Here, we demonstrate a relatively simple and straightforward procedure to assemble membrane-tethered actomyosin networks and study the dynamics using advanced light microscopic techniques. The key advantage is that our assay allows the sequential addition of proteins and small molecules without disturbing the membrane-tethered networks. This method can be easily adapted to study other cytoskeletal proteins or composite networks.
The most critical part of the protocol is to ensure that the supported lipid bilayer is formed correctly, so first, we had to master this step before starting to add the cytoskeletal proteins. To begin, take three to five rectangular glass coverslips and place them inside a coplin jar. Turn on the bath sonicator, and set the temperature to 65 degrees Celsius.
Fill the coplin jar with 2%cleaning solution to fully submerge the coverslips, and place it in the sonicator for 30 minutes at full pulse mode. Use blunt PTFE coated forceps to remove the coverslips one by one. Rinse them thoroughly with distilled water, and place them in another coplin jar filled with two normal sodium hydroxide.
Sonicate the coverslips for 20 minutes at full pulse mode. Remove the coverslips, rinse thoroughly with distilled water, and place in another coplin jar filled with distilled water. Before starting the experiment, take the jar in a chemical hood fitted with a nitrogen gas supply.
Use gloves and forceps to remove the coverslips to dry them under the nitrogen stream. Dry both the sides, and place them on a clean plastic grid with a cover. Place the box with the coverslips in a desiccator to avoid contact with dust particles, then take autoclaved PCR tubes and cut out their lids and lower conical halves with a sharp surgical blade.
Take the cylindrical half cut tubes, apply UV curable adhesive to the smooth rim of each cut tube, and place it inverted on a freshly cleaned coverslip such that the rim sits flat on the coverslip. Do not move the cylinder laterally once it is positioned on the coverslip to ensure the glue does not spill into the central space of the chamber. Put the chamber bearing coverslips inside a UV ozone cleaner with an oxygen supply and vacuum.
Turn on the UV light and illuminate for three to five minutes to allow the adhesive to polymerize. Take out the coverslips and test the chambers for leakage, and discard the leaky chambers. Wash each chamber with SLB formation buffer, leaving 100 microliters of buffer at the end.
Mark the level of the buffer at 100 microliters with a permanent marker, then add two microliters of 0.1 molar calcium chloride to the chamber, followed by eight microliters of the SUV solution to each chamber and incubate for 15 minutes at 25 degrees Celsius. Remove 50 microliters of the SLB formation buffer, leaving only 50 microliters in the sample chamber, then add 100 microliters of one XKMEH to the chamber and mix gently. Remove 100 microliters of the buffer without touching the bottom.
Repeat the washes eight to 10 times by adding 100 microliters of one XKMEH and removing 100 microliters. Add 10 microliters of one milligram per milliliter beta-casein to the bilayer, mix gently, and incubate for five to 10 minutes at room temperature. Wash off beta-casein thrice with one XKMEH, and bring the buffer level back to the 100 microliter mark.
During the beta-casein incubation, take out an aliquot of membrane-actin linker protein for minus 80 degrees Celsius thawed quickly at 37 degrees Celsius, and then keep it on ice. Dilute the aliquot with protein dilution buffer to a concentration of one micromolar. Add the linker protein to the solution at a defined final concentration and mix gently.
Incubate for 30 to 40 minutes at room temperature, and wash three to five times with one XKMEH buffer to remove the unbound HSE protein. Bring the buffer level in each chamber back to the 100 microliter mark. The sample is now ready for imaging.
Turn on the microscope, the excitation lasers, and the detection cameras. Ensure the laser is aligned, the objective is cleaned, and the software is ready to acquire images. Put oil on the 100 times objective, mount the sample on the microscope stage, and focus the objective on the bilayer.
Make sure the laser position is such that it undergoes total internal reflection on the sample. Use a 488 nanometer excitation. To determine the integrity of the bilayer, perform a quick FRAP assay by selecting a region of interest on the bilayer and recording a few images of the field of view using imaging conditions that provide a signal to noise ratio of five to one or higher.
Pause the recording, and close the field diaphragm of the TIRF microscope to focus a concentrated laser beam on a small circular region of the bilayer to locally bleach the fluorophores. Turn on the laser to its maximum output to photobleach the small region for three to 10 seconds, and then turn the laser off. Reopen the field diaphragm to its original radius, readjust the imaging condition back to pre-bleach settings, and immediately resume recording the recovery of fluorescent signal in the field of view.
Check if the bilayer is fluid, and save the images as 16 bit dot TIF files. Mix unlabeled and fluorescently labeled G-actin in a 10 to one molar ratio, and top it up with G-buffer so that the concentration of G-actin is 20 micromolar. Add one-tenth of 10 times ME buffer to the mix for a one-time solution, and incubate for two minutes at room temperature.
Next, thaw vial of capping protein stock quickly at 37 degrees Celsius, and then keep it on ice. Dilute with G-buffers such that the concentration of capping protein now is twice its desired final concentration, then add an equal volume of the diluted capping protein solution to the actin mix. Finally, add an equal volume of fresh two times target buffer to the reaction mix.
The final volume of the solution should be four times the volume of the actin mix. Ensure the final concentration of components is as described in the text manuscript. Incubate the samples in the dark at 25 degrees Celsius for 45 to 60 minutes to allow polymerization.
Gently pipette out five micromolar polymerized actin with a blunt ended pipette tip, and add it to a clean autoclaved PCR tube. Add one XKMEH to the tube to make the final volume more than 20 microliters, and mix gently to avoid shearing F-actin. From the mounted sample chamber, remove an equal volume of the buffer.
Add the polymerized actin solution to the chamber, and gently pipette up and down thrice without touching the bilayer at the bottom. Mount the sample on the TIRF microscope, and let it rest for 20 to 30 minutes. Record a few images from different fields of view after F-actin addition has reached a steady state.
After 30 minutes of actin incubation, mount the sample back on the microscope. Check the signal on the linker protein and F-actin channels. Adjust the imaging conditions if needed.
Select a good region for a long time-lapse recording. Record 10 to 15 frames at 0.1 to 0.2 hertz before myosin addition, and pause the recording. Pipette out the required volume of recycled muscle myosin two from the stock vial with a blunt ended pipette tip, and add to a clean autoclaved PCR tube.
Immediately add one XKMEH to the tube to make the volume more than 20 microliters, and mix gently. Carefully remove an equal volume of the KMEH buffer from the mounted sample chamber without disturbing it, and gently add the myosin solution to the sample chamber. Do not pipette up and down, as it will disturb the surface bound filaments.
Immediately resume the time-lapse recording, and save all the images, then take background images for all the channels using a buffer only sample. Save all the images as TIF files. The fitted value of the diffusion coefficient was 1.34 square micrometers per second, which closely agreed with the formula-based calculated value of 1.39 square micrometers per second.
The line profile after photobleaching and the recovery profile of the bleached region fit equations four and five respectively. The mobile fraction of the lipid bilayer representing the fraction of the bleached population that recovers back was greater than 0.9, indicating a good lipid bilayer. Myosin activity induced contractile actomyosin flows that emerged into astro-like structures at the steady state, driving local clustering of the coupled membrane component.
One can use a variety of microscopy techniques such as interferometric scattering microscopy to study the dynamics of unlabeled actomyosin networks without inducing any photo damage, or use super resolution fluorescence microscopy. This technique paves the way for researchers interested in understanding how membrane protein complexes interact with a dynamic actomyosin network to modulate their architecture and composition.
