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GFP-Grb2 Translocation Assay Using High-content Imaging to Screen for Modulators of EGFR-signaling
通过高含量成像观测GFP-Grb2易位筛查EGFR信号转导调控因子   

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Abstract

High-content screening is a useful tool to understand complex cellular processes and to identify genes, proteins or small molecule compounds that modulate such pathways. High-content assays monitor the function of a protein or pathway by visualizing a change in an image-based readout, such as a change in the localization of a reporter protein. Examples of this can be the translocation of a fluorescently tagged protein from the cytoplasm to the nucleus or to the plasma membrane. One protein that is known to undergo such translocation is the Growth Factor Receptor-bound protein 2 (GRB2) that is recruited to the plasma membrane upon stimulation of a growth factor receptor and subsequently undergoes internalization. We have used GFP-tagged Grb2 previously to identify genes that are involved in EGFR signaling (Petschnigg et al., 2017). Ultimately, the assay can be adapted to cDNA expression cloning (Freeman et al., 2012) and can be used in early stage drug discovery to identify compounds that modulate or inhibit EGFR signaling and internalization (Antczak and Djaballah, 2016).

Keywords: Grb2(Grb2), EGFR(EGFR), High-content imaging(高含量成像), cDNA(cDNA), si/shRNA(si/shRNA), Cancer signaling(癌症信号转导)

Background

Signal transduction by growth factor receptors is essential for cells to maintain proper function and thus requires tight control. Signal transduction by growth factor receptors is initiated by binding of an external ligand (e.g., Epidermal Growth Factor, EGF) to a transmembrane receptor such as the Epidermal Growth Factor Receptor (EGFR) and activation of downstream signaling cascades (Yao et al., 2015). A key regulator of EGFR-signaling is Growth Factor Receptor-bound protein 2 (Grb2), which is composed of an internal SH2 (Src homology 2) domain flanked by two SH3 domains. Grb2 binds to activated growth factor receptors at phosphorylated tyrosine residues through its SH2 domain, thus coupling receptor activation to SOS-Ras-MAPK (Mitogen-activated protein kinase) signaling cascades. The composition of Grb2 suggests that it can dock to a variety of receptors and transduce signals along multiple pathways. Mutations in signaling pathways frequently lead to the development of cancer. Hence, in order to better understand how aberrant signaling can lead to disease, it is important to identify novel signaling molecules in growth factor signaling. To accomplish this, we used the previously established microscopy-based GFP-Grb2 translocation assay that monitors the translocation of cytosolic GFP-tagged Grb2 to subcellular compartments upon expression of a cDNA library (Figure 1). We used this technique to identify novel proteins that can lead to translocation of GFP-Grb2 when overexpressed and in a second stage tested whether these proteins play a role in EGFR-signaling (Petschnigg et al., 2017). Examples that lead to punctate structures include TACC3, a novel EGFR-interactor, and AMPH (Figure 3). TACC3 led to induction of large GFP-Grb2 puncta, whereas AMPH results in formation of multiple small spots (Figure 3), pointing at potentially different mechanisms of those proteins in EGFR-signaling. In our recent study, we further characterized TACC3 and showed that TACC3 specifically binds to oncogenic EGFR variants and showed that TACC3 enhances EGFR-stability at the cell surface and increases EGFR-mediated signaling. The GFP-Grb2 assay can be expanded to multiple more applications. A siRNA/shRNA or CRISPR library could be co-expressed with GFP-Grb2 and translocation subsequently observed following EGF stimulation. As EGF stimulation would sequester GFP-Grb2 to endosomal structures and the plasma membrane, translocation from there upon siRNA/shRNA knockdown or CRISPR knockout could point at possible factors that ablate EGFR-Grb2 interactions and signaling. In a similar way, small molecule compound screens could be done to test for drugs that can specifically disrupt EGFR-Grb2 interactions upon EGF-stimulation (Figure 1). Other options could be to use mutated Grb2-variants that fail to bind to EGFR or other binding partners, thus the assay can give insight into which gene (when overexpressed or knock-downed) or which drug has an influence on specific binding domains of Grb2.


Figure 1. Schematic overview of the Grb2 translocation assay. Under non-stimulated or basal conditions, GFP-tagged Grb2 is mostly found in the cytosol (A), but can be recruited to localized interaction partners such as activated or endocytosed Epidermal Growth Factor Receptor, EGFR. Expression of a cDNA expression plasmid can lead to relocalization of GFP-Grb2 to the plasma membrane, endosomal structures or other sub-cellular locations by binding to Grb2 interaction partners or activation of Grb2-dependent cellular pathways (B). Stimulation with EGF recruits Grb2 to phosphotylated EGFR (C). Small molecule compounds or si/shRNA libraries can help identify genes or drugs that can disrupt Grb2 binding to the EGFR, impairing the recruitment to the receptor and thus predominant cytosolic localization of GFP-Grb2 (D). Since Grb2 can interact with other growth factor receptors as well, the assay can be adapted to monitor interaction/recruitment to other receptors as well.

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 ScientificTM, 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, GibcoTM, 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, GibcoTM, catalog number: 10500056 )
  10. Penicillin-streptomycin (10,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  11. Trypsin/EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: R001100 )
  12. GlutaMax (100x) (Thermo Fisher Scientific, GibcoTM, 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, InvitrogenTM, 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 ScientificTM, 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 st ably 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.

简介

高含量筛选是了解复杂细胞过程和鉴定调节这种途径的基因,蛋白质或小分子化合物的有用工具。高含量测定法通过显现基于图像的读数的变化来监测蛋白质或途径的功能,例如报告蛋白的定位的变化。其实例可以是将荧光标记的蛋白质从细胞质转移到细胞核或质膜。已知发生这种易位的一种蛋白质是生长因子受体结合蛋白2(GRB2),其在刺激生长因子受体并且随后经历内化后被招募到质膜。我们以前用GFP标记的Grb2来鉴定涉及EGFR信号的基因(Petschnigg等,2017)。最终,该测定可以适应于cDNA表达克隆(Freeman等人,2012),并且可用于早期药物发现以鉴定调节或抑制EGFR信号传导和内化的化合物(Antczak和Djaballah,2016)。
【背景】生长因子受体的信号转导对细胞维持正常功能至关重要,因此需要严格控制。生长因子受体的信号转导通过外部配体(例如,表皮生长因子,EGF)与跨膜受体(例如表皮生长因子受体(EGFR))和下游信号级联的活化(Yao等人,2015 )。 EGFR-信号传导的关键调节因子是生长因子受体结合蛋白2(Grb2),其由两个SH3结构域的内部SH2(Src同源性2)结构域组成。 Grb2通过其SH2结构域结合磷酸化酪氨酸残基上的活化生长因子受体,从而将受体活化与SOS-Ras-MAPK(丝裂原活化蛋白激酶)信号级联偶联。 Grb2的组成表明它可以对接多种受体并沿着多个途径转导信号。信号途径的突变常常导致癌症的发展。因此,为了更好地了解异常信号传导如何导致疾病,重要的是在生长因子信号传导中鉴定新的信号分子。为了实现这一点,我们使用了以前建立的基于显微镜的GFP-Grb2易位测定法,其在表达cDNA文库时监测细胞溶质GFP标记的Grb2向亚细胞区室的转位(图1)。我们使用这种技术来鉴定当过表达时可导致GFP-Grb2易位的新蛋白质,并且在第二阶段测试这些蛋白质是否在EGFR-信号传导中起作用(Petschnigg等,2017)。导致点状结构的实例包括TACC3,一种新型EGFR相互作用因子和AMPH(图3)。 TACC3导致大的GFP-Grb2斑点的诱导,而AMPH导致形成多个小斑点(图3),指出EGFR-信号传导中那些蛋白质的潜在不同机制。在我们最近的研究中,我们进一步表征了TACC3,并且显示TACC3特异性结合致癌EGFR变体,并且显示TACC3在细胞表面增强EGFR-稳定性并增加EGFR介导的信号传导。 GFP-Grb2测定可以扩展到多个应用。 siRNA / shRNA或CRISPR文库可以与GFP-Grb2共同表达,随后在EGF刺激后观察到易位。由于EGF刺激会将GFP-Grb2螯合至内体结构和质膜,因此在siRNA / shRNA敲低或CRISPR敲除后的易位可能指向可能消除EGFR-Grb2相互作用和信号传导的因素。以类似的方式,可以进行小分子化合物筛选,以测试能够在EGF刺激后特异性破坏EGFR-Grb2相互作用的药物(图1)。其他选择可能是使用不能与EGFR或其他结合配偶体结合的突变的Grb2变体,因此该测定可以了解哪种基因(何时过表达或敲低)或哪种药物对Grb2的特异性结合结构域有影响。

关键字:Grb2, EGFR, 高含量成像, cDNA, si/shRNA, 癌症信号转导

材料和试剂

  1. 移液器提示(可以使用任何标准无菌提示,无过滤或过滤)
  2. ViewPlate-96黑色,光学透明底部(PerkinElmer,目录号:6005225)
  3. 组织培养板:Nunc细胞培养培养皿(Thermo Fisher Scientific,Thermo Scientific TM,目录号:172931)
  4. 0.22μm过滤器
  5. HeLa细胞(ATCC,目录号:CCL-2)或HEK293T细胞(ATCC,目录号:CRL-3216)
  6. pMOS-GFP-Grb2质粒(Ketteler等人,2002)
  7. 3xFLAG-TACC3质粒(Petschnigg等人,2017)
  8. Dulbecco改良的Eagle培养基(DMEM)(Thermo Fisher Scientific,Gibco TM,目录号:61965026)
    注意:可以使用任何市售的DMEM,并且可以加入GlutaMax,但HeLa和HEK293T细胞不需要。
  9. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:10500056)
  10. 青霉素 - 链霉素(10,000U / ml)(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  11. 胰蛋白酶/ EDTA(Thermo Fisher Scientific,Gibco TM,目录号:R001100)
  12. GlutaMax(100x)(Thermo Fisher Scientific,Gibco TM,目录号:35050061)
  13. 四聚甲醛,4%(v / v)(Santa Cruz Biotechnologies,目录号:sc-281692)
    注意:应以-20°C储存,以便长期储存。解冻的等分试样可以在4℃下保存长达一个月。
  14. Hoechst 33342三盐酸盐,三水合物,1mg / ml储备液(Thermo Fisher Scientific,Invitrogen ,目录号:H3570)
  15. 聚乙烯亚胺(PEI),10mg / ml水溶液(Sigma-Aldrich,目录号:408727)
  16. 氯化钠(NaCl),AnalaR NORMAPUR(VWR,目录号:27810.295)
  17. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541)
  18. 二钠磷酸钠(Na 2 HPO 4),AnalaR NORMAPUR(VWR,目录号:102494C)
  19. 磷酸氢二钾(K 2 H 2 HPO 4)(Sigma-Aldrich,目录号:P8281)
  20. PEI解决方案(见配方)
  21. 1x磷酸缓冲盐水(PBS)(pH 7.4)(参见食谱)
  22. 10倍磷酸盐缓冲盐水(PBS)(见食谱)

设备

  1. 多通道移液器(Finnipipette,8通道P300,Thermo Fisher Scientific,Thermo Scientific TM,目录号:4661030N)
  2. 孵化器(Eppendorf,型号:Galaxy ® 170 R)
  3. 高含量筛选显微镜(PerkinElmer,Opera)

软件

  1. ImageJ的

程序

  1. 将PEI溶液(见食谱)制成10mg / ml的水。
    注意:也可以使用其他转染方法,因为PEI是廉价的,显示出良好的转染效率。
  2. 培养在37℃和5%CO 2的补充有10%FBS和青霉素 - 链霉素的DMEM中的HeLa或HEK293T细胞,直到70-80%汇合。
  3. 使用多通道移液管(总体积为50μl)胰蛋白酶化细胞并将2×10 4个细胞种子浸入PerkinElmer Viewplate的每个孔中。包括阴性和阳性对照的井(见注3)。对于弱粘附细胞,可能需要用明胶或聚-L-赖氨酸进行预处理。
  4. 在37℃和5%CO 2孵育细胞过夜。
  5. 在不含FBS和青霉素的DMEM(25μl)中制备100ng GFP-Grb2质粒DNA与100ng cDNA(例如,/或>,3xFLAG-TACC3作为阳性对照或其他对照,参见附注3)的混合物链霉素。
  6. 以4:1的比例添加PEI(10mg / ml溶液),即200ng总DNA添加0.8μlPEI。
  7. 旋转DMEM / DNA / PEI混合物,并在室温下放置15-20分钟
  8. 使用温和分配方法将DMEM / DNA / PEI-混合物滴加到细胞中。
    注意:或者,可以将转染混合物首先分配到平板中,然后将细胞添加到顶部(反向转染)。
  9. 转染后6 h,用新鲜的DMEM加FBS /青霉素 - 链霉素培养基代替转染混合物。
  10. 在37℃和5%CO 2孵育细胞过夜。
  11. 第二天,吸出媒体,用1x PBS洗一次细胞(见食谱)。
    注意:在此阶段,可以在PBS洗涤前进行诸如EGF刺激或药物治疗的治疗,这取决于孵育的持续时间(例如,10分钟EGF或几小时的药物)。 em>
  12. 在PBS中加入50μl4%PFA的PBS,并在室温下孵育10分钟 注意:测定也可以在活细胞上进行,在这种情况下可以省略步骤12和13。在这里,我们固定细胞,因为它们可以保存在PBS中几天,并且可以在稍后的时间点对板进行成像。然而,活细胞成像允许更多的实验灵活性,例如延时显微镜。
  13. 吸出PFA并用1x PBS洗一次。
  14. 加入Hoechst 33342(1x PBS中1:100稀释)10分钟
  15. 用1x PBS洗涤细胞一次,加入50μl1x PBS 注意:在这个阶段,板材可以在4°C下储存,直到成像,直到几天。
  16. 通过以下设置在合适的荧光显微镜(例如,Opera Phenix)上获取图像:GFP通道488 nm,100%激光功率,40x物镜,合并'1',曝光时间可能在500之间变化msec和2秒,具体取决于转染效率和亮度。图2显示了Opera-high-content筛选显微镜上的设置。示例图像(TACC3-过表达和AMPH过表达仅与GFP-Grb2相比)如图3所示。
  17. 进行图像分析或定性评估图像。


    图2.图像采集的仪器设置。 Opera LX图像采集页面的屏幕截图。箭头表示可以调整的设置,包括激光通道,相机,板类型,目标,激光功率,过滤器和曝光时间(从上到下)。其他仪器在布局和设置的设置上有所不同。


    图3. GFP-Grb2在TACC3或AMPH过表达时定位的变化示例图。箭头表示易位时的点状结构。刻度棒=20μm。

数据分析

使用免费获得的软件ImageJ分析荧光图像。对于我们的研究,单独分析结构,并通过眼睛评估不同的点状结构。然而,对于高通量屏幕,还可以使用定制的图像分析ImageJ管线来量化结构。更多细节请参见(Ketteler等人,,2017)。或者,可以使用其他软件,如CellProfiler可以使用。典型的高分辨率图像处理工作流程包括降噪,分割和特征提取步骤。通常,用中值滤波器对样本单元的原始核通道进行平滑,用k均值聚类算法进行分割,并选择和量化对象(Ketteler和Kriston-Vizi,2016)。

笔记

  1. 该测定是相当鲁棒和可重复的。然而,当进行测定时,确保细胞相当低通过(
  2. 转移效率可能在板/孔之间变化,因此稳定的细胞系的产生,例如表达GFP-Grb2的细胞可以改善变异性。如本协议所示,我们共转染了GFP-Grb2和推定的EGFR相互作用因子,如TACC3和AMPH。我们还产生稳定的TACC3细胞系和转染的GFP-Grb2并获得相同的结果。然而,对于大规模筛选实验,我们建议生成GFP-Grb2稳定的HeLa或HEK293T细胞系。
  3. 在测定中包括对照以确保易位测定工作是重要的。在这种情况下,阴性对照只是GFP-Grb2(应该显示为胞质模式),也可以包括阳性对照,如过表达TACC3(3xFLAG-TACC3),这应该导致大的点状GFP模式。其他阳性对照可以是已知诱导Grb2从细胞溶质转移的任何蛋白质,例如EGFR,其将Grb2螯合到质膜和/或内体。
  4. 该协议描述了易位测定用于筛选应用的用途,因此使用了高通量共聚焦显微镜。可以使用任何(共焦)荧光显微镜进行测定。因此,细胞也可以种植在盖玻片或特殊成像室上。播种后的协议基本相同。
  5. 该测定可以适应于背景技术部分所述的更多应用。药物孵育时间,EGF刺激或siRNA / shRNA表达需要由每个用户单独确定。
  6. HeLa和HEK293T细胞均已成功用于我们的检测。然而,应该注意的是,HeLa细胞在96孔板中更容易工作,因为它们在洗涤步骤(4%多聚甲醛固定之前)不倾向于从板上移出。如果使用低粘附细胞,则可以用明胶或聚-L-赖氨酸预处理平板。测定也可以在活细胞中进行,在这种情况下,用户可以省略固定步骤。

食谱

  1. PEI解决方案
    在无菌蒸馏水中制备10 mg / ml溶液,并过滤灭菌(0.22μm过滤器),并在4°C下储存
    注意:每月准备一次新鲜溶液,以确保转染效率相等。
  2. 1x磷酸缓冲盐水(PBS)(pH 7.4)
    137 mM NaCl
    10 mM磷酸盐
    2.7 mM KCl
    注意:准备10x PBS储存液,用无菌水稀释至1×PBS。
  3. 10倍磷酸缓冲盐水(PBS)
    80克NaCl
    2克KCl
    14.4g Na 2 HPO 4
    2.4g KH 2 PO 4
    溶解于800ml蒸馏水中的H 2 O / / 将pH调节至7.4
    填写1 L

致谢

这项工作得到英国医学研究委员会向MRC-UCL大学单位(MC_EX_G0800785)提供核心资金和BBSRC RK新研究员奖(BB / JO / 5881/1)的支持。 JP获得了EC-Marie Curie International Incoming Fellowship(FP7-PEOPLE-2013-IIF)的支持。该协议已经从(Petschnigg等人,2017)改编。

参考

  1. Antczak,C.and Djaballah,H。(2016)。&nbsp; 用于筛选EGFR功能调节剂的高含量测定法。 Methods Mol Biol 1360:97-106。
  2. Freeman,J.,Kriston-Vizi,J.,Seed,B.and Ketteler,R。(2012)。&lt; a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih (68)。
  3. Ketteler,R.,Freeman,J.,Ferraro,F.,Bata,N.,Cutler,DF and Kriston-Vizi,J.(2017)。&lt; a class ="ke-insertfile"href ="http: //www.ncbi.nlm.nih.gov/pubmed/28248923"target ="_ blank">基于图像的siRNA筛选鉴定使用电穿孔调节Weibel-Palade体型大小控制的激酶。科学数据< / em> 4:170022.
  4. Ketteler,R.,Glaser,S.,Sandra,O.,Martens,UM和Klingmuller,U.(2002)。&lt; a class ="ke-insertfile"href ="http://www.ncbi.nlm .nih.gov / pubmed / 11948372"target ="_ blank">通过优化的逆转录病毒杂交载体有效转导的原始造血祖细胞和胚胎干细胞中增强的转基因表达。 8):477-487。
  5. Ketteler,R.和Kriston-Vizi,J.(2016)。细胞生物学中的高含量筛选。 在Bradshaw,AR和Stahl,DP(Eds)中。 Encyclopedia of Cell Biology,第4卷。学术出版社 pp:234-244。
  6. 致癌EGFR相互作用伴侣的系统鉴定。分子生物 429(2):280- 294.
  7. Yao,Z.,Petschnigg,J.,Ketteler,R.and Stagljar,I.(2015)。&nbsp; 细胞信号传递方法的应用指南 Nat Chem Biol 11(6):387-397。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Petschnigg, J. and Ketteler, R. (2017). GFP-Grb2 Translocation Assay Using High-content Imaging to Screen for Modulators of EGFR-signaling. Bio-protocol 7(17): e2528. DOI: 10.21769/BioProtoc.2528.
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