This method use fluorescence to determine if a protein transfer to biologically important liqids between synthetic membrane and thus contribute to the distribution of these lipids inside eukaryotic cells. This technique makes it possible to the extraction and transport of a natural lipid by a protein, it requires and fluorescence cells and liposomes that are easy to make. This method can be used and modified to get insight into cellular biology by proteins behind the distribution of some essential lipids between organ and membranes.
Visual demonstration is critical to explain how to perform real-time measurements of lipid transfer by fluorescence. So demonstrating the procedure will also be Maud Magdeleine, an engineer for my laboratory. Begin by preparing fresh filtered and de-gassed HEPES potassium acetate buffer, supplemented with one millimolar magnesium chloride to form HKM buffer.
Then, prepare liposomes only made of PC or additionally doped with two molar percent PS or PI(4)P. Now, place the flask on a rotary evaporator to dry the lipid under vacuum. In one well, mix liposomes containing two molar percent PS with lipid sensor, NBD-C2 Lact, to a final concentration of 250 nanomolar and a volume of 100 microliters.
Fill a second well with the same amount of liposome and NBD-C2 lact mixed with three micromolar lipid transfer protein or LTP. Fill a third well with 250 nanomolar NBD-C2 lact mixed with pure 80 micromolar PC liposomes and a fourth well with pure 80 micromolar PC liposomes. Repeat these steps to prepare three additional series of four wells.
Then place the multi-well plate in the fluorescence reader. For each well record an NBD spectrum from 505 to 650 nanometers with a five nanometer bandwidth upon excitation at 490 nanometers at 25 degrees Celsius. For each series subtract the spectrum recorded with only liposomes from the other spectra.
For the PI(4)P extraction assay prepare liposomes doped with two molar percent phosphatidylinositol-4-phosphate and carry out measurements with the NBD-PH FAPP probe. Perform control experiments and determine the extraction percentage. After finishing extruding liposomes fill a tube With these extruded liposomes.
Prepare freshly de-gassed and filtered HKM buffer and keep the tubes containing extruded liposomes at room temperature. Wrap tubes containing liposomes with rhodamine labeled phosphatidylethanolamine in aluminum foil and store them in an opaque box to prevent any photo bleaching. Set the temperature between 25 and 37 degrees Celsius and adjust the excitation monochromemeters to 460 nanometers with a short bandwidth of one to three nanometers and emission to 530 nanometers with a large bandwidth of greater than 10 nanometers.
Set the acquisition time to twenty five minutes with the time resolution of at most one second. In the quartz cuvette dilute 30 microliters of the LA liposome suspension and NBD-C2 lact stock solution and prewarm HKM buffer to prepare a 570 microliter sample that contains 200 micromolar total lipids and 250 nanomolar or NBD-C2 lact. Add a small magnetic stirring bar and position the cuvette in the fluorometer holder.
Once the sample is thermally equilibrated trigger the measurement. After one minute, add 30 microliters of the LB liposome suspension to the sample. After three minutes inject LTP into the sample so that the final concentration of the LTP is 200 nanomolar, then acquire the signal for the remaining 21 minutes.
Carry out a parallel experiment to normalize the NBD signal. Mix 30 microliters of LA equilibrium liposome suspension with 250 nanomolar NBD-C2 lact in HKM buffer at a final volume of 570 microliters. After one minute, inject 30 microliters of LB equilibrium liposome suspension.
Convert the kinetic curves measured with an LTP of interest to determine the amount of PS transferred from LA to LB liposomes over time. Then normalize each data point of the curve. Perform the PI(4)P experiment using the same floor emitter settings and conditions as for the PS transfer assay.
In the cuvette, mix 30 microliters of lb liposome suspension and NBD-PH FAPP probe with pre warmed HKM buffer to obtain a final volume of 570 microliters. Once the thermal equilibration of the sample is reached, start the measurement. After one minute, inject 30 microliters of LA liposome suspension.
After three minutes, inject the LTP of interest and record the signal. Perform a second experiment to normalize the NBD signal. Mix 30 microliters of lb equilibrium liposome suspension with 250 nanomolar NBD-PH FAPP and 570 microliters of HKM buffer.
After one minute, inject 30 microliters of LA equilibrium liposome suspension. Convert the kinetic curves to determine the amount of PI(4)P transferred from LB to LA liposomes over time, then normalize each data point. To quantify the extent of which an LTP is efficient.
Perform a linear regression of the first data points of the transfer kinetics to obtain a slope. Divide the slope value by the LTP concentration in the reaction mixture to determine the number of lipid molecules transferred per protein per time unit. Efficient recovery of C2 lact from the beads was verified with SDS page analysis.
The ultraviolet visible absorbent spectrum of C2 lact labeled with NBD confirmed that all C2 lact molecules were labeled with an NBD group. The purity of NBD-C2 lact and its fluorescence were determined by SDS page analysis. The fluorescence of NBD-C2 lact or NBD-PH FAPP was maximal when only liposomes containing PS or PI(4)P were used for incubation, indicating that the sensor was membrane-bound.
A low fluorescence was seen when Osh6p was also present indicating that this protein efficiently extracted PS or PI(4)P from liposomes. The results from a PS transfer assay using Osh6p as an LTP are shown. The average kinetic curves of PS transfer from LA liposomes to LB liposomes, doped, or not doped with phosphatidylinositol-4-phosphate were calculated.
The mean initial PS transport rate was determined from three distinct experiments. Similarly, results from a PI(4)P transfer assay using Osh6p as an LTP are shown here. Along with the average kinetic curves obtained after signal normalization.
Mean transfer rates were measured with LA liposomes that were doped or not doped with PS.When performing this protocol use fresh buffer and prepare the same days. Minimize light exposition of florescent liposomes and proteins, use well purified NBD labeled protein and keep them on ice. can be confirmed by measuring PS and PI(4)P transfer between organelle inside the prokaryotic cell using genetically encoded fluorescent lipid sensors.
This technique paves a way to a better understanding of PS and PI(4)are distributed within the cell, and their activity undergo specificity of several LTPS.
