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.

The authors declare no conflict of interest.

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><tbody><tr><td>&lt;strong&gt;1 mL Syringe&lt;/strong&gt;</td><td>BD</td><td>309628</td><td></td></tr><tr><td>&lt;strong&gt;2&lt;/strong&gt; M CaCl&lt;sub&gt;2 &lt;/sub&gt;Solution</td><td>VWR</td><td>10128-558</td><td></td></tr><tr><td>&lt;strong&gt;2 M MgCl&lt;/strong&gt;&lt;sub&gt;2 &lt;/sub&gt;Solution</td><td>VWR</td><td>10128-298</td><td></td></tr><tr><td>&lt;strong&gt;27 Gauge Needle&lt;/strong&gt;</td><td>Becton Dickinson</td><td>309623</td><td></td></tr><tr><td>&lt;strong&gt;5 M NaCl Solution&lt;/strong&gt;</td><td>KD Medical</td><td>RGE-3270</td><td></td></tr><tr><td>&lt;strong&gt;Acetic Acid&lt;/strong&gt;</td><td>ThermoFisher Scientific</td><td>984303</td><td></td></tr><tr><td>&lt;strong&gt;Amyl Acetate&lt;/strong&gt;</td><td>Ladd Research Industries</td><td>10825</td><td></td></tr><tr><td>&lt;strong&gt;Anti-FLAG M2 Affinity Gel&lt;/strong&gt;</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>&lt;strong&gt;ATP&lt;/strong&gt;</td><td>Millipore Sigma</td><td>A7699</td><td></td></tr><tr><td>&lt;strong&gt;Biotinylated G-Actin&lt;/strong&gt;</td><td>Cytoskeleton, Inc.</td><td>AB07</td><td></td></tr><tr><td>&lt;strong&gt;Bovine Serum Albumin&lt;/strong&gt;</td><td>Millipore Sigma</td><td>5470</td><td></td></tr><tr><td>&lt;strong&gt;bPEG-silane&lt;/strong&gt;</td><td>Laysan Bio, Inc</td><td>Biotin-PEG-SIL-3400-1g</td><td></td></tr><tr><td>&lt;strong&gt;Bradford Reagent Concentrate&lt;/strong&gt;</td><td>Bio-Rad</td><td>5000006</td><td></td></tr><tr><td>&lt;strong&gt;Calmodulin&lt;/strong&gt;</td><td></td><td>PMID: 2985564</td><td></td></tr><tr><td>&lt;strong&gt;Catalase&lt;/strong&gt;</td><td>Millipore Sigma</td><td>C40</td><td></td></tr><tr><td>&lt;strong&gt;Cell Line (Sf9) in SF-900 II SFM&lt;/strong&gt;</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>&lt;strong&gt;Circular Filter Paper - Gliding Assay&lt;/strong&gt;</td><td>Millipore Sigma</td><td>WHA1001125</td><td></td></tr><tr><td>&lt;strong&gt;Circular Filter Paper - Inverted Assay&lt;/strong&gt;</td><td>Millipore Sigma</td><td>WHA1001090</td><td></td></tr><tr><td>&lt;strong&gt;cOmplete, EDTA-Free Protease Inhibitor Tablets&lt;/strong&gt;</td><td>Millipore Sigma</td><td>5056489001</td><td>This should be stored at 4 &amp;deg;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 &amp;deg;C for 1-2 weeks or at least 12 weeks at -20 &amp;deg;C.&amp;nbsp;</td></tr><tr><td>&lt;strong&gt;Concentrating Tubes (100,000 MWCO)&lt;/strong&gt;</td><td>EMD Millipore Corporation</td><td>UFC910024</td><td>The MWCO of the tube is not necessarily &quot;one size fits all,&quot; 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>&lt;strong&gt;Coomassie Brilliant Blue R-250 Dye&lt;/strong&gt;</td><td>ThermoFisher Scientific</td><td>20278</td><td></td></tr><tr><td>&lt;strong&gt;Coverslip Rack&lt;/strong&gt;</td><td>Millipore Sigma</td><td>Z688568-1EA</td><td></td></tr><tr><td>&lt;strong&gt;Coverslips: Gliding Acting Filament Assay&lt;/strong&gt;</td><td>VWR International</td><td>48366-227</td><td></td></tr><tr><td>&lt;strong&gt;Coverslips: Inverted Motility Assay&lt;/strong&gt;</td><td>Azer Scientific</td><td>ES0107052</td><td></td></tr><tr><td>&lt;strong&gt;Dialysis Tubing (3500 Dalton MCWO)&lt;/strong&gt;</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 &amp;deg;C.</td></tr><tr><td>&lt;strong&gt;DL-Dithiothreitol&lt;/strong&gt;</td><td>Millipore Sigma</td><td>D0632</td><td></td></tr><tr><td>&lt;strong&gt;Double-Sided Tape&lt;/strong&gt;</td><td>Office Depot</td><td>909955</td><td></td></tr><tr><td>&lt;strong&gt;DYKDDDDK Peptide&lt;/strong&gt;</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 NaN&lt;sub&gt;3&lt;/sub&gt;, and 10 mM MOPS (pH 7.2) to a final concentration of 50 mg/mL. This can be stored at -20 &amp;deg;C as 300 &amp;micro;L aliquots.&amp;nbsp;</td></tr><tr><td>&lt;strong&gt;EGTA&lt;/strong&gt;</td><td>Millipore Sigma</td><td>E4378</td><td></td></tr><tr><td>&lt;strong&gt;Elution Column&lt;/strong&gt;</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&amp;deg; C before use.</td></tr><tr><td>&lt;strong&gt;Ethanol&lt;/strong&gt;</td><td>Fischer Scientific</td><td>A4094</td><td></td></tr><tr><td>&lt;strong&gt;G-actin&lt;/strong&gt;</td><td></td><td>PMID: 4254541</td><td>G-actin stock can be stored at 200 &amp;mu;M in liquid N&lt;sub&gt;2&lt;/sub&gt;.</td></tr><tr><td>&lt;strong&gt;Glucose&lt;/strong&gt;</td><td>Millipore Sigma</td><td>G8270</td><td></td></tr><tr><td>&lt;strong&gt;Glucose Oxidase&lt;/strong&gt;</td><td>Millipore Sigma</td><td>G2133</td><td></td></tr><tr><td>&lt;strong&gt;Glycine Buffer Solution, 100 mM, pH 2-2.5, 1 L&lt;/strong&gt;</td><td>Santa Cruz Biotechnology</td><td>sc-295018</td><td></td></tr><tr><td>&lt;strong&gt;HaloTag&lt;/strong&gt;</td><td>Promega</td><td>G100A</td><td></td></tr><tr><td>&lt;strong&gt;HCl&lt;/strong&gt;</td><td>Millipore Sigma</td><td>320331</td><td></td></tr><tr><td>&lt;strong&gt;KCl&lt;/strong&gt;</td><td>Fischer Scientific</td><td>P217-500</td><td></td></tr><tr><td>&lt;strong&gt;Large-Orifice Pipet Tips&lt;/strong&gt;</td><td>Fischer Scientific</td><td>02-707-134</td><td></td></tr><tr><td>&lt;strong&gt;Leupeptin Protease Inhibitor&lt;/strong&gt;</td><td>ThermoFisher Scientific</td><td>78435</td><td></td></tr><tr><td>&lt;strong&gt;Mark12 Unstained Standard Ladder&lt;/strong&gt;</td><td>ThermoFisher Scientific</td><td>LC5677</td><td></td></tr><tr><td>&lt;strong&gt;Methanol&lt;/strong&gt;</td><td>Millipore Sigma</td><td>MX0482</td><td></td></tr><tr><td>&lt;strong&gt;Methylcellulose&lt;/strong&gt;</td><td>Millipore Sigma</td><td>M0512</td><td></td></tr><tr><td>&lt;strong&gt;Microscope Slides&lt;/strong&gt;</td><td>Fischer Scientific</td><td>12-553-10</td><td></td></tr><tr><td>&lt;strong&gt;MOPS&lt;/strong&gt;</td><td>Fischer Scientific</td><td>BP308-100</td><td></td></tr><tr><td>&lt;strong&gt;mPEG-silane&lt;/strong&gt;</td><td>Laysan Bio, Inc</td><td>MPEG-SIL-2000-1g</td><td></td></tr><tr><td>&lt;strong&gt;Myosin Light Chain Kinase&lt;/strong&gt;</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.&amp;nbsp;</td></tr><tr><td>&lt;strong&gt;NaN&lt;sub&gt;3&lt;/sub&gt;&lt;/strong&gt;</td><td>Millipore Sigma</td><td>S8032</td><td></td></tr><tr><td>&lt;strong&gt;NeutrAvidin&lt;/strong&gt;</td><td>ThermoFisher Scientific</td><td>31050</td><td></td></tr><tr><td>&lt;strong&gt;Nitrocellulose&lt;/strong&gt;</td><td>Ladd Research Industries</td><td>10800</td><td></td></tr><tr><td>&lt;strong&gt;NuPAGE 4 to 12% Bis-Tris Mini Protein Gel, 15-well&lt;/strong&gt;</td><td>ThermoFisher Scientific</td><td>NP0323PK2</td><td></td></tr><tr><td>&lt;strong&gt;NuPAGE LDS Sample Buffer (4X)&lt;/strong&gt;</td><td>ThermoFisher Scientific</td><td>NP0007</td><td></td></tr><tr><td>&lt;strong&gt;Phosphate-Buffered Saline, pH 7.4&lt;/strong&gt;</td><td>ThermoFisher Scientific</td><td>10010023</td><td></td></tr><tr><td>&lt;strong&gt;PMSF&lt;/strong&gt;</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 &amp;deg;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.&amp;nbsp;</td></tr><tr><td>&lt;strong&gt;Razor Blades&lt;/strong&gt;</td><td>Office Depot</td><td>397492</td><td></td></tr><tr><td>&lt;strong&gt;Rhodamine-Phalloidin&lt;/strong&gt;</td><td>ThermoFisher Scientific</td><td>R415</td><td>Stock can be diluted in 100% methanol to a final concentration of 200 &amp;mu;M.</td></tr><tr><td>&lt;strong&gt;Sf9 Media&lt;/strong&gt;</td><td>ThermoFisher Scientific</td><td>12658-027</td><td>This should be stored at 4&amp;deg; C. Its shelf life is 18 months from the date of manufacture.</td></tr><tr><td>&lt;strong&gt;Tissue Culture Dish - Gliding Assay&lt;/strong&gt;</td><td>Corning</td><td>353025</td><td>Each tissue culture dish can hold approximately nine coverslips.</td></tr><tr><td>&lt;strong&gt;Tissue Culture Dish - Inverted Assay&lt;/strong&gt;</td><td>Corning</td><td>353003</td><td>Each tissue culture dish can hold approximately four coverslips.</td></tr><tr><td>&lt;strong&gt;Smooth-sided 200 &amp;micro;L Pipette Tips&lt;/strong&gt;</td><td>Thomas Scientific</td><td>1158U38</td><td></td></tr><tr><td>&lt;strong&gt;EQUIPMENT&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Centrifuge&lt;/strong&gt;</td><td>ThermoFisher Scientific</td><td>75006590</td><td></td></tr><tr><td>&lt;strong&gt;Microscope&lt;/strong&gt;</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>&lt;strong&gt;Microscope Camera&lt;/strong&gt;</td><td>Andor</td><td>Model: iXon DU888 EMCCD camera (1024 x 1024 sensor format)</td><td></td></tr><tr><td>&lt;strong&gt;Microscope Environmental Control Box&lt;/strong&gt;</td><td>Tokai HIT</td><td>Custom Thermobox</td><td></td></tr><tr><td>&lt;strong&gt;Microscope Laser Unit&lt;/strong&gt;</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>&lt;strong&gt;Mid Bench Centrifuge&lt;/strong&gt;</td><td>ThermoFisher Scientific</td><td>Model: CR3i</td><td></td></tr><tr><td>&lt;strong&gt;Misonix Sonicator&lt;/strong&gt;</td><td>Misonix</td><td>XL2020</td><td></td></tr><tr><td>&lt;strong&gt;Optima Max-Xp Tabletop Ultracentrifuge&lt;/strong&gt;</td><td>Beckman Coulter</td><td>393315</td><td></td></tr><tr><td>&lt;strong&gt;Plasma-Cleaner&lt;/strong&gt;</td><td>Diener electronic GmbH + Co. KG</td><td>System Type: Zepto</td><td></td></tr><tr><td>&lt;strong&gt;Sonicator Probe (3.2 mm)&lt;/strong&gt;</td><td>Qsonica</td><td>4418</td><td></td></tr><tr><td>&lt;strong&gt;Standard Incubator&lt;/strong&gt;</td><td>Binder</td><td>Model: 56</td><td></td></tr><tr><td>&lt;strong&gt;Waverly Tube Mixer&lt;/strong&gt;</td><td>Waverly</td><td>TR6E</td><td></td></tr><tr><td>&lt;strong&gt;SOFTWARE&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;ImageJ FIJI&lt;/strong&gt;</td><td>https://imagej.net/Fiji/Downloads</td><td></td><td></td></tr><tr><td>&lt;strong&gt;FAST (Version 1.01)&lt;/strong&gt;</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>&lt;strong&gt;Image Stabilizer Plugin&lt;/strong&gt;</td><td>https://imagej.net/Image_Stabilizer</td><td></td><td></td></tr><tr><td>&lt;strong&gt;ImageJ TrackMate&lt;/strong&gt;</td><td>https://imagej.net/TrackMate</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Imaging Software&lt;/strong&gt;</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...

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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 ...

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5.8K Views.  National Institutes of Health. Dit protocol kan worden gebruikt om niet-spier myosine dynamiek op eiwitniveau te karakteriseren, die de beweeglijke eigenschappen kan informeren die bijdragen aan hun rol in cellen. Dit is een snelle, reproduceerbare en zeer gecontroleerde methode die kan worden gebruikt om de effecten van verschillende regulerende omstandigheden op de beweeglijkheid van myosine te bestuderen en hun cellulaire gedrag te informeren. Deze technieken kunnen worden gebruikt om te onderzoeken hoe ziekteverwekkend...

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

Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. Screening peptides affords an additional layer of complexity as conventional tip-based sample handling methods expose peptides to a large surface area of plasticware, providing an increased opportunity for peptide loss via adsorption. Preventing excessive exposure to plasticware reduces peptide loss via adherence to plastics and thus minimizes inaccuracies in potency prediction, and we have previously described the benefits of non-contact acoustic dispensing for in vitro high-throughput screening of peptide agonists1. Here we discuss a fully integrated automation solution for non-contact acoustic preparation of peptide serial dilutions in microtiter plates utilizing the example of screening for peptide agonists at the mouse glucagon-like peptide-1 receptor (GLP-1R). Our methods allow for high-throughput cell-based assays to screen for agonists and are easily scalable to support increased sample throughput, or to allow for increased numbers of assay plate copies (e.g., for a panel of more target cell lines).

Peptide adsorption to plasticware during traditional tip-based serial dilutions can significantly impact potency determination and confound the understanding of structure-activity relationships used for lead identification and lead optimization phases of drug discovery. Here methods for automated acoustic non-contact serial dilution of peptide samples are described.

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. ...

Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells

The mitotic bipolar kinesin-5 motors perform essential functions in spindle dynamics. These motors exhibit a homo-tetrameric structure with two pairs of catalytic motor domains, located at opposite ends of the active complex. This unique architecture enables kinesin-5 motors to crosslink and slide apart antiparallel spindle microtubules (MTs), thus providing the outwardly-directed force that separates the spindle poles apart. Previously, kinesin-5 motors were believed to be exclusively plus-end directed. However, recent studies revealed that several fungal kinesin-5 motors are minus-end directed at the single-molecule level and can switch directionality under various experimental conditions. The Saccharomyces cerevisiae kinesin-5 Cin8 is an example of such bi-directional motor protein: in high ionic strength conditions single molecules of Cin8 move in the minus-end direction of the MTs. It was also shown that Cin8 forms motile clusters, predominantly at the minus-end of the MTs, and such clustering allows Cin8 to switch directionality and undergo slow, plus-end directed motility. This article provides a detailed protocol for all steps of working with GFP-tagged kinesin-5 Cin8, from protein overexpression in S. cerevisiae cells and its purification to in vitro single-molecule motility assay. A newly developed method described here helps to differentiate between single molecules and clusters of Cin8, based on their fluorescence intensity. This method enables separate analysis of motility of single molecules and clusters of Cin8, thus providing the characterization of the dependence of Cin8 motility on its cluster size.

Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells

The bi-directional mitotic kinesin-5 Cin8 accumulates in clusters that split and merge during their motility. Accumulation in clusters also changes the velocity and directionality of Cin8. Here, a protocol for motility assays with purified Cin8-GFP and analysis of motile properties of single molecules and clusters of Cin8 is described.

The mitotic bipolar kinesin-5 motors perform essential functions in spindle dynamics. These motors exhibit a homo-tetrameric structure with two pairs of ...

Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved single-molecule studies and has gained immense popularity in detecting and understanding the dynamic behavior of fluorescent-tagged molecules. Here, we describe a detailed method for&#160;in vitro single-molecule studies of kinesin-3 family motors using Total Internal Reflection Fluorescence (TIRF) microscopy. Kinesin-3 is a large family that plays critical roles in cellular and physiological functions ranging from intracellular cargo transport to cell division to development. We have shown previously that constitutively active dimeric kinesin-3 motors exhibit fast and superprocessive motility with high microtubule affinity at the single-molecule level using cell lysates prepared by expressing motor in mammalian cells. Our lab studies kinesin-3 motors and their regulatory mechanisms using cellular, biochemical and biophysical approaches, and such studies demand purified proteins at a large scale. Expression and purification of these motors using mammalian cells would be expensive and time-consuming, whereas expression in a prokaryotic expression system resulted in significantly aggregated and&#160;inactive protein. To overcome the limitations posed by bacterial purification systems and mammalian cell lysate, we have established a robust Sf9-baculovirus expression system to express and purify these motors. The kinesin-3 motors are C-terminally tagged with 3-tandem fluorescent proteins (3xmCitirine or 3xmCit) that provide enhanced signals and decreased photobleaching. In vitro single-molecule and multi-motor gliding analysis of Sf9 purified proteins demonstrate that kinesin-3 motors are fast and superprocessive akin to our previous studies using mammalian cell lysates. Other applications using these assays include detailed knowledge of oligomer conditions of motors, specific binding partners paralleling biochemical studies, and their kinetic state.

This study details purification of KIF1A(1-393LZ), a member of kinesin-3 family, using Sf9-baculovirus expression system. In vitro single-molecule and multi-motor gliding analysis of these purified motors exhibited robust motility properties comparable to motors from mammalian cell lysate. Thus, Sf9-baculovirus system is amenable to express and purify motor protein of interest.

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved ...

Recombinant Human Myosin-7a Production and Functional Characterization Protocol

MultiBac System-Based Purification and Biophysical Characterization of Human Myosin-7a

Myosin-7a is an actin-based motor protein vital for auditory and visual processes. Mutations in myosin-7a lead to Usher syndrome type 1, the most common and severe form of deaf-blindness in humans. It is hypothesized that myosin-7a forms a transmembrane adhesion complex with other Usher proteins, essential for the structural-functional integrity of photoreceptor and cochlear hair cells. However, due to the challenges in obtaining pure, intact protein, the exact functional mechanisms of human myosin-7a remain elusive, with limited structural and biomechanical studies available. Recent studies have shown that mammalian myosin-7a is a multimeric motor complex consisting of a heavy chain and three types of light chains: regulatory light chain (RLC), calmodulin, and calmodulin-like protein 4 (CALML4). Unlike calmodulin, CALML4 does not bind to calcium ions. Both the calcium-sensitive, and insensitive calmodulins are critical for mammalian myosin-7a for proper fine-tuning of its mechanical properties. Here, we describe a detailed method to produce recombinant human myosin-7a holoenzyme using the MultiBac Baculovirus protein expression system. This yields milligram quantities of high-purity full-length protein, allowing for its biochemical and biophysical characterization. We further present a protocol for assessing its mechanical and motile properties using tailored in vitro motility assays and fluorescence microscopy. The availability of the intact human myosin-7a protein, along with the detailed functional characterization protocol described here, paves the way for further investigations into the molecular aspects of myosin-7a in vision and hearing.

Author Spotlight: Unraveling the Role of Myosin-7a and Usher Proteins in Hearing and Human Disease

This protocol details the procedures for recombinantly producing the human myosin-7a holoenzyme using the MultiBac Baculovirus system and for studying its motility using a tailored in vitro filament gliding assay.

Myosin-7a is an actin-based motor protein vital for auditory and visual processes. Mutations in myosin-7a lead to Usher syndrome type 1, the most common ...

Probing Myosin Ensemble Mechanics in Actin Filament Bundles Using Optical Tweezers

Myosins are motor proteins that hydrolyze ATP to step along actin filament (AF) tracks and are essential in cellular processes such as motility and muscle contraction. To understand their force-generating mechanisms, myosin II has been investigated both at the single-molecule (SM) level and as teams of motors in vitro using biophysical methods such as optical trapping.
These studies showed that myosin force-generating behavior can differ greatly when moving from the single-molecule level in a three-bead arrangement to groups of motors working together on a rigid bead or coverslip surface in a gliding arrangement. However, these assay constructions do not permit evaluating the group dynamics of myosin within viscoelastic structural hierarchy as they would within a cell. We have developed a method using optical tweezers to investigate the mechanics of force generation by myosin ensembles interacting with multiple actin filaments.
These actomyosin bundles facilitate investigation in a hierarchical and compliant environment that captures motor communication and ensemble force output. The customizable nature of the assay allows for altering experimental conditions to understand how modifications to the myosin ensemble, actin filament bundle, or the surrounding environment result in differing force outputs.

Formation of actomyosin bundles in vitro and measuring myosin ensemble force generation using optical tweezers is presented and discussed.

Myosins are motor proteins that hydrolyze ATP to step along actin filament (AF) tracks and are essential in cellular processes such as motility and muscle ...

Bioengineering

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (&#62;200 &#181;m), localization precision of 2-3 nm can be achieved.

Fluorescence Imaging with One-nanometer Accuracy FIONA

Single fluorophores can be localized with nanometer precision using FIONA. Here a summary of the FIONA technique is reported, and how to carry out FIONA experiments is described.

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...

Biology

Biophysical Characterization of Flagellar Motor Functions

The role of flagellar motors in bacterial motility and chemotaxis is well-understood. Recent discoveries suggest that flagellar motors are able to remodel in response to a variety of environmental stimuli and are among the triggers for surface colonization and infections. The precise mechanisms by which motors remodel and promote cellular adaptation likely depend on key motor attributes. The photomultiplier-based bead-tracking technique presented here enables accurate biophysical characterization of motor functions, including adaptations in motor speeds and switch-dynamics. This approach offers the advantage of real-time tracking and the ability to probe motor behavior over extended durations. The protocols discussed can be readily extended to study flagellar motors in a variety of bacterial species.

Recent findings suggest that bacterial flagellar motors sense a variety of environmental signals and remodel in response. The bead-assays discussed here are expected to help explain the role of remodeling in cellular adaptation to environmental stressors.

The role of flagellar motors in bacterial motility and chemotaxis is well-understood. Recent discoveries suggest that flagellar motors are able to remodel ...

Directly Measuring Forces Within Reconstituted Active Microtubule Bundles

Microtubule networks are employed in cells to accomplish a wide range of tasks, ranging from acting as tracks for vesicle transport to working as specialized arrays during mitosis to regulate chromosome segregation. Proteins that interact with microtubules include motors such as kinesins and dynein, which can generate active forces and directional motion, as well as non-motor proteins that crosslink filaments into higher-order networks or regulate filament dynamics. To date, biophysical studies of microtubule-associated proteins have overwhelmingly focused on the role of single motor proteins needed for vesicle transport, and significant progress has been made in elucidating the force-generating properties and mechanochemical regulation of kinesins and dyneins. However, for processes in which microtubules act both as cargo and track, such as during filament sliding within the mitotic spindle, much less is understood about the biophysical regulation of ensembles of the crosslinking proteins involved. Here, we detail our methodology for directly probing force generation and response within crosslinked microtubule minimal networks reconstituted from purified microtubules and mitotic proteins. Microtubule pairs are crosslinked by proteins of interest, one microtubule is immobilized to a microscope coverslip, and the second microtubule is manipulated by an optical trap. Simultaneous total internal reflection fluorescence microscopy allows for multichannel visualization of all the components of this microtubule network as the filaments slide apart to generate force. We also demonstrate how these techniques can be used to probe pushing forces exerted by kinesin-5 ensembles and how viscous braking forces arise between sliding microtubule pairs crosslinked by the mitotic MAP PRC1. These assays provide insights into the mechanisms of spindle assembly and function and can be more broadly adapted to study dense microtubule network mechanics in diverse contexts, such as the axon and dendrites of neurons and polar epithelial cells.

Here, we present a protocol for reconstituting microtubule bundles in vitro and directly quantifying the forces exerted within them using simultaneous optical trapping and total internal reflection fluorescence microscopy. This assay allows for nanoscale-level measurement of the forces and displacements generated by protein ensembles within active microtubule networks.

Microtubule networks are employed in cells to accomplish a wide range of tasks, ranging from acting as tracks for vesicle transport to working as ...

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

Source: Tripathi, A., et al. <a href="https://app.jove.com/v/62180">Myosin-Specific Adaptations of In vitro Fluorescence Microscopy-Based Motility Assays.</a> 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.

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

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

Source: Tripathi, A., et al. <a href="https://app.jove.com/v/62180">Myosin-Specific Adaptations of In vitro Fluorescence Microscopy-Based Motility Assays.</a> 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.

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

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