These methods can help answer key questions about the role of the pseudokinase mixed-lineage kinase-domain like or MLKL in necroptosis such as membrane translocation and permeabilization in lipid binding. The main advantage of these techniques is that they directly implicate MLKL as the executioner of plasma membrane rupture in necroptosis. Live-cell imaging has revolutionized the field of cell-death research, especially for MLKL-mediated necroptosis.
Nuclear magnetic resonance is a powerful technique for monitoring lipid binding to proteins as demonstrated here for MLKL. Begin by plating five times 10 to the fourth MLKL knockout mouse embryonic fibroblasts or MEFs expressing the executioner domain of MLKL fused to an oligomerization cassette and Venus in one milliliter of supplemented DMEM in 24-well plates for their overnight incubation in a cell-culture incubator. The next morning, treat the cells with doxycycline for 12 hours to induce expression of the transgene, followed by the addition of standard DMEM supplemented with FK506-binding protein dimerizer and membrane-impermeable green fluorescent dye to induce necroptosis.
To monitor the cell death, place the plate in an appropriate imaging unit housed in a mammalian-tissue culture incubator and open the imaging system software. Select the Schedule Upcoming Scans tab and select the tray, vessel, scan types, and scan pattern in the Physical Layout tab. Select fast fluorescence in the Properties tab and click on the Timeline window and the total number of time points and frequency of acquisition to add the scan time.
Then, click Apply to start the image acquisition. To quantify the necroptosis, select Tasks pane and hover the mouse cursor over the background of several images to set the object counting new analysis with fixed segmentation threshold above the background level. Then, select Previewing to examine the fluorescent objects in the current image and refine the threshold levels as needed.
Careful selection of the threshold could be performed observing multi wells on the plate and using the Preview feature on the software. Just be aware to use the same threshold setting for all the wells in the plate. To integrate the green fluorescence counts, under the Launch New Analysis tab select time range and the wells to be analyzed and enter the analysis job name.
Then, click Okay and double-click on the job name to export the data for additional analysis in an appropriate graphing software program. For lipid binding of the MLKL by nuclear magnetic resonance spectroscopy or NMR, use airtight syringes to aliquot one milligram per milliliter of the phospholipids of interest into clean glass vials for individual NMR titration series and place the vials under a gaseous stream of argon or nitrogen for five to 30 minutes. Dispensing lipids from volatile solvents such as chloroform or methanol must be performed quickly from stockpiles kept on dry ice in order to minimize inconsistencies in lipid concentrations between samples due to solvent evaporation.
At the end of the evaporation period, use large tweezers to arrange the vials at the bottom of a Buchner flask. Seal the flask with a rubber stopper and attach plastic tubing connected to a vacuum line to evacuate the vial contents under mild vacuum overnight to remove any residual organics. The next morning, add NMR buffer supplemented with detergent at the appropriate experimental concentration to each glass vial with a lipid film and sonicate the vial in a water bath for up to 30 minutes, stopping the sonication every 10 minutes to check the solubilization of the lipid film.
At the end of the sonication, allow the vials to cool for 15 minutes and perform a serial dilution of the lipid-detergent micelles in detergent-positive NMR buffer, sonicating the 1:1 mixtures for the desired concentration series. Prepare control and reference NMR samples of protein and additives in detergent-negative and detergent-positive NMR buffers respectively, adding protein and additives to each lipid dilution in detergent-positive buffer. Then, transfer the appropriate volume of samples to suitably-sized NMR tubes and load the samples onto the NMR instrument.
Complete necroptosis of MLKL-knockout MEFs can be observed within one hour of dimer-mediated oligomerization of doxycycline pre-incubated cells expressing the fused transgene. This fast kinetics necroptosis supports the role of MLKL as the putative executioner of plasma membrane rupture, resulting in the production of plasma-membrane-associated Venus puncta within one to two minutes of treatment with the dimer and Venus coding of the cell periphery and gradual cell rounding within two to three minutes. Under fast necroptosis, the cells change morphology from their normal elongated shape to rounded and swelled within five minutes of oligomerization, partially ruptured at 10 minutes, and extensively dismantled with vanished cytosol at 20 minutes.
In vitro lipid binding experiments monitored by NMR spectroscopy reveal major structural changes in the MLKL executioner domain protein upon lipid binding to displace the inhibitory brace region from the closed N-helical to the open and intrinsically disordered conformation as well as per-residue information on mixtures of both conformers. The percentages of closed and open conformers in a given sample can also be monitored, providing supporting evidence for the interaction of the MLKL executioner domain with phospholipids and suggesting a direct link between MLKL and the plasma membrane. So while attempting to quantify cell death using this procedure, it is important to know and remember that the DNA-binding dye have higher threshold fluorescence than normally genetically-encoding fluorescence protein.
For this reason, they can be used in parallel. Also note that micellar lipids are temperature sensitive and therefore the NMR sample temperature may need to be optimized. So following this procedure, other technique could be used like total internal reflection microscopy and electromicroscopy to address how fast the cell ruption happens on cells undergoing necroptosis and where the rupture are localized on the plasma membrane.
In addition, microscale thermophoresis can be performed to orthogonally validate MLKL binding to micellar lipids. The techniques described in this video paved the way for researchers in the field of necroptosis to explore the role of MLKL as the executioner of plasma membrane rupture. Don't forget that working with chloroform can be extremely hazardous and that precautions such as wearing gloves and working in a well-ventilated area should always be taken when performing this procedure.
