Inverted Motility Assay: An In Vitro Technique to Visualize Myosin Movement on Immobilized Actin Filaments

1. Protein purification

1. Cell lysis and protein extraction
   1. Prepare a 1.5x Extraction Buffer based on Table 1. Filter and store at 4 °C.
   2. Begin thawing the cell pellets on ice. While the pellets are thawing, supplement 100 mL of Extraction Buffer with 1.2 mM dithiothreitol (DTT), 5 µg/mL leupeptin, 0.5 µM phenylmethylsulfonyl fluoride (PMSF), and two protease inhibitor tablets. Keep on ice.
   3. Once the pellet has thawed, add 1 mL of the supplemented Extraction Buffer per 10 mL of cell culture. For example, if the cell pellets were formed from 500 mL of cell culture, then add 50 mL of supplemented Extraction Buffer to the pellet.
   4. Sonicate the cell pellets while keeping them on ice. For each pellet, use the following conditions: 5 s ON, 5 s OFF, duration of 5 min, power 4-5.
   5. Collect all the homogenized lysate into a beaker and add ATP (0.1 M stock solution; pH 7.0) such that the final concentration of ATP is 1 mM. Stir for 15 min in a cold room. The ATP dissociates active myosin from actin, allowing it to be separated in the following centrifugation step. It is, therefore, essential to proceed to the next step immediately to minimize the possibility of ATP depletion and rebinding to actin.
   6. Centrifuge the lysates at 48,000 x g for 1 h at 4 °C. While this is occurring, begin washing 1-5 mL of a 50% slurry of Anti-FLAG affinity resin (for a pellet formed from 1 L of cells) with 100 mL phosphate-buffered saline (PBS), according to the manufacturer's instructions. For example, for 5 mL of resin, wash 10 mL of a 50% slurry. In the final wash step, resuspend the resin with 1-5 mL of PBS with enough volume to create a 50% slurry.
   7. Following lysate centrifugation, combine the supernatant with the washed resin slurry and rock gently in the cold room for 1-4 h. While waiting, make the buffers described in Table 1 and keep them on ice.
2. FLAG affinity purification preparation
   1. Centrifuge the solution in step 1.7 at 500 x g for 5 min at 4 °C. The resin will be packed at the bottom of the tube. Without disturbing the resin, remove the supernatant.
   2. Resuspend the resin in 50 mL of Buffer A as detailed in Table 1 and centrifuge at 500 x g for 5 min at 4 °C. Without disturbing the resin, remove the supernatant.
   3. Resuspend the resin in 50 mL of Buffer B as detailed in Table 1 and centrifuge at 500 x g for 5 min at 4 °C. Repeat this step once more and resuspend the resin in 20 mL of Buffer B. Then, mix the resin and the buffer thoroughly by gently inverting the tube by hand approximately 10 times.
3. Protein elution and concentration
   1. Make 30 mL of Elution Buffer as described in Table 1 and let it chill on ice.
   2. Set up the elution column in a cold room. Gently pour the resin slurry into the column. Wash the column with 1-2 column volumes of Buffer B as the resin packs on the bottom, ensuring that the resin does not dry out.
   3. Flow 1 mL of the Elution Buffer through the resin and collect the flow-through in a 1.5 mL tube. Repeat such that 12, 1 mL fractions are collected.
   4. At this point, perform a crude Bradford test on the fractions to qualitatively determine which fractions are the most concentrated. On one row of a 96-well plate, pipette 60 µL 1x Bradford reagent. As fractions are collected, mix 20 µL of each fraction per well. A darker blue coloration indicates the more concentrated fractions.
   5. In a 50 mL tube, collect the remaining protein by gently pipetting the remaining Elution Buffer through the column, to release any remaining myosin bound to the resin in the column flowthrough. This flow-through will be concentrated in the next step. Ensure that the resin is then regenerated for reuse and stored according to the manufacturer's instructions.
   6. Pool the three most concentrated fractions and further concentrate the flow-through in the 50 mL tube as well as the remaining 1 mL fractions using a 100,000 MWCO concentrating tube. Load the pooled sample onto the concentrating tube and centrifuge at 750 x g for 15 min at 4 °C and repeat until all eluted protein has been concentrated to a final volume of approximately 0.5-1 mL.
      NOTE: This pore size allows for the retention of the myosin molecules, which have masses several times the molecular weight cutoff. The light chains remain tightly bound to the motor domains during this time course of concentration, as verified by performing SDS-PAGE gel electrophoresis on the final product.
4. Dialysis and flash-freezing
   1. Make 2 L of Dialysis Buffer, as described in Table 1. Load the sample in a dialysis bag or chamber and dialyze overnight in the cold room.
      NOTE: In the case of M5a-HMM, after the overnight dialysis, the protein will be sufficiently pure for use in subsequent assays. Further purification steps such as gel filtration or ionic exchange chromatography can be performed, if required.
5. Recovering myosin after dialysis
   1. For M5a-HMM, carefully collect the entire sample from the dialysis chamber and centrifuge at 4 °C for 15 min at 49,000 x g in case any unwanted aggregates are present. Take the supernatant.
6. Concentration determination and flash-freezing
   1. To determine the concentration of the product, measure the absorbance using a spectrophotometer at wavelengths 260, 280, 290, and 320 nm. Calculate the concentration in mg/mL (c_mg/mL) with Equation 1, where A₂₈₀ represents the absorption at 280 nm and A₃₂₀ represents the absorption at 320 nm. The resulting concentration in mg/mL can be converted into µM of myosin molecules with Equation 2, where M is the molecular weight of the entire protein (including the heavy chains, light chains, fluorophores, and all tags).
      c_mg/mL = (A₂₈₀ - A₃₂₀) / ε   (1)
      μM molecules = 1000c_mg/mL/M   (2)
      NOTE: If a dilution is necessary, then it must be done in a high ionic strength buffer. The extinction coefficient (ε) can be determined by importing the amino acid sequence of the protein into a program such as ExPASy. Typical yield for the M5a-HMM is approximately 0.5-1 mL of 1-5 mg/mL protein. The extinction coefficient for the M5a-HMM used in this paper was 0.671.
   2. Store the purified myosin in one of the two ways. Aliquot between 10-20 µL into a thin-walled tube, such as a polymerization chain reaction tube, and drop the tube into a container of liquid nitrogen for flash-freezing. Alternatively, directly pipette between 20-25 µL of myosin into liquid nitrogen and store the frozen beads of protein in sterile cryogenic tubes. In either case, the resulting tubes can be stored in -80 °C or liquid nitrogen for future use.
      NOTE: Since motility assay described below requires very small amounts of protein, storage in small aliquots, as described, is economical.

2. Single-molecule TIRF assay

1. Coverslip preparation
   1. Divide the stock powder into 10 mg aliquots (in 1.5 mL tubes) of methoxy-Peg-silane (mPEG) and 10 mg aliquots of biotin-Peg-silane (bPEG). Store at -20 °C in a sealed, moisture-free container and use within 6 months.
   2. Load eight No. 1.5H (high precision) thickness 22-mm square coverslips onto a rack and wash with 2-5 mL of 200-proof ethanol followed by 2-5 mL of distilled water. Repeat this washing step, ending with water. Then, dry the coverslips completely using an air-line or N₂ and plasma-clean with argon for 3 min.
   3. Place the clean coverslips on filter paper (90 mm) in a tissue culture dish (100 x 20 mm) and incubate in a 70 °C oven while performing the following steps.
      NOTE: The plasma cleaning can be replaced with other chemical cleaning methods.
   4. Prepare 80% ethanol solution with dH₂O and adjust the pH to 2.0 using HCl. Add 1 mL of this to a 10 mg aliquot of mPEG and 1 mL to a 10 mg aliquot of bPEG. Vortex to dissolve, which should not take more than 30 s.
   5. Take 100 µL of the bPEG solution and add 900 µL of 80% ethanol (pH 2.0). This solution is 1 mg/mL bPEG. Then, make a solution of both the PEGs as follows, mixing thoroughly.
      1. 200 µL of 10 mg/mL mPEG (final concentration: 2 mg/mL).
      2. 10 µL of the 1 mg/mL bPEG (final concentration: 10 µg/mL).
      3. 790 µL of the 80% ethanol (pH 2.0) solution.
   6. Take the coverslips out of the oven. Carefully dispense 100 µL of the PEG solution onto the center of each coverslip, ensuring that only the top surface is wet. Then, place the slips back in the oven and incubate for 20 to 30 min.
   7. When the coverslips begin to take on a holey appearance, with small circles apparent across the surface, remove them from the oven.
   8. Wash each coverslip with 100% ethanol, dry with an air line, and place back in the oven. Incubate only for the time required to create chambers in step 2.
2. Chamber preparation
   1. Clean a microscope slide for use in making the chamber. Cut two pieces of double-sided tape, approximately 2 cm in length.
   2. Place one piece along the middle of the long edge of the microscope slide. Ensure that the edge of the tape aligns with the edge of the slide. Place the second piece of tape roughly 2 mm below the first piece of tape such that the two are parallel and aligned.
   3. Take one of the functionalized coverslips from the oven (created in 2.1). Carefully stick the coverslip onto the tape such that the side coated with PEG is face down and making direct contact with the tape, as shown in Figure 1. Using a pipette tip, gently press down on the slide-tape interface to ensure that the coverslip has properly adhered to the slide.
   4. Cut the excess tape hanging over the slide with a razor blade. These chambers can be used immediately or placed pairwise into a 50 mL tube and stored in a -80 °C freezer for future use. It is important to store immediately or the surface will degrade.
3. Performing the myosin 5a TIRF microscopy assay
   1. Prepare the solutions for myosin 5a inverted motility assay described in Table 2 and keep them on ice.
   2. Wash the chamber with 10 µL of 50 mM MB with 1 mM DTT.
   3. Flow in 10 µL of the 1 mg/mL BSA in 50 mM MB with 1 mM DTT. Repeat this wash two more times and wait for 1 min after the third wash. Use the corner of a tissue paper or filter paper to wick the solution through the channel.
   4. Wash with 10 µL of 50 mM MB with 1 mM DTT. Repeat this wash two more times.
   5. Flow in 10 µL of the NeutrAvidin solution in 50 mM MB with 1 mM DTT and wait for 1 min.
   6. Wash with 10 µL of 50 mM MB with 1 mM DTT. Repeat this wash two more times.
   7. Flow in 10 µL of biotinylated rhodamine actin (bRh-Actin) containing 1 mM DTT in 50 mM MB and wait for 1 min. For this step, use a large-bored pipette tip and avoid pipetting up and down to minimize shearing of the fluorescent actin filaments to ensure that long actin filaments can be attached to the surface (20-30 µm or longer). An effective alternative is cutting the cone of a standard pipette tip (with an opening of ≈1-1.5 mm).
   8. Wash with 10 µL of 50 mM MB with 1 mM DTT. Repeat this wash two more times.
   9. Flow in 30 µL of Final Buffer with 10 nM myosin 5a added, then immediately load onto the TIRF microscope and record after finding the optimum focus for TIRF imaging modality. Exposure times between 100-200 ms are appropriate at 1.4 mW laser power for the actin and GFP-labeled myosin. An appropriate acquisition time for velocity analysis is 3 min.

Table 1: Buffers used in protein purification.

Table 2: Buffers used in TIRF assay.