The kinesin-13 MCAK has an unconventional ATPase cycle adapted for microtubule depolymerization

Unlike other kinesins, members of the kinesin-13 subfamily do not move directionally along microtubules but, instead, depolymerize them. To understand how kinesins with structurally similar motor domains can have such dissimilar functions, we elucidated the ATP turnover cycle of the kinesin-13, MCAK. In contrast to translocating kinesins, ATP cleavage, rather than product release, is the rate-limiting step for ATP turnover by MCAK; unpolymerized tubulin and microtubules accelerate this step. Further, microtubule ends fully activate the ATPase by accelerating the exchange of ADP for ATP. This tuning of the cycle adapts MCAK for its depolymerization activity: lattice-stimulated ATP cleavage drives MCAK into a weakly bound nucleotide state that reaches microtubule ends by diffusion, and end-specific acceleration of nucleotide exchange drives MCAK into a strongly bound state that promotes depolymerization. This altered cycle accounts well for the different mechanical behaviour of this kinesin, which depolymerizes microtubules from their ends, compared to translocating kinesins that walk along microtubules. Thus, the kinesin motor domain is a nucleotide-dependent engine that can be differentially tuned for transport or depolymerization functions.     

Structure of a kinesin microtubule depolymerization machine

With their ability to depolymerize microtubules (MTs), KinI kinesins are the rogue members of the kinesin family. Here we present the 1.6 A crystal structure of a KinI motor core from Plasmodium falciparum, which is sufficient for depolymerization in vitro. Unlike all published kinesin structures to date, nucleotide is not present, and there are noticeable differences in loop regions L6 and L10 (the plus-end tip), L2 and L8 and in switch II (L11 and helix4); otherwise, the pKinI structure is very similar to previous kinesin structures. KinI-conserved amino acids were mutated to alanine, and studied for their effects on depolymerization and ATP hydrolysis. Notably, mutation of three residues in L2 appears to primarily affect depolymerization, rather than general MT binding or ATP hydrolysis. The results of this study confirm the suspected importance of loop 2 for KinI function, and provide evidence that KinI is specialized to hydrolyze ATP after initiating depolymerization.     

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.      

Regulation of KinI kinesin ATPase activity by binding to the microtubule lattice

KinI kinesins are important in regulating the complex dynamics of the microtubule cytoskeleton. They are unusual in that they depolymerize, rather than move along microtubules. To determine the attributes of KinIs that distinguish them from translocating kinesins, we examined the ATPase activity, microtubule affinity, and three-dimensional microtubule-bound structure of a minimal KinI motor domain. Together, the kinetic, affinity, and structural data lead to the conclusion that on binding to the microtubule lattice, KinIs release ADP and enter a stable, low-affinity, regulated state, from which they do not readily progress through the ATPase cycle. This state may favor detachment, or diffusion of the KinI to its site of action, the microtubule ends. Unlike conventional translocating kinesins, which are microtubule lattice-stimulated ATPases, it seems that with KinIs, nucleotide-mediated modulation of tubulin affinity is only possible when it is coupled to protofilament deformation. This provides an elegant mechanistic basis for their unique depolymerizing activity.     

Speeding up kinesin-driven microtubule gliding in vitro by variation of cofactor composition and physicochemical parameters

So far, there has been a discrepancy between the velocities of kinesin-dependent microtubule motility measured in vitro and within cells. By changing ATP, Mg(2+), and kinesin concentrations, pH and ionic strength, we tried to find conditions that favour microtubule gliding across kinesin-covered glass surfaces. For porcine brain kinesin, we found that raising the molar Mg(2+)/ATP ratio can substantially elevate gliding velocity. Gliding became also faster after temperature elevation or lowering the number of kinesin molecules bound to the glass surface. The highest mean gliding velocity (1.8 microm/s+/-0.09 microm/s), approaching velocities measured for anterograde transport in vivo, was achieved by combination of favourable factors (2.5 m m ATP, 12.5 m m Mg(2+), 37 degrees C, 450 kinesin molecules/microm(2)).     

ARK2 stabilizes the plus-end of microtubules and promotes microtubule bundling in Arabidopsis

Microtubule dynamics and organization are important for plant cell morphogenesis and development. The microtubule-based motor protein kinesins are mainly responsible for the transport of some organelles and vesicles, although several have also been shown to regulate microtubule organization. The ARMADILLO REPEAT KINESIN (ARK) family is a plant-specific motor protein subfamily that consists of three members (ARK1, ARK2, and ARK3) in Arabidopsis thaliana. ARK2 has been shown to participate in root epidermal cell morphogenesis. However, whether and how ARK2 associates with microtubules needs further elucidation. Here, we demonstrated that ARK2 co-localizes with microtubules and facilitates microtubule bundling in vitro and in vivo. Pharmacological assays and microtubule dynamics analyses indicated that ARK2 stabilizes cortical microtubules. Live-cell imaging revealed that ARK2 moves along cortical microtubules in a processive mode and localizes both at the plus-end and the sidewall of microtubules. ARK2 therefore tracks and stabilizes the growing plus-ends of microtubules, which facilitates the formation of parallel microtubule bundles.     

Cryo-EM structure of a microtubule-bound parasite kinesin motor and implications for its mechanism and inhibition

Plasmodium parasites cause malaria and are responsible annually for hundreds of thousands of deaths. Kinesins are a superfamily of microtubule-dependent ATPases that play important roles in the parasite replicative machinery, which is a potential target for antiparasite drugs. Kinesin-5, a molecular motor that cross-links microtubules, is an established antimitotic target in other disease contexts, but its mechanism in Plasmodium falciparum is unclear. Here, we characterized P. falciparum kinesin-5 (PfK5) using cryo-EM to determine the motor''s nucleotide-dependent microtubule-bound structure and introduced 3D classification of individual motors into our microtubule image processing pipeline to maximize our structural insights. Despite sequence divergence in PfK5, the motor exhibits classical kinesin mechanochemistry, including ATP-induced subdomain rearrangement and cover neck bundle formation, consistent with its plus-ended directed motility. We also observed that an insertion in loop5 of the PfK5 motor domain creates a different environment in the well-characterized human kinesin-5 drug-binding site. Our data reveal the possibility for selective inhibition of PfK5 and can be used to inform future exploration of Plasmodium kinesins as antiparasite targets.     

Kinesin-2 is a motor for late endosomes and lysosomes

The bidirectional nature of late endosome/lysosome movement suggests involvement of at least two distinct motors, one minus-end directed and one plus-end directed. Previous work has identified dynein as the minus-end-directed motor for late endosome/lysosome localization and dynamics. Conventional kinesin (kinesin-1) has been implicated in plus-end-directed late endosome/lysosome movement, but other kinesin family members may also be involved. Kinesin-2 is known to drive the movement of pigment granules, a type of lysosomally derived organelle, and was recently found to be associated with purified late endosomes. To determine whether kinesin-2 might also power endosome movement in non-pigmented cells, we overexpressed dominant negative forms of the KIF3A motor subunit and KAP3 accessory subunit and knocked down KAP3 levels using RNAi. We found kinesin-2 to be required for the normal steady-state localization of late endosomes/lysosomes but not early endosomes or recycling endosomes. Despite the abnormal subcellular distribution of late endosomes/lysosomes, the uptake and trafficking of molecules through the conventional endocytic pathway appeared to be unaffected. The slow time-course of inhibition suggests that both kinesin-2 itself and its attachment to membranes do not turn over quickly.     

Fractionation and characterization of kinesin II species in vertebrate brain

Recent research on kinesin motors has outlined the diversity of the superfamily and defined specific cargoes moved by kinesin family (KIF) members. Owing to the difficulty of purifying large amounts of native motors, much of this work has relied on recombinant proteins expressed in vitro. This approach does not allow ready determination of the complement of kinesin motors present in a given tissue, the relative amounts of different motors, or comparison of their native activities. To address these questions, we isolated nucleotide-dependent, microtubule-binding proteins from 13-day chick embryo brain. Proteins were enriched by microtubule affinity purification, then subjected to velocity sedimentation to separate the 20S dynein/dynactin pool from a slower sedimenting KIF containing pool. Analysis of the latter pool by anion exchange chromatography revealed three KIF species: kinesin I (KIF5), kinesin II (KIF3), and KIF1C (Unc104/KIF1). The most abundant species, kinesin I, exhibited the expected long range microtubule gliding activity. By contrast, KIF1C did not move microtubules. Kinesin II, the second most abundant KIF, could be fractionated into two pools, one containing predominantly A/B isoforms and the other containing A/C isoforms. The two motor species had similar activities, powering microtubule gliding at slower speeds and over shorter distances than kinesin I.     

Engineering the processive run length of the kinesin motor

Conventional kinesin is a highly processive molecular motor that takes several hundred steps per encounter with a microtubule. Processive motility is believed to result from the coordinated, hand-over-hand motion of the two heads of the kinesin dimer, but the specific factors that determine kinesin's run length (distance traveled per microtubule encounter) are not known. Here, we show that the neck coiled-coil, a structure adjacent to the motor domain, plays an important role in governing the run length. By adding positive charge to the neck coiled-coil, we have created ultra-processive kinesin mutants that have fourfold longer run lengths than the wild-type motor, but that have normal ATPase activity and motor velocity. Conversely, adding negative charge on the neck coiled-coil decreases the run length. The gain in processivity can be suppressed by either proteolytic cleavage of tubulin's negatively charged COOH terminus or by high salt concentrations. Therefore, modulation of processivity by the neck coiled-coil appears to involve an electrostatic tethering interaction with the COOH terminus of tubulin. The ability to readily increase kinesin processivity by mutation, taken together with the strong sequence conservation of the neck coiled-coil, suggests that evolutionary pressures may limit kinesin's run length to optimize its in vivo function.     

From cilia and flagella to intracellular motility and back again: a review of a few aspects of microtubule-based motility

Ciliary or flagellar movement is the model of microtubule-dependent motility, the best studied at the molecular level. It is based on the relative sliding of outer doublets of microtubules that are linked at their proximal end to the basal structure and interconnected by associated proteins, among which dynein ATPase is at the origin of the movement. It is regulated from inside and outside media by various diffusible factors such as Ca2+, cyclic adenosine monophosphate (cAMP), polypeptides and so on (see other conferences presented during this meeting). Other motility processes are based on microtubules: vesicle and organelle transport through the cytoplasm (axonal flow in neurons, pigment granule movements in fish chromatophores, movements of particles along heliozoan axopods, etc.) could be mediated by microtubule motors such as kinesin or MAP 1C. Kinesin and MAP 1C, like dynein, are proteins that bind to microtubules and show an ATPase activity associated with force production. They differ from each other by their structure, and biochemical and pharmacological properties. The movements of chromosomes during mitosis and meiosis have long been studied, but are still poorly understood at the molecular level; this topic will be discussed in the light of recent data. Other constituents of the cytoskeleton are certainly involved in cellular motility: actin microfilaments and their motor myosin, intermediate filaments, non-actin filaments, all organized around the Microtubule Organizing Center (MTOC). As more information becomes available, it seems increasingly obvious that these various networks are closely interconnected and that each component probably modulates, resists, or favors properties of its partners, contributing to cellular and intracellular motility.     

Effect of temperature on kinesin-driven microtubule gliding and kinesin ATPase activity

DeCuevas et al. [J. Cell Biol. 116 (1992) 957-965] demonstrated by circular dichroism spectroscopy for the kinesin stalk fragment that shifting temperature from 25 to 30 degrees C caused a conformational transition. To gain insight into functional consequences of such a transition, we studied the temperature dependence of a full-length kinesin by measuring both the velocity of microtubule gliding across kinesin-coated surfaces and microtubule-promoted kinesin ATPase activity in solution. The corresponding Arrhenius plots revealed distinct breaks at 27 degrees C, corroborating the temperature-dependent conformational transition for a motility-competent full-length kinesin. Microtubules were found to glide up to 45 degrees C; at higher temperatures, kinesin was irreversibly damaged.     

Epimorphin Functions as a Key Morphoregulator for Mammary Epithelial Cells

Hepatocyte growth factor (HGF) and EGF have been reported to promote branching morphogenesis of mammary epithelial cells. We now show that it is epimorphin that is primarily responsible for this phenomenon. In vivo, epimorphin was detected in the stromal compartment but not in lumenal epithelial cells of the mammary gland; in culture, however, a subpopulation of mammary epithelial cells produced significant amounts of epimorphin. When epimorphin-expressing epithelial cell clones were cultured in collagen gels they displayed branching morphogenesis in the presence of HGF, EGF, keratinocyte growth factor, or fibroblast growth factor, a process that was inhibited by anti-epimorphin but not anti-HGF antibodies. The branch length, however, was roughly proportional to the ability of the factors to induce growth. Accordingly, epimorphin-negative epithelial cells simply grew in a cluster in response to the growth factors and failed to branch. When recombinant epimorphin was added to these collagen gels, epimorphin-negative cells underwent branching morphogenesis. The mode of action of epimorphin on morphogenesis of the gland, however, was dependent on how it was presented to the mammary cells. If epimorphin was overexpressed in epimorphin-negative epithelial cells under regulation of an inducible promoter or was allowed to coat the surface of each epithelial cell in a nonpolar fashion, the cells formed globular, alveoli-like structures with a large central lumen instead of branching ducts. This process was enhanced also by addition of HGF, EGF, or other growth factors and was inhibited by epimorphin antibodies. These results suggest that epimorphin is the primary morphogen in the mammary gland but that growth factors are necessary to achieve the appropriate cell numbers for the resulting morphogenesis to be visualized.     

The motor domain of testis-enriched kinesin KIF9 is essential for its localization in the mouse flagellum

Kinesin is a molecular motor that moves along microtubules. Testis-enriched kinesin KIF9 (Kinesin family member 9) is localized in the mouse sperm flagellum and is important for normal sperm motility and male fertility; however, it is unclear if the motor domain of KIF9 is involved in these processes. In this study, we substituted threonine of the ATP binding motif in the KIF9 motor domain to asparagine (T100N) in mice using the CRISPR/Cas9 system, which is known to impair kinesin motor activity. T100N mutant mice exhibit reduced sperm motility and male fertility consistent with Kif9 knockout mice. Further, KIF9 was depleted in the spermatozoa of T100N mutant mice although the amounts of KIF9 were comparable between wild-type and T100N mutant testes. These results indicate that the motor domain of KIF9 is essential for its localization in the sperm flagellum.     

Crystal structure of the Kar3-like kinesin motor domain from the filamentous fungus Ashbya gossypii

Kar3 kinesins are microtubule (MT) minus‐end‐directed motors with pleiotropic functions in mitotic spindle formation and nuclear movement in budding and fission yeasts. A Kar3‐like kinesin is also expressed by the filamentous fungus Ashbya gossypi, which exhibits different nuclear movement challenges from its yeast relatives. Presented here is a 2.35 Å crystal structure and enzymatic analysis of the AgKar3 motor domain (AgKar3MD). Compared to the previously published Saccharomyces cerevisiae Kar3MD structure (ScKar3MD), AgKar3MD displays differences in the conformation of some of its nucleotide‐binding motifs and peripheral elements. Unlike ScKar3MD, the salt bridge between Switch I and Switch II in AgKar3MD is broken. Most of the Switch I, and the adjoining region of helix α3, are also disordered instead of bending into the active site cleft as is observed in ScKar3MD. These aspects of AgKar3MD are highly reminiscent of the ScKar3 R598A mutant that disrupts the Switch I–Switch II salt bridge and impairs MT‐stimulated ATPase activity of the motor. Subtle differences in the disposition of secondary structure elements in the small lobe (β1a, β1b, and β1c) at the edge of the MD are also apparent even though it contains approximately the same number of residues as ScKar3. These differences may reflect the unique enzymatic properties we measured for this motor, which include a lower MT‐stimulated ATPase rate relative to ScKar3, or they could relate to its interactions with different regulatory companion proteins than its budding yeast counterpart. Proteins 2011;. © 2011 Wiley Periodicals, Inc.     

A pathway of neuregulin-induced activation of cofilin-phosphatase Slingshot and cofilin in lamellipodia

Cofilin mediates lamellipodium extension and polarized cell migration by stimulating actin filament dynamics at the leading edge of migrating cells. Cofilin is inactivated by phosphorylation at Ser-3 and reactivated by cofilin-phosphatase Slingshot-1L (SSH1L). Little is known of signaling mechanisms of cofilin activation and how this activation is spatially regulated. Here, we show that cofilin-phosphatase activity of SSH1L increases approximately 10-fold by association with actin filaments, which indicates that actin assembly at the leading edge per se triggers local activation of SSH1L and thereby stimulates cofilin-mediated actin turnover in lamellipodia. We also provide evidence that 14-3-3 proteins inhibit SSH1L activity, dependent on the phosphorylation of Ser-937 and Ser-978 of SSH1L. Stimulation of cells with neuregulin-1beta induced Ser-978 dephosphorylation, translocation of SSH1L onto F-actin-rich lamellipodia, and cofilin dephosphorylation. These findings suggest that SSH1L is locally activated by translocation to and association with F-actin in lamellipodia in response to neuregulin-1beta and 14-3-3 proteins negatively regulate SSH1L activity by sequestering it in the cytoplasm.     

Vaccinia virus proteins A36 and F12/E2 show strong preferences for different kinesin light chain isoforms

Vaccinia virus (VACV) utilizes microtubule‐mediated trafficking at several stages of its life cycle, of which virus egress is the most intensely studied. During egress VACV proteins A36, F12 and E2 are involved in kinesin‐1 interactions; however, the roles of these proteins remain poorly understood. A36 forms a direct link between virions and kinesin‐1, yet in its absence VACV egress still occurs on microtubules. During a co‐immunoprecipitation screen to seek an alternative link between virions and kinesin, A36 was found to bind isoform KLC1 rather than KLC2. The F12/E2 complex associates preferentially with the C‐terminal tail of KLC2, to a region that overlaps the binding site of cellular 14‐3‐3 proteins. F12/E2 displaces 14‐3‐3 from KLC and, unlike 14‐3‐3, does not require phosphorylation of KLC for its binding. The region determining the KLC1 specificity of A36 was mapped to the KLC N‐terminal heptad repeat region that is responsible for its association with kinesin heavy chain. Despite these differing binding properties F12/E2 can co‐operatively enhance A36 association with KLC, particularly when using a KLC1‐KLC2 chimaera that resembles several KLC1 spliceforms and can bind A36 and F12/E2 efficiently. This is the first example of a pathogen encoding multiple proteins that co‐operatively associate with kinesin‐1.      

Microtubule formation and kinesin-driven microtubule gliding in vitro in the presence of lipopolysaccharide

Lipopolysaccharide (LPS) is a main trigger substance for the development of septic shock and multiple organ failure. We showed by turbidity measurements that LPS inhibits microtubule formation in a pH-dependent manner. Inhibition was found to be not only due to sequestration of MAP2 by LPS, but also of MAP1 and tau MAPs, indicating that LPS is able to react with a broad variety of MAPs. LPS-induced inhibition of microtubule formation could be compensated by additional tau or by addition of taxol. Dot blots revealed that LPS binds directly to tau, but seems not to bind to tubulin. As tau is expressed in various tissue types involved in multiorgan failure, it might be regarded as a further target for LPS action. In contrast, kinesin-dependent microtubule gliding was not affected by LPS. The toxin neither blocked the cargo (vesicle) nor the microtubule binding site of kinesin, suggesting a certain specificity of LPS-MAP interaction.     

Combinatorial and antagonistic effects of tubulin glutamylation and glycylation on katanin microtubule severing

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A structural pathway for activation of the kinesin motor ATPase

Molecular motors move along actin or microtubules by rapidly hydrolyzing ATP and undergoing changes in filament-binding affinity with steps of the nucleotide hydrolysis cycle. It is generally accepted that motor binding to its filament greatly increases the rate of ATP hydrolysis, but the structural changes in the motor associated with ATPase activation are not known. To identify the conformational changes underlying motor movement on its filament, we solved the crystal structures of three kinesin mutants that decouple nucleotide and microtubule binding by the motor, and block microtubule-activated, but not basal, ATPase activity. Conformational changes in the structures include a disordered loop and helices in the switch I region and a visible switch II loop, which is disordered in wild-type structures. Switch I moved closer to the bound nucleotide in two mutant structures, perturbing water-mediated interactions with the Mg2+. This could weaken Mg2+ binding and accelerate ADP release to activate the motor ATPASE: The structural changes we observe define a signaling pathway within the motor for ATPase activation that is likely to be essential for motor movement on microtubules.