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.

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To explore whether methotrexate (MTX) prevents joint destruction and improves pain-related behaviors in the acute phase of knee osteoarthritis (OA) induced by monosodium iodoacetate (MIA) in a rat model. Methods: Twenty of 25 male Wistar rats (10-14 weeks old) received 3 mg MIA via intraarticular injection into their right knee and were then administered a vehicle control (n=10) or 3 mg/kg MTX orally weekly (n=10). We assessed differences in pain-related behavior, spontaneous lifting behavior, micro-computed tomography (CT), histopathology, and expression of pain-and inflammatory-related genes using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) between the two groups for 4 weeks. Five rats were used as untreated controls to assess pain-and inflammatory-related mRNA expression in the dorsal root ganglia (DRG) and knee joints using RT-qPCR. Results: Joint destruction and mechanical hyperalgesia were observed in the vehicle group. Decreases in mechanical pain thresholds for the knee joint and calf muscles were improved after MTX administration; however, joint damage assessed by micro-CT and histopathology was not significantly inhibited by MTX administration, while upregulation levels of transient receptor potential cation channel, subfamily V, member 1 (TRPV-1) (P<0.01) and brainderived neurotrophic factor (BDNF) (P=0.02) mRNA in the DRG and nerve growth factor NGF mRNA (P=0.03) in the affected knee joints were significantly suppressed in the MTX group compared with the vehicle group at week 4. Conclusion:Our results imply that MTX administration improves pain-related behaviors and suppresses expression of pain-related mRNAs in the DRG and knee joint; however, MTX is not expected to prevent cartilage degeneration in MIA-induced OA in rat knee.

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.