We combine fluorescence microscopy with microfluidics to study how actin-binding proteins regulate the assembly, and the disassembly, of actin filaments. With microfluidics, we are able to quantify reactions happening on tens of individual actin filaments simultaneously, while precisely controlling biochemical and mechanical conditions. Remember that solutions are first injected in parallel until they reach the chamber, and then, filaments are exposed sequentially to each solution by changing the pressure setting.
To start the preparation of polydimethylsiloxane, or PDMS, chamber assembly, preheat the hot plate to 100 degrees Celsius. Expose one cleaned PDMS chamber and one glass cover slip in a clean Petri dish to ultraviolet light for three to five minutes in a deep UV cleaner. When done, position the PDMS chamber over the cover slip such that the two surfaces directly exposed to UV are put in contact.
To remove air bubbles trapped at the PDMS cover slip interface, gently press the surface of the chamber with a finger. Press strongly over corners and sides, ensuring that the chamber's ceiling does not come in contact with the glass surface. Place the chamber with the glass bottom facing the hot plate at 100 degrees Celsius for five minutes.
Then use the chamber immediately, or store it in a clean Petri dish for a week. To rinse the inlet and outlet tubing, fill one 2 milliliter reservoir tube with 300 microliters of F-buffer, screw it on the microfluidic holder, and set the pressure to 300 millibars. Let five to eight drops of F-buffer go to waste, then set pressure to zero and repeat the procedure for each channel.
Next, set the pressure for the outlet on reservoir tube four to 50 millibars, once a droplet comes out from the tubing tip, connect the tubing to the outlet of the PDMS chamber. As the liquid fills the chamber and comes out of all inlets, set the outlet pressure to 20 millibars. Later, set the pressure for the reservoir tube to 1 to 50 millibars.
To avoid introducing air bubbles, ensure that a droplet comes out of the tubing and the PDMS inlet before connecting the tubing to inlet one. Set the pressure of the inlet to 30 millibars. After connecting the inlets two and three as demonstrated, set the pressure of all inlets to 20 millibars, and the outlet pressure to zero millibars.
Ensure that the flow rates in the inlets are roughly equal. When changing the reservoir, set all inlet pressures to 12 millibars and outlet pressure to 5 millibars. To rapidly inject a solution, set the pressure of the corresponding inlet to 150 millibars, and adjust the pressure in the other inlets to around 100 millibars, such that the resulting flow rate in the inlets is 500 nanoliters per minute.
To change the solution, fill 200 to 300 microliters of the solution in a new reservoir tube and set the pressure to the change setting. Then, unscrew the reservoir tube of the inlet. Once a small droplet has formed at the tubing tip, screw in the new tube with the fresh solution, change the pressure setting to high flow.
Depending on the microfluidic configuration and a chamber geometry, wait for three to five minutes for the solution to fill in the tubing and reach the chamber. The process can be followed by measuring the increase in fluorescence over time. For the surface functionalization, change solution three to 200 microliters of 50 picomolar spectrin-actin seeds in F-buffer, and inject the solution for two minutes with high flow three.
For the surface passivation, change tube three with 300 microliters of 5%BSA in F-buffer, then, inject for five minutes at high flow three, followed by five minutes at mid flow three, with reduced pressure of seven to eight millibars in channels one and two to get a counterflow of 100 nanoliters per minute. The entire chamber surface will be BSA passivated. Change tube three to F-buffer to rinse the channel for five minutes at high flow three.
Later, change tubes one to three with one micromolar actin profilin, 0.15 micromolar actin, and F buffer, respectively, as explained in the manuscript, and inject freshly prepared solutions using the high flow all preset for three to four minutes. Switch on the microscope and adjust the camera exposure time to 100 to 200 milliseconds, excitation laser to 10 to 20%power, and total internal reflection fluorescence, or TIRF depth, at 200 to 300 nanometers to capture the images with a 60X objective. Next, set the pressure setting to mid flow one for 10 minutes to record the filament polymerization at the rate of one frame every 20 seconds, followed by mid flow two for 15 minutes for filament aging.
Capture depolymerization in the epifluorescence mode at one frame every five seconds, after one to two frames, switch to mid flow three to record filaments depolymerizing at around 10 subunits per second. To reset the experiment, break all fluorescently labeled filaments by continuously exposing them to the laser at maximum power for two minutes. Test different conditions by changing solutions one, two, or three and injecting them at high flow for three to four minutes.
The outcome of the basic polymerization/depolymerization experiment is presented here. The resultant kymographs were used to quantify the polymerization and depolymerization rates. When the grown actin filaments were exposed to a fluorescently labeled cofilin solution, cofilin clusters were nucleated and grew toward the pointed and barbed ends.
When single filaments are exposed to fascin they form bundles that appear brighter and not perfectly aligned with the flow. It is very important to avoid introducing air bubbles in the system when changing tubes and pay attention not to get reservoir tubes empty. This technique was critical to apply pulling forces over tens of single filaments, look at formin and cofilin mechanical sensitivity, and ARP2/3 debranching.
