TITLE: Immunofluorescence-based assay to assess LRRK2 association with microtubules in HEK293 cells
AUTHORS: Elena Purlyte, Alexia  Kalogeropulou, Francesca Tonelli  , Dario R Alessi

[DESCRIPTION]
Previous studies using confocal fluorescence microscopy and cryo-electron microscopy reported that most pathogenic LRRK2 mutants, as well as pharmacological inhibition of LRRK2 kinase activity with Type-I inhibitors, cause relocalization of LRRK2 to filamentous structures that colocalize with microtubules (PMID: 22080837; PMID: 28453723; PMID: 32783917; PMID: 32814344). Here we describe our confocal immunofluorescence microscopy method for measuring LRRK2 association with microtubules in a cell-based assay. This method can be used to screen the impact that LRRK2 mutations have on LRRK2 microtubule binding, as well as the effect of any compound on LRRK2 association with microtubules.

[GUIDELINES]
This protocol includes our method for:
1) Reverse transfection of HEK293 cells with LRRK2 cDNA;
2) Sample preparation for immunofluorescence microscopy;
3) Imaging and cell counting.

Note:
In parallel with the preparation of samples for immunofluorescence microscopy, we recommend preparing samples for quantitative immunoblotting analysis (as described in dx.doi.org/10.17504/protocols.io.bsgrnbv6) to assess LRRK2 expression levels and efficient LRRK2 inhibition in samples treated with MLi-2.

  





[STEPS]
SECTION: Reverse transfection of HEK293 cells
1. Prepare a transfection mix by adding 0.6 µg of Flag-LRRK2 (or Flag-empty vector) cDNA and 1.8 µL of 1 mg/mL PEI solution into 150 µL of Opti-MEM for each well. Vortex for 20 s/30 s.
 



SECTION: Reverse transfection of HEK293 cells
2. Incubate the transfection mix for 20 min at Room temperature to allow the DNA/PEI complex to form.

SECTION: Reverse transfection of HEK293 cells
3. For each LRRK2 construct, add 150 µL of the transfection mix to each well.
 


SECTION: Reverse transfection of HEK293 cells
4. Remove culture medium from one flask of HEK293 cells.
SECTION: Reverse transfection of HEK293 cells
5. Briefly rinse the cell layer with 0.25% (w/v) Trypsin - 0.53 millimolar (mM) EDTA solution to remove all traces of serum.
SECTION: Reverse transfection of HEK293 cells
6. Add 2 mL of Trypsin-EDTA solution to the flask and incubate at 37 °C until the cell layer is dispersed.
SECTION: Reverse transfection of HEK293 cells
7. Add 8 mL of complete growth medium and resuspend cells by gently pipetting.
SECTION: Reverse transfection of HEK293 cells
8. Count the cells using the method of choice.
SECTION: Reverse transfection of HEK293 cells
9. Resuspend the cells to a concentration of 8x104 cells per ml of complete growth medium.
SECTION: Reverse transfection of HEK293 cells
10. Add 1 mL of cell suspension (8x104 cells) into the well containing the transfection mix. 
 

SECTION: Reverse transfection of HEK293 cells
11. Transfer the plates to a humidified incubator maintaining 37 °C and 5% (v/v) CO2.

SECTION: Sample preparation for immunoblotting analysis
12. 45 hours after transfection, treat cells with 100 nanomolar (nM) MLI-2 or 0.1% (v/v) DMSO (control vehicle) and incubate for 180 min at 37 °C in a humidified incubator maintaining 5% (v/v) CO2.

SECTION: Sample preparation for immunoblotting analysis
13. After treatment with MLi-2/DMSO, remove culture medium completely from each well using an aspirator.
SECTION: Sample preparation for immunoblotting analysis
14. Immediately add 50 µL of ice-cold complete lysis buffer to each well ensuring that the entire surface is covered by lysis buffer.

SECTION: Sample preparation for immunoblotting analysis
15. Transfer the plate on ice.

SECTION: Sample preparation for immunoblotting analysis
16. Scrape the cells on the dish using a cell lifter to ensure all cells are detached from the well.
SECTION: Sample preparation for immunoblotting analysis
17. Using a pipette, transfer the lysate to a 1.5 mL Eppendorf tube.
SECTION: Sample preparation for immunoblotting analysis
18. Leave samples on ice for 20/30 minutes to allow for efficient lysis.

SECTION: Sample preparation for immunoblotting analysis
19.  Spin down lysates at 17000 x g, 10 min, 4 °C.

SECTION: Sample preparation for immunoblotting analysis
20. Transfer supernatant to a new Eppendorf tube and discard the pellet.
SECTION: Sample preparation for immunoblotting analysis
21. Proceed to quantitative immunoblotting analysis as described in dx.doi.org/10.17504/protocols.io.bsgrnbv6 (Quantitative Immunoblotting Analysis of LRRK2 Signalling Pathway). 
 

SECTION: Sample preparation for immunofluorescence microscopy
22. 45 hours after transfection, treat cells with 100 nanomolar (nM) MLI-2 or 0.1% (v/v) DMSO (control vehicle) and incubate for 180 min at 37 °C in a humidified incubator maintaining 5% (v/v) CO2.

SECTION: Sample preparation for immunofluorescence microscopy
23. After treatment with MLi-2/DMSO, remove culture medium completely from each well using an aspirator.
SECTION: Sample preparation for immunofluorescence microscopy
24. Fix cells by adding 4% (v/v) PFA in PBS pre-warmed to 37 °C.
SECTION: Sample preparation for immunofluorescence microscopy
25. Incubate for 10 min at 37 Room temperature.

SECTION: Sample preparation for immunofluorescence microscopy
26. Remove PFA completely using a pipette and wash with PBS.
 

SECTION: Sample preparation for immunofluorescence microscopy
27. Permeabilise cells by incubating with 1% (v/v) NP-40 alternative in PBS for 10 min at 37 Room temperature.    

SECTION: Sample preparation for immunofluorescence microscopy
28. Remove the solution completely using an aspirator.
SECTION: Sample preparation for immunofluorescence microscopy
29. Block with 1% (w/v) BSA in PBS for 60 min at 37 Room temperature.

SECTION: Sample preparation for immunofluorescence microscopy
30. Prepare the primary antibody solution by diluting anti-Flag M2 antibody and anti-β-tubulin antibody in 0.2% (w/v) BSA in PBS (1:1000 and 1:500 dilution, respectively).
SECTION: Sample preparation for immunofluorescence microscopy
31. Incubate the samples with primary antibodies for 120 min at 37 °C in a humidified chamber. 
 


SECTION: Sample preparation for immunofluorescence microscopy
32. Wash the samples 3 times with 0.2% (w/v) BSA in PBS (10 minutes per wash).
SECTION: Sample preparation for immunofluorescence microscopy
33. Prepare the secondary antibody solution by diluting the secondary antibodies (Alexa Fluor 488 goat anti-mouse and Alexa Fluor 594 goat anti-rabbit) in 0.2% BSA in PBS (1:500 dilution). Add DAPI at 1 μg/ml final concentration to the secondary antibody solution.

SECTION: Sample preparation for immunofluorescence microscopy
34. Incubate the samples with the secondary antibodies and DAPI at 37 Room temperature for 60 min in the dark.

SECTION: Sample preparation for immunofluorescence microscopy
35. Wash the samples 3 times with 0.2% (w/v) BSA in PBS (10 minutes per wash).
 

SECTION: Laser confocal imaging
36. Image cells using a Zeiss LSM 710 or 880 laser scanning microscopes using the x40 EC Plan-Neofluar (NA 1.3) objective with a zoom of 0.6 and optical section thickness of 1.0 mm (image size 2048x2048 pixels, pixel size 0.173 μm).
SECTION: Laser confocal imaging
37. Image 50-100 cells from 4-6 randomly selected fields with Alexa Fluor 488-positive cells for each well (These are cells successfully transfected with Flag-LRRK2). 

SECTION: Cell counting and statistical analysis
38. Perform cell counting using the Image J Cell Counter feature.
 

SECTION: Cell counting and statistical analysis
39. Once cell counting is done, data is unblinded and analysed as percentage of cells for each category using GraphPad Prism and applying a 2-way ANOVA and the post-hoc Dunnett’s test to evaluate statistical significance between different experimental conditions.
SECTION: How to generate a Python code script to generate randomised file names
40. Copy the code below and change the folder directories according to where the data is located and where you would like the data with changed names to be located (the original data will not be changed).

 

SECTION: How to generate a Python code script to generate randomised file names
41. Select “Run” and the program will create the renamed copies for you to use.