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

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. Protein purification Cell lysis and protein extraction Prepare a 1.5x Extraction Buffer based on Table 1. Filter and store at 4 °C. 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. Once the pellet has thawed, add 1 m…

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 ove…

Materials

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