Microtubule Gliding Research Articles

Microtubules and motors

New tools and approaches are providing exciting new insights into the structure and function of motors and microtubules and their contributions to cell migration, mitosis, and neuronal function. Janel Titus, from the Wadsworth laboratory (University of Massachusetts, Amherst), discussed the regulation of the plus end–directed mitotic motor Eg5 by the spindle assembly factor, TPX2. Spindle formation in cells expressing truncated TPX2, which lacks the Eg5-binding region, was defective. Acute inhibition of the TPX2-Eg5 interaction in metaphase cells resulted in spindle lengthening, suggesting TPX2 functions to modulate or restrict Eg5 motor activity. To test this possibility, in vitro biophysical experiments with purified proteins were used to show that TPX2 reduces Eg5-dependent microtubule gliding and sliding and is important for targeting Eg5 to the microtubule. Microtubules are dynamic polymers that convert stochastically between growing and shrinking phases. Joe Howard, from the Max Planck Institute of Molecular Cell Biology and Genetics, reported the unexpected discovery that catastrophe, the conversion of a growing microtubule to a shrinking one, is a multistep process in which the rate of catastrophe increases with time. The budding yeast kinesin-8, Kip3, increases catastrophe frequency by increasing the rate of each step, whereas the mammalian kinesin-13 MCAK increases catastrophe frequency by decreasing the number of steps to catastrophe. By differentially regulating different aspects of the catastrophe mechanism, the kinesins are able to differentially control both microtubule length and its variance. In polarized, directionally migrating cells, microtubules show persistent growth at the leading edge, but the mechanism responsible for local control of microtubule dynamics is not established. Kenneth Myers, from Clare Waterman's laboratory (National Institutes of Health), used automated microtubule tracking to test the contribution of the microtubule depolymerase mitotic centromere-associated kinesin (MCAK) to this process. Knockdown of MCAK homogenized microtubule dynamic instability by promoting long-lived microtubule growth throughout the entire cell, resulting in the loss of directed cell migration. The effects of MCAK on microtubule dynamics were discovered to be dependent on the activity of Aurora A kinase, suggesting that active Aurora A regulates the depolymerase activity of MCAK in migrating cells. Expression of a constitutively active form of the small GTPase Rac1 promoted long-lived microtubule growth in an MCAK- and Aurora A–dependent manner, establishing a signaling pathway in which local inhibition of MCAK depolymerase activity downstream of Rac1 and Aurora A drives polarized microtubule growth and thereby promotes directed cell migration. Melissa Rolls (Pennsylvania State University), winner of the ASCB 2011 Women in Cell Biology Junior Award, described her work on the microtubule cytoskeleton of neurons and how axon injury triggers a microtubule-based pathway that protects dendrites. Working with Drosophila neurons, the Rolls group observed an increase in growing microtubules after injury. The increased microtubule dynamics were dependent on new nucleation, but were not required for axon regeneration. Rather, microtubule dynamics protected dendrites from degeneration following injury. The results indicate that a dynamic microtubule array protects dendrites while the cell is attempting to repair itself. Cytoplasmic dynein transports diverse cargoes in cells and can tether cargoes at specific destinations. Anthony Roberts, from the Reck-Peterson laboratory (Harvard Medical School), addressed the mechanism of dynein regulation, using purified yeast proteins and single-molecule microscopy. The data show that Lis1/Pac1 reduces dynein velocity and increases encounter time with the microtubule, inducing a novel anchored state of the enzyme without catalytic arrest. Structural work shows that Lis1 binds at the interface between dynein's ATPase domain and the microtubule-binding stalk. Taken together, the results suggest that Lis1 could alter communication between different motor domains, thus regulating motor function in cells. Antonina Roll-Mecak (National Institutes of Health) presented the first structural studies of tubulin tyrosine ligase (TTL), the enzyme responsible for the addition of tyrosine to the C-terminus of α-tubulin. Although tyrosination–detyrosination of tubulin has long been known to affect microtubule dynamics, the molecular mechanism of TTL is not established. The new results show that TTL engages the C-terminal tail of tubulin via low-affinity, high-specificity interactions. Additionally, the enzyme forms a tight complex with tubulin, which blocks the longitudinal tubulin interface and prevents tubulin addition onto the microtubule end. These insights suggest that the TTL interaction with tubulin could affect the polymerization-competent pool of tubulin in cells.

In Vitro Gliding Assays Indicate that Chlamydomonas Dynein Moves Microtubules Polymerized from Chlamydomonas Axonemal Tubulin Faster than those Polymerized from Porcine Brain Tubulin

The axonemal dyneins drive the motility of cilia and flagella. Dynein activity must be coordinated spatially and temporally within the axoneme to generate the regular, repeating waveforms characteristic of cilial motion. Recent studies have suggested that the mechanochemical properties of dynein, in particular the dependance of velocity and dissociation constants on force, may be sufficient to achieve coordination. Because the processes involving these properties require interactions between the dyneins and the microtubules, it is likely that they are dependent on the specific dynein-tubulin interactions. We investigated this possibility using microtubule gliding assays with outer arm dynein from Chlamydomonas. We found that that the translocation speed of microtubules polymerized from Chlamydomonas tubulin is 1.6 times faster than the speed of porcine brain microtubules. Additionally, we found that the source of the tubulin also influenced how translocation speed depended on microtubule length. Both of these results suggest that dynein from Chlamydomonas prefers Chlamydomonas axonemal tubulin despite greater than 85% similarity in primary structure. Finally, we found that the instantaneous translocation speed of microtubules in the gliding assay is unsteady. Our analysis suggests that the source of this unsteadiness may arise from the same mechanochemical properties of dynein that have been predicted to be sufficient for coordination. Together these results suggest that the interactions between dynein and tubulin are important factors in axonemal dynein coordination.

TPX2 regulates the localization and activity of Eg5 in the mammalian mitotic spindle

Mitotic spindle assembly requires the regulated activity of numerous spindle-associated proteins. In mammalian cells, the Kinesin-5 motor Eg5 interacts with the spindle assembly factor TPX2, but how this interaction contributes to spindle formation and function is not established. Using bacterial artificial chromosome technology, we generated cells expressing TPX2 lacking the Eg5 interaction domain. Spindles in these cells were highly disorganized with multiple spindle poles. The TPX2-Eg5 interaction was required for kinetochore fiber formation and contributed to Eg5 localization to spindle microtubules but not spindle poles. Microinjection of the Eg5-binding domain of TPX2 resulted in spindle elongation, indicating that the interaction of Eg5 with TPX2 reduces motor activity. Consistent with this possibility, we found that TPX2 reduced the velocity of Eg5-dependent microtubule gliding, inhibited microtubule sliding, and resulted in the accumulation of motor on microtubules. These results establish a novel function of TPX2 in regulating the location and activity of the mitotic motor Eg5.

Visualization of dynein-dependent microtubule gliding at the cell cortex: implications for spindle positioning

Dynein motors move along the microtubule (MT) lattice in a processive "walking" manner. In the one-cell Caenorhabditis elegans embryo, dynein is required for spindle-pulling forces during mitosis. Posteriorly directed spindle-pulling forces are higher than anteriorly directed forces, and this imbalance results in posterior spindle displacement during anaphase and an asymmetric division. To address how dynein could be asymmetrically activated to achieve posterior spindle displacement, we developed an assay to measure dynein's activity on individual MTs at the embryo cortex. Our study reveals that cortical dynein motors maintain a basal level of activity that propels MTs along the cortex, even under experimental conditions that drastically reduce anaphase spindle forces. This suggests that dynein-based MT gliding is not sufficient for anaphase spindle-pulling force. Instead, we find that this form of dynein activity is most prominent during spindle centering in early prophase. We propose a model whereby different dynein-MT interactions are used for specific spindle-positioning tasks in the one-cell embryo.

A New Microtubule Gliding Assay Analysis of Microtubule Persistence Length

Microtubules are cytoskeletal polymers which play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires microtubules that are stiff and straight enough to span a significant fraction of the cell diameter. As a result, the microtubule persistence length, a measure of stiffness, has been actively studied for the past 15 years. Curiously, short microtubules are 10-50 times less stiff than long microtubules. In order to study this phenomenon, we have developed a new kinesin-driven microtubule gliding assay analysis of persistence length. By combining sparse fluorescent labeling of individual microtubules with single particle tracking of individual fluorophores, we tracked microtubule gliding trajectories with nanometer-level precision. By varying the surface density of kinesin, we measured the persistence length of microtubules on length scales from 100s of nanometers to 10s of micrometers. Microtubule persistence length increased from about 200 micrometers to 3 mm across these scales, consistent with previous experiments at short or long length scales. The scale dependence of persistence length we measured supports models in which protofilaments are loosely coupled near microtubule tips, becoming tightly coupled far from the tip.

Synthesis of Photochromic ATP Analogue and its Interaction with Motor Protein

Azobenzene is one of the photochromic molecules, which undergoes rapid and reversible transitions between the cis isomer and trans isomer by visible and ultra-violet light irradiation. We have been trying to control the activities of motor proteins using the photochromic molecules as photo-regulatory devices. We have recently demonstrated that microtubules dependent ATPase activity of the kinesin modified by azobenzene derivative was regulated by UV-VIS light irradiation. However, it was not so easy to incorporate the photochromic molecules into the functional site of motor proteins without altering the native enzymatic properties. In the present study, we have designed the novel ATP analogues consist of photochromic molecules in order to photo-regulate the motor protein kinesin without their chemical modification. It is expected that the ATP analogues induce the reversible conformational change in the active site by alternate UV-VIS light irradiation. We have synthesized non-nucleotide ATP analogue composed of azobenzene derivative, Phenylazobenzoic-aminoethyl -triphosphate (PABATP). PABATP showed UV/VIS light absorption spectral change accompanied by transition between cis and trans in a similar manner observed in azobenzene. PABATP was hydrolyzed by conventional kinesin and the hydrolysis rate was activated by microtubules. It has been demonstrated that the cis isomer and trans isomer perform differently on a microtubule gliding motility assay. We have also examined the formation of kinesin-PABADP-Pi analogues (BeFn, AlF4-, Vi) which may mimic the transient states in ATPase cycle.

Ca 2+ Dependent Dimerization of Kinesin-CaM and Kinesin-M13 Fusion Proteins

Kinesin is known as a dimeric motor protein, which carries cellular cargoes along microtubules by hydrolyzing ATP. Calmodulin (CaM) is a calcium binding protein that participates in cellular regulatory processes. CaM undergoes a conformational change upon binding to calcium, which enables it to bind to specific proteins for a specific response. We have previously demonstrated that kinesin fused with CaM at the C-terminal binds reversibly to M13-Qdots in a calcium dependent manner. In this study, we tried to make the calcium dependent reversible dimerization of kinesin utilizing CaM- target peptide M13 binding system in order to control motility of kinesin. First we designed and prepared the cDNA of the truncated kinesin (355 amino acids) that does not form dimer. We prepared the cDNA encoding two kinesin chimeric proteins in which C-terminal of kinesin355 was fused with calmodulin (K355-CaM) and fused with M13-Cys (K355-M13-cys). The cDNAs of the kinesin chimeras were cloned into expression vector pET21a and transformed into E.coli BL21(DE3). The kinesin chimeras were successfully expressed and purified by Co-Chelate column. These kinesin chimeras showed normal ATPase activities. Furthermore, K355-CaM bound to M13-YFP in a calcium dependent manner. The calcium dependent interaction between K355-CaM and K355-M13-Cys was examined using Size-exclusion chromatography (SEC)-HPLC, whereas two chimeras did not bind in the absence of Ca2+. In vitro motility assay demonstrated that the dimerized chimera induced microtubule gliding in the presence of Ca2+.

Distinct roles of 1α and 1β heavy chains of the inner arm dynein I1 ofChlamydomonasflagella

The Chlamydomonas I1 dynein is a two-headed inner dynein arm important for the regulation of flagellar bending. Here we took advantage of mutant strains lacking either the 1α or 1β motor domain to distinguish the functional role of each motor domain. Single- particle electronic microscopic analysis confirmed that both the I1α and I1β complexes are single headed with similar ringlike, motor domain structures. Despite similarity in structure, however, the I1β complex has severalfold higher ATPase activity and microtubule gliding motility compared to the I1α complex. Moreover, in vivo measurement of microtubule sliding in axonemes revealed that the loss of the 1β motor results in a more severe impairment in motility and failure in regulation of microtubule sliding by the I1 dynein phosphoregulatory mechanism. The data indicate that each I1 motor domain is distinct in function: The I1β motor domain is an effective motor required for wild-type microtubule sliding, whereas the I1α motor domain may be responsible for local restraint of microtubule sliding.

Tau isoform‐specific modulation of kinesin‐driven microtubule gliding rates and trajectories as determined with tau‐stabilized microtubules

We have utilized tau-assembled and tau-stabilized microtubules (MTs), in the absence of taxol, to investigate the effects of tau isoforms with three and four MT binding repeats upon kinesin-driven MT gliding. MTs were assembled in the presence of either 3-repeat tau (3R tau) or 4-repeat tau (4R tau) at tau:tubulin dimer molar ratios that approximate those found in neurons. MTs assembled with 3R tau glided at 31.1 μm/min versus 25.8 μm/min for 4R tau, a statistically significant 17% difference. Importantly, the gliding rates for either isoform did not change over a fourfold range of tau concentrations. Further, tau-assembled MTs underwent minimal dynamic instability behavior while gliding and moved with linear trajectories. In contrast, MTs assembled with taxol in the absence of tau displayed curved gliding trajectories. Interestingly, addition of 4R tau to taxol-stabilized MTs restored linear gliding, while addition of 3R tau did not. The data are consistent with the ideas that (i) 3R and 4R tau-assembled MTs possess at least some isoform-specific features that impact upon kinesin translocation, (ii) tau-assembled MTs possess different structural features than do taxol-assembled MTs, and (iii) some features of tau-assembled MTs can be masked by prior assembly by taxol. The differences in kinesin-driven gliding between 3R and 4R tau suggest important features of tau function related to the normal shift in tau isoform composition that occurs during neural development as well as in neurodegeneration caused by altered expression ratios of otherwise normal tau isoforms.

Cargo Loading onto Kinesin Powered Molecular Shuttles

Cells have evolved sophisticated molecular machinery, such as kinesin motor proteins and microtubule filaments, to support active intracellular transport of cargo. While kinesins tail domain binds to a variety of cargoes, kinesins head domains utilize the chemical energy stored in ATP molecules to step along the microtubule lattice. The long, stiff microtubules serve as tracks for long-distance intracellular transport. These motors and filaments can also be employed in microfabricated synthetic environments as components of molecular shuttles. In a frequently used design, kinesin motors are anchored to the track surface through their tails, and functionalized microtubules serve as cargo carrying elements, which are propelled by these motors. These shuttles can be loaded with cargo by utilizing the strong and selective binding between biotin and streptavidin. The key components (biotinylated tubulin, streptavidin, and biotinylated cargo) are commercially available. Building on the classic inverted motility assay, the construction of molecular shuttles is detailed here. Kinesin motor proteins are adsorbed to a surface precoated with casein; microtubules are polymerized from biotinylated tubulin, adhered to the kinesin and subsequently coated with rhodamine-labeled streptavidin. The ATP concentration is maintained at subsaturating concentration to achieve a microtubule gliding velocity optimal for loading cargo. Finally, biotinylated fluorescein-labeled nanospheres are added as cargo. Nanospheres attach to microtubules as a result of collisions between gliding microtubules and nanospheres adhering to the surface. The protocol can be readily modified to load a variety of cargoes such as biotinylated DNA, quantum dots or a wide variety of antigens via biotinylated antibodies.

Cargo Loading onto Kinesin Powered Molecular Shuttles

Cells have evolved sophisticated molecular machinery, such as kinesin motor proteins and microtubule filaments, to support active intracellular transport of cargo. While kinesins tail domain binds to a variety of cargoes, kinesins head domains utilize the chemical energy stored in ATP molecules to step along the microtubule lattice. The long, stiff microtubules serve as tracks for long-distance intracellular transport. These motors and filaments can also be employed in microfabricated synthetic environments as components of molecular shuttles 1. In a frequently used design, kinesin motors are anchored to the track surface through their tails, and functionalized microtubules serve as cargo carrying elements, which are propelled by these motors. These shuttles can be loaded with cargo by utilizing the strong and selective binding between biotin and streptavidin. The key components (biotinylated tubulin, streptavidin, and biotinylated cargo) are commercially available. Building on the classic inverted motility assay 2, the construction of molecular shuttles is detailed here. Kinesin motor proteins are adsorbed to a surface precoated with casein; microtubules are polymerized from biotinylated tubulin, adhered to the kinesin and subsequently coated with rhodamine-labeled streptavidin. The ATP concentration is maintained at subsaturating concentration to achieve a microtubule gliding velocity optimal for loading cargo 3. Finally, biotinylated fluorescein-labeled nanospheres are added as cargo. Nanospheres attach to microtubules as a result of collisions between gliding microtubules and nanospheres adhering to the surface. The protocol can be readily modified to load a variety of cargoes such as biotinylated DNA4, quantum dots 5 or a wide variety of antigens via biotinylated antibodies 4-6.

Mitotic Kinesin CENP-E Promotes Microtubule Plus-End Elongation

Centromere protein CENP-E is a dimeric kinesin (Kinesin-7 family) with critical roles in mitosis, including establishment of microtubule (MT)-chromosome linkage and movement of mono-oriented chromosomes on kinetochore microtubules for proper alignment at metaphase [1-9]. We performed studies to test the hypothesis that CENP-E promotes MT elongation at the MT plus ends. A human CENP-E construct was engineered, expressed, and purified, and it yielded theCENP-E-6His dimeric motor protein. The results show that CENP-E promotes MT plus-end-directed MT gliding at 11 nm/s. The results from real-time microscopy assays indicate that 60.3% of polarity-marked MTs exhibited CENP-E-promoted MT plus-end elongation. The MT extension required ATP turnover, and MT plus-end elongation occurred at 1.48 μm/30 min. Immunolocalization studies revealed that 80.8% of plus-end-elongated MTs showed CENP-E at the MT plus end. The time dependence of CENP-E-promoted MT elongation in solution best fit a single exponential function (k(obs) = 5.1 s(-1)), which is indicative of a mechanism in which α,β-tubulin subunit addition is tightly coupled to ATP turnover. Based on these results, we propose that CENP-E, as part of its function in chromosome kinetochore-MT linkage, plays a direct role in MT elongation.

Strikingly fast microtubule sliding in bundles formed by Chlamydomonas axonemal dynein

Chlamydomonas axonemal extracts containing outer-arm dynein bundle microtubules when added in the absence of ATP. The bundles dissociate after addition of ATP (Haimo et al., Proc Natl Acad Sci USA 76:5759-5768, 1979). In the present study, we investigated the ATP-induced bundle dissociation process using caged ATP. Application of approximately 0.5 mM ATP induced microtubule sliding at approximately 30 microm.s(-1), which was 1.5 times faster than the microtubule sliding observed in protease-treated axonemes and five times faster than microtubule gliding on glass surfaces coated with outer-arm dynein. Bundles formed by mutant dynein molecules that lack one of the three heavy chains (HCs) displayed similar high-speed intermicrotubule sliding. These results suggest that Chlamydomonas outer-arm dynein molecules, when aligned, can translocate microtubules at high speed and that the high-speed sliding under load-free conditions does not require the complete set of the three HCs. It is likely that each of the three HCs has the ability to produce high-speed sliding, which should be an important property for their cooperation.

Microtubule Motility on Reconstituted Meiotic Chromatin

During cell division, correct positioning of chromosomes in mitotic and meiotic spindles depends on interactions of microtubules with kinetochores and, especially in higher eukaryotes, with the chromosome arms [1, 2]. Chromokinesins, highly concentrated on mitotic and meiotic chromatin, are thought to actively push the chromosome arms toward the spindle center, thereby contributing to chromosome alignment at the metaphase plate in early mitosis [1-9]. How many distinct classes of chromokinesins exist and how they cooperate to form a motile chromatin-microtubule interface are not known. Using a novel experimental assay with nonkinetochore chromatin reconstituted from Xenopus egg extract, we demonstrate that the microtubule motility generated on chromatin is continuous and plus-end directed. Using specific antibody depletions, we identify two distinct chromokinesins, kinesin-10 (Xkid) [8, 10, 11] and kinesin-4 (Xklp1) [12, 13], as the major activities mediating the interaction of meiotic chromatin with microtubules. Interestingly, we find that the slower motor, kinesin-10, more efficiently recruits microtubules and also dominates in collective microtubule transport both in the close-to-physiological environment of chromatin and also in a minimal in vitro assay. Our results provide an identification of the molecular activities involved in the generation of motor protein-mediated chromosome arm motility and yield mechanistic insight into the cooperation of the two major chromokinesins.

The Effects of Removal of C-termini of Tubulin for Mitotic Kinesin CENP-E Microtubule Interactions

The carboxyl-terminus of both a- and b- tubulin consists of helices H11 and H12 and are located on the outer surface of the microtubule (MT). H12 extends into a disordered region that is characterized by a highly negatively charged motif of approximately 10-18 residues (CTT). These MT CTTs have been shown to affect the MT interactions with kinesin molecular motors for function. The research presented here tests the hypothesis that the MT CTTs modulate the behavior of the kinesin motor CENP-E with the MT and affect ATP-promoted motility. Subtilisin was used to remove the CTTs of a,b-tubulin, and the impact of the loss of the CTTs was evaluated by MT-CENP-E cosedimentation and CENP-E promoted MT gliding assays. The cosedimentation results show a dramatic decrease in MT affinity to subtilisin-MTs in the presence of 1 mM MgAMPPNP although 100% CENP-E binding is achieved. In contrast, the 1 mM MgADP results demonstrate an increase in CENP-E binding to subtilisin-MTs and an increase in MT affinity in comparison to native MTs. The in vitro motility assays reveal an increase in the rate of CENP-E promoted MT gliding on subtilisin MTs at 7.2 nm/s relative to 5.1 nm/s on native MTs. However, after 15 min, most subtilisin-MTs detached from the coverslip suggesting almost complete dissociation of the subtilisin-MT-CENP-E complex. In contrast, CENP-E promoted native MT gliding for >60 min. This study provides further evidence for the importance of the electrostatic interactions mediated by the MT CTTs for CENP-E mitotic function.

Single Molecule Analysis of the Mitotic Kinesin Eg5

In vertebrates, proper formation of the mitotic spindle requires the activity of Eg5, a motor protein of the kinesin-5 family. Loss of Eg5 function leads to monopolar spindles and mitotic arrest. Eg5's homotetrameric configuration, in which two pairs of motor domains are connected by a central stalk, allows it to crosslink and slide microtubules. Previous single molecule fluorescence studies of Eg5 have shown that in physiological ionic strength its motion along single microtubules is predominantly diffusive and ATP-independent, but becomes predominantly directional upon crosslinking two microtubules. The structural and mechanistic basis of this allosteric regulation of Eg5's motor activity is poorly understood. One possibility is that Eg5's non-motor domains, including the central stalk and C-terminal tail, transmit signals of microtubule binding state across the Eg5 tetramer and regulate motor activity. To probe the roles of Eg5's non-motor domains and to establish structure-function relationships for these domains, we have generated a series of GFP-labeled Eg5 deletion constructs, including a dimeric version and tetrameric versions containing deletions of the motor domain and of the C-terminal tail domain. Each of the constructs, including one that lacks the kinesin motor domain, binds microtubules with varying affinities. In a multiple motor microtubule gliding assay, deletion of the tail domain results in a 50% increase in gliding velocity, from 15 nm/s for full-length Eg5, to 23 nm/s for tail-less Eg5. This indicates that the tail domain is capable of attenuating Eg5 motility. To understand the contributions of the Eg5's domains to its directional and diffusive movement, we are using single molecule fluorescence microscopy to characterize the motility along microtubules for each construct. These studies suggest a model wherein the non-motor domains of Eg5 modulate its motor activity and contribute to its ability to associate with microtubules.

“Popoffs” Under a Transverse Force Reveal the Number and Location of Active Kinesin Motors During Motility Assays

Determination of the number of active motors pulling a single MT or bead during motility assays has proven difficult. Traditional protein concentration assays, such as Bradford, cannot distinguish between active and inactive motors. We attach a superparamagnetic bead to the (+) end of a microtubule. When placed in a magnet with uniform magnetic field gradient, the bead pulls on the MT with a controllable 0-10 pN force. If the force is perpendicular to the gliding direction of the MT, a short section of the MT “pops off” the surface every 2 to 5 s, as shown in the diagram. This detachment is characterized by rapid motion of the superparamagnetic bead in the direction of higher magnetic field gradient followed by normal microtubule gliding velocity when the MT is pulled taut. The length of the short section between “popoffs” is the distance between active kinesins along a microtubule.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Interactions between Subunits in Heterodimeric Ncd Molecules

The nonprocessive minus-end-directed kinesin-14 Ncd is involved in the organization of the microtubule (MT) network during mitosis. Only one of the two motor domains is involved in the interaction with the MT. The other head is tethered to the bound one. Here we prepared, purified, and characterized mutated Ncd molecules carrying point mutations in one of the heads, thus producing heterodimeric motors. The mutations tested included substitutions in Switch I and II: R552A, E585A, and E585D; the decoupling mutant N600K; and a deletion in the motor domain in one of the subunits resulting in a single-headed molecule (NcN). These proteins were isolated by two sequential affinity chromatography steps, followed by measurements of their affinities to MT, enzymatic properties, and the velocity of the microtubule gliding test in vitro. A striking observation is a low affinity of the single-headed NcN for MT both without nucleotides and in the presence of 5'-adenylyl-beta,gamma-imidodiphosphate, implying that the tethered head has a profound effect on the structure of the Ncd-MT complex. Mutated homodimers had no MT-activated ATPase and no motility, whereas NcN had motility comparable with that of the wild type Ncd. Although the heterodimers had one fully active and one inactive head, the ATPase and motility of Ncd heterodimers varied dramatically, clearly demonstrating that interactions between motor domains exist in Ncd. We also show that the bulk property of dimeric proteins that interact with the filament with only one of its heads depends also on the distribution of the filament-interacting subunits.

Kinesin velocity increases with the number of motors pulling against viscoelastic drag

Although the properties of single kinesin molecular motors are well understood, it is not clear whether multiple motors pulling a single vesicle in a cell cooperate or interfere with one another. To learn how small numbers of motors interact, microtubule gliding assays were carried out with full-length Drosophila kinesin in a novel motility medium containing xanthan, a stiff, water-soluble polysaccharide. At 2 mg/ml xanthan, the zero-shear viscosity of this medium is 1,000 times the viscosity of water, similar to cellular viscosity. To mimic the rheological drag force on the motors when attached to a vesicle in a cell, we attached a 2 microm bead to one end of the microtubule (MT). During gliding assays in our novel medium, the moving bead exerted a drag force of 4-15 pN on the kinesins pulling the MT. The velocity of MTs with an attached bead increased with MT length and with kinesin concentration. The increase with MT length arose because the number of motors is directly proportional to MT length. Our results show that small numbers of kinesins cooperate constructively when pulling against a viscoelastic drag. In the absence of a bead but still in the viscous medium, MT velocity was independent of MT length and kinesin concentration because the thin MT, like a snake moving through grass, was able to move between xanthan molecules with little resistance. A minimal shared-load model in which the number of motors is proportional to MT length fits the observed dependence of gliding velocity on MT length and kinesin concentration.

Kinesin-Calmodulin fusion protein as a molecular shuttle

In this study, we developed a molecular shuttle with reversible cargo-loading system by using calmodulin (CaM) and M13 peptide. We designed a kinesin (K560) chimera protein with CaM fused at the C-terminal tail region of K560 (K560-CaM). K560-CaM was expressed using an Escherichia coli expression system and purified. Its ATPase activity and microtubule gliding velocity were almost in a similar range as those of the wild-type kinesin. Ca(2+)-dependent reversible binding of K560-CaM and M13 peptide was monitored by size-exclusion-HPLC. Rotary shadowing and electron microscopy revealed tetrameric configuration of K560-CaM in the absence of Ca(2+), while both dimeric and tetrameric configurations in the presence of Ca(2+). Further, Ca(2+)-dependent change in the configuration of K560-CaM was monitored by size-exclusion-HPLC and analytical ultracentrifugation. Finally, by total internal reflection fluorescence microscopy, we successfully observed that K560-CaM transported quantum dot-conjugated M13 peptide along the microtubule in the presence of Ca(2+).