Myosin-Specific Adaptations of In vitro Fluorescence Microscopy-Based Motility Assays

Summary

Presented here is a procedure to express and purify myosin 5a followed by a discussion of its characterization, using both ensemble and single molecule in vitro fluorescence microscopy-based assays, and how these methods can be modified for the characterization of nonmuscle myosin 2b.

Abstract

Myosin proteins bind and interact with filamentous actin (F-actin) and are found in organisms across the phylogenetic tree. Their structure and enzymatic properties are adapted for the particular function they execute in cells. Myosin 5a processively walks on F-actin to transport melanosomes and vesicles in cells. Conversely, nonmuscle myosin 2b operates as a bipolar filament containing approximately 30 molecules. It moves F-actin of opposite polarity toward the center of the filament, where the myosin molecules work asynchronously to bind actin, impart a power stroke, and dissociate before repeating the cycle. Nonmuscle myosin 2b, along with its other nonmuscle myosin 2 isoforms, has roles that include cell adhesion, cytokinesis, and tension maintenance. The mechanochemistry of myosins can be studied by performing in vitro motility assays using purified proteins. In the gliding actin filament assay, the myosins are bound to a microscope coverslip surface and translocate fluorescently labeled F-actin, which can be tracked. In the single molecule/ensemble motility assay, however, F-actin is bound to a coverslip and the movement of fluorescently labeled myosin molecules on the F-actin is observed. In this report, the purification of recombinant myosin 5a from Sf9 cells using affinity chromatography is outlined. Following this, we outline two fluorescence microscopy-based assays: the gliding actin filament assay and the inverted motility assay. From these assays, parameters such as actin translocation velocities and single molecule run lengths and velocities can be extracted using the image analysis software. These techniques can also be applied to study the movement of single filaments of the nonmuscle myosin 2 isoforms, discussed herein in the context of nonmuscle myosin 2b. This workflow represents a protocol and a set of quantitative tools that can be used to study the single molecule and ensemble dynamics of nonmuscle myosins.

Introduction

Myosins are motor proteins that exert force on actin filaments using the energy derived from adenosine triphosphate (ATP) hydrolysis¹. Myosins contain a head, neck, and tail domain. The head domain contains the actin-binding region as well as the site of ATP binding and hydrolysis. The neck domains are composed of IQ motifs, which bind to light chains, calmodulin, or calmodulin-like proteins^2,3. The tail region has several functions specific to each class of myosins, including but not limited to the dimerization of two heavy chains, binding of cargo molecules, and regulation of the myos....

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 E....

Results

The purification of myosin can be evaluated by performing reducing sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel-electrophoresis as shown in Figure 2. While this figure represents the final, post-dialyzed myosin, SDS-PAGE can be performed on aliquots from the various stages of the purification procedure to identify any products lost to the supernatant. Myosin 5a HMM has a band in the 120-130 kDa range and the full-length nonmuscle myosin 2b has a band in the 200-230 kDa range, corresp.......

Discussion

Presented here is a workflow for the purification and in vitro characterization of myosin 5a and nonmuscle myosin 2b. This set of experiments is useful for quantifying the mechanochemical properties of purified myosin constructs in a fast and reproducible manner. Although the two myosins shown here are just two specific examples out of the many possibilities, the conditions and techniques can be applied, with some tailoring, to most myosins and to many other motor proteins.

The protocols discu.......

Materials

Name	Company	Catalog Number	Comments
1 mL Syringe	BD	309628
2 M CaCl2 Solution	VWR	10128-558
2 M MgCl2 Solution	VWR	10128-298
27 Gauge Needle	Becton Dickinson	309623
5 M NaCl Solution	KD Medical	RGE-3270
Acetic Acid	ThermoFisher Scientific	984303
Amyl Acetate	Ladd Research Industries	10825
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 - Gliding Assay	Millipore Sigma	WHA1001125
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 NM2b herein was concentrated with a 100,000 MWCO tube and 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: Gliding Acting Filament Assay	VWR International	48366-227
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 NM2b used herein was dialyzed in an 18 mm dialysis tube. 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
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
Glycine Buffer Solution, 100 mM, pH 2-2.5, 1 L	Santa Cruz Biotechnology	sc-295018
HaloTag	Promega	G100A
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
Methylcellulose	Millipore Sigma	M0512
Microscope Slides	Fischer Scientific	12-553-10
MOPS	Fischer Scientific	BP308-100
mPEG-silane	Laysan Bio, Inc	MPEG-SIL-2000-1g
Myosin Light Chain Kinase		PMID: 23148220	FLAG-tagged MLCK can be purified the same way that the FLAG-tagged myosin was purified herein.
NaN3	Millipore Sigma	S8032
NeutrAvidin	ThermoFisher Scientific	31050
Nitrocellulose	Ladd Research Industries	10800
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 - Gliding Assay	Corning	353025	Each tissue culture dish can hold approximately nine coverslips.
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
SOFTWARE
ImageJ FIJI	https://imagej.net/Fiji/Downloads
FAST (Version 1.01)	http://spudlab.stanford.edu/fast-for-automatic-motility-measurements	FAST is available for Mac OSX and Linux based systems.
Image Stabilizer Plugin	https://imagej.net/Image_Stabilizer
ImageJ TrackMate	https://imagej.net/TrackMate
Imaging Software	NIS Elements (AR package)
	http://www.cs.cmu.edu/~kangli/code/Image_Stabilizer.html
	File:TrackMate-manual.pdf
	https://github.com/turalaksel/FASTrack
	https://github.com/turalaksel/FASTrack/blob/master/README.md