GFP-Grb2 Translocation Assay Using High-content Imaging to Screen for Modulators of EGFR-signaling

Materials and Reagents

1. Pipette tips (any standard sterile tips can be used, either non-filtered or filtered)
   
2. ViewPlate-96 Black, Optically Clear Bottom (PerkinElmer, catalog number: 6005225 )
   
3. Tissue culture plates: Nunc cell culture Petri dishes (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 172931 )
   
4. 0.22 µm filter
   
5. HeLa cells (ATCC, catalog number: CCL-2 ) or HEK293T cells (ATCC, catalog number: CRL-3216 )
   
6. pMOS-GFP-Grb2 plasmid (Ketteler et al., 2002)
   
7. 3xFLAG-TACC3 plasmid (Petschnigg et al., 2017)
   
8. Dulbecco modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, Gibco^TM, catalog number: 61965026 )
   Note: Any commercially available DMEM can be used, and GlutaMax can be added, but is not required for HeLa and HEK293T cells.
   
9. Fetal bovine serum (FBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 10500056 )
   
10. Penicillin-streptomycin (10,000 U/ml) (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140122 )
    
11. Trypsin/EDTA (Thermo Fisher Scientific, Gibco^TM, catalog number: R001100 )
    
12. GlutaMax (100x) (Thermo Fisher Scientific, Gibco^TM, catalog number: 35050061 )
    
13. Paraformaldehyde, 4% (v/v) (Santa Cruz Biotechnologies, catalog number: sc-281692 )
    Note: Should be stored at -20 °C in aliquots for long-term storage. Thawed aliquots can be kept at 4 °C for up to a month.
    
14. Hoechst 33342 trihydrochloride, trihydrate, 1 mg/ml stock (Thermo Fisher Scientific, Invitrogen^TM, catalog number: H3570 )
    
15. Polyethylenimine (PEI), 10 mg/ml stock in water (Sigma-Aldrich, catalog number: 408727 )
    
16. Sodium chloride (NaCl), AnalaR NORMAPUR (VWR, catalog number: 27810.295 )
    
17. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
    
18. Sodium phosphate dibasic (Na₂HPO₄), AnalaR NORMAPUR (VWR, catalog number: 102494C )
    
19. Potassium phosphate dibasic (K₂HPO₄) (Sigma-Aldrich, catalog number: P8281 )
    
20. PEI solution (see Recipes)
    
21. 1x phosphate buffered saline (PBS) (pH 7.4) (see Recipes)
    
22. 10x phosphate buffered saline (PBS) (see Recipes)
    

Equipment

1. Multi-channel pipette (Finnpipette, 8-channel P300, Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 4661030N )
   
2. Incubator (Eppendorf, model: Galaxy^® 170 R )
   
3. High-content screening microscope (PerkinElmer, Opera)
   

Software

1. ImageJ

Procedure

1. Prepare PEI solution (see Recipes) as 10 mg/ml in water.
   Note: Other transfection methods can be used as well, PEI is used here due to being inexpensive and showing good transfection efficiency.
   
2. Cultivate HeLa or HEK293T cells in DMEM supplemented with 10% FBS and penicillin-streptomycin at 37 °C and 5% CO₂ until 70-80% confluent.
   
3. Trypsinize cells and seed 2 x 10⁴ cells into each well of a PerkinElmer Viewplate using a multi-channel pipette (total volume of 50 µl). Include wells for negative and positive controls (see Note 3). For weakly adherent cells, pre-treatment with gelatin or poly-L-lysine may be required.
   
4. Incubate cells overnight at 37 °C and 5% CO₂.
   
5. Prepare a mix of 100 ng GFP-Grb2 plasmid DNA with 100 ng of cDNA (e.g., 3xFLAG-TACC3 as positive control or other controls, see Note 3) in DMEM (25 µl) without FBS and penicillin-streptomycin.
   
6. Add PEI (10 mg/ml solution) at a ratio of 4:1, i.e., for 200 ng total DNA add 0.8 µl PEI.
   
7. Vortex the DMEM/DNA/PEI-mix and leave at room temperature for 15-20 min.
   
8. Add the DMEM/DNA/PEI-mix dropwise to cells using a gentle dispensing method.
   Note: Alternatively, the transfection mix can be dispensed first into plates and then cells added on top (reverse transfection).
   
9. 6 h after transfection, replace the transfection mix with fresh DMEM plus FBS/penicillin-streptomycin media.
   
10. Incubate cells overnight at 37 °C and 5% CO₂.
    
11. The next day, aspirate off the media and wash the cells once with 1x PBS (see Recipes).
    Note: At this stage, treatments such as EGF stimulation or drug treatment can be performed before the PBS-wash, depending on the duration of the incubation (e.g., 10 min EGF, or a couple of hours of drugs).
    
12. Add 50 µl of 4% PFA in PBS to each well and incubate for 10 min at room temperature.
    Note: The assay can also be performed on live cells, in which case steps 12 and 13 can be omitted. Here, we fixed the cells as they can be kept in PBS for a couple of days and plates can be imaged at a later time-point. Yet, live-cell imaging allows for more experimental flexibility, such as time-lapse microscopy.
    
13. Aspirate off the PFA and wash once with 1x PBS.
    
14. Add Hoechst 33342 (diluted 1:10,000 in 1x PBS) for 10 min.
    
15. Wash cells once with 1x PBS and add 50 µl 1x PBS.
    Note: At this stage, plates can be stored at 4 °C until imaging, up to a couple of days.
    
16. Acquire images on a suitable fluorescence microscope (e.g., Opera Phenix) with the following settings: Channel 488 nm for GFP, 100% laser power, 40x objective, binning ‘1’, exposure time may vary between 500 msec and 2 sec, depending on transfection efficiency and brightness. Figure 2 shows the settings on the Opera-high-content screening microscope. Example images (TACC3-overexpression and AMPH-overexpression compared to GFP-Grb2 only) are shown in Figure 3.
    
17. Perform image analysis or qualitatively assess images by eye.
    
    
    Figure 2. Instrument settings for image acquisition. A screenshot of the Opera LX image acquisition page. Arrows indicate settings that can be adjusted including laser channels, cameras, plate types, objectives, laser power, filters and exposure times (from top to bottom). Other instruments vary in the layout and setup of settings.
    
    
    Figure 3. Example images of changes in localization of GFP-Grb2 upon overexpression of TACC3 or AMPH. Arrows indicate punctate structures upon translocation. Scale bar = 20 µm.
    

Data analysis

Fluorescence images were analysed using the freely available software ImageJ. For our study, structures were analysed individually and different punctate structures were assessed by eye. However, for a high-throughput screen, structures can furthermore be quantified using a custom-made image analysis ImageJ-pipeline. For more details see as an example (Ketteler et al., 2017). Alternatively, other software can be used such as CellProfiler can be used. A typical high-content analysis image processing workflow consists of noise reduction, segmentation, and feature extraction steps. Typically, the original nuclear channel of a sample cell is smoothened with a median filter, segmented with k-means clustering algorithm and the objects are selected and quantified (Ketteler and Kriston-Vizi, 2016).

Notes

1. The assay is quite robust and reproducible. However, when performing the assay, it is crucial to ensure that cells are quite low passage (< P30) and regularly passaged (at 70-80% confluency), as this can affect transfection efficiency.
   
2. Transfection efficiency can be variable between plates/wells, hence the generation of stable cell lines, such as cells stably expressing GFP-Grb2, could improve variability. As shown in this protocol, we co-transfected GFP-Grb2 and putative EGFR-interactors such as TACC3 and AMPH. We have also generated stable TACC3-cell lines and transfected GFP-Grb2 and get the same results. Yet, for large-scale screening experiments, we recommend generating GFP-Grb2 stable HeLa or HEK293T cell lines.
   
3. It is important to include controls in the assay to ensure the translocation assay works. In this case, negative control is GFP-Grb2 only (which should appear as cytosolic pattern), and a positive control can be included as well, such as overexpressing TACC3 (3xFLAG-TACC3), which should result in big, punctate GFP-patterns. Other positive controls can be any proteins that are known to induce translocation of Grb2 from the cytosol, such as EGFR, which would sequester Grb2 to the plasma membrane and/or endosomes.
   
4. This protocol describes the use of the translocation assay for screening applications, hence the high-throughput confocal microscope Opera is used. The assay can be performed using any (confocal) fluorescence microscope. Thus, cells can also be seeded onto cover slips or special imaging chambers. The protocol is substantially the same after seeding.
   
5. The assay can be adapted to many more applications as stated in the background section. Time of drug incubations, EGF-stimulation or siRNA/shRNA expression needs to be determined individually by each user.
   
6. Both HeLa and HEK293T cells have been successfully used for our assay. Yet, it should be noted that HeLa cells are easier to work within 96-well plates as they do not tend to dislodge from the plates during the washing steps (before the 4% paraformaldehyde fixing). If using low-adherent cells, plates can be pretreated with gelatin or poly-L-lysine. The assay can also be done in live cells, in which case the user can omit the fixing steps.

Recipes

1. PEI solution
   Prepare a 10 mg/ml solution in sterile distilled water and filter-sterilize (0.22 µm filter) and store at 4 °C
   Note: Prepare fresh solution about once a month to ensure equal transfection efficiency.
   
2. 1x phosphate buffered saline (PBS) (pH 7.4)
   137 mM NaCl
   10 mM phosphate
   2.7 mM KCl
   Note: Prepare a 10x PBS stock and dilute with sterile water to 1x PBS.
   
3. 10x phosphate buffered saline (PBS)
   80 g NaCl
   2 g KCl
   14.4 g Na₂HPO₄
   2.4 g KH₂PO₄
   Dissolved in 800 ml distilled H₂O
   Adjust pH to 7.4
   Fill up to 1 L
   

Acknowledgments

This work was supported by the UK Medical Research Council core funding to the MRC-UCL University Unit (MC_EX_G0800785) and a BBSRC New Investigator Award to RK (BB/JO/5881/1). JP was supported by an EC-Marie Curie International Incoming Fellowship (FP7-PEOPLE-2013-IIF). This protocol has been adapted from (Petschnigg et al., 2017).

References

1. Antczak, C. and Djaballah, H. (2016). A High-content assay to screen for modulators of EGFR function. Methods Mol Biol 1360: 97-106.
   
2. Freeman, J., Kriston-Vizi, J., Seed, B. and Ketteler, R. (2012). A high-content imaging workflow to study Grb2 signaling complexes by expression cloning. J Vis Exp (68).
   
3. Ketteler, R., Freeman, J., Ferraro, F., Bata, N., Cutler, D. F. and Kriston-Vizi, J. (2017). Image-based siRNA screen to identify kinases regulating Weibel-Palade body size control using electroporation. Sci Data 4: 170022.
   
4. Ketteler, R., Glaser, S., Sandra, O., Martens, U. M. and Klingmuller, U. (2002). Enhanced transgene expression in primitive hematopoietic progenitor cells and embryonic stem cells efficiently transduced by optimized retroviral hybrid vectors. Gene Ther 9(8): 477-487.
   
5. Ketteler, R. and Kriston-Vizi, J. (2016). High-content screening in cell biology. In: Bradshaw, A. R. and Stahl, D. P. (Eds). Encyclopedia of Cell Biology, Vol 4. Academic Press pp: 234-244.
   
6. Petschnigg, J., Kotlyar, M., Blair, L., Jurisica, I., Stagljar, I. and Ketteler, R. (2017). Systematic identification of oncogenic EGFR interaction partners. J Mol Biol 429(2): 280-294.
   
7. Yao, Z., Petschnigg, J., Ketteler, R. and Stagljar, I. (2015). Application guide for omics approaches to cell signaling. Nat Chem Biol 11(6): 387-397.