Supported Cell Membrane Sheets to Monitor Protein Assembly

[Abstract] Studying protein-protein and protein-lipid interactions in their native environment is highly desirable, yet, the heterogeneity and complexity of cellular systems limits the repertoire of experimental methods available. In cells, interactions are often taking place in confined microenvironments where factors such as avidity, hindered diffusion, reduced dimensionality, crowding etc . strongly influence the binding kinetics and therefore it can be problematic to equate binding affinities obtained by bulk in-solution methods ( e.g. , Fluorescence Polarization, Isothermal titration calorimetry, Microscale thermophoresis) with those occurring in real cellular environments. The Supported Cell Membrane Sheet method presented here, addresses these issues by allowing access to the inner leaflet of the apical plasma membrane. The method is a highly versatile, near-native platform for both qualitative and quantitative studies of protein-protein and protein-lipid interactions occurring directly in or on the plasma membrane.

The method was originally developed by Perez et al. (2006), to qualitatively investigate the mobility of GPCRs and their association to G-proteins, and has also been used for both FRET based stoichiometry (Miles et al., 2013) and super resolution microscopy based cluster analysis (Scarselli et al., 2012). A related study by Roizard et al. (2011) also managed to produce supported cell membrane sheets on porous beads (~80 μm in diameter) leaving both the intracellular and the solvent exposed extracellular side of the membrane accessible.
Here, we have adapted the SCMS method for also measuring protein-protein binding affinities, which is particularly useful in cases where one or more interaction partners are membrane bound or embedded.   a. 488 nm laser line from an argon-krypton laser, and a 505-550 nm bandpass filter b. 568 was excited at 543 nm with a helium-neon laser, detected using a 585 nm long-pass filter c. 647 was excited at 642 nm with a helium-neon laser, detected using a 650 nm long-pass filter www.bio-protocol.org/e3368  The protein used for the binding studies below should be pure and stable at concentrations 10x above the expected KD for at least 1 day at 10 °C after purification. In our experiments, we purify and label a new batch of protein for each independent experimental repetition. As maleimide or NHS labeling might interfere with membrane binding, we suggest using SNAP-tagged constructs for the initial binding studies. We use an N-terminal GST-SNAP-tag with a thrombin cleavage site between the GST and SNAP tag. 4. Next day, equilibrate Nap-5 gel filtration columns using PBS buffer (three column volumes).

5.
Bring the reaction volume up to 200 μl with PBS (add 90 μl) and gently mix.
6. Add the entire volume to the center of the column.
7. Allow the sample to enter the column and after the last drip, add 550 μl PBS to wash.

A3. Degree of labeling
Determine protein concentration and degree of labeling of all constructs. We use a TECAN M200 Infinite Pro instrument for this. The degree of labeling is determined as follows: First, calculate the protein concentration considering the contribution from the fluorophore: Then calculate the moles of dye per mole protein: object glasses. Immediately after cleaning the glasses are transferred to six well plate wells and covered with 0.3 mM poly-L-ornithine hydrobromide. After 30 min, the poly-L-ornithine hydrobromide solution is removed and cover glasses are washed once in approximately 2 ml water in the well and finally 1 ml water is added to keep glasses covered.
2. Typically, 24 glasses are prepared. Make a few more than needed-some will break.
3. Prepare 'Sheet buffer' (see Recipes) by adding 50 mg of fresh BSA to 50 ml buffer in a 50 ml Falcon tube and keep it on ice. Also, put 2 x 50 ml of MilliQ water on ice. 4. Prepare tissue paper box by wetting Whatman paper and cover it with parafilm. Make grids and write labels with a permanent marker. Do not use a red marker as this will dissolve during PFA fixation below (Figure 2).

Put cells M1 labeled cells on ice and start ultracentrifugation of protein preps (see B12).
Note: You now have (see Figure 2A): a. Fluorescently labeled protein.

b. Cold M1 labeled cells (with labeled M1 still on).
c. Cleaned and poly-L-ornithine hydrobromide treated cover glasses in water approx. 24 pieces depending on desired curves and sample points. d. BioAssay dish with moistened Whatman paper and marked up parafilm (Figure 2A). e. 50 ml cold sheet buffer with 1 mg/ml BSA.
g. In addition, you need tweezers, flat syringe pistil and bend needle (Figure 2A). Also, you need a P1000 pipette, a clock and suction.
h. At this point also put ~700 μl per sheet 4% PFA and 100 ml PBS on ice for later use.

C2. Production of Inverted membrane sheets
To prepare sheets, do as follows for each well with cells: 1. Aspirate medium containing M1 antibody ( Figure 2B). Figure 2B).

Add approximately 2 ml of cold water to wash cells and start clock (
3. Replace water with clean water to allow osmotic swell. 4. After 60 s, remove water, pick up cover glass and put on top of the cells with poly-L-ornithine hydrobromide treated surface facing the cells (Figures 2D-2E). Now press hard on the cover glass with the pistil ( Figure 2F). Apply constant pressure while moving around the pistil on the glass for 1 min. It might take a few tries to get the pressure right without breaking the cover glass. To get the pressure right, make an initial series in which the applied pressure is gradually increased and assess the number of sheets using the confocal microscope (see below).
The pressure required is more than you think. An alternative way to produce the inverted membrane sheets is to firmly press down the cover glass on the cells for 10 s and then remove the water completely. Make sure the cover glass is fully attached to the cells in the bottom of the well. Then wait for 120 s-the air pressure/surface tension between the cover glass and the well surface will also be sufficient to produce inverted membrane sheets. 9 www.bio-protocol.org/e3368

Next, again use the bended needle and tweezers to pick up the cover glass and transfer it to
the BioAssay dish with the surface containing the sheets facing upwards. Be patient, it might take a few tries to learn how to pick up the cover glass with the needle. If sufficient pressure has been applied the cover glass will be sticking quite well to the cell surface.
6. Immediately after transfer, cover the sheets with approximately 700 μl sheet buffer with BSA to prevent drying and to coat surface with BSA ( Figure 2G). 7. Repeat process for all sheet preps.
8. Leave last prepared sheets in the cold and dark for 20 min to allow BSA to coat the sheets. 9. Meanwhile, prepare protein dilutions as calculated above by diluting protein in sheet buffer.
Initial dilution in protein buffer is done for the lower conc. Prepare 1 ml per condition.
10. Aspirate sheet buffer from sheets and replace it with protein containing sheet buffer. Preferably approx. 700-1,000 μl, no less than 500 μl. Put on plastic cell dishes as lids in addition to box lids and incubate for typically 2 h or more for concentration series and leave at desired temperature in the dark. 11. After incubation, remove buffer and wash sheets on the parafilm surface with 1x 700 μl sheet buffer with BSA and two times with cold PBS (no incubation in wash buffer, just start removal in the same order after the last sheet has been covered). Use soft rubber pipettes.
12. Next, fix sheets for 40 min in 700 μl 4% cold PFA and in the dark. At this point take out antifade mounting media to allow this to reach room temp.
13. After fixation, wash sheets three times in PBS and leave in the last wash. For a few coverslips at the time, remove PBS and wash glasses in a beaker containing ~400 ml water. Remove drops from edge using paper towels and mount glasses on the object glass on a drop of antifade ( Figure 2H). Excess antifade can be sucked away. Preps. are kept in the dark at room temperature. After drying they should be stored at 4 °C.
www.bio-protocol.org/e3368  3. 547 SNAP-surface labeled species were excited at 543 nm with a helium-neon laser, and the emitted light was detected using a 560-615 nm band pass filter. The Alexa Fluor 647 was excited at 633 nm with another helium-neon laser, and the emitted light was detected using a 650 nm long pass filter. 4. The samples will contain mostly single layer sheets but also undisrupted cell and cell debris.
The membrane sheets can be differentiated from cells and debris by being very flat and thin.
Cells and debris are often visible in the direct transmission, whereas single layer membrane sheets are not ( Figure 3A).

Data analysis
Resulting images are then analyzed using ImageJ and Excel (or similar). Please see Video 1. We recommend using Graphpad Prism (or similar) for subsequent plotting and fitting.
1. Make sure Images from the different channels are perfectly aligned. There are microscope specific procedures for this-please consult your local expert microscopist if in doubt. The images should be stacked to ease the analysis.

Video 1. Quantification of binding using ImageJ and Excel
2. When a flat membrane sheet is found, define a region of interest (ROI) and use the measure tool (in ImageJ) to measure the mean intensity of the various channels. Also, measure the background intensity for all channels for all images. Areas with oversaturated pixels or mean intensities below background + 2σ (the background intensity + two times the standard deviation of the background intensity) should be discarded ( Figure 3A). 4. Next, the intensities need to be corrected for degree of labeling and in some cases laser gain settings. Correction of measured channel intensities can be calculated using the following equation: where, Iobs is mean intensity of sheet (Imembrane protein in Figure 3A) or ligand (Ilabeled protein in Figure   3A and Video 1), Iback is mean intensity of background and L is the moles of dye per mole of protein or receptor (see section A3). We do not recommend changing PMT gain settings between independent n's, however, if necessary, this can be corrected by multiplying by G(V) (will be 1 if gain settings are similar). G(V) is calculated as follows: is Conductance of dynodes of PMT and n the number of dynodes inside the PMT.
and n are specific for the PMTs on your microscopes.
5. Finally, the fractional binding can calculate using: All binding curves for direct comparison should be performed in parallel, and in presence of positive and negative (non-binding) controls.
6. An example of a concentration series and quantified binding can be found in Figures 3B-3C.  Scale bars: 10 μm.

Notes
The method is not restricted to any particular cell type and in principle any adherent cell type can be used. If sufficient signal-to-noise ratio can be obtained, we can also recommend using stable www.bio-protocol.org/e3368 transfected cell lines (or even native cell lines, with a good monoclonal antibody). This will provide an even more realistic view on the effective concentrations of receptors, their clustering and potential avidity effects.
The SCMS are native cell plasma membranes and they contain most, if not all, of the native cellular receptors, transporters, ion channels etc. as well as the cytoskeleton and smaller membrane associated organelles such as vesicles. For this reason, protein ligands can have specific binding to many of these components, as well as non-specific binding to the membrane (this is referred to as the background). In transiently transfected cell cultures, overexpression of the receptor of interest will neglect the native "background" binding. In Erlendsson et al. (2019) the non-specific binding, tested by introducing a single but completely disruptive point mutation in the ligand-binding domain, is not detectable. Specific binding tested using a single but completely disruptive point mutation in the receptor binding epitope is 15-20% of binding observed with the wild-type receptor. Please note that background binding is highly system dependent and should be tested in parallel with any binding experiments.