The Gliding Actin Filament Assay: An In Vitro Motility Assay to Study Translocation of Actin Filaments on Immobilized Myosins

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. Gliding actin filament assay

1. Coverslip preparation
   1. Make a 1% nitrocellulose solution in amyl acetate.
   2. Obtain a tissue culture dish (150 x 25 mm) and add a circular filter paper (125 mm diameter) to the bottom of the dish.
   3. Load eight No. 1.5 thickness 22 mm square coverslips onto a rack and wash with approximately 2-5 mL of 200-proof ethanol followed by 2-5 mL of distilled water (dH₂O). Repeat this washing step, ending with water. Then, dry the coverslips completely using a filtered air line or N₂-line.
   4. Take one coverslip and slowly pipette 10 µL of the 1% nitrocellulose solution along one edge of the slip. Then, in one smooth motion, smear it across the rest of the coverslip using the side of a smooth-sided 200 µL pipette tip. Place this coverslip on the tissue culture dish with the nitrocellulose side up. Repeat for the remaining coverslips and allow them to dry while preparing the remaining reagents, and use coverslips within 24 h after coating.
2. Chamber preparation
   1. Wipe a microscope slide with an optical lens paper to clean off large debris. 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. This creates a flow chamber that can hold approximately 10 µL of solution (see Figure 1).
   3. Take one of the nitrocellulose-coated coverslips from Part 1. Carefully stick the coverslip onto the tape such that the side coated with nitrocellulose is making direct contact with the tape, (see 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. Cut the excess tape hanging over the edge of the slide with a razor blade.
3. Actin preparation
   1. Make 20 µM F-actin by polymerizing globular actin (G-actin) in polymerization buffer (50 mM KCl, 2 mM MgCl₂, 1 mM DTT, 25 mM MOPS (pH 7.0) at 4 °C overnight.
   2. Dilute F-actin to 5 µM in motility buffer (20 mM MOPS, 5 mM MgCl₂, 0.1 mM EGTA, 1 mM DTT (pH 7.4)). Label with at least 1.2x molar excess of rhodamine-phalloidin. Leave (covered in aluminum foil) for at least 2 h on ice. This can be used for up to 1-2 months, and stored on ice.
4. Performing the myosin 5a gliding actin filament assay
   NOTE: In this section, the details of the myosin 5a (HMM) gliding assay are provided.
   1. Prepare the solutions for myosin 5a described in Table 1 and keep them on ice.
   2. Flow in 10 µL of the myosin 5a (50-100 nM) through the flow chamber and wait for 1 min.
   3. Flow in 10 µL of the 1 mg/mL BSA in 50 mM MB with 1 mM DTT ("low salt" buffer). 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 by gently placing the corner of the paper at the flow chamber exit.
   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 black actin solution (5 µM F-actin, 1 µM calmodulin, and 1 mM ATP in 50 mM MB with 1 mM DTT) to eliminate "dead heads".
      1. Pipette the solution with a 1 mL syringe and 27 G needle to shear the actin filaments before introducing the solution to the chamber. Repeat this step two more times and wait for 1 min after the third time. Approximately 20 pipetting events are sufficient.
      2. To perform the "dead head" spin, add a stoichiometric amount of F-actin to myosin in the presence of 1 mM ATP and 1 mM MgCl₂ at a salt concentration of 500 mM. Then ultracentrifuge at 480,000 x g for 15 min at 4 °C. The dead myosin will be in the pellet.
   6. Flow in 50 µL of 50 mM MB with 1 mM DTT and 1 mM ATP to deplete the chamber of free actin filaments.
   7. Wash with 10 µL of 50 mM MB with 1 mM DTT. Repeat this wash two more times to deplete the chamber of any ATP.
   8. Flow in 10 µL of 20 nM rhodamine actin (Rh-Actin) solution containing 1 mM DTT in 50 mM MB and wait for 1 min to allow rigor binding of actin filaments to the myosin 5a attached to the surface of the coverslip.
   9. Wash with 10 µL of 50 mM MB with 1 mM DTT to wash away Rh-Actin filaments not bound to the surface. Repeat this wash two more times.
   10. Flow in 30 µL of Final Buffer.
   11. Record images on a fluorescence microscope using an excitation wavelength of 561 nm to visualize Rh-Actin. An appropriate exposure time is 200 ms at 1.4 mW laser power for a total acquisition duration of 0.5-1 min.
       NOTE: Ensure that the acquisition rate is scaled appropriately to the speed of the moving filaments. An important consideration before collecting data for use with tracking programs is the acquisition frame rate. Subpixel movements between frames will result in an overestimate of the velocity, and movements of several hundred nanometers are required to obtain accurate values. An optimal acquisition rate features actin gliding for at least a one-pixel distance between frames. In the case of the TIRF microscope used for the imaging here, this threshold translates to 130 nm; therefore, a myosin expected to travel 1 µm/s must be imaged at a rate of 5 frames/s (0.2 s interval) to achieve 200 nm of movement while a myosin expected to travel 10 nm/s requires 0.05 frames/s (20 s intervals). Data can therefore be downsampled at this stage if necessary.

Table 1: Buffers used in gliding assay

Buffer Name	Composition (M5a)	Composition (NM2b)	Step(s) Used (M5a/NM2b)	Comments
4X Motility Buffer (4X MB)	80 mM MOPS, pH 7.2	80 mM MOPS, pH 7.2		Vacuum filter and store in 4°C
	20 mM MgCl2	20 mM MgCl2
	0.4 mM EGTA	0.4 mM EGTA
	pH 7.4	pH 7.4
50 mM Salt Motility Buffer (50 mM MB)	25% v/v 4X MB	25% v/v 4X MB		Vacuum filter and store in 4°C
	50 mM KCl	50 mM NaCl
	Raise to volume with dH2O	Raise to volume with dH2O
500 mM Salt Motility Buffer (500 mM MB)	N/A	25% v/v 4X MB		Vacuum filter and store in 4°C
		500 mM NaCl
		Raise to volume with dH2O
Myosin	0.05-0.1 µM myosin	0.2 µM myosin	4.2	Keep on ice.
	1 mM DTT	1 mM DTT
	Dilute in 50 mM MB	Dilute in 500 mM MB
1 mg/mL Bovine Serum Albumin (BSA)	1 mg/mL BSA	1 mg/mL BSA	4.3	Keep on ice.
	Dilute in 50 mM MB	Dilute in 500 mM MB
	1 mM DTT	1 mM DTT
5 µM Unlabeled F-actin in 50 mM MB (black actin)	5 µM unlabeled F-actin	5 µM unlabeled F-actin	4.5	Keep on ice. Shear actin by pipetting up and down 5-10 times, or by using a syringe.
	1 μM calmodulin (CaM)	1 mM ATP
	1 mM ATP	0.2 mM CaCl2
	Dilute in 50 mM MB	1 μM CaM
		1–10 nM myosin light chain kinase (MLCK)
		Dilute in 50 mM MB
MB with 1 mM DTT and 1 mM ATP	1 mM DTT	1 mM DTT	4.6	Keep on ice.
	1 mM ATP	1 mM ATP
	Dilute in 50 mM MB	Dilute in 50 mM MB
MB with DTT	1 mM DTT	1 mM DTT	4.4, 4.7, 4.9	Keep on ice.
	Dilute in 50 mM MB	Dilute in 50 mM MB
20 nM Rhodamine-Phalloidin F-actin (Rh-Actin)	20 nM Rhodamine-phalloidin F-actin	20 nM Rhodamine-phalloidin F-actin	4.8	Keep on ice. Do not vortex.
	1 mM DTT	1 mM DTT
	Dilute in 50 mM MB	Dilute in 50 mM MB
Final Buffer	50 mM KCl	0.7% methylcellulose (optional)	4.10	Add in the glucose, glucose oxidase, and catalase immediately before performing the experiment. Keep on ice.
	20 mM MOPS, pH 7.2	50 mM NaCl
	5 mM MgCl2	20 mM MOPS, pH 7.2
	0.1 mM EGTA	5 mM MgCl2
	1 mM ATP	0.1 mM EGTA
	50 mM DTT	1 mM ATP
	1 μM calmodulin	50 mM DTT
	2.5 mg/mL glucose	1–10 nM MLCK
	100 μg/mL glucose oxidase	0.2 mM CaCl2
	40 μg/mL catalase	1 μM calmodulin
		2.5 mg/mL glucose
		100 μg/mL glucose oxidase
		40 μg/mL catalase

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 (i.e., nitrocellulose) 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
Amyl Acetate	Ladd Research Industries	10825
ATP	Millipore Sigma	A7699
Bovine Serum Albumin	Millipore Sigma	5470
Calmodulin		PMID: 2985564
Catalase	Millipore Sigma	C40
Circular Filter Paper - Gliding Assay	Millipore Sigma	WHA1001125
Coverslip Rack	Millipore Sigma	Z688568-1EA
Coverslips: Gliding Acting Filament Assay	VWR International	48366-227
DL-Dithiothreitol	Millipore Sigma	D0632
Double-Sided Tape	Office Depot	909955
EGTA	Millipore Sigma	E4378
Ethanol	Fischer Scientific	A4094
G-actin		PMID: 4254541	G-actin stock can be stored at 200 μM in liquid N2.
Glucose	Millipore Sigma	G8270
Glucose Oxidase	Millipore Sigma	G2133
KCl	Fischer Scientific	P217-500
Large-Orifice Pipet Tips	Fischer Scientific	02-707-134
Methylcellulose	Millipore Sigma	M0512
Microscope Slides	Fischer Scientific	12-553-10
MOPS	Fischer Scientific	BP308-100
Nitrocellulose	Ladd Research Industries	10800
Razor Blades	Office Depot	397492
Rhodamine-Phalloidin	ThermoFisher Scientific	R415	Stock can be diluted in 100%  methanol to a final concentration of 200 μM.
Tissue Culture Dish - Gliding Assay	Corning	353025	Each tissue culture dish can hold approximately nine 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
Optima Max-Xp Tabletop Ultracentrifuge	Beckman Coulter	393315