mutant SM α-actin filaments with SM myosin. Polymerization studies using total internal reflection fluorescence microscopy showed enhanced nucleation of mutant SM α-actin by formin, which correlated with disorganized and reduced SM α-actin filaments in Acta2R149C/+ smooth muscle cells (SMCs). However, the most prominent molecular defect was the increased retention of mutant SM α-actin in the chaperonin-containing t-complex polypeptide folding complex, which was associated with reduced levels of mutant compared with WT SM α-actin in Acta2R149C/+ SMCs. These data indicate that Acta2R149C/+ mice do not develop thoracic aortic disease despite decreased contraction of aortic segments and disrupted SM α-actin filament formation and function in Acta2R149C/+ SMCs. Enhanced binding of mutant SM α-actin to chaperonin-containing t-complex polypeptide decreases the mutant actin versus WT monomer levels in Acta2R149C/+ SMCs, thus minimizing the effect of the mutation on SMC function and potentially preventing aortic disease in the Acta2R149C/+ mice.

The actomyosin interface contains an evolutionary conserved core and an ancillary interface involved in specificity.

Plasmodium falciparum, the causative agent of malaria, moves by an atypical process called gliding motility. Actomyosin interactions are central to gliding motility. However, the details of these interactions remained elusive until now. Here, we report an atomic structure of the divergent Plasmodium falciparum actomyosin system determined by electron cryomicroscopy at the end of the powerstroke (Rigor state). The structure provides insights into the detailed interactions that are required for the parasite to produce the force and motion required for infectivity. Remarkably, the footprint of the myosin motor on filamentous actin is conserved with respect to higher eukaryotes, despite important variability in the Plasmodium falciparum myosin and actin elements that make up the interface. Comparison with other actomyosin complexes reveals a conserved core interface common to all actomyosin complexes, with an ancillary interface involved in defining the spatial positioning of the motor on actin filaments.

Full-length Plasmodium falciparum myosin A and essential light chain PfELC structures provide new anti-malarial targets.

Parasites from the genus Plasmodium are the causative agents of malaria. The mobility, infectivity, and ultimately pathogenesis of Plasmodium falciparum rely on a macromolecular complex, called the glideosome. At the core of the glideosome is an essential and divergent Myosin A motor (PfMyoA), a first order drug target against malaria. Here, we present the full-length structure of PfMyoA in two states of its motor cycle. We report novel interactions that are essential for motor priming and the mode of recognition of its two light chains (PfELC and MTIP) by two degenerate IQ motifs. Kinetic and motility assays using PfMyoA variants, along with molecular dynamics, demonstrate how specific priming and atypical sequence adaptations tune the motor's mechano-chemical properties. Supported by evidence for an essential role of the PfELC in malaria pathogenesis, these structures provide a blueprint for the design of future anti-malarials targeting both the glideosome motor and its regulatory elements.

Actin R256 Mono-methylation Is a Conserved Post-translational Modification Involved in Transcription.

Nuclear actin has been elusive due to the lack of knowledge about molecular mechanisms. From actin-containing chromatin remodeling complexes, we discovered an arginine mono-methylation mark on an evolutionarily conserved R256 residue of actin (R256me1). Actin R256 mutations in yeast affect nuclear functions and cause diseases in human. Interestingly, we show that an antibody specific for actin R256me1 preferentially stains nuclear actin over cytoplasmic actin in yeast, mouse, and human cells. We also show that actin R256me1 is regulated by protein arginine methyl transferase-5 (PRMT5) in HEK293 cells. A genome-wide survey of actin R256me1 mark provides a landscape for nuclear actin correlated with transcription. Further, gene expression and protein interaction studies uncover extensive correlations between actin R256me1 and active transcription. The discovery of actin R256me1 mark suggests a fundamental mechanism to distinguish nuclear actin from cytoplasmic actin through post-translational modification (PTM) and potentially implicates an actin PTM mark in transcription and human diseases.

Coiled-coil registry shifts in the F684I mutant of Bicaudal D result in cargo-independent activation of dynein motility.

The dynein adaptor Drosophila Bicaudal D (BicD) is auto-inhibited and activates dynein motility only after cargo is bound, but the underlying mechanism is elusive. In contrast, we show that the full-length BicD/F684I mutant activates dynein processivity even in the absence of cargo. Our X-ray structure of the C-terminal domain of the BicD/F684I mutant reveals a coiled-coil registry shift; in the N-terminal region, the two helices of the homodimer are aligned, whereas they are vertically shifted in the wild-type. One chain is partially disordered and this structural flexibility is confirmed by computations, which reveal that the mutant transitions back and forth between the two registries. We propose that a coiled-coil registry shift upon cargo-binding activates BicD for dynein recruitment. Moreover, the human homolog BicD2/F743I exhibits diminished binding of cargo adaptor Nup358, implying that a coiled-coil registry shift may be a mechanism to modulate cargo selection for BicD2-dependent transport pathways.

Multiple kinesins induce tension for smooth cargo transport.

How cargoes move within a crowded cell-over long distances and at speeds nearly the same as when moving on unimpeded pathway-has long been mysterious. Through an in vitro force-gliding assay, which involves measuring nanometer displacement and piconewtons of force, we show that multiple mammalian kinesin-1 (from 2 to 8) communicate in a team by inducing tension (up to 4 pN) on the cargo. Kinesins adopt two distinct states, with one-third slowing down the microtubule and two-thirds speeding it up. Resisting kinesins tend to come off more rapidly than, and speed up when pulled by driving kinesins, implying an asymmetric tug-of-war. Furthermore, kinesins dynamically interact to overcome roadblocks, occasionally combining their forces. Consequently, multiple kinesins acting as a team may play a significant role in facilitating smooth cargo motion in a dense environment. This is one of few cases in which single molecule behavior can be connected to ensemble behavior of multiple motors.

The inside of a cell is a crowded space, full of proteins and other molecules. Yet, the molecular motors that transport some of those molecules within the cell move at the same speed as they would in pure water ­ about one micrometer per second. How the molecular motors could achieve such speeds in crowded cells was unclear. Nevertheless, Tjioe et al. suspected that the answer might be related to how multiple motors work together. Molecular motors move by walking along filaments inside the cell and pulling their cargo from one location to another. Other molecules that bind to the filaments should, in theory, act like "roadblocks" and impede the movement of the cargo. Tjioe et al. studied a motor protein called kinesin, which walks on filaments called microtubules. But instead of looking at these motors moving along microtubules inside a cell, Tjioe et al. used a simpler system where the cell was eliminated, and all parts were purified. Specifically, Tjioe et al. tethered purified motors to a piece of glass and then observed them under an extremely accurate microscope as they moved free-floating, fluorescently labelled microtubules. The microtubules, in this scenario, were acting like cargoes, where many kinesins could bind. Each kinesin motor also had a small chemical tag that could emit light. By following the movement of the lights, it was possible to calculate what each kinesin was doing and how the cargo moved. When more than one kinesin molecule was acting, the tension and speed of one kinesin affected the movement of the others. In any group of kinesins, about two-thirds of kinesin pulled the cargo, and unexpectedly, about one-third tended to resist and slow the cargo. These latter kinesins were moved along with the group without actually driving the cargo. These resisting kinesins did come off more rapidly than the driving kinesins, meaning the cargo should be able to quickly bypass roadblocks. This would help to keep the whole group travelling in the right direction at a steady pace.

Unusual dynamics of the divergent malaria parasite PfAct1 actin filament.

Gliding motility and host cell invasion by the apicomplexan parasite Plasmodium falciparum (Pf), the causative agent of malaria, is powered by a macromolecular complex called the glideosome that lies between the parasite plasma membrane and the inner membrane complex. The glideosome core consists of a single-headed class XIV myosin PfMyoA and a divergent actin PfAct1. Here we use total internal reflection fluorescence microscopy to visualize growth of individual unstabilized PfAct1 filaments as a function of time, an approach not previously used with this actin isoform. Although PfAct1 was thought to be incapable of forming long filaments, filaments grew as long as 30 µm. Polymerization occurs via a nucleation-elongation mechanism, but with an ∼4 µM critical concentration, an order-of-magnitude higher than for skeletal actin. Protomers disassembled from both the barbed and pointed ends of the actin filament with similar fast kinetics of 10 to 15 subunits/s. Rapid treadmilling, where the barbed end of the filament grows and the pointed end shrinks while maintaining an approximately constant filament length, was visualized near the critical concentration. Once ATP has been hydrolyzed to ADP, the filament becomes very unstable, resulting in total dissolution in <40 min. Dynamics at the filament ends are suppressed in the presence of inorganic phosphate or more efficiently by BeFX A chimeric PfAct1 with a mammalian actin D-loop forms a more stable filament. These unusual dynamic properties distinguish PfAct1 from more canonical actins, and likely contribute to the difficultly in visualizing PfAct1 filaments in the parasite.

Plasmodium myosin A drives parasite invasion by an atypical force generating mechanism.

Plasmodium parasites are obligate intracellular protozoa and causative agents of malaria, responsible for half a million deaths each year. The lifecycle progression of the parasite is reliant on cell motility, a process driven by myosin A, an unconventional single-headed class XIV molecular motor. Here we demonstrate that myosin A from Plasmodium falciparum (PfMyoA) is critical for red blood cell invasion. Further, using a combination of X-ray crystallography, kinetics, and in vitro motility assays, we elucidate the non-canonical interactions that drive this motor's function. We show that PfMyoA motor properties are tuned by heavy chain phosphorylation (Ser19), with unphosphorylated PfMyoA exhibiting enhanced ensemble force generation at the expense of speed. Regulated phosphorylation may therefore optimize PfMyoA for enhanced force generation during parasite invasion or for fast motility during dissemination. The three PfMyoA crystallographic structures presented here provide a blueprint for discovery of specific inhibitors designed to prevent parasite infection.

MAP7 regulates organelle transport by recruiting kinesin-1 to microtubules.

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.

Myosin Va transport of liposomes in three-dimensional actin networks is modulated by actin filament density, position, and polarity.

The cell's dense 3D actin filament network presents numerous challenges to vesicular transport by teams of myosin Va (MyoVa) molecular motors. These teams must navigate their cargo through diverse actin structures ranging from Arp2/3-branched lamellipodial networks to the dense, unbranched cortical networks. To define how actin filament network organization affects MyoVa cargo transport, we created two different 3D actin networks in vitro. One network was comprised of randomly oriented, unbranched actin filaments; the other was comprised of Arp2/3-branched actin filaments, which effectively polarized the network by aligning the actin filament plus-ends. Within both networks, we defined each actin filament's 3D spatial position using superresolution stochastic optical reconstruction microscopy (STORM) and its polarity by observing the movement of single fluorescent reporter MyoVa. We then characterized the 3D trajectories of fluorescent, 350-nm fluid-like liposomes transported by MyoVa teams (∼10 motors) moving within each of the two networks. Compared with the unbranched network, we observed more liposomes with directed and fewer with stationary motion on the Arp2/3-branched network. This suggests that the modes of liposome transport by MyoVa motors are influenced by changes in the local actin filament polarity alignment within the network. This mechanism was supported by an in silico 3D model that provides a broader platform to understand how cellular regulation of the actin cytoskeletal architecture may fine tune MyoVa-based intracellular cargo transport.

Hypertrophic cardiomyopathy R403Q mutation in rabbit ß-myosin reduces contractile function at the molecular and myofibrillar levels.

In 1990, the Seidmans showed that a single point mutation, R403Q, in the human ß-myosin heavy chain (MHC) of heart muscle caused a particularly malignant form of familial hypertrophic cardiomyopathy (HCM) [Geisterfer-Lowrance AA, et al. (1990) Cell 62:999-1006.]. Since then, more than 300 mutations in the ß-MHC have been reported, and yet there remains a poor understanding of how a single missense mutation in the MYH7 gene can lead to heart disease. Previous studies with a transgenic mouse model showed that the myosin phenotype depended on whether the mutation was in an α- or ß-MHC backbone. This led to the generation of a transgenic rabbit model with the R403Q mutation in a ß-MHC backbone. We find that the in vitro motility of heterodimeric R403Q myosin is markedly reduced, whereas the actin-activated ATPase activity of R403Q subfragment-1 is about the same as myosin from a nontransgenic littermate. Single myofibrils isolated from the ventricles of R403Q transgenic rabbits and analyzed by atomic force microscopy showed reduced rates of force development and relaxation, and achieved a significantly lower steady-state level of isometric force compared with nontransgenic myofibrils. Myofibrils isolated from the soleus gave similar results. The force-velocity relationship determined for R403Q ventricular myofibrils showed a decrease in the velocity of shortening under load, resulting in a diminished power output. We conclude that independent of whether experiments are performed with isolated molecules or with ordered molecules in the native thick filament of a myofibril, there is a loss-of-function induced by the R403Q mutation in ß-cardiac myosin.

Recruitment of two dyneins to an mRNA-dependent Bicaudal D transport complex.

We investigated the role of full-length Drosophila Bicaudal D (BicD) binding partners in dynein-dynactin activation for mRNA transport on microtubules. Full-length BicD robustly activated dynein-dynactin motility only when both the mRNA binding protein Egalitarian (Egl) and K10 mRNA cargo were present, and electron microscopy showed that both Egl and mRNA were needed to disrupt a looped, auto-inhibited BicD conformation. BicD can recruit two dimeric dyneins, resulting in faster speeds and longer runs than with one dynein. Moving complexes predominantly contained two Egl molecules and one K10 mRNA. This mRNA-bound configuration makes Egl bivalent, likely enhancing its avidity for BicD and thus its ability to disrupt BicD auto-inhibition. Consistent with this idea, artificially dimerized Egl activates dynein-dynactin-BicD in the absence of mRNA. The ability of mRNA cargo to orchestrate the activation of the mRNP (messenger ribonucleotide protein) complex is an elegant way to ensure that only cargo-bound motors are motile.

Magnetic Cytoskeleton Affinity Purification of Microtubule Motors Conjugated to Quantum Dots.

We develop magnetic cytoskeleton affinity (MiCA) purification, which allows for rapid isolation of molecular motors conjugated to large multivalent quantum dots, in miniscule quantities, which is especially useful for single-molecule applications. When purifying labeled molecular motors, an excess of fluorophores or labels is usually used. However, large labels tend to sediment during the centrifugation step of microtubule affinity purification, a traditionally powerful technique for motor purification. This is solved with MiCA, and purification time is cut from 2 h to 20 min, a significant time-savings when it needs to be done daily. For kinesin, MiCA works with as little as 0.6 µg protein, with yield of ∼27%, compared to 41% with traditional purification. We show the utility of MiCA purification in a force-gliding assay with kinesin, allowing, for the first time, simultaneous determination of whether the force from each motor in a multiple-motor system drives or hinders microtubule movement. Furthermore, we demonstrate rapid purification of just 30 ng dynein-dynactin-BICD2N-QD (DDB-QD), ordinarily a difficult protein-complex to purify.