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

Overview

In this video, we perform an in vitro motility assay to analyze the actin-myosin interactions. The gliding movement of actin filaments on immobilized myosin molecules is visualized using fluorescence microscopy.

Protocol

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.

Buffer Name	Composition	Step(s) Used	Comments
M5a Extraction Buffer	0.3 M NaCl	1.1	Keep on ice.
	15 mM MOPS, pH 7.2
	15 mM MgCl2
	1.5 mM EGTA
	4.5 mM NaN3
Buffer A	0.5 M NaCl	2.2	Keep on ice.
	10 mM MOPS, pH 7.2
	0.1 mM EGTA
	3 mM NaN3
	1 mM ATP
	1 mM DTT
	5 mM MgCl2
Buffer B	0.5 M NaCl	2.3	Keep on ice.
	10 mM MOPS, pH 7.2
	0.1 mM EGTA
	3 mM NaN3
	1 mM DTT
Elution Buffer	0.5 M NaCl	3.1	Keep on ice.
	0.5 mg/mL FLAG peptide
	10 mM MOPS, pH 7.2
	0.1 mM EGTA
	3 mM NaN3
	pH 7.2
M5a Dialysis Buffer	500 mM KCl	4.1	Use cold dH2O to bring to volume.
	10 mM MgCl2
	10 mM MOPS, pH 7.2
	0.1 mM EGTA
	1 mM DTT

Table 2: Buffers used in TIRF assay.

Buffer Name	Composition	Step(s) Used	Comments
4X Motility Buffer (4X MB)	80 mM MOPS, pH 7.2		Vacuum filter and store in 4°C
	20 mM MgCl2
	0.4 mM EGTA
	pH 7.4
50 mM salt Motility Buffer (50 mM MB)	25% v/v 4X MB		Vacuum filter and store in 4°C
	50 mM KCl
	Raise to volume with dH2O
150 mM salt Motility Buffer (150 mM MB)			Vacuum filter and store in 4°C
Myosin		See "Final Buffer" Recipe	Keep on ice.
2 mg/mL NeutrAvidin	2 mg/mL NeutrAvidin	3.5	Keep on ice.
	1 mM DTT
	Dilute in 50 mM MB
1 mg/mL bovine serum albumin (BSA)	1 mg/mL BSA	3.3	Keep on ice.
	1 mM DTT
	Dilute in 50 mM MB
200 nM rhodamine-phalloidin biotinylated F-actin (bRh-Actin)	200 nM rhodamine-phalloidin biotinylated F-actin	3.7	Avoid shearing by not vortexing or pipetting up and down. To mix, gently invert.
	1 mM DTT
	Dilute in 50 mM MB
MB with DTT	50 mM DTT	3.2, 3.4, 3.6, 3.8	Keep on ice.
	Dilute in 50 mM MB
Final Buffer	50 mM KCl	3.9	Add in the glucose, glucose oxidase, and catalase immediately before performing the experiment. Keep on ice.
	20 mM MOPS, pH 7.2
	5 mM MgCl2
	0.1 mM EGTA
	1 mM ATP
	50 mM DTT
	1 μM calmodulin
	2.5 mg/mL glucose
	100 μg/mL glucose oxidase
	40 μg/mL catalase
	10 nM myosin

Representative Results

Figure 1: Preparation of functionalized flow-cell chambers. (A) Begin with a cleaned microscope slide, two pieces of double-sided tape cut to approximately 2 cm, and a functionalized coverslip. (B) Add the tape to the center of the microscope slide. (C) Attach the coverslip to the tape with the coating facing down and gently press on the overlapping regions with the tape using a plastic pipette tip to ensure that the coverslip has adhered to the chamber.

Materials

Name	Company	Catalog Number	Comments
2 M MgCl2 Solution	VWR	10128-298
5 M NaCl Solution	KD Medical	RGE-3270
Acetic Acid	ThermoFisher Scientific	984303
Anti-FLAG M2 Affinity Gel	Millipore Sigma	A2220	https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/a2220bul.pdf
ATP	Millipore Sigma	A7699
Biotinylated G-Actin	Cytoskeleton, Inc.	AB07
Bovine Serum Albumin	Millipore Sigma	5470
bPEG-silane	Laysan Bio, Inc	Biotin-PEG-SIL-3400-1g
Bradford Reagent Concentrate	Bio-Rad	5000006
Calmodulin		PMID: 2985564
Catalase	Millipore Sigma	C40
Cell Line (Sf9) in SF-900 II SFM	ThermoFisher Scientific	11496015	http://tools.thermofisher.com/content/sfs/manuals/bevtest.pdf https://tools.thermofisher.com/content/sfs/manuals/bactobac_man.pdf
Circular Filter Paper - Inverted Assay	Millipore Sigma	WHA1001090
Complete, EDTA-Free Protease Inhibitor Tablets	Millipore Sigma	5056489001	This should be stored at 4 °C. The tablets can be used directly or can be reconstituted as a 25x stock solution by dissolving 1 tablet in 2 mL of distilled water. The resulting solution can be stored at 4 °C for 1-2 weeks or at least 12 weeks at -20 °C.
Concentrating Tubes (100,000 MWCO)	EMD Millipore Corporation	UFC910024	The MWCO of the tube is not necessarily "one size fits all," as long as the MWCO is less than the total molecular weight of the protein being purified. The M5a was concentrated with a 30,000 MWCO tube.
Coomassie Brilliant Blue R-250 Dye	ThermoFisher Scientific	20278
Coverslip Rack	Millipore Sigma	Z688568-1EA
Coverslips: Inverted Motility Assay	Azer Scientific	ES0107052
Dialysis Tubing (3500 Dalton MCWO)	Fischer Scientific	08-670-5A	The diameter of the dialysis tube can vary, but the MWCO should be the same. The tubes can be stored in 20% alcohol solution at 4 °C.
DL-Dithiothreitol	Millipore Sigma	D0632
Double-Sided Tape	Office Depot	909955
DYKDDDDK Peptide	GenScript	RP10586	This can be dissolved in a buffer containing 0.1 M NaCl, 0.1 mM EGTA, 3 mM NaN3, and 10 mM MOPS (pH 7.2) to a final concentration of 50 mg/mL. This can be stored at -20 °C as 300 µL aliquots.
EGTA	Millipore Sigma	E4378
Elution Column	Bio-Rad	761-1550	These can be reused. To clean, rinse the column with 2-3 column volumes of PBS and distilled water. Chill the column at 4° C before use.
Ethanol	Fischer Scientific	A4094
Glucose	Millipore Sigma	G8270
Glucose Oxidase	Millipore Sigma	G2133
Glycine Buffer Solution, 100 mM, pH 2-2.5, 1 L	Santa Cruz Biotechnology	sc-295018
HCl	Millipore Sigma	320331
KCl	Fischer Scientific	P217-500
Large-Orifice Pipet Tips	Fischer Scientific	02-707-134
Leupeptin Protease Inhibitor	ThermoFisher Scientific	78435
Mark12 Unstained Standard Ladder	ThermoFisher Scientific	LC5677
Methanol	Millipore Sigma	MX0482
Microscope Slides	Fischer Scientific	12-553-10
MOPS	Fischer Scientific	BP308-100
mPEG-silane	Laysan Bio, Inc	MPEG-SIL-2000-1g
NaN3	Millipore Sigma	S8032
NeutrAvidin	ThermoFisher Scientific	31050
NuPAGE 4 to 12% Bis-Tris Mini Protein Gel, 15-well	ThermoFisher Scientific	NP0323PK2
NuPAGE LDS Sample Buffer (4X)	ThermoFisher Scientific	NP0007
Phosphate-Buffered Saline, pH 7.4	ThermoFisher Scientific	10010023
PMSF	Millipore Sigma	78830	PMSF can be made as a 0.1 M stock solution in isopropanol and stored in 4 °C. Isopropanol addition results in crystal precipitation, which can be dissolved by stirring at room temperature. Immediately before use, PMSF can be added dropwise to a rapidly stirring solution to a final concentration of 0.1 mM.
Razor Blades	Office Depot	397492
Rhodamine-Phalloidin	ThermoFisher Scientific	R415	Stock can be diluted in 100% methanol to a final concentration of 200 μM.
Sf9 Media	ThermoFisher Scientific	12658-027	This should be stored at 4° C. Its shelf life is 18 months from the date of manufacture.
Tissue Culture Dish - Inverted Assay	Corning	353003	Each tissue culture dish can hold approximately four coverslips.
Smooth-sided 200 µL Pipette Tips	Thomas Scientific	1158U38
EQUIPMENT
Centrifuge	ThermoFisher Scientific	75006590
Microscope	Nikon	Model: Eclipse Ti with H-TIRF system with 100x TIRF Objective (N.A. 1.49)
Microscope Camera	Andor	Model: iXon DU888 EMCCD camera (1024 x 1024 sensor format)
Microscope Environmental Control Box	Tokai HIT	Custom Thermobox
Microscope Laser Unit	Nikon	LU-n4 four laser unit with solid state lasers for 405nm, 488nm, 561nm,and 640nm
Mid Bench Centrifuge	ThermoFisher Scientific	Model: CR3i
Misonix Sonicator	Misonix	XL2020
Optima Max-Xp Tabletop Ultracentrifuge	Beckman Coulter	393315
Plasma-Cleaner	Diener electronic GmbH + Co. KG	System Type: Zepto
Sonicator Probe (3.2 mm)	Qsonica	4418
Standard Incubator	Binder	Model: 56
Waverly Tube Mixer	Waverly	TR6E