Our research is focused on investigation of plasma membrane repair mechanisms in both living cells, but also in biomimetic systems called the giant unilamellar vesicles. We are particularly interested in understanding the role of proteins like annexins in facilitating surface repair. These intricate processes are explored using our innovative thermoplasmonic puncture technique.
Currently, cell repair is being investigated using pulse lasers combined with molecular biology to identify proteins that are recruited to the site of injury. The drawback of this approach is that it's hard to control the extent of damage caused by using pulse lasers. Currently, our experiments come with a few challenges.
We're working on fine tuning the alignment of our laser focus with a nanoparticle, which is crucial for precision. And although it can be a bit tricky, we're also actively working on minimizing the formation of nano bubbles during the heating process. Our research demonstrated that annexin proteins swiftly respond to calcium influx, playing a key role in membrane repair and reveal diverse behaviors of individual annexins.
To better understand the mechanisms involved, we turn to biomimetic systems, allowing us to precisely measure how annexins influence membrane bending near a membrane hole. Our protocol meets the need for precise localized membrane injured in healthy cells. It provides valuable insight into live cell membrane repair mechanisms.
Furthermore, it enable the study of the biophysical role of membrane proteins recruited to the anura region near pore heads in biometric membranes. To begin, coat a microwell dish with 150 microliters of 0.01%to 0.1%cell attachment solution before incubating. Once the dish has air dried, add 80 microliters of gold nanoparticle solution drop-wise over the dry surface.
The next day, remove any added culture medium from the microwell. Then add two milliliters of the transfected cells on top of the immobilized gold nanoparticles on the glass surface. Incubate the plate before the start of the experimentation.
Use a confocal microscope with an argon laser set to 488 nanometers to view the GFP fluorescence from the annexin protein. Set the helium neon laser to 633 nanometers to observe the gold nanoparticle reflection. Choose single cells instead of cell clusters to prevent overlap of plasma membranes.
Ensure that the immobilized gold nanoparticles are present as single particles and adequately spaced to prevent increased thermal gradient. Use the optical tweezer to irradiate the gold nanoparticle for one second to disrupt the plasma membrane. To generate giant unilamellar vesicles, or GUV, apply 90 microliters of warmed 5%PVA gel on a glass slide.
Spread the gel uniformly over the slide. Then let the slide dry in a heating cabinet at 50 degrees Celsius for 50 minutes. Next, use a glass syringe to apply 30 microliters of lipid mixture.
With the needle edge, spread the mixture into a thin film. Apply a gentle flow of nitrogen gas to evaporate the chloroform of the lipid mixture. Then dry the slides under vacuum for 1.5 to two hours.
In a separate two milliliter tube, add 400 microliters of growing buffer. Then add the recombinant protein of interest to a final concentration of 500 nanomolar. Pipette 400 microliters of the diluted protein to the assembled in-house chamber, and wrap the chamber in parafilm.
After a one hour incubation, transfer 400 microliters of the chamber content into a two milliliter tube. Add one milliliter of observation buffer into the collected solution to remove any non-encapsulated proteins. Then centrifuge the solution at 600 G for 10 minutes at 13 degrees Celsius.
Next, replace one milliliter of the supernatant with observation buffer and gently pipette to disperse the GUVs. Refrigerate until the start of the experimentation. To puncture the GUV, first coat the surface of a 35 millimeter glass bottom dish with beta-casein.
Next, add five microliters of 150 nanometer gold nano shells to the GUV mixture containing calcium chloride. Transfer the mixture to the chamber and mount it on the microscope stage. Use the optical tweezers to trap an individual gold nano shell at the GUV surface.
When the trapped nano shell is in close contact with the GUV membrane, increase the laser power to puncture the target site. Nanoparticle irradiation resulted in a membrane injury and caused rapid recruitment of annexins to the injury site, following calcium influx to form a ring-like structure. In the GUV experiments, the membrane punctures were rapidly resealed in the absence of annexins.
The unique biophysical characteristics of annexin A4 protein led to the bursting of the GUV due to its ability to roll membranes. The presence of annexin in GUVs caused a rapid accumulation at the injury site, following membrane puncture. Analysis of the annexin A5 rings in the cell experiments showed that they remained constant over time and space.
Plasma membrane disruption using thermoplasmonics caused elevated calcium ion levels. The cells reached a maximum calcium intensity at 6.6 seconds, a time point that is presumed to correspond to the time of wound closure.
