Two Tetrahymena kinesin-9 family members exhibit slow plus-end-directed motility in vitro.

The kinesin-9 family comprises two subfamilies specific to ciliated eukaryotic cells, and has recently attracted considerable attention because of its importance in ciliary bending and formation. However, only scattered data are available on the motor properties of kinesin-9 family members; these properties have not been compared under identical experimental conditions using kinesin-9 motors from the same species. Here, we report the comprehensive motor properties of two kinesin-9 molecules of Tetrahymena thermophila, TtK9A (Kif9/Klp1 ortholog) and TtK9B1 (Kif6 ortholog), using microtubule-based in vitro assays, including single-motor and multi-motor assays and microtubule-stimulated ATPase assays. Both subfamilies exhibit microtubule plus-end-directed, extremely slow motor activity, both in single and multiple molecules. TtK9A shows lower processivity than TtK9B1. Our findings indicate that the considerable slow movement of kinesin-9 that corresponds to low ATP hydrolysis rates is a common feature of the ciliary kinesin-9 family.

Tether-scanning the kinesin motor domain reveals a core mechanical action.

Natural kinesin motors are tethered to their cargoes via short C-terminal or N-terminal linkers, whose docking against the core motor domain generates directional force. It remains unclear whether linker docking is the only process contributing directional force or whether linker docking is coupled to and amplifies an underlying, more fundamental force-generating mechanical cycle of the kinesin motor domain. Here, we show that kinesin motor domains tethered via double-stranded DNAs (dsDNAs) attached to surface loops drive robust microtubule (MT) gliding. Tethering using dsDNA attached to surface loops disconnects the C-terminal neck-linker and the N-terminal cover strand so that their dock-undock cycle cannot exert force. The most effective attachment positions for the dsDNA tether are loop 2 or loop 10, which lie closest to the MT plus and minus ends, respectively. In three cases, we observed minus-end-directed motility. Our findings demonstrate an underlying, potentially ancient, force-generating core mechanical action of the kinesin motor domain, which drives, and is amplified by, linker docking.

Myosin-induced F-actin fragmentation facilitates contraction of actin networks.

Mechanical forces play a crucial role in diverse physiological processes, such as cell migration, cytokinesis, and morphogenesis. The actin cytoskeleton generates a large fraction of the mechanical forces via molecular interactions between actin filaments (F-actins) and myosin motors. Recent studies have shown that the common tendency of actomyosin networks to contract into a smaller structure deeply involves F-actin buckling induced by motor activities, fragmentation of F-actins, and the force-dependent unbinding of cross-linkers that inter-connect F-actins. The fragmentation of F-actins was shown to originate from either buckling or tensile force from previous single-molecule experiments. While the role of buckling in network contraction has been studied extensively, to date, the role of tension-induced F-actin fragmentation in network contraction has not been investigated. In this study, we employed in vitro experiments and an agent-based computational model to illuminate when and how the tension-induced F-actin fragmentation facilitates network contraction. Our experiments demonstrated that F-actins can be fragmented due to tensile forces, immediately followed by catastrophic rupture and contraction of networks. Using the agent-based model, we showed that F-actin fragmentation by tension results in distinct rupture dynamics different from that observed in networks only with cross-linker unbinding. Moreover, we found that tension-induced F-actin fragmentation is particularly important for the contraction of networks with high connectivity. Results from our study shed light on an important regulator of the contraction of actomyosin networks which has been neglected. In addition, our results provide insights into the rupture mechanisms of polymeric network structures and bio-inspired materials.

Effect of temperature on actin filament corkscrewing driven by nonprocessive myosin IC.

Myosin family proteins are ATP-driven, actin filament-based motor proteins that generate force along actin filaments. In in vitro actin filament gliding assays, certain myosins generate rotation of gliding actin filaments around their long axes. In this study, we assessed the effects of temperature on the corkscrewing motion of actin filaments, including factors like gliding and rotational velocities and corkscrewing pitch. The corkscrewing motion was driven by a nonprocessive, full-length single-headed Drosophila myosin IC attached to an antibody adsorbed onto a cover glass. We performed an in vitro actin filament corkscrewing assay at temperatures ranging from 25 °C to 35 °C. We found that the gliding and rotational velocities and the pitch of corkscrewing actin filaments generated by myosin IC molecules increased with increasing temperature. Since the pitch is determined by dividing the gliding velocity by the rotational velocity, an increase in the pitch indicates that the gliding velocity increased faster than the rotational velocity with increasing temperature. These results suggest that temperature has distinct effects on the gliding and rotational forces produced by myosin IC, with implications for interpreting the temperature effect on torque-generation mechanisms driven by myosins on actin filaments at physiological temperatures.

Membrane-bound myosin IC drives the chiral rotation of the gliding actin filament around its longitudinal axis.

Myosin IC, a single-headed member of the myosin I family, specifically interacts with anionic phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2) in the cell membrane via the pleckstrin homology domain located in the myosin IC tail. Myosin IC is widely expressed and physically links the cell membrane to the actin cytoskeleton; it plays various roles in membrane-associated physiological processes, including establishing cellular chirality, lipid transportation, and mechanosensing. In this study, we evaluated the motility of full-length myosin IC of Drosophila melanogaster via the three-dimensional tracking of quantum dots bound to actin filaments that glided over a membrane-bound myosin IC-coated surface. The results revealed that myosin IC drove a left-handed rotational motion in the gliding actin filament around its longitudinal axis, indicating that myosin IC generated a torque perpendicular to the gliding direction of the actin filament. The quantification of the rotational motion of actin filaments on fluid membranes containing different PI(4,5)P2 concentrations revealed that the rotational pitch was longer at lower PI(4,5)P2 concentrations. These results suggest that the torque generated by membrane-bound myosin IC molecules can be modulated based on the phospholipid composition of the cell membrane.

Motor generated torque drives coupled yawing and orbital rotations of kinesin coated gold nanorods.

Kinesin motor domains generate impulses of force and movement that have both translational and rotational (torque) components. Here, we ask how the torque component influences function in cargo-attached teams of weakly processive kinesins. Using an assay in which kinesin-coated gold nanorods (kinesin-GNRs) translocate on suspended microtubules, we show that for both single-headed KIF1A and dimeric ZEN-4, the intensities of polarized light scattered by the kinesin-GNRs in two orthogonal directions periodically oscillate as the GNRs crawl towards microtubule plus ends, indicating that translocating kinesin-GNRs unidirectionally rotate about their short (yaw) axes whilst following an overall left-handed helical orbit around the microtubule axis. For orientations of the GNR that generate a signal, the period of this short axis rotation corresponds to two periods of the overall helical trajectory. Torque force thus drives both rolling and yawing of near-spherical cargoes carrying rigidly-attached weakly processive kinesins, with possible relevance to intracellular transport.

Fission yeast Dis1 is an unconventional TOG/XMAP215 that induces microtubule catastrophe to drive chromosome pulling.

The shortening of microtubules attached to kinetochores is the driving force of chromosome movement during cell division. Specific kinesins are believed to shorten microtubules but are dispensable for viability in yeast, implying the existence of additional factors responsible for microtubule shortening. Here, we demonstrate that Dis1, a TOG/XMAP215 ortholog in fission yeast, promotes microtubule shortening to carry chromosomes. Although TOG/XMAP215 orthologs are generally accepted as microtubule polymerases, Dis1 promoted microtubule catastrophe in vitro and in vivo. Notably, microtubule catastrophe was promoted when the tip was attached to kinetochores, as they steadily anchored Dis1 at the kinetochore-microtubule interface. Engineered Dis1 oligomers artificially tethered at a chromosome arm region induced the shortening of microtubules in contact, frequently pulling the chromosome arm towards spindle poles. This effect was not brought by oligomerised Alp14. Thus, unlike Alp14 and other TOG/XMAP215 orthologs, Dis1 plays an unconventional role in promoting microtubule catastrophe, thereby driving chromosome movement.

Torque generating properties of Tetrahymena ciliary three-headed outer-arm dynein.

Eukaryotic cilia/flagella are cellular bio-machines that drive the movement of microorganisms. Molecular motor axonemal dyneins in the axoneme, which consist of an 9 + 2 arrangement of microtubules, play an essential role in ciliary beating. Some axonemal dyneins have been shown to generate torque coupled with the longitudinal motility of microtubules across an array of dyneins fixed to the coverglass surface, resulting in a corkscrew-like translocation of microtubules. In this study, we performed three-dimensional tracking of a microbead coated with axonemal outer-arm dyneins on a freely suspended microtubule. We found that microbeads coated with multiple outer-arm dyneins exhibited continuous right-handed helical trajectories around the microtubule. This unidirectional helical motion differs from that of other types of cytoplasmic dyneins, which exhibit bidirectional helical motility. We also found that, in an in vitro microtubule gliding assay, gliding microtubules driven by outer-arm dyneins tend to turn to the left, causing a curved path, suggesting that the outer-arm dynein itself is able to rotate on its own axis. Two types of torque generated by the axonemal dyneins, corresponding to the forces used to rotate the microtubule unidirectionally with respect to the long and short axes, may regulate ciliary beating with complex waveforms.

Anchoring geometry is a significant factor in determining the direction of kinesin-14 motility on microtubules.

Kinesin-14 microtubule-based motors have an N-terminal tail attaching the catalytic core to its load and usually move towards microtubule minus ends, whilst most other kinesins have a C-terminal tail and move towards plus ends. Loss of conserved sequences external to the motor domain causes kinesin-14 to switch to plus-end motility, showing that an N-terminal attachment is compatible with plus-end motility. However, there has been no systematic study on the role of attachment position in minus-end motility. We therefore examined the motility of monomeric kinesin-14s differing only in their attachment point. We find that a C-terminal attachment point causes kinesin-14s to become plus-end-directed, with microtubule corkscrewing rotation direction and pitch in motility assays similar to that of kinesin-1, suggesting that both C-kinesin kinesins-14 and N-kinesin kinesin-1 share a highly conserved catalytic core function with an intrinsic plus-end bias. Thus, an N-terminal attachment is one of the requirements for minus-end motility in kinesin-14.

Three-dimensional tracking of the ciliate Tetrahymena reveals the mechanism of ciliary stroke-driven helical swimming.

Helical swimming in free-space is a common behavior among microorganisms, such as ciliates that are covered with thousands hair-like motile cilia, and is thought to be essential for cells to orient directly to an external stimulus. However, a direct quantification of their three-dimensional (3D) helical trajectories has not been reported, in part due to difficulty in tracking 3D swimming behavior of ciliates, especially Tetrahymena with a small, transparent cell body. Here, we conducted 3D tracking of fluorescent microbeads within a cell to directly visualize the helical swimming exhibited by Tetrahymena. Our technique showed that Tetrahymena swims along a right-handed helical path with right-handed rolling of its cell body. Using the Tetrahymena cell permeabilized with detergent treatment, we also observed that influx of Ca2+ into cilia changed the 3D-trajectory patterns of Tetrahymena swimming, indicating that the beating pattern of cilia is the determining factor in its swimming behavior.

Characterization of the motility of monomeric kinesin-5/Cin8.

Cin8, the Saccharomyces cerevisiae kinesin-5, has an essential role in mitosis. In in vitro motility assays, tetrameric and dimeric Cin8 constructs showed bidirectional motility in response to ionic strength or Cin8 motor density. However, whether property-switching directionality is present in a monomeric form of Cin8 is unknown. Here we engineered monomeric Cin8 constructs with and without the Cin8-specific ∼99 residues in the loop 8 domain and examined the directionality of these constructs using an in vitro polarity-marked microtubule gliding assay within the range of the motor density or ionic strength. We found that both monomeric constructs showed only plus end-directed activity over the ranges measured, which suggested that minus end-directed motility driven by Cin8 is necessary for at least dimeric forms. Using an in vitro microtubule corkscrewing assay, we also found that monomeric Cin8 corkscrewed microtubules around their longitudinal axes with a constant left-handed pitch. Overall, our results imply that plus-end-directed and left-handed motor activity comprise the intrinsic properties of the Cin8 motor domain as with other monomeric N-kinesins.

CYK4 relaxes the bias in the off-axis motion by MKLP1 kinesin-6.

Centralspindlin, a complex of the MKLP1 kinesin-6 and CYK4 GAP subunits, plays key roles in metazoan cytokinesis. CYK4-binding to the long neck region of MKLP1 restricts the configuration of the two MKLP1 motor domains in the centralspindlin. However, it is unclear how the CYK4-binding modulates the interaction of MKLP1 with a microtubule. Here, we performed three-dimensional nanometry of a microbead coated with multiple MKLP1 molecules on a freely suspended microtubule. We found that beads driven by dimeric MKLP1 exhibited persistently left-handed helical trajectories around the microtubule axis, indicating torque generation. By contrast, centralspindlin, like monomeric MKLP1, showed similarly left-handed but less persistent helical movement with occasional rightward movements. Analysis of the fluctuating helical movement indicated that the MKLP1 stochastically makes off-axis motions biased towards the protofilament on the left. CYK4-binding to the neck domains in MKLP1 enables more flexible off-axis motion of centralspindlin, which would help to avoid obstacles along crowded spindle microtubules.

N-terminal ß-strand of single-headed kinesin-1 can modulate the off-axis force-generation and resultant rotation pitch.

In in vitro microtubule gliding assays, most kinesins drive the rotation of gliding microtubules around their longitudinal axes in a corkscrew motion. The corkscrewing pitch is smaller than the supertwisted protofilament pitch of microtubules, indicating that the corkscrewing pitch is an inherent property of kinesins. To elucidate the molecular mechanisms through which kinesins corkscrew the microtubule, we performed three-dimensional tracking of a quantum dot bound to a microtubule translocating over a surface coated with single-headed kinesin-1 s under various assay conditions to alter the interactions between the kinesin and microtubule. Although alternations in kinesin concentration, ionic strength, and ATP concentration changed both gliding and rotational velocities, the corkscrewing pitch remained left-handed and constant at ~0.3 µm under all tested conditions apart from a slight increase in pitch at a low ATP concentration. We then used our system to analyze the effect of point mutations in the N-terminal ß-strand protruding from the kinesin motor core and found mutations that decreased the corkscrewing pitch. Our findings confirmed that the corkscrewing motion of microtubules is caused by the intrinsic properties of the kinesin and demonstrates that changes in the active or retarding force originating from the N-terminal ß-strand in the head modulate the pitch.

Visualizing dynamic actin cross-linking processes driven by the actin-binding protein anillin.

Anillin is a type of actin filament cross-linking protein that stabilizes the actin-based contractile ring during cytokinesis. To elucidate the underlying intermolecular interactions between actin filaments and anillin, we utilized total internal reflection fluorescence microscopy (TIRFM) and high-speed atomic force microscopy (Hs-AFM). Single-molecule imaging of anillin using TIRFM showed that anillin exists as monomers with relatively low binding affinity for actin filaments. Real-time imaging of actin filament cross-linking dynamics induced by anillin using Hs-AFM revealed that anillin monomers cross-link with actin filaments at a distance of 8 nm and that the polarity of those filaments is both parallel and antiparallel. These results are consistent with anillin playing a role in actin ring transition in vivo, where it might be responsible for thinning the ring-shaped apolar actin bundles.

Structural Basis of Backwards Motion in Kinesin-1-Kinesin-14 Chimera: Implication for Kinesin-14 Motility.

Kinesin-14 is a unique minus-end-directed microtubule-based motor. A swinging motion of a class-specific N-terminal neck helix has been proposed to produce minus-end directionality. However, it is unclear how swinging of the neck helix is driven by ATP hydrolysis utilizing the highly conserved catalytic core among all kinesins. Here, using a motility assay, we show that in addition to the neck helix, the conserved five residues at the C-terminal region in kinesin-14, namely the neck mimic, are necessary to give kinesin-1 an ability to reverse its directionality toward the minus end of microtubules. Our structural analyses further demonstrate that the C-terminal neck mimic, in cooperation with conformational changes in the catalytic core during ATP binding, forms a kinesin-14 bundle with the N-terminal neck helix to swing toward the minus end of microtubules. Thus, the neck mimic plays a crucial role in coupling the chemical ATPase reaction with the mechanical cycle to produce the minus-end-directed motility of kinesin-14.