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

Abstract

Source: Tripathi, A., et al. Myosin-Specific Adaptations of In vitro Fluorescence Microscopy-Based Motility Assays. J. Vis. Exp. (2021).

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% nitrocellu....

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. (

Materials

List of materials used in this article

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