In our work, we aim to reproduce cellular processes in artificial cell-like models that require the precise localization of proteins in a given spot at a certain time. Here, as an example, we demonstrate the formation of dynamic protein patterns on model lipid membranes with high precision and high spatial temporal control using photoswitchable proteins and visible light. One of the current experimental challenge consists of reproducing dynamic protein patterns on monolipid membranes.
Here, we need to manipulate these protein patterns fast and traversibly with high precision in a biocompatible and non-invasive way. We use photoswitchable proteins to recruit and pattern proteins of interest on support lipid by layers in giant unilamellar vesicles. We achieve rabbit and localized protein recruitment, and the protein patterning is fully reversible in the dark and can be repeated multiple times.
In our protocol, we employ visible light to form protein patterns. Light is an attractive trigger to control interactions with dye, special resolution, and tunability. Light is also not invasive and doesn't damage biomolecules.
To begin, take a micro slide 18-well glass bottom chamber. Add 150 microliters of sodium hydroxide into each well of the chamber and incubate for one hour at room temperature. Then, remove the sodium hydroxide solution and wash the wells three to five times with deionized water, followed by three washes with buffer containing 10 millimolar calcium chloride.
Next, add 15 microliters of freshly prepared SUVs into the wells containing 150 microliters buffer with 10 millimolar calcium chloride. After 30 minutes of incubation at room temperature, wash the SLBs at least seven times with a buffer that does not contain calcium chloride. For the functionalization of the biotinelated SLBs with streptavidin, add a solution of streptavidin, then wash the SLBs at least five times with buffer to remove excess streptavidin.
Next, add b-disiLID to a final concentration of one micromolar to the well. After 30 minutes of incubation at room temperature, wash the excess protein with buffer at least five times. Then, add mOrange-Nano to a final concentration of 200 nanomolar.
Cover the sample with aluminum foil and keep in the dark. Afterwards, place the micro slide under the fluorescence microscope. Set the 552 nanometer laser to visualize mOrange-Nano.
Fluorescence microscopy revealed mOrange-Nano patterned on the SLBs functionalized with b-disiLID. After photo activation, there is a rapid increase in fluorescence signal in the orange channel within the region of interest. Fluorescence of mOrange-Nano increases after each photo activation, saturates in 120 seconds, and then decreases to background levels in the following 120 seconds.
To begin, prepare a 5%solution of polyvinyl alcohol with 100 millimolar sucrose in ultrapure water. Mix overnight at 80 degrees Celsius while shaking at 400 RPM. Then, prepare a lipid solution in chloroform with the desired composition.
To prepare GUVs, use a pipette tip to spread a thin layer of 40 microliters of the prepared polyvinyl alcohol solution on a glass slide. Dry this layer at 50 degrees Celsius for 30 minutes. Then, using a needle, spread five microliters of the lipid solution on the polyvinyl alcohol layer and let it dry at 30 degrees Celsius for one hour.
Now, use a spacer and a second glass slide to assemble a chamber on the functionalized glass slide. Add one milliliter of rehydration buffer into the chamber and incubate for one hour at room temperature to form GUVs. Then, invert the chamber and gently tap on the glass surfaces.
Carefully remove the glass slide on one side to open the built chamber and harvest the GUVs with a pipette. Place the solution in a plastic tube and let the GUVs settle for two hours. To begin, take the tube of freshly harvested GUVs.
Add streptavidin solution and let it sit for 30 minutes at room temperature. Then, add one micromolar of b-disiLID to the GUV solution. Cover the sample with aluminum foil and keep it in the dark.
Pre-treat the micro slide 18-well glass bottom chamber with 150 microliters of BSA solution for 10 minutes. Then, remove the BSA solution and wash the wells three times with 150 microliters of water. Next, add 145 microliters of 200 nanomolar mOrange-Nano in buffer to the well.
Add five microliters of GUVs decorated with a b-disiLID to the solution. Cover the sample with aluminum foil and wait approximately 15 minutes for the vesicles to settle. Afterwards, place the micro slide under the confocal microscope.
Excite the sample at 552 nanometers for MOrange visualization, and at 638 nanometers for DID in the GUV's membranes. The mOrange-Nano GUVs failed to exhibit fluorescence in the dark. The mOrange intensity quantified at the GUV membrane showed fast and effective protein recruitment with full reversibility.
