参见作者原研究论文

本实验方案简略版
May 2018
Advertisement

本文章节


 

Proximity Ligation Assay for the Investigation of the Intramolecular Interaction of ELMO1
邻位连接技术用于ELMO1分子内相互作用研究    

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

Intramolecular interaction is a common mechanism that regulates protein activities. Conventionally, such interactions are investigated by classical in vitro biochemical assays. Here, we describe a protocol for studying the intramolecular interaction of cell motility and engulfment 1 (ELMO1) in mammalian cells by using proximity ligation assay (PLA). PLA is a specific and sensitive method that allows the observation of interacting proteins by target-specific antibody detection coupled to rolling circle amplification. ELMO1 is the regulatory subunit of ELMO1-dedicator of cytokinesis 180 (DOCK180) bipartite Rac1 guanine nucleotide exchange factor (GEF) which adopts a closed autoinhibitory conformation via an intramolecular interaction of its N-terminal ELMO inhibitory domain (EID) and C-terminal ELMO autoregulatory domain (EAD). In the assay, PLA signals are detected in cells transfected with ELMO11-315 and ELMO1315-727 fragments. Moreover, overexpression of FE65, a neuronal adaptor which has been shown to disrupt ELMO1 intramolecular interaction, reduces the PLA signals of the two ELMO1 fragments significantly. Together, our results demonstrate that PLA can be employed for studying protein intramolecular interaction.

Keywords: Autoinhibition (自身抑制), Engulfment and cell motility 1 (吞噬与细胞运动蛋白1), Fluorescence (荧光 ), Intramolecular interaction (分子内相互作用 ), PLA (邻位连接技术)

Background

The activities of proteins in cells can be regulated by various mechanisms including intramolecular interaction (Miernyk and Thelen, 2008). Several protein-protein interaction assays have been adopted for studying intramolecular interaction such as co-immunoprecipitation and protein complementation assays (PCAs). Despite being widely employed, there are known limitations of these conventional assays. For instance, co-immunoprecipitation is not ideal for detecting low-affinity interaction. Even though PCAs provide good sensitivity, the reporter fragments may hinder the interaction of interest (Ohad et al., 2007; Miernyk and Thelen, 2008). Proximity ligation assay (PLA) is an innovative technology that allows in situ detection of protein-protein interactions. The protein targets are first labeled by their specific primary antibodies from different species, followed by the recognition of species-specific secondary antibodies conjugated to oligonucleotides (PLA probes). Proximity ligation of the oligonucleotides generates the templates for single-stranded rolling circle amplification. The resultant products are detected with fluorescence-labeled complementary oligonucleotide detection probes. PLA offers exceptional specificity and sensitivity as target-specific antibodies and single-stranded rolling circle amplification are involved. The resultant products are detected with fluorescence-labeled complementary oligonucleotide detection probes, and PLA signals can be visualized and quantified by using fluorescence microscopy (Weibrecht et al., 2010). Although the technology has been available for over a decade, the possibility of using PLA for investigating protein intramolecular interaction has not been explored.

Engulfment and cell motility 1 (ELMO1) is the regulatory subunit of the bipartite ELMO1-dedicator of cytokinesis 180 (DOCK180) guanine nucleotide exchange factor (GEF) for the activation of the small GTPase Rac1. ELMO1 possesses several distinct domains: a Ras binding domain (RBD), ELMO inhibitory domain (EID), ELMO domain, pleckstrin homology (PH) domain, ELMO autoregulatory domain (EAD) and a PXXP motif (Figure 1). Similar to many other GEFs, the activity of ELMO1 is modulated by the intramolecular interaction between its EID and EAD which allows ELMO1 to adopt a closed autoinhibitory conformation at the basal state (Patel et al., 2010) (Figure 1). The relief of ELMO1 autoinhibition is required for the targeting of the complex of ELMO1-DOCK180 to the plasma membrane where it stimulates Rac1 (Patel et al., 2011). However, the mechanism that alleviates the autoinhibition of ELMO1 remains elusive. We have recently reported that the neuronal adaptor protein FE65 interacts with ELMO1 EAD and to disrupt the interaction of ELMO11-315 (comprises of ELMO1 amino acid residues 1-315) and ELMO1315-727 (comprises of ELMO1 amino acid residues 315-727) fragments, which possess the EID and EAD respectively, by using both classical protein binding assay and PLA (Li et al., 2018). Therefore, we demonstrate for the first time that PLA can be applied for examining protein intramolecular interaction.

Here, we describe how to investigate the intramolecular interaction of ELMO1 EID and EAD by using PLA. The method may also be adopted for the investigation of other protein intramolecular interactions.


Figure 1. Schematic diagram shows the subdomains and the intramolecular interaction of ELMO1. At basal stage, the EAD and EID interact intramolecularly (indicated with double headed arrow) to form an autoinhibitory conformation. The disruption of the intramolecular interaction alleviates the autoinhibition for the subsequence Rac1 activation. Numbers denote the amino acid residue number in ELMO1. RBD: Ras-binding domain; EID: ELMO inhibitory domain; ELMO: ELMO domain; PH: Pleckstrin homology domain; EAD: ELMO autoregulatory domain; PXXP: PXXP motif.

Materials and Reagents

  1. SterilinTM Standard 90 mm Petri Dish (Thermo Scientific, catalog number: 101VIRR), stored at room temperature
  2. 12-well cell culture plate (SPL, catalog number: 30012), stored at room temperature
  3. Cover glass 18-mm circles (Thermo-Menzel, catalog number: CB00180RA1), stored at room temperature
  4. Hemocytometer (HBG, catalog number: 9010-01), stored at room temperature
  5. Microscope slides (Thermo Scientific, catalog number: 6776214), stored at room temperature
  6. HEK293 cells (ATCC, catalog number: CRL-1573), stored in liquid nitrogen
  7. Goat anti-ELMO1 antibody (Santa Cruz Biotechnology, catalog number: sc-21651), stored at 4 °C
  8. Mouse anti-FLAG antibody (Sigma-Aldrich, catalog number: F1804), stored at -20 °C
  9. Rabbit anti-β-tubulin antibody (Abcam, catalog number: AB6046)
  10. Donkey anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor 488 (Invitrogen, catalog number: A21206), stored at 4 °C
  11. Fetal bovine serum (HyClone, catalog number: SV30160.03), stored at -20 °C
  12. pCMV-Tag2B vector (Aligent, catalog number: 211172), stored at -20 °C
  13. T4 DNA Ligase (1 U/μl) (Thermo Scientific, catalog number: 15224025), stored at -20 °C
  14. BamHI (10 U/μl) (Thermo Scientific, catalog number: ER0055), stored at -20 °C
  15. XhoI (10 U/μl) (Thermo Scientific, catalog number: ER0691), stored at -20 °C
  16. TIANprep Mini Plasmid Kit (TIANGEN, catalog number: DP103), stored at room temperature
  17. QuikChange II Site-Directed Mutagenesis Kit (Agilent, catalog number: 200523), stored at -20 °C
  18. Phusion High-Fidelity PCR Kit (Thermo Scientific, catalog number: F553S), stored at -20 °C
  19. UltraPureTM Agarose (Invitrogen, catalog number: 16500500), stored at room temperature
  20. TAE Buffer (Tris-acetate-EDTA) (50x) (Thermo Scientific, catalog number: B49), stored at room temperature
  21. UltraPureTM Ethidium Bromide, 10 mg/ml (Invitrogen, catalog number: 15585011), stored at room temperature
  22. 1 kb Plus DNA Ladder (New England Biolabs, catalog number: N3200), stored at -20 °C
  23. Luria Broth Base, powder (Invitrogen, catalog number: 12795084), stored at room temperature
  24. Clear nail polish
  25. LB Agar, powder (Invitrogen, catalog number: 22700041), stored at room temperature
  26. Ampicillin sodium salt (Sigma-Aldrich, catalog number: A0166), stored at 4 °C
  27. Kanamycin sulfate (Sigma-Aldrich, catalog number: 60615), stored at 4 °C
  28. Poly-D-lysine hydrobromide (Sigma-Aldrich, catalog number: P7280), stored at -20 °C
  29. DMEM (HyClone, catalog number: SH30021.01), stored at 4 °C
  30. Trypsin-EDTA 0.05% (Gibco, catalog number: 25300062), stored at -20 °C
  31. Opti-MEM reduced serum medium (Gibco, catalog number: 31985088), stored at 4 °C
  32. X-tremeGENETM 9 DNA transfection reagent (Sigma-Aldrich, catalog number: 6365779001), stored at 4 °C
  33. PBS (Gibco, catalog number: 10010023), stored at room temperature
  34. Paraformaldehyde (Sigma-Aldrich, catalog number: P6148), stored at 4 °C
  35. Triton X-100 (Anatrace, catalog number: T1001500ML), stored at room temperature
  36. Duolink® In Situ Red Starter Kit Mouse/Goat (Sigma-Aldrich, catalog number: DUO92103), stored at -20 °C
  37. 1x TAE buffer (see Recipes)
  38. Poly-D-lysine solution (see Recipes)
  39. 4% paraformaldehyde (freshly prepared) (see Recipes)
  40. 1x Ligation buffer (see Recipes)
  41. 1x Amplification red (see Recipes)
  42. Wash buffer A (see Recipes)
  43. Wash buffer B (see Recipes)

Equipment

  1. Water bath (Labnet, model: W1106)
  2. Heat block (Labnet, model: D1301-230V)
  3. UV transilluminator (Accuris, model: E3000)
  4. T100TM Thermal Cycler (Bio-rad) 
  5. HerathermTM Compact Microbiological Incubators (37 °C, Thermo Scientific)
  6. FormaTM Series II 3110 Water-Jacketed CO2 Incubators (37 °C, 5% CO2, Thermo Scientific)
  7. Fluorescence microscope (Nikon, model: Eclipse Ni-U) equipped with QI2 high-resolution microscope camera (Nikon)

Software

  1. NIS-Elements research basic (Nikon)
  2. Prism (https://www.graphpad.com/scientific-software/prism/)

Procedure

  1. Generation of mammalian expression construct of ELMO11-315
    1. Perform site-directed mutagenesis to introduce a stop codon after ELMO1 residue 315 in pcDNA3-N-Myc-Elmo1 by using QuikChange II Site-Directed Mutagenesis Kit (Figure 2). The mutagenesis primers used are listed in Table 1.


      Figure 2. Schematic diagram of ELMO1 fragment constructs. Site-directed mutagenesis is performed to create a stop codon after ELMO1 residue 315 in pcDNA3-N-Myc-Elmo1 to generate ELMO11-315 fragment. cDNA of ELMO1315-727 is amplified from pcDNA3-N-Myc-Elmo1 by using PCR and the product is inserted to pCMV-Tag2B for the expression of N-terminal FLAG tagged protein. The asterisk indicates the mutation site. Numbers denote the amino acid residue number in ELMO1.

      Table 1. Primers used for generating ELMO1 fragment constructs. Primers 1 and 2 are used for the site-directed mutagenesis to introduce a stop codon to the pcDNA3-N-Myc-Elmo1. Primers 3 and 4 are used for the amplification of ELMO1315-727 fragment by using pcDNA3-N-Myc-Elmo1 as a template.


    2. Transform the mutagenesis product to DH5α competent E. coli cells by heat shock for 1 min at 42 °C.
    3. Plate the transformants on LB agar plates with 100 μg/ml ampicillin. 
    4. Incubate the plates overnight in 37 °C incubator.
    5. Inoculate isolated colonies in 5 ml LB with 100 μg/ml ampicillin and grow overnight at 37 °C.
    6. Isolate the plasmid DNA by using TIANprep Mini Plasmid Kit.
    7. Perform DNA sequencing to confirm the introduction of the stop codon.

  2. Generation of mammalian expression construct of FLAG-ELMO1315-727
    1. Perform polymerase chain reaction (PCR) to obtain cDNA encoded for ELMO1315-727 fragment by using Phusion High-Fidelity PCR Kit with pcDNA3-N-Myc-Elmo1 as a template. Primers used are listed in Table 1.
    2. Resolve the PCR product by a 1% agarose gel with 0.2 μg/ml ethidium bromide in 1x TAE buffer. 
    3. Visualize the PCR product by using a UV transilluminator and excise the PCR product (~1.2 kb) with a razor blade after electrophoresis.
    4. Purify the PCR product from gel by using MEGAquick-spinTM Plus Total Fragment DNA Purification Kit.
    5. Digest the purified ELMO1315-727 PCR product and pCMV-Tag2B, a vector with an N-terminal FLAG tag, with BamHI and XhoI for 2 h in a 37 °C water bath.
    6. Purify the digestion products by using MEGAquick-spinTM Plus Total Fragment DNA Purification Kit.
    7. Ligate the digested ELMO1315-727 fragment and linearized pCMV-Tag2B vector with T4 DNA ligase overnight in a 16 °C water bath.
    8. Transform the ligation product as stated in Steps A2-A6. 50 μg/ml Kanamycin should be used for selection.
    9. Perform DNA sequencing to confirm the successful insertion of the ELMO1315-727 encoding cDNA into pCMV-Tag2B.

  3. Cell preparation and transfection
    1. Coat 18-mm cover glasses with 5 μg/ml poly-D-lysine solution in 12-well cell culture plate overnight in a 37 °C incubator.
      Note: Sterilize the cover glass before use.
    2. Wash the cover glasses once with distilled water.
    3. Seed HEK293 cells on the pre-coated 18-mm cover glasses at 0.4 x 105 cells in 1 ml DMEM medium supplemented with 10% FBS.
    4. After 24 h, transfect the cells with mammalian expression constructs of myc-ELMO11-315 and FLAG-ELMO1315-727 together with either empty vector (EV), FE65 or FE65 K48A/R51A (FE65m, an ELMO1 binding defective FE65 mutant) using X-tremeGENETM 9 DNA transfection reagent in Opti-MEM reduced serum medium according to the manufacturer’s instruction (Table 2).

      Table 2 Transfection combinations for studying the effect of FE65 in ELMO1 intramolecular interaction. HEK293 cells are co-transfected with either EV, FE65 or FE65m together with ELMO11-315 and ELMO1315-727 constructs in 1:1:1 ratio. All transfections receive the same amount of DNA. “+” denotes the presence of the plasmid while “-” denotes the absence of the plasmid.


  4. Cell fixing and proximity ligation assay
    Note: Wash Buffer A, Wash Buffer B, PLA probe anti-goat MINUS, PLA probe anti-mouse PLUS, Antibody Diluent, Ligase, 5x Ligation Buffer, Polymerase, 5x Amplification Red and Duolink In Situ Mounting Media with DAPI are provided within Duolink® In Situ Red Starter Kit Mouse/Goat. Please see Figure 3 for the workflow of PLA.


    Figure 3. Timeline illustrates the workflow of PLA of ELMO1 fragments using Duolink® In Situ Red Starter Kit Mouse/Goat

    1. After 24 h transfection, wash the cells once with PBS and fix the cells with 4% paraformaldehyde for 10 min at room temperature.
    2. Wash the fixed cells three times with PBS.
    3. Permeabilize the cells with 0.1% Triton X-100/PBS for 15 min at room temperature.
    4. Block the cells with 5% FBS/PBS for 30 min at room temperature.
    5. Dilute the goat anti-ELMO1 (recognizes myc-ELMO11-315) (1:100), mouse anti-FLAG (recognizes FLAG-ELMO1315-727) (1:1000) and rabbit anti-β-tubulin (1:200) antibodies in 5% FBS/PBS and stain the cells with 80 μl of diluted antibody mix for 1 h at room temperature.
      Note: The concentrations of primary antibodies used are critical for obtaining high-quality PLA images and should be optimized. 
    6. Wash the cells five times with PBS.
    7. Dilute the Alexa Fluor 488 conjugated Donkey anti-rabbit IgG (1:1000) antibody in 5% FBS/PBS and stain the cells for 1 h in the dark at room temperature.
    8. Wash the cells five times with PBS.
    9. Wash the cells twice with Wash Buffer A for 5 min in the dark at room temperature.
    10. Add 80 μl PLA probe reaction mix to each cover glass and incubate in a humidified 37 °C incubator for 1 h.
      Note: For each reaction, mix 16 μl of PLA probe anti-goat MINUS (Donkey anti-goat secondary antibody conjugated to oligonucleotide MINUS) with 16 μl of PLA probe anti-mouse PLUS (Donkey anti-mouse secondary antibody conjugated to oligonucleotide PLUS) in 48 μl of Antibody Diluent and incubate for 20 min at room temperature before use.
    11. Wash the cover glasses twice with Wash Buffer A for 5 min in the dark at room temperature.
    12. Add 80 μl Ligation reaction mix to each cover glass and incubate in a humidified 37 °C incubator for 30 min.
      Note: For each reaction, add 2 μl Ligase to 78 μl 1x Ligation buffer.
    13. Wash the cover glasses twice with Wash Buffer A for 5 min in the dark at room temperature.
    14. Add 80 μl Amplification reaction mix to each cover glass and incubate in humidified 37 °C incubator for 100 min.
      Note: For each reaction, add 1 μl Polymerase to 79 μl 1x Amplification Red.
    15. Wash the cover glasses twice with Wash Buffer B for 10 min in the dark at room temperature.
    16. Wash the cover glasses once with 0.01x Wash Buffer B for 1 min in the dark at room temperature.
    17. Mount the cover glasses on microscope slides with a minimal amount of Duolink In Situ Mounting Media with DAPI.
    18. Seal the edge of the mounted cover glasses with clear nail polish.
    19. Capture the cell images with a 60x water immersion lens using a Nikon fluorescent microscope.
      Note: Store the slides in the dark at 4 °C if necessary.

Data analysis

Microscope slides are imaged by a Nikon fluorescent microscope with a 60x water immersion lens. FITC and TRITC filter sets are used to visualize β-tubulin and PLA signals, respectively. The number of discrete fluorescent PLA spots inside the cells is counted. The average numbers of PLA signals per cell in different transfections are compared to investigate the change of interaction between the two fragments. The PLA signals from at least 60 cells are counted, and three independent experiments are performed. Statistical analysis is performed by using one-way analysis of variance (ANOVA) with Bonferroni post-hoc test.

Representative data
Recently, we demonstrated that the interaction of ELMO11-315 and ELMO1315-727, which contain its EID and EAD, respectively, in cells by PLA. Moreover, the number of PLA signals reduced significantly in cells co-transfected with the wildtype FE65 but not the binding defective mutant (i.e., FE65m) (Figure 4). These findings from the PLAs are in line with the result from the classical protein binding assay (Li et al., 2018).


Figure 4. Representative images and quantification of PLA signals. HEK293 cells were transfected with myc-ELMO11-315 and FLAG-ELMO1315-727 together with either empty vector (EV), FE65 or FE65m. After 24 h transfection, the cells were fixed and subjected to PLA. The number of PLA signals from at least 60 cells was counted in each transfection and three independent experiments were performed. The bar chart shows the relative PLA signal (fold change to EV) in different transfections. *P < 0.001. Error bars are SEM. Significant reduction in PLA signals was observed only in FE65-transfected cells but not in EV- or FE65m-transfected cells. β-tubulin and nucleus were stained to serve as a morphological and nuclear marker, respectively. Scale bar = 10 μm. (This research was originally published in the Journal of Biological Chemistry. Li W, Tam KMV, Chan WWR, Koon AC, Ngo JCK, Chan HYE and Lau KF. Neuronal adaptor FE65 stimulates Rac1-mediated neurite outgrowth by recruiting and activating ELMO1. J. Biol. Chem. 2018; 293(20): 7674-7688. © the American Society for Biochemistry and Molecular Biology)

Recipes

  1. 1x TAE buffer
    Dilute the 50x stock solution to 1x with distilled water
  2. Poly-D-lysine solution
    5 mg poly-D-lysine
    5 ml distilled water
    Dissolve 5 mg poly-D-lysine in 5 ml distilled water to obtain a stock solution (200x)
    Store the stock solution in aliquots at -20 °C
  3. 4% paraformaldehyde (freshly prepared)
    0.4 g paraformaldehyde powder
    10 ml PBS
    1. Add 0.4 g paraformaldehyde powder to 10 ml PBS
    2. Incubate in a 70 °C water bath until the powder dissolved
    3. Cooldown the solution at room temperature
    4. Filter the solution with a 0.22 μm syringe filter
  4. 1x Ligation buffer
    For each reaction, dilute 16 μl 5x Ligation buffer with 64 μl distilled water
  5. 1x Amplification red
    For each reaction, dilute 16 μl 5x Amplification Red with 64 μl distilled water
  6. Wash buffer A
    0.01 M Tris
    0.15 M NaCl
    0.05% Tween 20
    pH 7.4
  7. Wash buffer B
    0.2 M Tris
    0.1 M NaCl
    pH 7.5

Acknowledgments

This work was supported by funds from the Research Grants Council Hong Kong, Health and Medical Research Fund (Hong Kong), CUHK direct grant scheme and the United College endowment fund. This protocol was adapted from previous work. We thank Prof Jean-François Côté (Montreal Clinical Research Institute) for the mammalian expression construct of pcDNA3-N-Myc-Elmo1.

Competing interests

The authors declare that they have no conflicts of interest with the contents of this article.

References

  1. Li, W., Tam, K. M. V., Chan, W. W. R., Koon, A. C., Ngo, J. C. K., Chan, H. Y. E. and Lau, K. F. (2018). Neuronal adaptor FE65 stimulates Rac1-mediated neurite outgrowth by recruiting and activating ELMO1. J Biol Chem 293(20): 7674-7688. 
  2. Miernyk, J. A. and Thelen, J. J. (2008). Biochemical approaches for discovering protein-protein interactions. Plant J 53(4): 597-609. 
  3. Ohad, N., Shichrur, K. and Yalovsky, S. (2007). The analysis of protein-protein interactions in plants by bimolecular fluorescence complementation. Plant Physiol 145(4): 1090-1099. 
  4. Patel, M., Margaron, Y., Fradet, N., Yang, Q., Wilkes, B., Bouvier, M., Hofmann, K. and Cote, J. F. (2010). An evolutionarily conserved autoinhibitory molecular switch in ELMO proteins regulates Rac signaling. Curr Biol 20(22): 2021-2027. 
  5. Patel, M., Pelletier, A. and Cote, J. F. (2011). Opening up on ELMO regulation: New insights into the control of Rac signaling by the DOCK180/ELMO complex. Small GTPases 2(5): 268-275. 
  6. Weibrecht, I., Leuchowius, K. J., Clausson, C. M., Conze, T., Jarvius, M., Howell, W. M., Kamali-Moghaddam, M. and Soderberg, O. (2010). Proximity ligation assays: a recent addition to the proteomics toolbox. Expert Rev Proteomics 7(3): 401-409.

简介

分子内相互作用是调节蛋白质活性的常见机制。通常,通过经典的体外生化分析研究这种相互作用。在这里,我们描述了一种协议,用于通过使用邻近结扎分析(PLA)研究哺乳动物细胞中细胞运动和吞噬1(ELMO1)的分子内相互作用。 PLA是一种特异而灵敏的方法,它允许通过结合滚动环扩增的靶标特异性抗体检测来观察相互作用的蛋白质。 ELMO1是细胞分裂180(DOCK180)二分体Rac1鸟嘌呤核苷酸交换因子(GEF)的ELMO1专用调节子,其N端ELMO抑制域(EID)和C端ELMO的分子内相互作用采用封闭的自抑制构象自动调节域(EAD)。在测定中,在用ELMO1 1-315 和ELMO1 315-727 片段转染的细胞中检测到PLA信号。此外,过表达的FE65是一种神经元衔接子,已被证明能破坏ELMO1分子间的相互作用,可显着降低两个ELMO1片段的PLA信号。在一起,我们的结果表明PLA可用于研究蛋白质分子内相互作用。


【背景】
细胞中蛋白质的活性可以通过多种机制调节,包括分子内相互作用(Miernyk和Thelen,2008)。已经采用了几种蛋白质-蛋白质相互作用测定法来研究分子内相互作用,例如免疫共沉淀和蛋白质互补测定法(PCA)。尽管被广泛采用,但是这些常规测定法存在已知的局限性。例如,共免疫沉淀法不是检测低亲和力相互作用的理想选择。即使PCA具有良好的敏感性,报告片段也可能会阻碍感兴趣的相互作用(Ohad et al。,2007; Miernyk和Thelen,2008)。邻近结扎测定法(PLA)是一项创新技术,允许对蛋白质相互作用进行原位检测。首先用来自不同物种的特异性一抗标记蛋白质靶标,然后识别与寡核苷酸(PLA探针)偶联的物种特异性二抗。寡核苷酸的邻近连接产生用于单链滚环扩增的模板。用荧光标记的互补寡核苷酸检测探针检测所得产物。由于涉及靶标特异性抗体和单链滚环扩增,因此PLA提供了卓越的特异性和敏感性。用荧光标记的互补寡核苷酸检测探针检测所得产物,并可以使用荧光显微镜对PLA信号进行可视化和量化(Weibrecht et al。,2010)。尽管这项技术已经使用了十多年,但尚未探索使用PLA研究蛋白质分子内相互作用的可能性。

吞噬和细胞运动1(ELMO1)是胞质分裂180(DOCK180)鸟嘌呤核苷酸交换因子(GEF)的二聚体ELMO1专用调节剂的调节亚基,用于激活小GTPase Rac1。ELMO1具有几个不同的域:Ras结合域(RBD),ELMO抑制域(EID),ELMO域,pleckstrin同源性(PH)域,ELMO自调节域(EAD)和PXXP基序(图1)。与许多其他GEF相似,ELMO1的活性受其EID和EAD之间的分子内相互作用调节,从而使ELMO1在基础状态下采取封闭的自抑制构象(Patel et al。,2010)(图1)。为了将ELMO1-DOCK180的复合物靶向质膜并刺激Rac1,需要解除ELMO1自抑制作用(Patel et al。,2011)。但是,减轻ELMO1自抑制的机制仍然难以捉摸。最近,我们报道了神经元衔接蛋白FE65与ELMO1 EAD相互作用,并破坏ELMO1 1-315 (由ELMO1氨基酸残基1-315组成)和ELMO1 315-727的相互作用(由ELMO1氨基酸残基315-727组成)片段,分别通过经典蛋白质结合测定和PLA进行鉴定,分别具有EID和EAD(Li et al。,2018)。因此,我们首次证明PLA可用于检查蛋白质分子内相互作用。

在这里,我们描述如何通过使用PLA研究ELMO1 EID和EAD的分子内相互作用。该方法还可以用于研究其他蛋白质分子内相互作用。

“”
图1.示意图显示了ELMO1的亚域和分子内相互作用。在基础阶段,EAD和EID在分子内相互作用(用双头箭头指示),形成自抑制构象。分子内相互作用的破坏减轻了对亚序列Rac1激活的自动抑制。数字表示ELMO1中的氨基酸残基编号。RBD:Ras结合域;EID:ELMO抑制域;ELMO:ELMO域;PH:Pleckstrin同源结构域;EAD:ELMO自动调节域;PXXP:PXXP主题。

关键字:自身抑制, 吞噬与细胞运动蛋白1, 荧光 , 分子内相互作用 , 邻位连接技术

材料和试剂

  1. Sterilin TM 标准90 mm培养皿(Thermo Scientific,目录号:101VIRR),在室温下保存
  2. 室温保存的12孔细胞培养板(SPL,目录号:30012)
  3. 盖玻片18毫米圈(Thermo-Menzel,目录号:CB00180RA1),在室温下保存
  4. 血细胞计数器(HBG,目录号:9010-01),在室温下保存
  5. 显微镜载玻片(Thermo Scientific,目录号:6776214),在室温下保存
  6. HEK293细胞(ATCC,目录号:CRL-1573),存储在液氮中
  7. 山羊抗ELMO1抗体(Santa Cruz Biotechnology,目录号:sc-21651),在4°C下储存
  8. 小鼠抗FLAG抗体(Sigma-Aldrich,目录号:F1804),储存在-20°C
  9. 兔抗β-微管蛋白抗体(Abcam,目录号:AB6046)
  10. 驴抗兔IgG(H + L)高度交叉吸附的二抗Alexa Fluor 488(Invitrogen,目录号:A21206),在4°C下存储
  11. 胎牛血清(HyClone,目录号:SV30160.03),在-20°C下保存
  12. pCMV-Tag2B向量(Aligent,目录号:211172),存储在-20°C
  13. T4 DNA连接酶(1 U /μl)(Thermo Scientific,目录号:15224025),储存在-20°C
  14. BamHI(10 U /μl)(Thermo Scientific,目录号:ER0055),储存在-20°C
  15. XhoI(10 U /μl)(Thermo Scientific,目录号:ER0691),储存在-20°C
  16. TIANprep微型质粒试剂盒(TIANGEN,目录号:DP103),在室温下保存
  17. QuikChange II定点诱变试剂盒(安捷伦,目录号:200523),储存在-20°C下
  18. Phusion高保真PCR试剂盒(Thermo Scientific,目录号:F553S),储存在-20°C
  19. UltraPure TM 琼脂糖(Invitrogen,目录号:16500500),在室温下保存
  20. TAE缓冲液(Tris-acetate-EDTA)(50x)(Thermo Scientific,目录号:B49),在室温下保存
  21. UltraPure TM 溴化乙锭,10 mg / ml(Invitrogen,目录号:15585011),在室温下保存
  22. 1 kb Plus DNA梯子(New England Biolabs,目录号:N3200),在-20°C下保存
  23. Luria Broth Base,粉末(Invitrogen,目录号:12795084),在室温下保存
  24. 透明指甲油
  25. LB琼脂,粉末(Invitrogen,目录号:22700041),在室温下保存
  26. 氨苄西林钠盐(Sigma-Aldrich,目录号:A0166),储存于4°C
  27. 硫酸卡那霉素(Sigma-Aldrich,目录号:60615),储存于4°C
  28. 聚-D-赖氨酸氢溴酸盐(Sigma-Aldrich,目录号:P7280),储存在-20°C
  29. DMEM(HyClone,目录号:SH30021.01),储存在4°C
  30. 胰蛋白酶-EDTA 0.05%(Gibco,目录号:25300062),在-20°C下保存
  31. Opti-MEM还原的血清培养基(Gibco,目录号:31985088),在4°C下储存
  32. X-tremeGENE TM 9 DNA转染试剂(Sigma-Aldrich,目录号:6365779001),在4°C下保存
  33. PBS(Gibco,目录号:10010023),在室温下保存
  34. 多聚甲醛(Sigma-Aldrich,目录号:P6148),储存于4°C
  35. Triton X-100(Anatrace,目录号:T1001500ML),在室温下保存
  36. Duolink ®原位红色入门套件鼠标/山羊(Sigma-Aldrich,目录号:DUO92103),在-20°C下存储
  37. 1个TAE缓冲区(请参阅食谱)
  38. 聚-D-赖氨酸溶液(请参阅食谱)
  39. 4%多聚甲醛(新鲜配制)(请参阅食谱)
  40. 1个连接缓冲液(请参阅配方)
  41. 1x放大红色(请参阅食谱)
  42. 洗涤缓冲液A(请参阅食谱)
  43. 洗涤缓冲液B(请参见食谱)

设备

  1. 水浴(Labnet,型号:W1106)
  2. 加热块(Labnet,型号:D1301-230V)
  3. 紫外线透射仪(Accuris,型号:E3000)
  4. T100 TM 热循环仪(Bio-rad)&nbsp;
  5. Heratherm TM 紧凑型微生物培养箱(37°C,Thermo Scientific)
  6. Forma TM 系列II 3110带水套的CO 2 培养箱(37°C,5%CO 2 ,Thermo Scientific)
  7. 配备QI2 高分辨率显微镜相机(尼康)的荧光显微镜(尼康,型号:Eclipse Ni-U )

软件

  1. NIS-Elements基础研究(Nikon)
  2. 棱镜( https://www.graphpad.com/scientific-software/prism/ )

程序

  1. ELMO1 1-315 的哺乳动物表达构建体的产生
    1. 使用QuikChange II定点诱变试剂盒(图2),进行定点诱变以在pcDNA3-N-Myc-Elmo1中的ELMO1残基315之后引入终止密码子。表1列出了使用的诱变引物。


      图2. ELMO1片段构建体的示意图。进行定点诱变,在pcDNA3-N-Myc-Elmo1中的ELMO1残基315之后产生终止密码子,从而生成ELMO1 1-315 < / sup>片段。通过PCR从pcDNA3-N-Myc-Elmo1中扩增出ELMO1 315-727 的cDNA,并将该产物插入pCMV-Tag2B中以表达N端FLAG标记的蛋白。星号表示突变位点。数字表示ELMO1中的氨基酸残基编号。

      表1。用于产生ELMO1片段构建体的引物。引物1和2用于定点诱变,将终止密码子引入pcDNA3-N-Myc-Elmo1。以pcDNA3-N-Myc-Elmo1为模板,使用引物3和4扩增ELMO1 315-727 片段。


    2. 将诱变产物转化为DH5α感受态 E。42°C通过热休克1分钟来培养大肠杆菌细胞。
    3. 用100μg/ ml氨苄青霉素将转化体接种在LB琼脂平板上。
    4. 将板在37°C的培养箱中孵育过夜。
    5. 用100μg/ ml氨苄青霉素在5 ml LB中接种分离的菌落,并在37°C下生长过夜。
    6. 使用TIANprep Mini Plasmid Kit分离质粒DNA。
    7. 进行DNA测序以确认终止密码子的引入。

  2. FLAG-ELMO1 315-727 的哺乳动物表达构建体的产生
    1. 以pcDNA3-N-Myc-Elmo1为模板,通过Phusion高保真PCR试剂盒进行聚合酶链反应(PCR),以获得编码为ELMO1 315-727 片段的cDNA。表1列出了使用的引物。
    2. 通过在1x TAE缓冲液中的含0.2μg/ ml溴化乙锭的1%琼脂糖凝胶解析PCR产物。
    3. 电泳后,使用紫外线透射仪观察PCR产物,并用剃须刀切除PCR产物(〜1.2 kb)。
    4. 使用MEGAquick-spin TM Plus总片段DNA纯化试剂盒从凝胶中纯化PCR产物。
    5. 在37°C水浴中,将纯化的ELMO1 315-727 PCR产物和pCMV-Tag2B(带有N端FLAG标签的载体)与BamHI和XhoI一起消化2小时。
    6. 使用MEGAquick-spin TM Plus总片段DNA纯化试剂盒纯化消化产物。
    7. 在16°C水浴中,用T4 DNA连接酶将消化的ELMO1 315-727 片段和线性化的pCMV-Tag2B载体连接过夜。
    8. 按照步骤A2-A6所述转化连接产物。选择应使用50μg/ ml卡那霉素。
    9. 进行DNA测序以确认将ELMO1 315-727 编码cDNA成功插入pCMV-Tag2B。

  3. 细胞制备和转染
    1. 在37℃培养箱中,在12孔细胞培养板中用5μg/ ml聚D-赖氨酸溶液涂覆18 mm盖玻片过夜。
      注意:使用前要消毒盖玻片。
    2. 用蒸馏水清洗盖玻片一次。
    3. 在预涂的18毫米盖玻片上以0.4 x 105个细胞的密度将HEK293细胞接种在补充有10%FBS的1 ml DMEM培养基中。
    4. 24小时后,将myc-ELMO1 1-315 和FLAG-ELMO1 315-727 的哺乳动物表达构建体与空载体(EV),FE65或FE65一起转染细胞。根据制造商的说明,在Opti-MEM还原的血清培养基中使用X-tremeGENE TM 9 DNA转染试剂对FE65 K48A / R51A(FE65m,ELMO1结合缺陷型FE65突变体)进行了稀释(表2)。 >
      表2转染组合用于研究FE65在ELMO1分子内相互作用中的作用。将HEK293细胞与EV,FE65或FE65m以及ELMO1 1-315 和ELMO1共转染 315-727 以1:1:1的比例构建。所有转染均接受相同量的DNA。”+”表示质粒的存在,而“-”表示质粒的不存在。


  4. 细胞固定和邻近连接测定
    注:洗涤缓冲液A,洗涤缓冲液B,PLA探针抗山羊MINUS,PLA探针抗小鼠PLUS,抗体稀释剂,连接酶,5x连接缓冲液,聚合酶,5x扩增红色和带有DAPI的Duolink原位安装介质是在Duolink ®原位红色入门工具包鼠标/山羊中提供。请参见图3了解PLA的工作流程。


    图3.时间线说明了使用Duolink ® 原位 的ELMO1片段的PLA工作流程。 em> 红色入门套件鼠标/山羊

    1. 转染24小时后,用PBS洗涤细胞一次,并在室温下用4%多聚甲醛固定细胞10分钟。
    2. 用PBS洗涤固定的细胞3次。
    3. 在室温下用0.1%Triton X-100 / PBS透化细胞15分钟。
    4. 在室温下用5%FBS / PBS封闭细胞30分钟。
    5. 稀释山羊抗ELMO1(识别myc-ELMO1 1-315 )(1:100),小鼠抗FLAG(识别FLAG-ELMO1 315-727 )(1 :1000)和含5%FBS / PBS的兔抗β-微管蛋白(1:200)抗体,并在室温下用80μl稀释的抗体混合物对细胞染色1小时。
      注意:所用的一抗浓度对于获得高质量的PLA图像至关重要,应进行优化。
    6. 用PBS洗涤细胞五次。
    7. 用5%FBS / PBS稀释Alexa Fluor 488偶联的驴抗兔IgG(1:1000)抗体,在室温下于黑暗中将细胞染色1小时。
    8. 用PBS洗涤细胞五次。
    9. 在室温下于黑暗中用洗涤缓冲液A洗涤细胞两次,每次5分钟。
    10. 向每个盖玻片中加入80μlPLA探针反应混合物,并在37°C的潮湿培养箱中孵育1小时。
      注:对于每个反应,将16μlPLA探针抗山羊MINUS(与寡核苷酸MINUS缀合的驴抗山羊二抗)与16μlPLA探针抗小鼠PLUS(与之共轭的驴抗小鼠二抗)混合寡核苷酸PLUS)加入48μl抗体稀释液中,并在室温下孵育20分钟,然后使用。
    11. 在室温下于黑暗中用洗涤缓冲液A洗涤盖玻片两次,持续5分钟。
    12. 将80μl连接反应混合物添加到每个盖玻片中,并在37°C湿润的培养箱中孵育30分钟。
      注意:对于每个反应,将2μl连接酶添加到78μl1x连接缓冲液中。
    13. 在室温下于黑暗中用洗涤缓冲液A洗涤盖玻片两次,持续5分钟。
    14. 向每个盖玻片中加入80μl扩增反应混合物,并在加湿的37°C恒温箱中孵育100分钟。
      注意:对于每个反应,将1μl聚合酶添加到79μl1x扩增红中。
    15. 在室温下于黑暗中,用洗涤缓冲液B清洗盖玻片两次,持续10分钟。
    16. 在室温下,在黑暗中,用0.01x洗涤缓冲液B洗涤盖玻片一次,持续1分钟。
    17. 使用最少的带有DAPI的Duolink Insitu 固定介质将盖玻片固定在显微镜载玻片上。
    18. 用透明的指甲油密封已安装的防护玻璃的边缘。
    19. 使用尼康荧光显微镜,使用60倍水浸镜头捕获细胞图像。
      注意:如果需要,将载玻片在黑暗中于4°C下保存。

数据分析

显微镜载玻片由带有60倍水浸镜头的尼康荧光显微镜成像。FITC和TRITC过滤器分别用于可视化β-微管蛋白和PLA信号。计算细胞内离散的荧光PLA点的数量。比较不同转染中每个细胞的PLA信号平均数,以研究两个片段之间相互作用的变化。计数来自至少60个细胞的PLA信号,并进行三个独立的实验。统计分析是通过Bonferroni事后检验使用单方差分析(ANOVA)进行的。

代表数据
最近,我们证明了PLA在细胞中ELMO1 1-315 和ELMO1 315-727 的相互作用,它们分别包含其EID和EAD。此外,在与野生型FE65共转染的细胞中,PLA信号的数量显着减少,但结合缺陷型突变体( ie ,FE65m)却没有降低(图4)。PLA的这些发现与经典蛋白质结合测定的结果一致(Li et al。,2018)。


图4.代表性图像和PLA信号量化。用myc-ELMO1 1-315 和FLAG-ELMO1 315-727 转染HEK293细胞以及空向量(EV),FE65或FE65m。转染24小时后,将细胞固定并进行PLA。在每次转染中计数来自至少60个细胞的PLA信号数,并进行三个独立的实验。条形图显示了不同转染中的相对PLA信号(相对于EV的倍数变化)。* P &lt; 0.001。误差棒是SEM。仅在转染FE65的细胞中观察到PLA信号的显着降低,而在EV或FE65m转染的细胞中未观察到。β微管蛋白和细胞核分别染色,作为形态学和核标记。比例尺= 10μm。(这项研究最初发表在《生物化学杂志》上。LiW,Tam KMV,Chan WWR,Koon AC,Ngo JCK,Chan HYE和Lau KF。神经元衔接子FE65 通过募集和激活ELMO1 刺激Rac1介导的神经突生长。J 。生物化学.2018; 293(20):7674-7688。©美国生物化学与分子生物学学会)

菜谱

  1. 1个TAE缓冲区
    用蒸馏水将50倍原液稀释至1倍
  2. 聚-D-赖氨酸溶液
    5毫克聚D-赖氨酸
    5毫升蒸馏水
    将5 mg聚D-赖氨酸溶解在5 ml蒸馏水中以得到储备溶液(200x)
    将储备溶液等分储存在-20°C
  3. 4%多聚甲醛(新鲜配制)
    0.4克低聚甲醛粉末
    10毫升PBS
    1. 将0.4 g低聚甲醛粉末加到10 ml PBS中
    2. 在70°C水浴中孵育直至粉末溶解
    3. 在室温下冷却溶液
    4. 用0.22μm注射器过滤器过滤溶液
  4. 1个连接缓冲液
    对于每个反应,用64μl蒸馏水稀释16μl5x连接缓冲液
  5. 1x放大红色
    对于每个反应,用64μl蒸馏水稀释16μl5x扩增红色
  6. 洗涤缓冲液A
    0.01 M Tris
    0.15 M氯化钠
    0.05%吐温20
    pH值7.4
  7. 洗涤缓冲液B
    0.2 M Tris
    0.1 M NaCl
    pH 7.5

致谢

这项工作得到了香港研究资助局,香港健康与医学研究基金会,中大直接资助计划和联合大学捐赠基金的资助。该协议改编自以前的工作。感谢Jean-FrançoisCôté教授(蒙特利尔临床研究所)提供pcDNA3-N-Myc-Elmo1的哺乳动物表达构建体。

利益争夺

作者声明,他们与本文的内容没有利益冲突。

参考文献

  1. Li W.,Tam,KMV,Chan,WWR,Koon,AC,Ngo,JCK,Chan,HYE和Lau,KF(2018)。神经元适配器FE65通过募集并激活ELMO1来刺激Rac1介导的神经突生长。 J Biol Chem 293(20):7674-7688。
  2. Miernyk,JA和Thelen,JJ(2008)。发现蛋白质间相互作用的生化方法。 植物J 53(4):597-609。
  3. Ohad,N.,Shichrur,K.和Yalovsky,S.(2007)。通过双分子荧光互补分析植物中蛋白质与蛋白质的相互作用。 植物生理学杂志145(4):1090-1099。
  4. Patel,M.,Margaron,Y.,Fradet,N.,Yang,Q.,Wilkes,B.,Bouvier,M.,Hofmann,K.和Cote,JF(2010)。 ELMO蛋白中一种进化上保守的自抑制分子开关调节Rac信号。 Curr生物学 20(22):2021-2027。
  5. Patel,M.,Pelletier,A.和Cote,JF(2011)。对ELMO法规开放:DOCK180 / ELMO复合体控制Rac信号的新见解。 小型GTPases 2(5):268-275。
  6. I. Weibrecht,KJ Leuchowius,CM Clausson,Conze,T.,Jarvius,M.,Howell,WM,Kamali-Moghaddam,M.和Soderberg,O.(2010)。邻近结扎分析:蛋白质组学工具箱中的最新功能。 Expert Rev蛋白质组学 7(3):401-409。
登录/注册账号可免费阅读全文
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Chan, W. W. R., Chau, D. L. D., Li, W. and Lau, K. (2019). Proximity Ligation Assay for the Investigation of the Intramolecular Interaction of ELMO1. Bio-protocol 9(23): e3449. DOI: 10.21769/BioProtoc.3449.
  2. Li, W., Tam, K. M. V., Chan, W. W. R., Koon, A. C., Ngo, J. C. K., Chan, H. Y. E. and Lau, K. F. (2018). Neuronal adaptor FE65 stimulates Rac1-mediated neurite outgrowth by recruiting and activating ELMO1. J Biol Chem 293(20): 7674-7688. 
提问与回复
提交问题/评论即表示您同意遵守我们的服务条款。如果您发现恶意或不符合我们的条款的言论,请联系我们:eb@bio-protocol.org。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。