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

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.