A Nanobar-Supported Lipid Bilayer System for the Study of Membrane Curvature Sensing Proteins in vitro

This nanobar-supported lipid bilayer system can be used to study the role of membrane curvature in regulating the dynamics and distribution of protein and lipids during cell activities. This technique offers both high sub root and bad in the membrane curvature generation by forming a continuous lipid bilayer on patent nanobar errors with membrane curvature predefined by high resolution nanofabrication. To begin, place the nano chip in a 10 milliliter beaker with the pattern side facing up.

Carefully add one milliliter of 98%sulfuric acid to the beaker and ensure the acid fully covers the front and back side of the chip. Slowly rotate the beaker and add 200 microliters of 30%hydrogen peroxide drop by drop until the whole beaker becomes hot. Ensure that sulfuric acid and hydrogen peroxide are well-mixed to form Piranha solution for the removal of organic molecules from the nano chip.

Place the beaker in a secondary glass container and keep the nano chip immersed in the Piranha solution overnight to clean the impurities thoroughly. Take the beaker out and carefully pipette the Piranha solution into an acid waste container. Load five milliliters of deionized water into the beaker to dilute the residual acid and discard it into the acid waste.

Repeat this step five times. Grab the chip with tweezers and wash with a continuous stream of deionized water to remove residual acid thoroughly. Blow dry the chip with 99.9%nitrogen gas for SLB formation.

Sonicate the lipid mixture for 30 minutes. Freeze the lipid mixture in liquid nitrogen for 20 seconds and then thaw at 42 degrees Celsius for two minutes in a water bath. Repeat the freeze/thaw cycles 30 times.

After that, the lipid mixture looks like a clear liquid. Pass the lipid mixture through the mini extruder and extrude back and forth 20 times. Collect the SUVs from the syringe at the other side to reduce the contamination with larger particles or foreign material, and transfer it into a 1.5 milliliter centrifuge tube.

Take out the cleaned nano chip from deionized water carefully with a pair of tweezers and blow dry with 99.9%nitrogen gas. Perform surface cleaning of the nano chip with air plasma treatment for one hour. Assemble the nano chip in a PDMS chamber.

Start with placing the chip on a clean surface with the pattern facing up. Gently cover the middle PDMS with the chip and make sure the whole pattern is exposed to the central area of the large oval-shaped opening in the middle PDMS. Cover the top PDMS with the middle PDMS and keep its two small holes within the region of the large oval hole of the middle PDMS.

Then, the PDMS chamber is assembled. Load the SUVs into the PDMS channel from one of the two small holes in the top PDMS with a pipette and incubate for 15 minutes at room temperature to form the SLB. Gently pipette the PBS buffer into the PDMS channel from one side of the small hole and remove the waste with a cotton bud from the other hole to wash away the unbound SUVs.

Then acquire the SLB formed on the nano chip. Set up the laser scanning confocal microscopy using a 100 x oil objective. Open the Zen software to select the excitation laser power that can excite the fluorescence of the lipid and protein.

Choose Acquisition Mode. Then click Smart Setup, followed by Texas Red. Adjust the focus with the focus knob to locate the nano bars on the chip until the nano bar edges are sharp under the lipid channel.

Set the scanning parameters to obtain a control image of the lipid channel before adding protein. Conduct the FRAP assay by bleaching with fluorescent labeled lipid bilayer on a random single nanobar area. Select the Experiment Regions and Bleaching check boxes.

Draw a circular area of five micrometers diameter which can include the whole nanobar at the center, and add to the experiment regions. Input TimeLapse imaging parameters. Choose Time Series.

Then click Duration. Select 100 cycles and interval equal to two seconds. Input bleaching parameters.

Select the laser check boxes that perform FRAP and change the power to 100%Click Start Experiment for the FRAP experiment. Load the protein solution into the PDMS channel and incubate for five minutes at room temperature to allow the binding of the protein on the SLB. Refocus the nanobars and set the scanning parameters to take images of both lipid and protein channels for protein curvature sensing detection.

Select Experiment Regions and conduct the FRAP assay on both lipid and protein channels, and perform TimeLapse imaging to characterize the mobility of curvature sensing protein. Both show increased accumulation on the end of SLB coated individual nanobars with 300 nanometers width. Here the SLB contains 10%PS to electrostatically enhance the protein binding.

Both proteins have a higher end to center ratio than lipids, which is around one. When comparing the IDR FBP17 and F-Bar, the higher end to center ratio of IDR FBP17 indicates stronger curvature sensing than F-Bar. All three proteins showed preferential accumulation at the nanobar end curved membrane sites when the curvature decreased below 400 nanometers in diameter.

Among these three protein domains, IDR FBP17 gives the highest nano bar and density, indicating its strongest curvature sensing ability, while F-Bar shows the lowest value. Protein binding curve can be plotted by gradually increasing the protein concentration which shows that IDR FBP17 has a strong cooperative curvature sensing ability. Compared to the fast recovery of lipid signals on nanobars, the F-Bar cannot recover within two minutes, suggesting its significantly decreased membrane mobility and association dynamic at the curved membrane sites.

Surprisingly, different from the behavior of F-Bar, IDR FBP17 signals showed obvious recovery within the same timeframe, indicating the dynamic nature of IDR FBP17 accumulated at the curved membranes. However, after washing away the unbound IDR FBP17 in the solution, the same recovery in the IDR FBP17 channel couldn't be observed as before. The SLB quality is very important when studying the interaction between curved membrane and protein.

So the FRAP test is necessary in our experiment to validate the membrane mobility. This system will help the researchers to study various parameters affecting the protein interaction with the curved membrane, such as the curvature sensing domains, and the lipid composition as well as to conduct the dynamic studies on curved membrane such as face separation behaviors.