Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors

1. Sf9 culture, transfection, and virus generation

NOTE: Maintain Sf9 cells in 30 mL of Sf-900/SFM medium in 100 mL sterile, disposable conical flask without any antibiotic/antimycotic at 28 °C. Keep the suspension culture in an orbital shaker at 90 rpm. Supply of CO₂ and humidity maintenance is not required. Cells are usually subcultured every fourth day by inoculating 0.5 x 10⁶ cells/mL to reach 2.0 x 10⁶ cells/mL density on the fourth day.

1. P0 virus stock generation
   1. For transfection, seed cells into a 35 mm dish with 4.5 x 10⁵ cells/mL confluency and maintain at 28 °C without shaking.
   2. After 24 h, once cells are attached and look healthy, proceed for transfection as described below.
      1. Tube A: Mix 1 µg of bacmid DNA encoding for constitutively active KIF1A(1-393LZ)-3xmCit-FLAG specific kinesin-3 motor with 100 µL of unsupplemented Grace's media.
      2. Tube B: Mix 6 µL of transfection reagent with 100 µL of unsupplemented Grace's media.
      3. Carefully transfer the content of Tube A into Tube B and mix thoroughly by pipetting up and down (approximately 20 times).
      4. Incubate the mixture for ~45 min at room temperature.
   3. After completing the incubation, add 0.8 mL of unsupplemented Grace's media to the above mixture and mix slowly by pipetting.
   4. Gently aspirate the Sf-900/SFM media from cells (to remove any traces of serum that may affect transfection efficiency).
   5. Add the transfection mixture from step 1.1.3 dropwise on the top of the cells and incubate the plate for 6 h at 28 °C.
   6. After the incubation, carefully remove the transfection mixture, add 2 mL of Sf-900/SFM media and incubate further for 48 h at 28 °C.
   7. Check for the motor protein expression under an inverted fluorescence microscope. The motor protein is tagged with a fluorescent protein, mCitrine, a variant of yellow fluorescent protein.
      NOTE: Transfected cells were visualized under an inverted microscope equipped with differential interference contrast (DIC) and epifluorescence illumination with a 20x objective (200 times magnification), mercury lamp and an EM-CCD camera. Check the efficiency of virus generation and infection by monitoring the expression of mCitrine-tagged motors in cells through mCitrine excitation and emission filter cube. In addition, check for morphological changes of infected cells, such as enlarged cells/nuclei (Figure 1A-C).
   8. Again, check the cells 72 h post-infection. Usually, cells start detaching from the surface (Figure 2).
   9. If >5% of cells detached from the surface, harvest the media with infected cells in 1.5 mL sterile microcentrifuge tubes and spin for 5 min at 500 x g.
   10. Collect the supernatant and snap freeze aliquots of 1 mL in liquid nitrogen and store as P0 stock at -80 °C or use it to generate P1 virus stock.
2. P1 virus stock generation
   1. To further amplify the P0 baculovirus stock and confirm protein expression, grow Sf9 cells in liquid suspension culture.
   2. In a sterile 100 mL conical flask, add 10 mL of Sf-900/SFM media with a cell density of 2 x 10⁶ cells/mL and 1 mL of P0 virus stock. Incubate at 28 °C with constant shaking at 90 rpm.
   3. After 72 h of infection, check for the protein expression as described earlier (step 1.1.7). If the protein expression is good (>90% of cells show a bright mCitrine fluorescence signal) and shows significant cell death (approximately 10%-15%), spin down the cells in a 15 mL sterile conical tube at 500 x g for 5 min.
   4. Collect the supernatant, snap freeze aliquots of 1 mL (P1 stock) in liquid nitrogen and store at -80°C or proceed for large-scale infection and protein purification.
3. Large-scale infection
   1. For large-scale protein expression, infect 30 mL of the suspension culture at 2 x 10⁶ cells/mL density with 1 mL of P1 virus stock and incubate at 28 °C with constant shaking at 90 rpm.
   2. After ~72 h post-infection, check for the protein expression (in general, maximum protein expression is achieved between 65-75h post-infection).
   3. If >90% of cells show a bright fluorescent signal with minimal cell death (<5%), collect the cells in a sterile 50 mL conical tube and spin down at 500 x g at 4 °C for 15 min.
      ​NOTE: There should be minimal cell death (<5%), because dead cells release the cellular contents into the media, which leads to loss of expressed protein.
   4. Discard the supernatant, collect the cell pellet, and proceed with protein purification.

2. Sf9 purification of kinesin-3 motors

1. To the above cell pellet, add 3 mL of ice-cold lysis buffer (Supplemental Table 1) freshly supplemented with 5 mM DTT, 5 µg/mL of aprotinin, 5 µg/mL of leupeptin, and 5 µg/mL of PMSF and lyse the cells by pipetting 20-25 times without generating any air bubbles. For all the buffer compositions and reagents, please refer to Supplemental Table 1.
2. Spin the cell lysate at 150,000 x g for 30 min at 4 °C.
3. Collect the supernatant into a fresh, sterile tube and mix with ~40 µL of 50% anti-FLAG M2 affinity resin. Incubate the mixture for 3 h at 4 °C with end-to-end tumbling.
4. After incubation, pellet the FLAG resin by spinning at 500 x g for 1 min at 4 °C. Gently aspirate the supernatant without disturbing the pellet with a 26 G needle and discard.
5. Wash the FLAG resin pellet three times with ice-cold wash buffer (Supplemental Table 1) freshly supplemented with 2 mM DTT, 5 µg/mL of aprotinin, 5 µg/mL of leupeptin, and 5 µg/mL of PMSF. Pellet the beads by spinning at 500 x g for 1 min at 4 °C in each wash.
6. After the third wash, carefully drain the wash buffer as much as possible without disturbing the pellet. For protein elution, add ~70 µL of wash buffer containing 100 µg/mL FLAG peptide to the resin pellet and incubate overnight at 4 °C with end-to-end tumbling.
7. On the subsequent day, spin down the resin at 500 x g for 1 min at 4 °C. Collect the supernatant containing purified protein into a fresh tube and supplement with 10% glycerol. Snap freeze aliquots of 5 µL in liquid nitrogen and store at -80 °C until further use.
8. Run the SDS-PAGE gel to determine the protein concentration and yield. Along with purified protein of interest, a standard protein control, BSA of known concentrations ranging from 0.2 µg, 0.4 µg, 0.6 µg, 0.8 µg, and 1 µg are loaded to generate a standard curve. Stain the gel with Coomassie Brilliant blue (Figure 3).
9. Analyze the gel using a built-in gel quantification tool in ImageJ software. First, measure the band intensity of known concentrations of BSA and generate the standard curve. Then, measure the intensity of the purified protein band and determine the protein concentration. To do so open the Image J software, click on the Analyze option in the menu bar and select Gels in the drop-down menu.

3. In vitro single-molecule motility assay using Sf9-purified kinesin-3 motors

NOTE: The Sf9-purified kinesin-3 motors can be used to study biochemical and biophysical properties such as ATP turnover rate, microtubule affinity, velocity, run length, step size, and force generation. Here, a detailed protocol for in vitro single-molecule motility analysis of KIF1A(1-393LZ) using Total Internal Reflection Fluorescence (TIRF) microscopy is described. For all the buffers composition and reagents, please refer to Supplemental Table 1.

1. Microtubule polymerization
   1. In a pre-chilled 0.5 mL microcentrifuge tube, prepare a polymerization mix by pipetting in the following order: 12.0 µL of BRB80 buffer, pH 6.9; 0.45 µL of 100 mM MgCl₂, 1 µL of 25 mM GTP.
   2. Take out a 10 µL aliquot of 10 mg/mL tubulin stored in liquid nitrogen, thaw immediately, and add into the above polymerization mixture. Mix gently by pipetting 2-3 times without creating any air bubbles.
      NOTE: Perform the above steps quickly and strictly on ice.
   3. Let the above mixture sit on ice for 5 min.
      NOTE: This is a critical step to prevent denaturing of tubulin or to avoid the formation of short microtubule seeds.
   4. Transfer the tube into a pre-warmed 37 °C heat block/water bath and incubate for 30 min for polymerization of microtubules.
   5. While microtubules are polymerizing, thaw an aliquot of P12 buffer and bring it to room temperature.
   6. Before completing 30 min of incubation, start preparing MT stabilization buffer by pipetting 100 µL of P12 buffer into a fresh microcentrifuge tube. To this, add 1 µL of 1 mM taxol and immediately vortex the mixture.
      NOTE: Start preparing the MT stabilization buffer approximately 5 min before completing the incubation in step 3.1.4. Taxol stabilizes the polymerized microtubules by binding to β-tubulin.
   7. Warm the microtubule stabilization buffer for 2-3 min at 37 °C and gently add it to the polymerized microtubules without disturbing the polymerization mixture at the bottom.
      ​NOTE: Warming the stabilization buffer will bring it to the same temperature as the polymerization mixture.
   8. Do not tap or pipette the mixture and incubate further at 37 °C for 5 min.
   9. Gently tap the mixture and mix it with a beveled cut tip (200 µL capacity) slowly using the pipette.
      NOTE: From this point onward, always handle microtubules with a beveled cut tip to avoid microtubule shearing.
   10. Take 10-15 µL of polymerized microtubules in a flow cell to check proper microtubule polymerization.
       NOTE: One can visualize the unlabeled microtubules using a DIC microscopy setup.
   11. If microtubules are concentrated, dilute them in P12 buffer supplemented with 10 µM taxol.
2. Preparation of motility flow cell chamber
   1. Prepare a motility chamber using a glass slide, double-sided tape and glass coverslip (22 mm x 30 mm).
   2. Take a glass slide, place a drop (~70 µL) of deionized distilled water in the middle and wipe it with a lint-free tissue paper.
   3. Cut two strips of double-sided tape (~35 x 3 mm) and firmly stick them to the glass slide parallelly, leaving an ~4-5 mm gap between two strips to create a narrow passage.
   4. Next, take a coverslip and add a drop (~20 µL) of deionized distilled water in the middle. Place a strip of lint-free lens cleaning tissue paper on the water drop until it absorbs the water. Then, slide it slowly toward one end of the coverslip.
      NOTE: The coverslip should be completely dry. No water should be visible on the coverslip.
   5. Place the coverslip on the double-sided strips stuck on the slide and press the coverslip evenly along the strips to stick firmly.
      NOTE: Please make sure that the cleaned side of the coverslip faces the glass slide.
   6. Ensure that together this creates a narrow chamber of 10-15 µL capacity for performing motility assay (Figure 4A).
3. In vitro single-molecule motility assay
   NOTE: To study the microtubule-based single-molecule motility properties of motors, microtubules need to be adsorbed onto the coverslip surface in the motility chamber.
   1. Dilute the polymerized taxol-stabilized microtubules in P12 buffer supplemented with 10 µM taxol at 1:5 ratios and mix by pipetting slowly with a beveled tip.
   2. Keep the flow chamber in a slant position (~15-20°). Flow 30 µL of diluted microtubule solution through the flow chamber from the upper end while keeping a lint-free tissue paper at the lower end to absorb the liquid. This creates a shear force to align the microtubule in the flow direction and helps to adsorb the microtubules straight and align parallel.
   3. Leave a small drop of liquid on both ends of the chamber and keep the flow cell in an inverted position (coverslip facing the bottom) in a closed, moist chamber to prevent drying of the motility chamber.
   4. Let it sit for ~30 min so that microtubules adsorb to the surface of the coverslip inside the motility chamber.
   5. In the meantime, prepare blocking buffer by mixing 500 µL P12-BSA buffer with 5 µL of 1 mM taxol.
   6. Flow 40-50 µL of blocking buffer and incubate the slide in an inverted position for 10 min in a moist chamber.
   7. Prepare the motility mixture by pipetting the following components into a 500 µL capacity sterile microcentrifuge tube in the following order: 25 µL of P12 buffer with taxol, 0.5 µL of 100 mM MgCl₂, 0.5 µL of 100 mM DTT, 0.5 µL of 20 mg/mL Glucose oxidase, 0.5 µL of 8 mg/mL Catalase, 0.5 µL of 2.25 M Glucose, and 1.0 µL of 100 mM ATP.
      NOTE: Fluorescence imaging has been widely used for biological applications^{22,23,24,25,26}. Photoexcitation of fluorescent proteins generates reactive oxygen species (ROS), which can cause photobleaching of the fluorescent proteins and damage to the biological samples^27,28. Oxygen scavengers such as glucose, glucose oxidase and catalase are routinely used in motility assays to limit photodamage and prolong the bleaching time of fluorescent proteins.
   8. Finally, add 1 µL of the purified motor to the above motility mix and mix well before flowing into the motility chamber.
   9. Seal both the ends of the motility chamber with liquid paraffin wax and immediately image under TIRF illumination using 100X TIRF objective of 1.49 NA with 1.5x magnification.
   10. In order to focus the coverslip surface, first, focus on one of the inner edges of double-sided tape in the motility chamber under differential interference contrast (DIC) illumination.
       NOTE: The bright and uneven surface will be visible.
   11. Then, move the focus into the motility chamber. Using fine adjustment, focus the coverslip surface and look for the microtubules adsorbed on the coverslip surface.
   12. Once the microtubules are focused, switch to TIRF illumination with a 488 nm excitation laser and adjust the illumination depth by changing the excitation beam angle to get the best and uniform TIRF illumination (Figure 4B).
   13. Focus the individual mCitrine-tagged motors moving processively along the microtubule surface with 100 ms exposure and record the motion using an EM-CCD camera.
       NOTE: Although the motility assays were performed on unlabeled microtubules, the maximum intensity z-projection function can be used to reveal the outline of the microtubule track. Preferentially, events on long microtubule tracks were considered for tracking analysis.
   14. Manually track the position of the fluorescently tagged individual motors walking on long microtubule tracks frame-by-frame using a custom-written plugin in ImageJ (nih.gov) software as described previously²⁹.
   15. Generate the histograms of velocity and run length for the motor population by plotting the number of events in each bin. Fit these histograms to a single Gaussian peak function to obtain average velocity and run length¹² (Figure 4C-E).

4. In vitro microtubule gliding assay

NOTE: To understand the collective behavior of kinesin-3 motors, in vitro microtubule gliding assay was performed^18,30,31. Where motors are immobilized onto the coverslip in an inverted position and upon adding microtubules into the chamber, microtubules land on motors and glide along as the motors try to walk on them (Figure 5A,B).

1. Fluorescent microtubule polymerization: Polymerize the microtubules following the protocol described previously except for mixing rhodamine labeled tubulin (3 mg/mL) with unlabeled tubulin (10 mg/mL) in a ratio 1:10.
2. After 30 min of polymerization, gently add 30 µL of pre-warmed microtubule stabilization buffer and incubate further for 5 min at 37 °C.
3. Next, shear the microtubules by pipetting with the capillary-loading tip (~25-30 times).
4. Prepare the motility flow chamber as described previously, flow 50 µL of Sf9-purified GFP nanobodies (2.5 µL of 100 nM diluted in 50 µL of P12 buffer) and incubate for 30 min at room temperature with the coverslip facing downward in a moist chamber.
   NOTE: The motors are C-terminally tagged with mCitrine. GFP nanobodies are used to immobilize the motors due to their low dissociation constant³².
5. Block the coverslip surface by flowing 50 µL of block buffer into the flow chamber to prevent nonspecific protein adsorption and incubate further for 5 min.
6. Prepare the motor mix by pipetting 50 µL of block buffer, 1 µL of 100mM ATP, and 5 µL of 100 nM Sf9-purified kinesin-3 motors. Mix gently before flowing into the chamber and incubate for 30 min at room temperature in a moist chamber.
7. Wash the chamber twice with 50 µL of P12 casein.
8. In a sterile microcentrifuge tube, prepare the gliding assay mixture in the following order, 45 µL of P12-casein with 10 µM taxol, 1 µL of 100 mM ATP, 0.5 µL of 8 mg/mL catalase, 0.5 µL of 20 mg/mL glucose oxidase, 0.5 µL of 2.25 M glucose and 1 µL of sheared fluorescent microtubules. Gently mix the content before flowing into the motility chamber and seal the ends of the chamber with liquid paraffin wax.
9. Image microtubule gliding under TIRF illumination at 100 ms exposure and capture the images with attached EM-CCD camera.
   NOTE: The average microtubule gliding velocity was determined by manually tracking approximately 100 individual microtubules frame-by-frame using a custom-written plugin in ImageJ²⁹. Determine the average microtubule gliding velocity by generating a histogram and fitting to a Gaussian function (Figure 5C,D).