Reconstitution of LRRK2 membrane recruitment onto planar lipid bilayers

Clean Lab-TeKII 8 chamber, No. 1.5 borosilicate coverglasses (Fisher) using Piranha solution by 0h 30m 0s incubation followed by extensive washing in Milli-Q water.

Before the experiments, dry the chambers and clean further using a Harrick Plasma PDC-32C plasma cleaner for 0h 10m 0s at 18W under ambient air.

Form the substrate supported lipid bilayers (SLB) with 65% (v/v) DOPC, 29% (v/v) DOPS, 5% (v/v) DOGS-NTA [Ni²], 1% (v/v) PI(4)P, 0.1% (v/v) DiD (Avanti Polar Lipids; ThermoFisher) to mimic the composition of the trans Golgi network (Thomas et al., 2016).

Dry the above-mentioned lipid mixture (in chloroform) under nitrogen flow in a glass vial and then maintained under house vacuum for at least 1h 0m 0s.

Resuspend the dried lipids in SLB buffer (20millimolar (mM) Hepes 8, 150millimolar (mM) potassium acetate, 1millimolar (mM) MgCl₂) and vigorously vortex to produce multilamellar vesicles (MLVs).

Prepare the large unilamellar vesicles (LUVs) by two, 0h 0m 10s cycles of bath sonication followed by extrusion through a 100 nm polycarbonate membrane, 21 times (Avestin) and store at -20°C.

Form the supported lipid bilayers in cleaned reaction chambers by addition of liposomes to the glass surfaces at a final concentration of 3millimolar (mM) liposomes in SLB buffer.

Induce the fusion of the LUVs by addition of 1millimolar (mM) CaCl₂ followed by incubation for 0h 45m 0s at 37°C.

Remove the unfused vesicles by extensive washing with Milli-Q water and STD buffer (20millimolar (mM) Hepes 8, 150millimolar (mM) NaCl, 5millimolar (mM) MgCl₂).

Plate 293T cells onto 2 x 15cm dishes to achieve 60% confluency the next day.

Transfect cells with FLAG-LRRK2 expression construct:

Tube#1: Dilute 16µg of DNA into a final volume of 700µL of Optimem.

Tube #2: Add 64µL of 1mg/mL PEI and 636µL of Optimem.

Add tube #1 to tube #2 and incubate for 0h 15m 0s at Room temperature.

Add the mixture dropwise onto cells and incubate at 37°C for 48h 0m 0s.

Harvest cells by pipetting vigorously with ~10mL - 20mLof 1xPBS for each plate.

Collect cells into a conical tube.

Spin cells at 1000rpm,4°C.

Remove supernatant and resuspend with PBS.

Repeat 3X to wash away serum.

Spin cells at 1000rpm,4°C. Remove supernatant and resuspend with PBS. (1/3).

Spin cells at 1000rpm,4°C. Remove supernatant and resuspend with PBS. (2/3).

Spin cells at 1000rpm,4°C. Remove supernatant and resuspend with PBS. (3/3).

Resuspend the pellet using 30mL lysis buffer (50millimolar (mM) Tris 8, 150millimolar (mM) NaCl, 5millimolar (mM) MgCl₂, 20micromolar (µM) GTP, 0.5% (v/v) Triton X 100, 10% (v/v) glycerol, 1x protease inhibitor tablet).

Rotate lysate end-over-end for 0h 10m 0s at 4°C.

Spin down cell debris at 12.500rpmusing a Fiberlite F15 rotor.

Collect supernatant and filter through a 0.2 μm filter; hold On ice.

Meanwhile, equilibrate anti-FLAG M2 resin (~200µL resin, capacity >0.6mg/mL Millipore Sigma #A2220) with 10 column volumes H₂O followed by 10 column volumes of lysis buffer.

Apply sample (~20mL) to resin and circulate lysate with resin for 4h 0m 0s in the cold with an open top column and peristaltic pump.

Wash resin 3 X 400µLlysis buffer.

Wash resin with 3 X 400µL Elution Buffer (NO PEPTIDE ADDED YET) (50millimolar (mM)Tris 8, 150millimolar (mM) NaCl, 5millimolar (mM)MgCl₂, 20micromolar (µM) GTP, 10% (v/v) glycerol).

Prepare FLAG peptide-containing Elution Buffer (0.25mg/mL peptide [stock is 5mg/mL] in 5 column volumes).

Remove as much liquid as possible from resin, add 250µL FLAG peptide in elution buffer and incubate in the cold room for 0h 5m 0s. Then open the stopper and collect eluate.

Repeat 4X

Remove as much liquid as possible from resin, add 250µL FLAG peptide in elution buffer and incubate in the cold room for 0h 5m 0s. Then open the stopper and collect eluate. (1/4).

Remove as much liquid as possible from resin, add 250µL FLAG peptide in elution buffer and incubate in the cold room for 0h 5m 0s. Then open the stopper and collect eluate. (2/4).

Remove as much liquid as possible from resin, add 250µL FLAG peptide in elution buffer and incubate in the cold room for 0h 5m 0s. Then open the stopper and collect eluate. (3/4).

Remove as much liquid as possible from resin, add 250µL FLAG peptide in elution buffer and incubate in the cold room for 0h 5m 0s. Then open the stopper and collect eluate. (4/4).

Load 20µL of each fraction onto an SDS-PAGE gel.

Detect the protein by Immunoblot using mouse anti-LRRK2 N241A/34 primary antibody ((Neuromab #75-253) diluted 1:1000 in 5% (v/v) milk/TBS-T (Tris buffered saline with 0.05% (v/v) Tween-20) overnight at 4°C, followed by secondary antibody (donkey anti-mouse IRDye680 (LI-COR #926-68072) for 1h 0m 0s, and image using a LICOR detector.

Determine the yield by Bradford Assay (Bradford, 1976); assess purity by SDS PAGE in conjunction with Instant Blue stain (Abcam #ab119211).

Label the purified FLAG-LRRK2 protein (in a non-primary amine-containing buffer) with CF633 succinimidyl ester (Biotium # 92217). Incubate the protein in the dark with the dye for 1h 0m 0sat Room temperature followed by removal of free dye by Pierce Dye Removal Columns (Thermo Scientific #22858). Labeling efficiency is determined by multiplying the dye extinction coefficient by molar protein concentration and then dividing the sample absorbances at 633nm by the product (as per labeling kit instructions). Labeling efficiencies of 2-3 moles dye per mole protein were used in experiments.

For a 100µLlabeling reaction, use 300µL of 1:1 resin slurry. First add slurry to supplier-provided column and remove storage buffer by spinning at 1000x g.

Add sample to column and briefly vortex; centrifuge at 1000x gto collect protein.

Check protein concentration after dye removal by Bradford Assay. Labeling efficiency is determined using the dye extinction coefficient;labeling efficiencies of 2-3 moles dye per mole LRRK2 are ideal.

Evaluate the possible aggregation of fluorescent LRRK2 by allowing the labeled protein to bind to Poly-D-lysine coated coverslips and visualizing the protein using TIRF microscopy as described in Section - TIRF Microscopy .

Dissolve 10mg Poly-D-lysine (MPBio # SKU:02150175-CF) in 1mL sterile Milli-Q water as a 1% (v/v) stock solution. Dilute the stock solution two fold in PBS to prepare a 1X coating solution. Add the coating solution (200µL) to the reaction chamber and incubate for 0h 5m 0s at 37°C. Remove the coating solution by rinsing the chamber thoroughly with sterile Milli-Q water and equilibrate with reaction buffer.

Apply FLAG-LRRK2 (14nanomolar (nM)) to a Poly-D-lysine coated chamber instead of a lipid bilayer, and evaluate behavior of the labeled protein by TIRF as described in Section 6 below. Conditions are otherwise identical to bilayer binding reactions including nucleotides and nucleotide regenerating system.

Add the C-terminally His-tagged eGFP Rab10 to supported lipid bilayers at a final concentration of 2.5micromolar (µM) in STD buffer and incubate for 0h 30m 0s at 37°C

Wash the Rab10-coated lipid surfaces horoughly with STD buffer and then equilibrate with 200µL of reaction buffer and nucleotide regeneration system (20millimolar (mM) Hepes 8, 150millimolar (mM) NaCl, 5millimolar (mM) MgCl₂, 4millimolar (mM) ATP, 20micromolar (µM) GTP, 20millimolar (mM) creatine phosphate, 30U creatine phosphokinase).

Dilute CF633-FLAG-LRRK2 to 14nanomolar (nM)concentration in reaction buffer and allow to equilibrate to Room temperature for 0h 5m 0s.

After 0h 0m 50s imaging, remove 100µL of the 200µL buffer in the reaction chamber.

At 0h 1m 0s, add 100µL of 14nanomolar (nM) CF-FLAG-LRRK2 to a final concentration of 7nanomolar (nM) and imaging continues for 0h 20m 0s.

Prepare control bilayers with Rab11-His that does not bind LRRK2.

Videos of LRRK2 recruitment onto Rab10-decorated, supported lipid bilayers are recorded at 25°Cat a frame capture rate interval of 500ms using a Nikon Ti-E inverted microscope with an Andor iXon+EMCCD camera model DU885, with PerfectFocus and a Nikon TIRF Apo 100X 1.46 NA oil immersion objective. Imaging is done with 300 EM camera gain and 50 ms exposure times at 200µW laser intensity.

Analyze the data from TIRF microscopy movies using TrackIt (Kuhn T., et al., 2021) to obtain spot densities of bilayer-bound LRRK2 from the videos over time. Export data from TrackIt using a Mathworks MATLAB (version R2021a) script (Appendix 1); normalize raw values by assigning the highest value as 1 in Excel to enable comparison of different videos.

#MATLAB script to extract data from TrackIt. 
function T = spots_export(btch, outfilename)
%eval('btch = currentBatch')
spots = getfield(getfield(btch, 'results'),'spotsAll');
spots_total = 0;
for i=1:length(spots)
	n = size(spots{i});
	spots_total= spots_total + n(1);	
end 
out = zeros(spots_total, 8);  
spots_total = 0;
for i=1:length(spots)
	%disp(i)
	n = size(spots{i});
	n = n(1);
	if n>0
    	spt = spots{i};
        out((spots_total+1):(spots_total+n),1) = spots_total+(1:n);
        out((spots_total+1):(spots_total+n),2) = i;
        out((spots_total+1):(spots_total+n),3:7) = spt(:,1:5);
        out((spots_total+1):(spots_total+n),8) =  sum(spt(:,3));
    	spots_total = spots_total+n;
	end
end 
T = array2table(out, 'VariableNames', {'SpotID','Frame','X','Y','A','BG','Sigma','Atotal_frame'}) 
writetable(T, outfilename)
end