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Protocol

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Acknowledgements

Materials

References

Biochemistry

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

Published: February 4th, 2021

DOI:

10.3791/62180

1Laboratory of Molecular Physiology, Cell and Developmental Biology Center, National Heart, Lung and Blood Institute, National Institutes of Health, 2Department of Physiology, Perelman School of Medicine, University of Pennsylvania

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.

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.

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 proteins2,3. 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....

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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 E.......

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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.......

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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.......

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We thank Dr. Fang Zhang for technical assistance with the preparation of the reagents used for collecting this data. This work was supported by the NHLBI/NIH Intramural Research Program funds HL001786 to J.R.S.

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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

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