Multiple sequence alignment and phylogenetic tree of the kinesin-9 family
Multiple sequence alignments and a phylogenetic tree of the full-length kinesin-9 family amino acid sequences were generated using SeaView version 5.0.5[42]. Multiple sequence alignment was performed using Clustal Omega[43] with the default parameters. Maximum likelihood phylogenetic trees were constructed using PhyML version 3.1[44] with the LG model[45]. The tree was internally validated using bootstrapping resampling (n = 500). The following protein sequences of kinesin-9 family members were used: Homo sapiens (NCBI Protein Database accession numbers NP_001400904.1 and NP_659464.3), Mus musculus (NP_001157041.1 and NP_796026.2), Xenopus laevis (XP_018124453.1 and XP_018118191.1), Danio rerio (XP_001922460.1 and NP_001410600.1), Strongylocentrotus purpuratus (XP_030830023.1 and XP_030854411.1), Tetrahymena thermophila (XP_001022313.1, XP_001024804.2, XP_001025897.4, and XP_001020926.2), Chlamydomonas reinhardtii (P46870.1 and XP_042928646.1), and Trypanosoma brucei (XP_846252.1 and XP_846346.1).
Plasmid construction
To construct C-terminally truncated Tetrahymena kinesin-9A and kinesin-9B dimers, coiled-coil predictions were calculated using Marcoil[26], and the genes from 1–473 amino acids of TtK9A (XP_001022313.1) and 1–506 amino acids of TtK9B1 (XP_001024804.2) were cloned into different pColdIII (Takara Bio Inc., Shiga, Japan) vectors: pColdIII-AviHis, pColdIII-mEGFP-AviHis, and pColdIII-mStayGold-AviHis (only for TtK9A_473). AviHis refers to the Avi-tag (GLNDIFEAQKIEWHE), which is biotinylated by BirA in Escherichia coli[46] and 6× His-tag. The sequence of mStayGold, a monomeric green fluorescent protein derived from StayGold[28] with the E138D mutation[29], was used. All the constructs were verified using DNA sequencing.
Kinesin expression and purification
All expression plasmids were transformed into Escherichia coli strain BL21 Star (DE3) cells (Invitrogen, Thermo Fisher Scientific, MA, USA) using the pBirAcm plasmid, which encoded BirA. Kinesin expression was induced via adding 0.1 mM isopropyl β- d-1-thiogalactopyranoside and > 50 µM biotin at 15°C, and cells were harvested after 24 h[47]. The cells were pelleted, resuspended in lysis buffer (500 mM NaCl, 80 mM PIPES-KOH, 1 mM MgCl2, 1 mM EGTA, 5 mM ATP, 1 mM DTT, 0.1% CHAPS, 0.1% Tween20, 10% glycerol, and protease inhibitors; pH 6.8), and sonicated on ice. After centrifuging the lysate, Ni-NTA affinity chromatography was performed on a HisTrap HP column (Cytiva, MA, USA) using an AKTA system (Cytiva), followed by buffer exchange into the desalting buffer (80 mM NaCl, 20 mM potassium phosphate, 1 mM MgCl2, 1 mM DTT, 20 µM ATP; pH 7.4) using a HiTrap Desalting column (Cytiva). Paclitaxel-stabilized microtubules were mixed with purified kinesins supplemented with 1 mM AMP-PNP and incubated for 15 min at 25°C. After removing the unbound kinesins via centrifugation (20 min, 305,000 × g, 23°C), the microtubule-bound kinesins were eluted with an ATP-containing buffer (10 mM ATP, 13 mM MgCl2, 200 mM potassium acetate, 20 µM paclitaxel, 16 mM PIPES-KOH, 1 mM EGTA; pH 7.4) via incubating for 10 min at 25°C. Microtubules were removed using centrifugation (20 min, 245,000 × g, 23°C)[48]. Supernatants were snap-frozen and stored in liquid nitrogen. The concentration of each kinesin was estimated using sodium dodecyl-sulfate polyacrylamide gel electrophoresis on 10% acrylamide gels using bovine serum albumin (BSA) as standards (Thermo Fisher Scientific) loaded on the same gel. Gels were stained with Quick-CBB PLUS (FUJIFILM Wako Pure Chemical Corporation, Tokyo, Japan) and imaged using a CCD camera (CSFX36BC3, Toshiba-Teli Corporation, Tokyo, Japan). Bands containing kinesins and BSA standards were quantified using ImageJ software (NIH)[49].
Tubulin purification
Tubulin was purified from porcine brains via four cycles of temperature-controlled polymerization and depolymerization in a high-molar PIPES buffer to remove the contaminating microtubule-associated proteins[50]. Purified tubulin in BRB80 buffer (80 mM PIPES-KOH, 1 mM MgCl2, and 1 mM EGTA; pH 6.8) was snap-frozen and stored in liquid nitrogen.
Cy5-labeled microtubule polymerization
The purified tubulin was polymerized and labeled with Cy5 mono-reactive NHS ester (Cytiva). Only polymerizable tubulin was collected after depolymerization, polymerization, and depolymerization cycles. The collected Cy5-labeled tubulin was snap-frozen and stored in liquid nitrogen. To polymerize the Cy5-labeled microtubules, 70% unlabeled tubulin and 30% Cy5-labeled tubulin were incubated in BRB80 buffer containing 7.5 mM GTP and 15 mM MgCl2 at 37°C for 30 min. Paclitaxel was added at a molar ratio of at least 10 times the tubulin quantity and incubated for another 10 min. Cy5-labeled microtubules were diluted to 10 µM in BRB80 buffer containing 20 µM paclitaxel, snap-frozen, and stored in liquid nitrogen. Cy5-labeled polarity-marked microtubules were prepared as described[51]. First, bright short microtubules (labeled tubulin: unlabeled tubulin = 1:2) were polymerized with 0.5 mM GMP-CPP (a non-hydrolyzable GTP analog, Jena Bioscience, Jena, Germany). The dim long segments were elongated on the plus-end (labeled tubulin: unlabeled tubulin = 1:9) via including N-ethyl maleimide-treated tubulin, which inhibited minus-end polymerization.
Microtubule gliding assays
Microtubule gliding assays were performed in flow chambers assembled from plasma-cleaned coverslips (24 × 36 mm and 18 × 18 mm, Matsunami Glass, Osaka, Japan) attached to each other using double-sided tape (Scotch W-12, 3M)[47]. For gliding assays, the flow chamber comprised 1 flow chamber volume (5 µL) of diluted (5,000-fold) 0.1-µm diameter microbeads (carboxylate-modified, red fluorescent [580/605], Thermo Fisher Scientific), which was incubated for 30 s for drift correction. A concentration of 1 flow chamber volume of 5 mg mL− 1 biotinylated-BSA (Sigma-Aldrich, MO, USA) was nonspecifically absorbed onto the glass surface, incubated for 5 min, and then rinsed with 4 volumes of BRB80 buffer. The chamber surface was sequentially coated with 1 volume of 1 mg mL− 1 streptavidin (FUJIFILM Wako Pure Chemical Corporation) for 5 min, 4 volumes of 1 mg mL− 1 casein (Nacalai Tesque Inc., Kyoto, Japan) for 3 min, 1 volume of either 0.05 µM TtK9A_473 or 0.1 µM TtK9B1_506 for 5 min, 4 volumes of 1 mg mL− 1 casein for 3 min, and then 4 volumes of 50 nM Cy5-labeled or polarity-marked microtubules in BRB80 buffer containing 0.4 mg mL− 1 casein and 20 µM paclitaxel for 3 min. Finally, 4 volumes of motility buffer (BRB80 buffer containing 3 mM ATP, 3 mM MgCl2, 20 µM paclitaxel, ATP regeneration system, oxygen scavenger system, 10 mM DTT, and 0.4 mg mL− 1 casein) were applied to the chamber. All chambers were sealed with grease (Apiezon M Grease; M&I Materials Ltd., Manchester, UK).
Assays were performed at 25°C with temperature-control equipment[52]. Microtubule gliding was observed using a fluorescence microscope (Eclipse Ti-E equipped with a Perfect Focus System, Nikon Corporation, Tokyo, Japan) with a stable stage (KS-N, Chukousya-Seisakujo, Tokyo, Japan) and a stage controller (QT-CM2-35, Chuo Precision Industrial Co., Ltd., Tokyo, Japan) using illumination from an LED light source (D-LEDI, Nikon Corporation), 100 × /1.49 NA, Plan-Apochromat objective lenses (Nikon Corporation), and a Cy5 filter set (Semrock, NY, USA). The images were acquired using an EM-CCD camera (iXon X3 DU897E; Andor Technologies, Belfast, UK). Images of TtK9A and TtK9B1 were acquired every 100 s for 80 min and every 10 s for 6 min 40 s, respectively.
Non-polarity-marked microtubules were tracked using Fluorescence Image Evaluation Software for Tracking and Analysis (FIESTA)[53]. Tracking data were corrected for drift using red beads as references. Only gliding microtubules that were > 2 µm in length, moved > 2 µm, and did not cross each other were analyzed. Microtubule gliding velocities were calculated via linearly fitting the travel distance versus time plots. Polarity-marked microtubules were analyzed to determine directionality. For TtK9A, out of 154 polarity-marked microtubules, 149 showed plus-end and 5 showed minus-end directionality. For TtK9B1, out of 135 polarity-marked microtubules, 128 showed plus-end and 7 showed minus-end directionality. Two independent measurements were performed for each construct and microtubules.
Single-molecule motility assays
Single-molecule motility assays were performed in the same chamber as the gliding assay. One flow chamber volume of 1 mg mL− 1 Protein G (Sigma-Aldrich) was non-specifically absorbed onto the glass surface, incubated for 5 min, and then rinsed with 4 volumes of P12 buffer (12 mM PIPES-KOH, 4 mM MgCl2, 1 mM EGTA; pH 6.8). The chamber surface was sequentially coated with 2 volumes of 5 µg mL− 1 anti-β-tubulin antibody solution (monoclonal, Santa Cruz Biotechnology, TX, USA) for 5 min, 4 volumes of 15 mg mL− 1 BSA (Sigma-Aldrich) blocking solution for 5 min, 4 volumes of diluted Cy5-labeled polarity-marked microtubules in BRB80 buffer containing 0.4 mg mL− 1 casein and 20 µM paclitaxel for 5 min, and then 4 volumes of blocking solution containing 20 µM paclitaxel for 5 min. Finally, 4 volumes of the kinesin solution (3–10 pM TtK9A_473-mStayGold or TtK9B1_506-mEGFP in P12 buffer containing 3 mM ATP, 20 µM paclitaxel, ATP regeneration system, oxygen scavenger system, 10 mM DTT, 6 mg mL− 1 BSA, and 0.4 mg mL− 1 casein) were applied to the chamber. All chambers were sealed with grease. The assays were performed at room temperature (23–24°C) with a lens heater (25°C; Tokai Hit, Shizuoka, Japan).
Fluorescent images of kinesins and Cy5-labeled polarity-marked microtubules were captured using a TIRF microscope (IX71 with a TIRF illumination module IX2-RFAEVA-2, Olympus Corporation, Tokyo, Japan) equipped with 100 × /1.45 NA, Plan-Apochromat objective lenses (Olympus) and a lens heater (Tokai Hit, Japan). Kinesins were illuminated using a 488-nm laser (OBIS 488 − 15 LS FP 15 mW, Coherent, PA, USA), which was further attenuated using neutral density filters with a total internal reflection angle. Microtubules were illuminated with epi-illumination using a 632-nm laser (632 05-LHP-991 30 mW, Melles Griot Inc., NY, USA). Kinesin and microtubule images were acquired simultaneously using an EM-CCD camera (iXon DV887DCS-BV, Andor Technologies) with a custom-designed optical path splitter[54]. Images were acquired at 1 frame s− 1 for 600–900 s. For TtK9B1_506-mEGFP, the 488-nm laser intensity was twice that of TtK9A_473-mStayGold because of its lower intensity.
Single molecules were tracked using FIESTA[53]. Tracking data were corrected for drift using fixed microtubules as references. Only the tracking data that bound to the microtubule for 5 s, moved > 100 nm towards the microtubule plus end, did not reach the plus end, and did not cross each other were analyzed. For TtK9A_473-mStayGold, out of 808 molecules, 86 moved > 100 nm towards the plus end, 9 molecules moved > 100 nm towards the minus end, and the others did not move > 100 nm (713 molecules). For TtK9B1_506-mEGFP, out of 125 molecules, 80 moved > 100 nm towards the plus end, 1 molecule moved > 100 nm towards the minus end, and the others did not move > 100 nm (44 molecules). Single-molecule velocities of the kinesin-9 motors were calculated via linearly fitting the distance to the origin versus time plot. The means of the dwell times and run lengths were calculated via fitting the histogram exponential distributions. Assays for each construct were performed with at least four independent measurements.
Photobleaching assays
Photobleaching assays were performed in the same chambers using a TIRF microscope as the single-molecule motility assays[55]. A concentration of 1 flow chamber volume of 1 mg mL− 1 Protein G was non-specifically absorbed on the glass surface, incubated for 5 min, and then rinsed with four volumes of P12 buffer. The chamber surface was sequentially coated with 1 volume of 5 µg mL− 1 anti-penta-His antibody solution (monoclonal, Qiagen, Hilden, Germany) for 5 min and 4 volumes of 15 mg mL− 1 BSA blocking solution for 5 min. Finally, 1 volume of kinesin solution (12.5 pM TtK9A_473-mStayGold or TtK9A_473-mEGFP in P12 buffer containing an oxygen scavenger system, 10 mM DTT, 6 mg mL− 1 BSA and 0.4 mg mL− 1 casein) were applied to the chamber. All chambers were sealed with grease. Each kinesin on the glass surface was illuminated using the same intense laser irradiation (5–10 times higher than that used in single-molecule motility assays). Images were acquired at 2 frame s− 1 for 150 s. An intensity of 5 × 5 pixels was averaged to center the brightness centroid, and an intensity time profile was created. The number of photobleaching steps and the photobleaching time were analyzed from the profiles.
Measuring microtubule-stimulated ATPase rates
ATPase measurements of kinesin-9 molecules were performed using a pyruvate kinase/lactate dehydrogenase-linked assay as previously described. The decrease in NADH (Sigma-Aldrich) absorbance at 340 nm via the catalytic reaction of pyruvate kinase/lactic dehydrogenase enzymes (Sigma-Aldrich) with phosphoenol-pyruvate (Sigma-Aldrich) and ADP produced by ATPase activity of kinesins in the presence of microtubules (0.2–18.5 µM) was measured. Assays were performed in buffer BRB80 containing 3 mM ATP, 3 mM MgCl2, and 97 nM TtK9A_473 or 31 nM TtK9B1_506. Assays were performed at 25°C with a temperature-control equipment (Shimadzu, Kyoto, Japan). Three independent measurements were conducted for the assays in each construct.
Statistics and reproducibility
Tracking data for microtubules were obtained from 2 independent experiments, tracking data for single molecules were obtained from at least 4 independent experiments, microtubule stimulated ATPase rates were obtained from 3 independent experiments, and sample sizes are indicated in detail in the text or the Methods section.