TITLE: Reconstitution of LRRK2 membrane recruitment onto planar lipid bilayers
AUTHORS: Ayan Adhikari, Edmundo  Vides, Suzanne  Pfeffer

[DESCRIPTION]
Supported lipid bilayers have emerged as an ideal model system to study the interaction of proteins with cellular membranes. We describe here a method to monitor the recruitment of purified LRRK2 kinase onto planar lipid bilayers containing lipid-anchored Rab10 protein using Total Internal Reflection Fluorescence (TIRF) Microscopy.This method utilizes purified, FLAG-tagged, full length LRRK2 labeled with CF633 succinimidyl ester (Biotium) and bacterially expressed eGFP-Rab10-His tagged protein. LRRK2 recruitment is captured in real time at 25°C 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.

[GUIDELINES]


REFERENCES
 

 

 


 


[STEPS]
SECTION: Method for cleaning glass coverslips
1. 
 
Clean Lab-TeKII 8 chamber, No. 1.5 borosilicate coverglasses (Fisher) using Piranha solution by 30 min incubation followed by extensive washing in Milli-Q water.
 

SECTION: Method for cleaning glass coverslips
2. Before the experiments, dry the chambers and clean further using a Harrick Plasma PDC-32C plasma cleaner for 10 min at 18W under ambient air.

SECTION: Preparation of Large Unilamellar Vesicles (LUVs)
3. 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).

SECTION: Preparation of Large Unilamellar Vesicles (LUVs)
4. Dry the above-mentioned lipid mixture (in chloroform) under nitrogen flow in a glass vial and then maintained under house vacuum for at least 60 min.

SECTION: Preparation of Large Unilamellar Vesicles (LUVs)
5. Resuspend the dried lipids in SLB buffer (20 millimolar (mM) Hepes pH 8, 150 millimolar (mM) potassium acetate, 1 millimolar (mM) MgCl2) and vigorously vortex to produce multilamellar vesicles (MLVs).

SECTION: Preparation of Large Unilamellar Vesicles (LUVs)
6. Prepare the large unilamellar vesicles (LUVs) by two, 10 s cycles of bath sonication followed by extrusion through a 100 nm polycarbonate membrane, 21 times (Avestin) and store at -20 °C.

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

SECTION: Preparation of Supported Lipid Bilayers
8. Induce the fusion of the LUVs by addition of 1 millimolar (mM) CaCl2 followed by incubation for 45 min at 37 °C. 

SECTION: FLAG-LRRK2 Purification
10. Plate 293T cells onto 2 x 15cm dishes to achieve 60% confluency the next day.
SECTION: FLAG-LRRK2 Purification
11. Transfect cells with FLAG-LRRK2 expression construct:

SECTION: FLAG-LRRK2 Purification
12. Harvest cells by pipetting vigorously with ~10 mL - 20 mLof 1xPBS for each plate.
 

SECTION: FLAG-LRRK2 Purification
17. Resuspend the pellet using 30 mL lysis buffer (50 millimolar (mM) Tris pH 8, 150 millimolar (mM) NaCl, 5 millimolar (mM) MgCl2, 20 micromolar (µM) GTP, 0.5 % (v/v) Triton X 100, 10 % (v/v) glycerol, 1x protease inhibitor tablet).

SECTION: FLAG-LRRK2 Purification
18. Rotate lysate end-over-end for 10 min at 4 °C.

SECTION: FLAG-LRRK2 Purification
19. Spin down cell debris at 12.500 rpm, 20 minusing a Fiberlite F15 rotor.

SECTION: FLAG-LRRK2 Purification
20. Collect supernatant and filter through a 0.2 μm filter; hold on ice.

SECTION: FLAG-LRRK2 Purification
21. Meanwhile, equilibrate anti-FLAG M2 resin (~200 µL resin, capacity >0.6 mg/mL Millipore Sigma #A2220)  with 10 column volumes H2O followed by 10 column volumes of lysis buffer.
 

SECTION: FLAG-LRRK2 Purification
22. Apply sample (~20 mL) to resin and circulate lysate with resin for 240 min in the cold with an open top column and peristaltic pump.
SECTION: FLAG-LRRK2 Purification
23. Wash resin 3 X 400 µLlysis buffer.
SECTION: FLAG-LRRK2 Purification
24. Wash resin with 3 X 400 µL Elution Buffer (NO PEPTIDE ADDED YET) (50 millimolar (mM)Tris pH 8, 150 millimolar (mM) NaCl, 5 millimolar (mM)MgCl2, 20 micromolar (µM) GTP, 10 % (v/v) glycerol).

SECTION: FLAG-LRRK2 Purification
25. Prepare FLAG peptide-containing Elution Buffer (0.25 mg/mL peptide [stock is 5 mg/mL] in 5 column volumes).

SECTION: FLAG-LRRK2 Purification
26. Remove as much liquid as possible from resin, add 250 µL FLAG peptide in elution buffer and incubate in the cold room for 5 min. Then open the stopper and collect eluate.

SECTION: FLAG-LRRK2 Purification
28. Load 20 µL of each fraction onto an SDS-PAGE gel.

SECTION: FLAG-LRRK2 Purification
30. Determine the yield by Bradford Assay (Bradford, 1976); assess purity by SDS PAGE in conjunction with Instant Blue stain (Abcam #ab119211).

SECTION: Fluorescent Tagging and analysis of Fluorescent FLAG-LRRK2:
31. 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 60 minat 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.
SECTION: Fluorescent Tagging and analysis of Fluorescent FLAG-LRRK2:
35. 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.
SECTION: Fluorescent Tagging and analysis of Fluorescent FLAG-LRRK2:
35.1. Dissolve 10 mg  Poly-D-lysine (MPBio # SKU:02150175-CF) in 1 mL 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 (200 µL) to the reaction chamber and incubate for 5 min at 37 °C. Remove the coating solution by rinsing the chamber thoroughly with sterile Milli-Q water and equilibrate with reaction buffer.

SECTION: Fluorescent Tagging and analysis of Fluorescent FLAG-LRRK2:
35.2. Apply FLAG-LRRK2 (14 nanomolar (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.

SECTION: Rab10 dependent recruitment of LRRK2 onto membranes
36. Add the C-terminally His-tagged eGFP Rab10 to supported lipid bilayers at a final concentration of 2.5 micromolar (µM) in STD buffer and incubate for 30 min at 37 °C

SECTION: Rab10 dependent recruitment of LRRK2 onto membranes
37. Wash the Rab10-coated lipid surfaces horoughly with STD buffer and then equilibrate with 200 µL of reaction buffer and nucleotide regeneration system (20 millimolar (mM) Hepes pH 8, 150 millimolar (mM) NaCl, 5 millimolar (mM) MgCl2, 4 millimolar (mM) ATP, 20 micromolar (µM) GTP, 20 millimolar (mM) creatine phosphate, 30U creatine phosphokinase).


SECTION: TIRF Microscopy
38. Dilute CF633-FLAG-LRRK2 to 14 nanomolar (nM)concentration in reaction buffer and allow to equilibrate to Room temperature for 5 min.

SECTION: TIRF Microscopy
38.1. After 50 s imaging, remove  100 µL of the 200 µL buffer in the reaction chamber. 
SECTION: TIRF Microscopy
38.2. At 1 min, add 100 µL of 14 nanomolar (nM) CF-FLAG-LRRK2 to a final concentration of 7 nanomolar (nM) and imaging continues for 20 min.

SECTION: TIRF Microscopy
38.3. Prepare control bilayers with Rab11-His that does not bind LRRK2.
SECTION: TIRF Microscopy
39. 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.

SECTION: TIRF Microscopy
40. 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.
 

SECTION: Preparation of Supported Lipid Bilayers
9. Remove the unfused vesicles by extensive washing with Milli-Q water and STD buffer (20 millimolar (mM) Hepes pH 8, 150 millimolar (mM) NaCl, 5 millimolar (mM) MgCl2).
SECTION: FLAG-LRRK2 Purification
11.1. Tube#1: Dilute  16 µg of DNA into a final volume of 700 µL of Optimem.
 

SECTION: FLAG-LRRK2 Purification
11.2. Tube #2: Add 64 µL of 1 mg/mL PEI and 636 µL of Optimem.
SECTION: FLAG-LRRK2 Purification
11.3. Add tube #1 to tube #2 and incubate for 15 min at Room temperature. 

SECTION: FLAG-LRRK2 Purification
11.4. Add the mixture dropwise onto cells and incubate at 37 °C for 2880 min.
SECTION: FLAG-LRRK2 Purification
13. Collect cells into a conical tube.
SECTION: FLAG-LRRK2 Purification
15. Remove supernatant and resuspend with PBS.

SECTION: FLAG-LRRK2 Purification
14. Spin cells at 1000 rpm, 5 min, 4 °C. 

SECTION: FLAG-LRRK2 Purification
29. 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 60 min, and image using a LICOR detector.

SECTION: Appendix
41. 
 

SECTION: FLAG-LRRK2 Purification
16. Repeat 3X to wash away serum.
SECTION: FLAG-LRRK2 Purification
16.1. Spin cells at 1000 rpm, 5 min, 4 °C. Remove supernatant and resuspend with PBS. (1/3).
SECTION: FLAG-LRRK2 Purification
16.2. Spin cells at 1000 rpm, 5 min, 4 °C. Remove supernatant and resuspend with PBS. (2/3).
SECTION: FLAG-LRRK2 Purification
16.3. Spin cells at 1000 rpm, 5 min, 4 °C. Remove supernatant and resuspend with PBS. (3/3).
SECTION: FLAG-LRRK2 Purification
27. Repeat 4X
SECTION: FLAG-LRRK2 Purification
27.1. Remove as much liquid as possible from resin, add 250 µL FLAG peptide in elution buffer and incubate in the cold room for 5 min. Then open the stopper and collect eluate. (1/4).
SECTION: FLAG-LRRK2 Purification
27.2. Remove as much liquid as possible from resin, add 250 µL FLAG peptide in elution buffer and incubate in the cold room for 5 min. Then open the stopper and collect eluate. (2/4).
SECTION: FLAG-LRRK2 Purification
27.4. Remove as much liquid as possible from resin, add 250 µL FLAG peptide in elution buffer and incubate in the cold room for 5 min. Then open the stopper and collect eluate. (4/4).
SECTION: FLAG-LRRK2 Purification
27.3. Remove as much liquid as possible from resin, add 250 µL FLAG peptide in elution buffer and incubate in the cold room for 5 min. Then open the stopper and collect eluate. (3/4).
SECTION: Fluorescent Tagging and analysis of Fluorescent FLAG-LRRK2:
32. For a 100 µLlabeling reaction, use 300 µL of 1:1 resin slurry. First add slurry to supplier-provided column and remove storage buffer by spinning at 1000 x g, 30 s. 
SECTION: Fluorescent Tagging and analysis of Fluorescent FLAG-LRRK2:
33. Add sample to column and briefly vortex; centrifuge at 1000 x g, 30 sto collect protein.
SECTION: Fluorescent Tagging and analysis of Fluorescent FLAG-LRRK2:
34. 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.