Name: myosin-specific adaptations of in vitro fluorescence microscopy-based motility assays
Uploaded: 2021-02-04T14:30:00
Description: Watch this Scientific Journal Video about Myosin-Specific Adaptations of In vitro Fluorescence Microscopy-Based Motility Assays at JoVE.com

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.

1. Protein purification
<ol>
	<li>Cell lysis and protein extraction
	<ol>
		<li>Prepare a 1.5x Extraction Buffer based on Table 1. Filter and store at 4 &#176;C.</li>
		<li>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 &#181;g/mL leupeptin, 0.5 &#181;M phenylmethylsulfonyl fluoride (PMSF) and two protease inhibitor tablets. Keep on ice.</li>
		<li>Once the pellet has thawed, add 1 mL of the supplemented E...

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...

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 myosin via autoinhibitory interactions with the head domains1.
The motile properties of myosin vary greatly between classes. Some of these properties include duty ratio (the fraction of myosin&#39;s mechanical cycle in which the myosin is bound to actin) and processivity (the ability of a motor to make multiple steps on its track before detachment)4. The over 40 classes of myosins were determined based on sequence analyses5,6,7,8. The class 2 myosins are classified as &#34;conventional&#34; since they were the first to be studied; all other classes of myosins are, therefore, classified as &#34;unconventional.&#34;
Myosin 5a (M5a) is a class 5 myosin and is a processive motor, meaning that it can take multiple steps along actin before dissociating. It has a high duty ratio, indicating that it spends a large part of its mechanical cycle bound to actin9,10,11,12,13,14. In common with other myosins, the heavy chain contains an N-terminal motor domain that includes both an actin-binding and an ATP hydrolysis site followed by a neck region that serves as a lever-arm, with six IQ motifs that bind to essential light chains (ELC) and calmodulin (CaM)15. The tail region contains &#945;-helical coiled-coils, which dimerize the molecule, followed by a globular tail region for binding cargo. Its kinetics reflect its involvement in the transport of melanosomes in melanocytes and of the endoplasmic reticulum in Purkinje neurons16,17. M5a is considered the prototypical cargo transport motor18.
Class 2 myosins, or the conventional myosins, include the myosins that power contraction of skeletal, cardiac, and smooth muscle in addition to the nonmuscle myosin 2 (NM2) isoforms, NM2a, 2b, and 2c19. The NM2 isoforms are found in the cytoplasm of all cells and have shared roles in cytokinesis, adhesion, tissue morphogenesis, and cell migration19,20,21,22. This paper discusses conventional myosin protocols in the context of nonmuscle myosin 2b (NM2b)23. NM2b, in comparison to M5a, has a low duty ratio and is enzymatically slower with a Vmax of 0.2 s-1 23 compared to M5a&#39;s Vmax of &#8776;18 s-1 24. Notably, truncated NM2b constructs with two heads do not readily move processively on actin; rather, each encounter with actin results in a power stroke followed by dissociation of the molecule25.
NM2b contains two myosin heavy chains, each with one globular head domain, one lever-arm (with one ELC and one regulatory light chain (RLC)), and an &#945;-helical coiled-coil rod/tail domain, approximately 1,100 amino acids long, that dimerizes these two heavy chains. The enzymatic activity and structural state of NM2b are regulated by phosphorylation of the RLC23. Unphosphorylated NM2b, in the presence of ATP and physiological ionic strengths (approximately 150 mM salt), adopts a compact conformation wherein the two heads make participate in an asymmetric interaction and the tail folds back over the heads in two places23. In this state, the myosin does not interact strongly with actin and has very low enzymatic activity. Upon RLC phosphorylation by calmodulin-dependent myosin light chain kinase (MLCK) or Rho-associated protein kinase, the molecule extends and associates with other myosins through the tail region to form bipolar filaments of approximately 30 myosin molecules23. The aforementioned phosphorylation of the RLC also leads to increased actin-activated ATPase activity of NM2b by approximately four times26,27,28. This bipolar filament arrangement, featuring many myosin motors at each end, is optimized for roles in contraction and tension maintenance, where actin filaments with opposing polarities can be moved relative to each other23,29. Accordingly, NM2b has been shown to act as an ensemble of motors when interacting with actin. The large number of motors within this filament allow NM2b filaments to move processively on actin filaments, making in vitro&#160;filament processivity possible to characterize29.
While progress has been made in understanding the role of myosins in the cell, there is a need to understand their individual characteristics at the protein level. To understand actomyosin interactions at a simple protein-protein interaction level, rather than inside of a cell, we can express and purify recombinant myosins for use in in vitro studies. The results of such studies then inform mechanobiologists about the biophysical properties of specific myosins that ultimately drive complex cellular processes12,13,14,25,29. Typically, this is accomplished by adding an affinity tag to a full-length or truncated myosin construct and purifying via affinity chromatography29,30,31. Additionally, the construct can be engineered to include a genetically encodable fluorophore or a tag for protein labeling with a synthetic fluorophore. By adding such a fluorescent label, single molecule imaging studies can be performed to observe myosin mechanics and kinetics.
Following purification, the myosin can be characterized in several ways. ATPase activity can be measured by colorimetric methods, providing insight into the overall energy consumption and actin affinity of the motor under different conditions32. To learn about the mechanochemistry of its motility, further experiments are required. This paper details two in vitro fluorescence microscopy-based methods that can be used to characterize the motile properties of a purified myosin protein.
The first of these methods is the gliding actin filament assay, which can be used to quantitatively study the ensemble properties of myosin motors, as well as qualitatively study the quality of a batch of purified protein33. Although this paper discusses the use of total internal reflection fluorescence (TIRF) microscopy for this assay, these experiments can be effectively performed using a wide-field fluorescence microscope equipped with a digital camera, commonly found in many labs34. In this assay, a saturating layer of myosin motors is attached to a coverslip. This can be accomplished using nitrocellulose, antibodies, membranes, SiO2-derivatized surfaces (such as trimethylchlorosilane), among others29,33,35,36,37,38. Fluorescently labeled actin filaments are passed through the coverslip chamber, upon which the actin binds to the myosin attached to the surface. Upon addition of ATP (and kinases in the study of NM2), the chamber is imaged to observe the translocation of actin filaments by the surface-bound myosins. Tracking software can be used to correlate the velocity and length of each gliding actin filament. Analysis software can also provide a measure of the number of both moving and stationary actin filaments, which can be useful to determine the quality of a given myosin preparation. The proportion of stalled filaments can also be intentionally modulated by surface tethering of actin to other proteins and measured to determine the load dependence of the myosin39. Because each actin filament can be propelled by a large number of available motors, this assay is very reproducible, with the final measured velocity being robust to perturbations such as alterations in the starting myosin concentration or the presence of additional factors in the solution. This means it can be easily modified to study myosin activity under different conditions, such as altered phosphorylation, temperature, ionic strength, solution viscosity, and the effects of load induced by surface tethers. Although factors such as strong-binding myosin &#34;dead heads&#34; incapable of ATP hydrolysis can cause stalled actin filaments, multiple methods exist to mitigate such issues and allow for accurate measurements. The kinetic properties of myosin vary greatly across classes and, depending on the specific myosin used, the speed of actin filament gliding in this assay can vary from under 20 nm/s (myosin 9)40,41, and up to 60,000 nm/s (Characean myosin 11)42.
The second assay inverts the geometry of the gliding actin filament assay12. Here, the actin filaments are attached to the coverslip surface and the movement of single molecules of M5a or of individual bipolar filaments of NM2b are visualized. This assay can be used to quantify the run lengths and velocities of single myosin molecules or filaments on actin. A coverslip is coated with a chemical compound that blocks non-specific binding and simultaneously functionalizes the surface, such as biotin-polyethylene glycol (biotin-PEG). The addition of modified avidin derivatives then primes the surface and biotinylated actin is passed through the chamber, resulting in a layer of F-actin stably bound to the bottom of the chamber. Finally, activated and fluorescently labeled myosin (typically 1-100 nM) is flowed through the chamber, which is then imaged to observe myosin movement over the stationary actin filaments.
These modalities represent fast and reproducible methods that can be employed to examine the dynamics of both nonmuscle and muscle myosins. This report outlines the procedures to purify and characterize both M5a and NM2b, representing unconventional and conventional myosins, respectively. This is followed by a discussion of some of the myosin-specific adaptations, which can be performed to achieve successful capturing of motion in the two types of the assay.
Expression and molecular biology 
The cDNA for the myosin of interest must be cloned onto a modified pFastBac1 vector that encodes for either a C-terminal FLAG-tag (DYKDDDDK) if expressing M5a-HMM, or an N-terminal FLAG-tag if expressing the full-length molecule of NM2b23,43,44,45,46. C-terminal FLAG-tags on NM2b results in a weakened affinity of the protein for the FLAG-affinity column. In contrast, the N-terminally FLAG-tagged protein usually binds well to the FLAG-affinity column23. The N-terminally tagged protein retains enzymatic activity, mechanical activity and phosphorylation-dependent regulation23.
In this paper, a truncated mouse M5a heavy meromyosin (HMM)-like construct with a GFP between the FLAG-tag and the C-terminus of the myosin heavy chain was used. Note that unlike NM2b, M5a-HMM can be successfully tagged and purified with either N- or C-terminal FLAG tags and in both cases the resulting construct will be active. The M5a heavy chain was truncated at amino acid 1090 and contains a three amino acid linker (GCG) between the GFP and the coiled-coil region of the M5a47. No additional linker was added between the GFP and FLAG-tag. M5a-HMM was co-expressed with calmodulin. The full-length human NM2b construct was co-expressed with ELC and RLC. The N-termini of the RLC was fused with a GFP via a linker of five amino acids (SGLRS). Directly attached to the FLAG-tag was a HaloTag. Between the HaloTag and the N-terminus of the myosin heavy chain was a linker made of two amino acids (AS).
Both myosin preparations were purified from one liter of Sf9 cell culture infected with baculovirus at a density of approximately 2 x 106 cells/mL. The volumes of the baculovirus for each subunit depended on the virus&#39;s multiplicity of infection as determined by the manufacturer&#39;s instructions. In the case of M5a, cells were co-infected with two different baculoviruses-one for calmodulin, and one for M5a heavy chain. In the case of the NM2b, cells were co-infected with three different viruses-one for ELC, one for RLC, and one for NM2b heavy chain. For labs working with a diversity of myosins (or other multi-complex proteins), this approach is efficient since it allows for many combinations of heavy and light chains and commonly used light chains such as calmodulin can be co-transfected with many different myosin heavy chains. All cell work was completed in a biosafety cabinet with proper sterile technique to avoid contamination.
For the expression of both M5a and NM2b, the Sf9 cells producing the recombinant myosins were collected 2-3 days post-infection, via centrifugation, and stored at -80 &#176;C. Cell pellets were obtained by centrifuging the co-infected Sf9 cells at 4 &#176;C for 30 min at 2,800 x g. The protein purification process is detailed below.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL Syringe</td>
			<td>BD</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr>
			<td>2 M CaCl2 Solution</td>
			<td>VWR</td>
			<td>10128-558</td>
			<td></td>
		</tr>
		<tr>
			<td>2 M MgCl2 Solution</td>
			<td>VWR</td>
			<td>10128-298</td>
			<td></td>
		</tr>
		<tr>
			<td>27 Gauge Needle</td>
			<td>Becton Dickinson</td>
			<td>309623</td>
			<td></td>
		</tr>
		<tr>
			<td>5 M NaCl Solution</td>
			<td>KD Medical</td>
			<td>RGE-3270</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>ThermoFisher Scientific</td>
			<td>984303</td>
			<td></td>
		</tr>
		<tr>
			<td>Amyl Acetate</td>
			<td>Ladd Research Industries</td>
			<td>10825</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-FLAG M2 Affinity Gel</td>
			<td>Millipore Sigma</td>
			<td>A2220</td>
			<td>https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/a2220bul.pdf</td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Millipore Sigma</td>
			<td>A7699</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated G-Actin</td>
			<td>Cytoskeleton, Inc.</td>
			<td>AB07</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Millipore Sigma</td>
			<td>5470</td>
			<td></td>
		</tr>
		<tr>
			<td>bPEG-silane</td>
			<td>Laysan Bio, Inc</td>
			<td>Biotin-PEG-SIL-3400-1g</td>
			<td></td>
		</tr>
		<tr>
			<td>Bradford Reagent Concentrate</td>
			<td>Bio-Rad</td>
			<td>5000006</td>
			<td></td>
		</tr>
		<tr>
			<td>Calmodulin</td>
			<td></td>
			<td>PMID: 2985564</td>
			<td></td>
		</tr>
		<tr>
			<td>Catalase</td>
			<td>Millipore Sigma</td>
			<td>C40</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Line (Sf9) in SF-900 II SFM</td>
			<td>ThermoFisher Scientific</td>
			<td>11496015</td>
			<td>http://tools.thermofisher.com/content/sfs/manuals/bevtest.pdf https://tools.thermofisher.com/content/sfs/manuals/bactobac_man.pdf</td>
		</tr>
		<tr>
			<td>Circular Filter Paper - Gliding Assay</td>
			<td>Millipore Sigma</td>
			<td>WHA1001125</td>
			<td></td>
		</tr>
		<tr>
			<td>Circular Filter Paper - Inverted Assay</td>
			<td>Millipore Sigma</td>
			<td>WHA1001090</td>
			<td></td>
		</tr>
		<tr>
			<td>cOmplete, EDTA-Free Protease Inhibitor Tablets</td>
			<td>Millipore Sigma</td>
			<td>5056489001</td>
			<td>This should be stored at 4 &#176;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 &#176;C for 1-2 weeks or at least 12 weeks at -20 &#176;C.&#160;</td>
		</tr>
		<tr>
			<td>Concentrating Tubes (100,000 MWCO)</td>
			<td>EMD Millipore Corporation</td>
			<td>UFC910024</td>
			<td>The MWCO of the tube is not necessarily &#34;one size fits all,&#34; 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.</td>
		</tr>
		<tr>
			<td>Coomassie Brilliant Blue R-250 Dye</td>
			<td>ThermoFisher Scientific</td>
			<td>20278</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip Rack</td>
			<td>Millipore Sigma</td>
			<td>Z688568-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips: Gliding Acting Filament Assay</td>
			<td>VWR International</td>
			<td>48366-227</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips: Inverted Motility Assay</td>
			<td>Azer Scientific</td>
			<td>ES0107052</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis Tubing (3500 Dalton MCWO)</td>
			<td>Fischer Scientific</td>
			<td>08-670-5A</td>
			<td>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 &#176;C.</td>
		</tr>
		<tr>
			<td>DL-Dithiothreitol</td>
			<td>Millipore Sigma</td>
			<td>D0632</td>
			<td></td>
		</tr>
		<tr>
			<td>Double-Sided Tape</td>
			<td>Office Depot</td>
			<td>909955</td>
			<td></td>
		</tr>
		<tr>
			<td>DYKDDDDK Peptide</td>
			<td>GenScript</td>
			<td>RP10586</td>
			<td>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 &#176;C as 300 &#181;L aliquots.&#160;</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Millipore Sigma</td>
			<td>E4378</td>
			<td></td>
		</tr>
		<tr>
			<td>Elution Column</td>
			<td>Bio-Rad</td>
			<td>761-1550</td>
			<td>These can be reused. To clean, rinse the column with 2-3 column volumes of PBS and distilled water. Chill the column at 4&#176; C before use.</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fischer Scientific</td>
			<td>A4094</td>
			<td></td>
		</tr>
		<tr>
			<td>G-actin</td>
			<td></td>
			<td>PMID: 4254541</td>
			<td>G-actin stock can be stored at 200 &#956;M in liquid N2.</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Millipore Sigma</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose Oxidase</td>
			<td>Millipore Sigma</td>
			<td>G2133</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine Buffer Solution, 100 mM, pH 2-2.5, 1 L</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-295018</td>
			<td></td>
		</tr>
		<tr>
			<td>HaloTag</td>
			<td>Promega</td>
			<td>G100A</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Millipore Sigma</td>
			<td>320331</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fischer Scientific</td>
			<td>P217-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Large-Orifice Pipet Tips</td>
			<td>Fischer Scientific</td>
			<td>02-707-134</td>
			<td></td>
		</tr>
		<tr>
			<td>Leupeptin Protease Inhibitor</td>
			<td>ThermoFisher Scientific</td>
			<td>78435</td>
			<td></td>
		</tr>
		<tr>
			<td>Mark12 Unstained Standard Ladder</td>
			<td>ThermoFisher Scientific</td>
			<td>LC5677</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Millipore Sigma</td>
			<td>MX0482</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylcellulose</td>
			<td>Millipore Sigma</td>
			<td>M0512</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Slides</td>
			<td>Fischer Scientific</td>
			<td>12-553-10</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Fischer Scientific</td>
			<td>BP308-100</td>
			<td></td>
		</tr>
		<tr>
			<td>mPEG-silane</td>
			<td>Laysan Bio, Inc</td>
			<td>MPEG-SIL-2000-1g</td>
			<td></td>
		</tr>
		<tr>
			<td>Myosin Light Chain Kinase</td>
			<td></td>
			<td>PMID: 23148220</td>
			<td>FLAG-tagged MLCK can be purified the same way that the FLAG-tagged myosin was purified herein.&#160;</td>
		</tr>
		<tr>
			<td>NaN3</td>
			<td>Millipore Sigma</td>
			<td>S8032</td>
			<td></td>
		</tr>
		<tr>
			<td>NeutrAvidin</td>
			<td>ThermoFisher Scientific</td>
			<td>31050</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose</td>
			<td>Ladd Research Industries</td>
			<td>10800</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAGE 4 to 12% Bis-Tris Mini Protein Gel, 15-well</td>
			<td>ThermoFisher Scientific</td>
			<td>NP0323PK2</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAGE LDS Sample Buffer (4X)</td>
			<td>ThermoFisher Scientific</td>
			<td>NP0007</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline, pH 7.4</td>
			<td>ThermoFisher Scientific</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Millipore Sigma</td>
			<td>78830</td>
			<td>PMSF can be made as a 0.1 M stock solution in isopropanol and stored in 4 &#176;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.&#160;</td>
		</tr>
		<tr>
			<td>Razor Blades</td>
			<td>Office Depot</td>
			<td>397492</td>
			<td></td>
		</tr>
		<tr>
			<td>Rhodamine-Phalloidin</td>
			<td>ThermoFisher Scientific</td>
			<td>R415</td>
			<td>Stock can be diluted in 100% methanol to a final concentration of 200 &#956;M.</td>
		</tr>
		<tr>
			<td>Sf9 Media</td>
			<td>ThermoFisher Scientific</td>
			<td>12658-027</td>
			<td>This should be stored at 4&#176; C. Its shelf life is 18 months from the date of manufacture.</td>
		</tr>
		<tr>
			<td>Tissue Culture Dish - Gliding Assay</td>
			<td>Corning</td>
			<td>353025</td>
			<td>Each tissue culture dish can hold approximately nine coverslips.</td>
		</tr>
		<tr>
			<td>Tissue Culture Dish - Inverted Assay</td>
			<td>Corning</td>
			<td>353003</td>
			<td>Each tissue culture dish can hold approximately four coverslips.</td>
		</tr>
		<tr>
			<td>Smooth-sided 200 &#181;L Pipette Tips</td>
			<td>Thomas Scientific</td>
			<td>1158U38</td>
			<td></td>
		</tr>
		<tr>
			<td>EQUIPMENT</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>ThermoFisher Scientific</td>
			<td>75006590</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Nikon</td>
			<td>Model: Eclipse Ti with H-TIRF system with 100x TIRF Objective (N.A. 1.49)</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Camera</td>
			<td>Andor</td>
			<td>Model: iXon DU888 EMCCD camera (1024 x 1024 sensor format)</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Environmental Control Box</td>
			<td>Tokai HIT</td>
			<td>Custom Thermobox</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Laser Unit</td>
			<td>Nikon</td>
			<td>LU-n4 four laser unit with solid state lasers for 405nm, 488nm, 561nm,and 640nm</td>
			<td></td>
		</tr>
		<tr>
			<td>Mid Bench Centrifuge</td>
			<td>ThermoFisher Scientific</td>
			<td>Model: CR3i</td>
			<td></td>
		</tr>
		<tr>
			<td>Misonix Sonicator</td>
			<td>Misonix</td>
			<td>XL2020</td>
			<td></td>
		</tr>
		<tr>
			<td>Optima Max-Xp Tabletop Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>393315</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma-Cleaner</td>
			<td>Diener electronic GmbH + Co. KG</td>
			<td>System Type: Zepto</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator Probe (3.2 mm)</td>
			<td>Qsonica</td>
			<td>4418</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard Incubator</td>
			<td>Binder</td>
			<td>Model: 56</td>
			<td></td>
		</tr>
		<tr>
			<td>Waverly Tube Mixer</td>
			<td>Waverly</td>
			<td>TR6E</td>
			<td></td>
		</tr>
		<tr>
			<td>SOFTWARE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ FIJI</td>
			<td>https://imagej.net/Fiji/Downloads</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FAST (Version 1.01)</td>
			<td>http://spudlab.stanford.edu/fast-for-automatic-motility-measurements</td>
			<td>FAST is available for Mac OSX and Linux based systems.</td>
			<td></td>
		</tr>
		<tr>
			<td>Image Stabilizer Plugin</td>
			<td>https://imagej.net/Image_Stabilizer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ TrackMate</td>
			<td>https://imagej.net/TrackMate</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging Software</td>
			<td>NIS Elements (AR package)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>http://www.cs.cmu.edu/~kangli/code/Image_Stabilizer.html</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>File:TrackMate-manual.pdf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>https://github.com/turalaksel/FASTrack</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>https://github.com/turalaksel/FASTrack/blob/master/README.md</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

myosin-specific adaptations of in vitro fluorescence microscopy-based motility assays

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&#160;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.

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...

Watch this Scientific Journal Video about Myosin-Specific Adaptations of In vitro Fluorescence Microscopy-Based Motility Assays at JoVE.com

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

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 ...

This protocol can be used to characterize non-muscle myosin dynamics at the protein level, which can inform the motile properties that contribute to their roles in cells. This is a fast, reproducible, and highly controlled method that can be used to study the effects of various regulatory conditions on myosin motility, informing their cellular behaviors. These techniques can be used to investigate how disease-causing mutations in non-muscle myosins affect their behaviors at the single molecule and ensemble level.The general approach of its methodology can be applied to various other cytoskeletal systems, such as the characterization of purified kinesins and dyneins with microtubules. This protocol's versatility can raise questions about which fluorophores and chemical conditions are most appropriate, but the method can be optimized relatively quickly. Understanding how to set up and coat the flow chambers makes for a strong foundation upon which one can adapt to various conditions for these experiments.To begin, prepare a one percent nitrocellulose solution in amyl acetate and place a circular filter paper with a 125 millimeter diameter on the bottom of a tissue culture plate. Load eight square cover slips onto a rack and thoroughly wash them with approximately two to five milliliters of 200-proof ethanol followed by two to five milliliters of distilled water. Then drive the cover slips completely using a filtered air line or nitrogen line.Slowly pipette 10 microliters of one percent nitrocellulose solution along one edge of one cover slip. Smear it across the rest of the cover slip using the side of a 200 microliter pipette tip in one smooth motion. Then place this cover slip on the tissue culture dish with the nitrocellulose side up.Repeat this for the remaining cover slips and allow them to dry. Wipe a microscope slide with an optical lens paper to clean off large debris. Cut two pieces of double-sided tape, approximately two centimeters in length, and place one piece along the middle of the long edge of the microscope slide.Place the second piece of tape roughly two millimeters below the first piece of tape such that the two are parallel creating a flow chamber that can hold approximately 10 microliters of solution. Carefully stick one of the nitrocellulose cover slips onto the tape so that the side coated with nitrocellulose is making direct contact with the tape. Using a pipette tip, gently press down on the slide tape interface to ensure that the cover slip is properly adhered to the slide.Then cut the excess tape hanging over the edge of the slide with a razor blade. Prepare the solutions for Myosin 5a and keep them on ice. Flow in 10 microliters of the Myosin 5a through the slide flow chamber and wait for one minute.Then flow in 10 microliters of one milligram per milliliter BSA in motility buffer with DTT. Repeat this twice and wait a minute 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.Then wash with 10 microliters of motility buffer with DTT. Pipette the black actin solution with a one milliliter syringe and a 27-gauge needle to shear the actin filaments before introducing that solution to the chamber. Add the black actin to the chamber in the presence of one millimolar ATP in 50 millimolar MB with one millimolar DTT.Next, flow in 50 microliters of motility buffer with DTT and one millimolar ATP to deplete the chamber of free actin filaments. Wash with 10 microliters of motility buffer with DTT three times to deplete the chamber of any ATP. Flow in 10 microliters of 20 nanomolar RH actin solution containing motility buffer with DTT and wait for one minute to allow rigor binding of actin filaments to the Myosin 5a attached to the surface of the cover slip.Wash with 10 microliters of 50 millimolar motility buffer with DTT twice to remove unbound RH actin filaments. Flow in 30 microliters of final buffer. Record images on a fluorescence microscope using an excitation wavelength of 561 nanometers to visualize the RH actin.Prepare the solutions for Myosin 5a inverted motility assay and keep them on ice. Wash them chamber with 10 microliters of motility buffer with DTT. Flow in 10 microliters of one milligram per milliliter BSA in motility buffer with DTT.Repeat this twice, waiting a minute after the third wash. Use tissue or filter paper to wick the solution through the channel by gently placing the corner of the paper at the flow chamber exit. Then, wash the chamber with 10 microliters of motility buffer with DTT three times.Flow in 10 microliters of the NeutrAvidin solution in motility buffer with DTT and wait for one minute. Then wash with 10 microliters of that solution three times. Flow in 10 microliters of BRH actin containing motility buffer with DTT using a large-bored pipette tip.And wait for one minute. Then, wash with 10 microliters of motility buffer with DTT three times. Flow in 30 microliters of final buffer with 10 nanomolar Myosin 5a.And immediately load onto the total internal reflection fluorescence microscope. Begin recording once the optimum focus for TIRF imaging is found. The purification of non-muscle Myosin 2b, essential light chains, and regulatory light chains, as well as the Myosin 5a and calmodulin was evaluated by performing SDS-PAGE.The gliding actin filament assay shows the characteristics of an ideal and trackable movie featuring smooth movements of labeled actin filaments. The black actin wash ensured that the dead myosin heads were removed from the measurement field further contributing to the overall smooth movement of the actin filaments. Filament tracking output images from the fast track program were obtained for non-muscle Myosin 2b and Myosin 5a.A representative histogram shows that the Actomyosin 2b gliding velocity is 77 nanometers per second. And that of Actomyosin 5a is 515 nanometers per second. In the absence of methylcellulose, the actin filaments do not remain as closely associated by the myosin-coated surface, causing it's flopping close to the surface of the non-muscle Myosin 2b-coated cover slips.Myosin movement was observed upon surface-tethered fluorescent actin filaments using the inverted motility assay. It is important to shear the unlabeled actin before introducing it to the flow cell for the black actin wash which will allow for the smooth translocation of actin. Additional experiments can investigate how myosin motility is affected by myosin mutations, load-inducing proteins, ionic strength, and regulatory proteins.Various cellular conditions can be reconstituted for future studies. These methods allow for the biochemical and biophysical investigations into how various myosin isoforms vary in their motile and mechanical properties at the single molecule and ensemble level.

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