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Apr 2020

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Live Cell FRET Analysis of the Conformational Changes of Human P-glycoprotein
人P-糖蛋白构象变化的活细胞FRET分析   

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Abstract

The molecular mechanisms of P-glycoprotein (P-gp; also known as MDR1 or ABCB1) have been mainly investigated using artificial membranes such as lipid-detergent mixed micelles, artificial lipid bilayers, and membrane vesicles derived from cultured cells. Although these in vitro experiments help illustrate details about the molecular mechanisms of P-gp, they do not reflect physiological membrane environments in terms of lateral pressure, curvature, constituent lipid species, etc. The protocol presented here includes a detailed guide for analyzing the conformational change of human P-gp in living HEK293 cells by using intramolecular fluorescence resonance energy transfer (FRET), in which excitation of the donor fluorophore is transferred to the acceptor without emission of a photon when two fluorescent proteins are in close proximity. Combining FRET analysis with membrane permeabilization, the contribution of small molecules such as nucleotides to the conformational change can be evaluated in living cells.

Keywords: Live cell analysis (活细胞分析), Intramolecular FRET (分子内荧光共振能量转移), Conformational change (构型改变), P-glycoprotein (p-糖蛋白), ABC transporter (ABC转运蛋白), Membrane protein (膜蛋白质)

Background

P-glycoprotein (P-gp) is an ATP-driven multidrug transporter that extrudes various hydrophobic toxic compounds to the extracellular space. P-gp consists of two transmembrane domains (TMDs) that form the substrate translocation pathway and two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP. At least two P-gp states are required for the transport. In the inward-facing (pre-drug transport) conformation, the two NBDs are separated, and the two TMDs are open to the intracellular side; in the outward-facing (post-drug transport) conformation, the NBDs are dimerized, and the TMDs are slightly open to the extracellular side (Kodan et al., 2020). Since the discovery of P-gp (Juliano and Ling, 1976; Chen et al., 1986; Ueda et al., 1986), numerous studies have been conducted to clarify its transport mechanism. Most have utilized artificial membrane environments such as lipid-detergent mixed micelles (Kodan et al., 2014 and 2019; Verhalen et al., 2017), artificial lipid bilayers (Verhalen et al., 2012; Moeller et al., 2015; Zoghbi et al., 2017; Dastvan et al., 2019), or membrane vesicles derived from cells overexpressing P-gp (Liu and Sharom, 1996; Qu and Sharom, 2001). However, these conditions do not reflect physiological membrane environments in terms of lateral pressure, curvature, or constituent lipid species. Because these environmental factors can affect the function of P-gp, the detailed transport mechanism should be investigated in living cells. Accordingly, a conformation-sensitive monoclonal antibody, UIC2, has been used (Bársony et al., 2016). However, because UIC2 binds to the extracellular loops of the inward-facing structure of P-gp, the conformational change of the intracellular side has not been revealed. The distances between the C termini of NBD1 and NBD2 are estimated to be about 30 and 11 Å in the inward-facing and outward-facing structures, respectively (Futamata et al., 2020) and this difference in distance is assumed to be one of the largest between the inward-facing and outward-facing structures. Therefore, we monitored the distance of the two NBDs by FRET analysis. When the donor and acceptor were attached to different domains of the macromolecule, strong FRET occurred when the two domains were in close proximity. The big advantage of FRET analysis is that it can be performed in living cells in real time, which is not true of antibody-based analyses. In this study, two fluorescent proteins, monomeric (m)Cerulean and monomeric (m)Venus, were fused to the N- and C-terminal regions of NBDs, respectively. A change in distance between the two NBDs was evaluated by investigating the sensitized-emission of mVenus (acceptor) elicited during the excitation of mCerulean (donor) in living cells in real time. While the protocol described focuses on human P-gp in living HEK293 cells, it is also applicable to other membrane proteins.


Materials and Reagents

  1. 35 mm glass-based dish (IWAKI, catalog number: 3971-035)

  2. 100 mm cell culture dish (Falcon, catalog number: 353003)

  3. 1.5 ml tube (Watson, catalog number: 131-815C)

  4. 15 ml tube (Corning, catalog number: CLS430791)

  5. 0.22 μm filter (Millipore, catalog number: SLGP033RS)

  6. Mammalian expression vector that encodes FRET probe (see the beginning of the section Procedure below)

  7. HEK293 cells (ACTT® CRL-1573TM)

  8. Poly-L-lysine (PLL) solution 0.01% (Sigma-Aldrich, catalog number: P4707-50ML)

  9. DMEM (Nacalai Tesque, catalog number: 08458-16)

  10. 0.5% Trypsin-EDTA (10×) (Gibco, catalog number: 15400-054)

  11. Fetal Bovine Serum (Gibco, catalog number: 10270-106)

  12. Lipofectamine LTX with PLUS Reagent (Thermo Fisher Scientific, catalog number: 15338100)

  13. Opti-MEM (1×) (Gibco, catalog number: 2151680)

  14. FluoroBrite DMEM (Gibco, catalog number: A1896701)

  15. Sodium pyruvate (100 mM) (Gibco, catalog number: 11360-070)

  16. GlutaMAX (100×) (Gibco, catalog number: 35050061)

  17. Verapamil chloride (Wako, catalog number: 228-00783)

  18. PEI MAX (MW = 40,000) (Polysciences, catalog number: 24765-1)

  19. Adenosine 5’-triphosphate (ATP) disodium salt (Oriental Yeast, catalog number: 45142000)

  20. Streptolysin O (SLO) (BioAcademia, catalog number: 01-531)

  21. DAPI (Sigma-Aldrich, catalog number: D9542-1MG)

  22. NaCl (Nacalai Tesque, catalog number: 31320-05)

  23. Na2HPO4·12H2O (Nacalai Tesque, catalog number: 31723-35)

  24. KCl (Nacalai Tesque, catalog number: 28514-75)

  25. KH2PO4 (Nacalai Tesque, catalog number: 28721-55)

  26. HEPES (Nacalai Tesque, catalog number: 17514-15)

  27. KOH (Nacalai Tesque, catalog number: 28616-45)

  28. CH3COOK (Wako catalog number: 160-03175)

  29. MgCl2·6H2O (Nacalai Tesque, catalog number: 20909-55)

  30. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D2650)

  31. NaOH (Nacalai Tesque, catalog number: 31511-05)

  32. PLL solution (see Recipes)

  33. 10× PBS(-) (see Recipes)

  34. 1× PBS(-) (see Recipes)

  35. 10× transport buffer (see Recipes)

  36. 1× transport buffer (see Recipes)

  37. 0.05% Trypsin-EDTA (see Recipes)

  38. 100 mM ATP stock solution (see Recipes)

  39. 50 mM verapamil stock solution (see Recipes)

  40. 1 mg/ml DAPI stock solution (see Recipes)

  41. 100 mM NaN3 stock solution (see Recipes)

  42. 1 M MgCl2 stock solution (see Recipes)

  43. 1 mg/ml PEI-MAX (see Recipes)

Equipment

  1. Autoclave (TOMY, model: LSX-500)

  2. Vortex mixer (Scientific Industries, model: VORTEX-GENIE 2)

  3. Temperature bath (TAITEC, model: SDminiN)

  4. CO2 incubator (Thermo Fisher Scientific, model: Forma 310 Direct Heat CO2 incubator)

  5. Centrifuge (TOMY, model: LC-200, rotor: TS-7LB)

  6. Cell counter (DeNovix, model: Cell Drop BF)

  7. Confocal laser scanning microscope (Carl-Zeiss, model: LSM700) operated by Zen 2012 and equipped with an objective lens (Plan-Apochromato ×63/1.4 NA oil immersion), an incubation chamber, temperature module S, and CO2 module S.

Software

  1. ImageJ-based Fiji (version 1.52p) (https://imagej.net/Fiji) (Schindelin et al., 2012)

  2. FRET and Colocalization Analyzer

    (https://imagej.nih.gov/ij/plugins/fret-analyzer/fret-analyzer.htm) (Hachet-Haas et al., 2006)

Procedure

For this protocol, we used human P-gp FRET probes (Futamata et al., 2020). Schematic representations of the fluorescent protein-tagged P-gps are shown in Figure 1. Human P-gp FRET probes are available from the corresponding author, Kazumitsu Ueda (ueda.kazumitsu.7w@kyoto-u.ac.jp).



Figure 1. Schematic representations of fluorescent protein-tagged P-gps. We generated a FRET construct, human P-gp–FRET, in which mCerulean (donor) was inserted after NBD1 and mVenus (acceptor) was fused after NBD2. Another FRET construct, P-gp–VsCn, in which mVenus and mCerulean were fused tandemly after NBD2, was predicted to show a high level of FRET despite the conformation. P-gp–mCerulean, in which mCerulean was inserted after NBD1, and P-gp–mVenus, in which mVenus was fused after NBD2, were constructed as negative controls. TMD, trans-membrane domain. NBD, nucleotide-binding domain, a.a., amino acid.

Note: HEK293 cells are maintained in DMEM supplemented with 10% FBS (10%FBS-DMEM) in a 100 mm cell culture dish at 37 °C under 5% CO2. For incubation, a CO2 incubator was used.


  1. Intact cells

    1. Transient expression of fluorescent protein-tagged P-gps

      1. Day 1 Add 100 μl PLL solution to the 35 mm glass-based dish and incubate for 30 min at room temperature.

        Note: PLL solution was used to promote cell adhesion to the dish.

      2. Wash with 1.5 ml 1× PBS(-) twice.

      3. Prepare HEK293 cells cultured in the 100 mm cell culture dish and discard the culture medium.

      4. Wash with 1.5 ml 1× PBS(-) twice

      5. Add 1 ml 0.05% EDTA-Trypsin and incubate for 2 min at 37 °C under 5% CO2.

      6. Add 9 ml 10% FBS-DMEM and transfer to a 15 ml tube.

      7. Centrifuge at 1,100 × g for 2 min at room temperature.

      8. Discard the supernatant and add 10 ml 10% FBS-DMEM.

      9. Count the number of cells using CellDrop following the manufacturer’s protocol.

      10. Dilute the cells to 1.5 × 105 cells/ml.

      11. Seed HEK293 cells to the glass-based dish at 3.0 × 105 cells/well and incubate for 24 h at 37 °C under 5% CO2.

      12. Day 2 Prepare the DNA-lipid complex.

        Note: The minimum plasmid DNA set is P-gp–FRET, two negative controls (P-gp–mCerulean and P-gp–mVenus), and one positive control (P-gp–VsCn).

        1. Mix 2.5 μg plasmid DNA and 2.5 μl PLUS Reagent in 500 μl Opti-MEM and mix with the vortex mixer.

        2. Incubate for 5 min at room temperature.

        3. Add 6.25 μl Lipofectamine LTX and mix with the vortex mixer.

        4. Incubate for 30 min at room temperature.

      13. Replace the medium with 500 μl DNA-lipid complex and 1.5 ml 10% FBS-DMEM and incubate for 23 h at 37 °C under 5% CO2.

    2. Image acquisition

      1. Day 3 Wash with 2 ml FluoroBrite DMEM twice.

      2. Replace the medium with 1 ml FluoroBrite DMEM supplemented with 10% FBS, 1 mM sodium pyruvate, and 1× GlutaMAX and incubate for 1 h.

      3. Transfer the dish to the incubation chamber equipped to the microscope.

      4. Transfer 500 μl medium to a 1.5 ml tube and mix with 2 μl verapamil chloride stock solution (final 100 μM).

        Notes:

        1. Do not add verapamil chloride stock solution to the glass-based dish directly because HEK293 cells can be peeled off by pipetting.

        2. Verapamil is a P-gp substrate.

      5. Return the medium to the glass-based dish and further incubate for 5 min.

      6. Acquire images of the mVenus, mCerulean, and FRET signals according to the parameters below and save as .lsm or .czi format.

        mVenus is excited at 488 nm, and the fluorescence emission is collected using a band pass filter (521-600 nm) (Figure 2). mCerulean is excited at 445 nm, and the fluorescence emission is collected using a short-pass filter (490 nm). For the FRET signal image, the laser is set to 445 nm, and the sensitized emission is collected using a band pass filter (521-600 nm) (Figure 3).

        Note: Representative raw images of every channel of all four plasmids are shown in Figure 4.



    Figure 2. Zen software settings for mVenus imaging



    Figure 3. Zen software settings for mCerulean imaging and FRET signal imaging



    Figure 4. Representative raw images of every channel for all four plasmids. Scale bars = 10 μm.


  2. Semi-intact cells

    Note: Membrane permeabilization is suitable for manipulating the concentration of small molecules such as nucleotides.

    1. Transient expression of the fluorescent protein tagged P-gp constructs.

      1. Day 1 Seed the HEK293 cells as described in Steps A1a-A1k.

      2. Day 2 Prepare the DNA-lipid complex as below.

        Note: Do not use Lipofectamine LTX when performing membrane permeabilization because it inhibits the pore formation by SLO.

        1. Mix 2 μg plasmid DNA and 98 μl Opti-MEM in a 1.5 ml tube and mix with the vortex mixer.

        2. Mix 10 μl 1 mg/ml PEI-MAX and 90 μl Opti-MEM in another 1.5 ml tube and mix with the vortex mixer.

        3. Mix the DNA and PEI-MAX and mix with the vortex mixer.

        4. Incubate at room temperature for 30 min.

      3. Replace the medium with 200 μl DNA-PEI complex and 1.8 ml 10% FBS-DMEM and incubate for 24 h at 37 °C under 5% CO2.

    2. Membrane permeabilization and image acquisition

      Note: We recommend performing this step using one or two samples at a time in order to avoid the cells permeabilizing for a long time.

      1. Day 3 Wash with 2 ml ice-cold 1× PBS(-).

      2. Add 1 ml serum-free DMEM containing 50 ng/μl SLO and incubate for 5 min on ice.

        Notes:

        1. Dilute SLO just before use.

        2. SLO is a streptococcal toxin which forms 25-30 nm aqueous pores within the plasma membrane, which allows the free passage of ions and small molecules.

      3. Wash with 2 ml ice-cold 1× PBS(-) three times.

      4. Add 1 ml 1× transport buffer pre-heated at 37 °C supplemented with 2 mM MgCl2, 5 mM ATP disodium salt, and 2 μg/ml DAPI. For ATP depletion, add 1× transport buffer supplemented with 2 mM MgCl2, 2 μg/ml DAPI, and 10 mM NaN3.

      5. Incubate for 10 min at 37 °C under 5% CO2.

      6. Wash twice with 2 ml 1× transport buffer pre-heated at 37 °C.

      7. Add 1 ml of the pre-heated 1× transport buffer supplemented with 2 mM MgCl2, 5 mM ATP disodium salt, and 100 μM verapamil chloride.

      8. Transfer the dish to the incubation chamber equipped to the microscope and incubate for 5 min at 37 °C under 5% CO2.

      9. Acquire images of the mVenus, mCerulean, and FRET signals of DAPI-positive cells according to the parameters described in Step A2f.

Data analysis

The FRET efficiency (proximity ratio) was calculated using Equation 1.


where IDA is the FRET signal or the sensitized emission of the acceptor during donor excitation (excitation 445 nm/emission 521-600 nm), IDD is the donor fluorescence during donor excitation (excitation 445 nm/emission 490 nm), and IAA is the acceptor fluorescence during acceptor excitation (excitation 488 nm/emission 521-600 nm). a and d are the bleed-through of the acceptor and donor, respectively.


Segmentation of the plasma membrane and calculation of the proximity ratio:

  1. Open the .lsm or .czi format images with ImageJ and convert to .tiff format.

  2. Split the image to each channel (Image menu > Color > Split Channels).

  3. Calculate the donor and acceptor bleed-through from the images expressing P-gp–mCerulean and P-gp–mVenus, respectively, using “FRET and Colocalization Analyzer”.

    1. Download “FRET and Colocalization Analyzer” from https://imagej.nih.gov/ij/plugins/fret-analyzer/fret-analyzer.htm to the plugins folder.

    2. Restart ImageJ to add the "Fret Analyzer" command to the Plugins menu.

    3. Open “FRET Analyzer” from the Plugins menu.

    4. For the donor bleed-through evaluation, input the number of controls (up to 10 controls).

    5. Select the donor channel image and the FRET signal image of the first control.

    6. Click the “Get” button.

    7. Perform (c) and (d) for all controls and acquire the mean of the donor bleed-through.

    8. Calculate the acceptor bleed-through the same way as the donor bleed-through.

  4. If necessary, crop the images so that only a single cell is visible.

  5. Save the images of the mCerulean, mVenus, FRET signals to separate folders.

  6. Subtract the donor and acceptor bleed-through from the FRET signal image and create a corrected FRET (cFRET) image. This procedure is automatically performed by the home-made ImageJ macro described below.

    Notes:

    1. To use the following ImageJ macro, save the macro text as .txt format in the Macro subfolder in the ImageJ directory. Install the macro (Plugins > Macros > Install). Run the macro (Plugins > Macros > macro name).

    2. Before executing this ImageJ macro, uncheck “Scale when converting” (Edit menu > Options > Conversions… ).


    setBatchMode(true); //Batchmode ON

    // setBatchMode(false); //Batchmode OFF


    dir1 = getDirectory("Select mCerulean folder");

    //Select the folder in which the mCerulean images are saved

    dir2 = getDirectory("Select mVenus folder");

    //Select the folder in which the mVenus images are saved

    dir3 = getDirectory("Select FRET signal folder");

    //Select the folder in which the FRET images are saved

    dir4 = getDirectory("Select cFRET folder");

    //Select the folder in which the cFRET images are to be saved


    list1 = getFileList(dir1);

    list2 = getFileList(dir2);

    list3 = getFileList(dir3);


    A = getNumber("Enter donor BT",0);

    B = getNumber("Enter acceptor BT",0);


    for (i = 0; i < list1.length; i++){

    open(dir1 + list1[i]);

    c = getTitle();

    run("32-bit");

    //convert 8 bit image to 32 bit image

    run("Duplicate...", "title=mc");

    run("Select All");

    setColor(A);

    fill();

    imageCalculator("Multiply", "mc", c);

    open(dir2 + list2[i]);

    v = getTitle();

    run("32-bit");

    run("Duplicate...", "title=mv");

    run("Select All");

    setColor(B);

    fill();

    imageCalculator("Multiply", "mv", v);

    open(dir3 + list3[i]);

    f = getTitle();

    run("32-bit");

    imageCalculator("Subtract", f, "mc");

    imageCalculator("Subtract", f, "mv");

    run("8-bit");

    retstr = split(f,"_");

    Save_name = dir4 + retstr[0] + "_cF";

    saveAs("Tiff", Save_name);

    run("Close All");

    }

  7. Segment the plasma membrane and evaluate the proximity ratio on the plasma membrane. This procedure is automatically performed by the home-made ImageJ macro described below.

    Notes:

    1. Representative images of the segmented plasma membrane are shown in Figure 5.

    2. Representative results of quantification of the proximity ratio are shown in Figure 6.



    Figure 5. Representative images of the original membrane and segmented plasma membrane. Scale bars = 5 μm.



    Figure 6. Representative results of the quantification of FRET efficiency. Representative data from cells expressing one type of fluorescent protein-tagged P-gp in the absence or presence of the transport substrate verapamil. Each value was obtained from a single cell. Average ratio indicates the proximity ratio.


    setBatchMode(true); //Batchmode ON

    //setBatchMode(false); //Batchmode OFF


    dir1 = getDirectory("Select mVenus folder");

    //Select the folder in which the mVenus images are saved

    dir2 = getDirectory("Select mCerulean folder");

    //Select the folder in which the mCerulean images are saved

    dir3 = getDirectory("Select cFRET folder");

    //Select the folder in which the cFRET images are saved

    dir4 = getDirectory("Select ROI folder");

    //Select the folder in which the ROI information is to be saved

    dir5 = getDirectory("Select PM folder");

    //Select the folder in which the segmented plasma membranes are to be saved

    dir6 = getDirectory("Select ratio image folder");

    //Select the folder in which the ratio images are to be saved


    list1 = getFileList(dir1);

    list2 = getFileList(dir2);

    list3 = getFileList(dir3);


    //Segmentation of the plasma membrane from a mVenus image


    for (i = 0; i < list1.length; i++){

    open(dir1 + list1[i]);

    Ori_name = getTitle(); //Get name of the image


    run("Duplicate...", Ori_name);

    //For segmentation of the plasma membrane, duplicate the original image

    run("Subtract Background...", "rolling=10");

    //Subtract background. ***Rolling ball radius should be optimized to avoid removing any objects***

    run("Enhance Contrast...", "saturated=0.4 normalize");

    run("Auto Local Threshold", "method=Phansalkar radius=15 parameter_1=0 parameter_2=0 white");

    //***To choose the most suitable method when making a binary image, try all methods (Image > Adjust > Auto Local Threshold > Method “Try all”)***

    run("Options...", "iterations=1 count=3 black do=Open");

    //Smoothen the plasma membrane by "Open (Dilute after Erode)"

    run("Analyze Particles...", "size=1-100 pixel include add");

    setForegroundColor(0,0,0);

    roiManager("deselect");

    roiManager("Fill");

    // Delete objects less than 100 pixels

    setOption("BlackBackground", true);

    run("Erode");

    roiManager("Delete");

    run("Analyze Particles...", "size=50-Infinity pixel circularity=0.00-0.3 add");

    // Select objects with size more than 50 pixels and circularity less than 0.3

    run("Select All");

    //Select all ROI

    roiManager("Combine");

    //Combine all ROI

    setBackgroundColor(0, 0, 0);

    run("Clear Outside");

    //Delete outside of the ROI in mVenus image

    Save_name = dir5 + "mem_" + Ori_name;

    saveAs("Tiff", Save_name);

    close();


    selectImage(1);

    setOption("Show All",true);

    roiManager("Measure");

    Save_name = dir4 + "ROI_" + Ori_name + ".zip";

    roiManager("Save", Save_name); //Save ROI information

    close();

    roiManager("Delete");

    }


    list4 = getFileList(dir4);


    //Evaluation of the proximity ratio from mCerulean and cFRET images


    for (i = 0; i < list2.length; i++){

    open(dir2 + list2[i]);

    //Open mCerulean image

    Ori_name = getTitle();

    open(dir3 + list3[i]);

    //Open cFRET image

    run("Images to Stack", "name=Stack title=[] use keep");

    setSlice(2);

    run("Add Slice");

    setSlice(1); //Select mCerulean image

    roiManager("Open", dir4 + list4[i]);

    run("Select All");

    roiManager("Combine");

    setBackgroundColor(0, 0, 0);

    run("Clear Outside","Slice");

    //Delete outside of the ROI in the mCerulean image

    for(ii = 0; ii < getWidth(); ii ++){

    for(iii = 0; iii < getHeight(); iii ++){

    val = getPixel(ii,iii);

    if(val > 0){

    setSlice(2);

    val2 = getPixel(ii,iii);

    ratio = val2 / val;

    //Calculate the proximity ratio (pixel value of cFRET image / pixel value of mCerulean image)

    sum += ratio;

    count += 1;

    setSlice(3);

    ratiop = ratio * 100;

    setPixel(ii, iii, ratiop);

    setSlice(1);

    }

    }

    }

    updateDisplay();

    Save_name = dir6 + "ratio_" + Ori_name;

    saveAs("Tiff", Save_name);

    ratiom = sum / count;

    //Divide total proximity ratio by the counted pixel number

    ar = newArray(sum, count, ratiom);

    Array.print(ar);

    //show sum of the proximity ratio, pixel numbers of the plasma membrane, and average of the proximity ratio on the Log window


    sum = 0;

    count = 0;

    run("Close All");

    roiManager("Delete");

    }

Recipes

  1. PLL solution

    0.01% PLL solution             1 ml

    1× PBS(-)                            29 ml

    Store at 4 °C

  2. 10× PBS(-)

    Note: (-) means without magnesium or calcium.

    NaCl                                         80 g  

    Na2HPO4·12H2O                    29 g

    KCl                                       2 g

    KH2PO4                                    2 g

    Fill up to 1 L with milliQ. Store at room temperature

  3. 1× PBS(-)

    10× PBS(-)     100 ml

    MilliQ             900 ml

    Sterilize by autoclave

    Store at 4 °C

  4. 10× transport buffer

    1 M Hepes-KOH (pH 7.4)   125 ml

    CH3COOK                             56.44 g

    MgCl2·6H2O                         2.54 g

    Fill up to 500 ml with MilliQ and sterilize with 0.22 μm filter

    Store at room temperature.

  5. 1× transport buffer

    10× transport buffer                    100 ml

    MilliQ              900 ml

    Store at 4 °C

  6. 0.05% Trypsin-EDTA

    0.5% Trypsin-EDTA (10×)  10 ml

    1× PBS(-)                                90ml

    Store at 4 °C

  7. 100 mM ATP stock solution

    ATP disodium salt 551.1 mg

    Fill up to 10 ml with MilliQ

    Adjust pH to 7 with NaOH

    Sterilize with 0.22 μm filter

    Store at -30 °C

  8. 50 mM verapamil stock solution

    Note: Prepare before use.

    Verapamil chloride                 24.6 mg

    DMSO                                        1 ml

    Sterilize with 0.22 μm filter

    Store at -30 °C

  9. 1 mg/ml DAPI stock solution

    DAPI                      1 mg

    DMSO                       1 ml

    Sterilize with 0.22 μm filter

    Store at -30 °C

  10. 100 mM NaN3 stock solution

    NaN3                   6.501 mg

    MilliQ                  1 ml

    Sterilize with 0.22 μm filter

    Store at -30 °C

  11. 1 M MgCl2 stock solution

    MgCl2·6H2O 20.33 g

    Fill up to 100 ml with MilliQ

    Sterilize by autoclave

    Store at 4 °C

  12. 1 mg/ml PEI-MAX

    PEI-MAX                 10 mg

    1× PBS(-)                10 ml

    Sterilize with 0.22 μm filter

    Store at 4 °C

Acknowledgments

This work was supported by JSPS KAKENHI Grant Numbers 18H05269.

Competing interests

The authors declare no competing financial interests.

References

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简介

[摘要] P-糖蛋白(P-gp;也称为MDR1或ABCB1)的分子机制已主要使用人造膜进行研究,例如脂质去污剂混合胶束,人造脂质双层和源自培养细胞的膜囊泡。尽管这些体外实验有助于阐明有关P-gp分子机制的细节,但它们在侧向压力,曲率,脂质成分等方面并未反映出生理膜环境。 此处提供的协议包括一个详细的指南,该指南用于通过使用分子内荧光共振能量转移(FRET)分析活HEK293细胞中人P-gp的构象变化,其中供体荧光团的激发被转移到受体上而不发射光子当两个荧光蛋白非常接近时。将FRET分析与膜通透性相结合,可以在活细胞中评估小分子(如核苷酸)对构象变化的贡献。

[背景] P-糖蛋白(P-gp)的是ATP驱动药转运该压出各种疏水有毒化合物到细胞外空间。P-gp由形成底物转运途径的两个跨膜结构域(TMD)和结合并水解ATP的两个核苷酸结合结构域(NBD)组成。传输至少需要两个P-gp状态。在向内(药物转运前)构型中,两个NBD分开,两个TMD向细胞内侧开放;在向外(药物转运)构象中,NBD是二聚体的,而TMD在细胞外侧略微开放(Kodan et al。,2020 )。自从发现P-gp (Juliano和Ling,1976; Chen等,1986; Ueda等,1986 )以来,已经进行了大量研究以阐明其转运机制。大多数使用了人工膜环境,例如脂质去污剂混合胶束(Kodan等人,2014和2019; Verhalen等人,2017 ),人工脂质双层(Verhalen等人,2012; Moeller等人,2015; Zoghbi等人,2017; Dastvan等人,2019 ),或衍生自过量表达P-gp的细胞的膜囊泡(Liu和Sharom,1996; Qu和Sharom,2001 )。但是,这些条件在侧向压力,曲率或脂质成分方面均不能反映生理膜环境。由于这些环境因素会影响P-gp的功能,因此应在活细胞中研究详细的转运机制。因此,已经使用了构象敏感的单克隆抗体UIC2 (Bársony等,2016 )。但是,由于UIC2与P-gp的向内结构的胞外环结合,因此尚未揭示胞内侧的构象变化。Ť他NBD1和NBD2的C-末端之间的距离估计为约在面向内的和向外的结构,分别为30和11埃(二俣等人,2020 )和在距离该差值被假定为一个在向内和向外结构之间最大的一个。因此,我们通过FRET分析监测了两个NBD的距离。当供体和受体连接到大分子的不同域时,当两个域非常接近时,会发生强FRET。FRET分析的最大优势在于它可以在活细胞中实时执行,而基于抗体的分析却并非如此。在这项研究中,两个荧光蛋白,单体(m)Cerulean和单体(m)Venus,分别融合到NBD的N和C端区域。通过实时研究活细胞中mCerulean(供体)激发过程中引起的mVenus(受体)的敏化发射,评估了两个NBD之间的距离变化。尽管所描述的协议专注于活HEK293细胞中的人P-gp,但它也适用于其他膜蛋白。

关键字:活细胞分析, 分子内荧光共振能量转移, 构型改变, p-糖蛋白, ABC转运蛋白, 膜蛋白质

材料和试剂
1. 35毫米玻璃皿(IWAKI,目录号:3971-035)      
2. 100毫米细胞培养皿(Falcon,目录号:353003)      
3. 1.5米升吨UBE(沃森,目录号:131-815C)      
4. 15米升管中(Corning,目录号:CLS430791)      
5. 0.22μm过滤器(Millipore,目录号:SLGP033RS)      
6.编码FRET探针的哺乳动物表达载体(请参见下面过程部分的开头)      
7. HEK293细胞(ACTT ® CRL-1573 TM )      
8.聚-L-赖氨酸(PLL)溶液0.01%(Sigma-Aldrich,目录号:P4707-50ML)      
9. DMEM(Ñ acalai Ť式的,目录号:08458-16)      
10. 0.5%胰蛋白酶-EDTA(10 ×)(Gibco,目录号:15400-054)   
11.胎牛血清(Gibco,目录号:10270-106)   
12.带有PLUS试剂的Lipofectamine LTX(Thermo Fisher Scientific,目录号:15338100)   
13. Opti-MEM(1 × )(Gibco,目录号:2151680)   
14. FluoroBrite DMEM(Gibco,目录号:A1896701)   
15.丙酮酸钠(100 mM)(Gibco,目录号:11360-070)   
16. GlutaMAX(100 × )(Gibco,目录号:35050061)   
17.氯化维拉帕米(Wako,目录号:228-00783)   
18. PEI MAX(MW = 40,000)(Polysciences,目录号:24765-1)   
19. 5'-t磷酸腺苷(ATP)二钠盐(东方Y东,目录号:45142000)   
20.链球菌溶血素O(SLO)(生物科学院,目录号:01-531)   
21. DAPI(Sigma-Aldrich,目录号:D9542-1MG)   
22. NaCl(N acalai T esque,目录号:31320-05)   
23. Na 2 HPO 4 · 12H 2 O(Nacalai Tesque ,目录号:31723-35)   
24. KCl(Nacalai Tesque ,目录号:28514-75)   
25. KH 2 PO 4 (Nacalai Tesque ,目录号:28721-55)   
26. HEPES(Nacalai Tesque ,目录号:17514-15)   
27. KOH(Nacalai Tesque ,目录号:28616-45)   
28. CH 3 COOK(和光目录号:160-03175)   
29.氯化镁2    · 6H 2 O(Nacalai Tesque ,目录号:20909-55)
30.二甲基亚砜(DMSO)(西格玛奥德里奇,目录号:D2650)   
31. NaOH(Nacalai Tesque ,目录号:31511-05)   
32. PLL解决方案(请参阅食谱)                 
33. 10 × PBS(-)(请参阅食谱)   
34. 1 × PBS(-)(请参阅食谱)   
35. 10 ×传输缓冲区(请参阅食谱)   
36. 1 ×传输缓冲区(请参阅食谱)   
37. 0.05%胰蛋白酶-EDTA(请参阅食谱)   
38. 100 mM ATP储备溶液(请参阅食谱)   
39. 50 mM维拉帕米储备溶液(请参阅食谱)   
40. 1 mg / m l DAPI储备溶液(请参阅食谱)   
41. 100 mM NaN 3储备溶液(请参阅食谱)   
42. 1 M MgCl 2储备溶液(请参阅配方)   
43. 1 mg / m l PEI-MAX(请参阅食谱)   
设备
自动拍(TOMY,型号:LSX-500)
涡旋混合器(科学工业公司,型号:VORTEX-GENIE 2)
温度浴(TAITEC,型号:SDminiN)
CO 2培养箱(Thermo Fisher Scientific,型号:Forma 310 Direct Heat CO 2培养箱)
离心机(TOMY,型号:LC-200,转子:TS-7LB)
细胞计数器(DeNovix,型号:Cell Drop BF)
共焦激光扫描显微镜(Carl-Zeiss,型号:LSM700)由Zen 2012操作,并配有物镜(Plan-Apochromato × 63 / 1.4 NA油浸),孵育室,温度模块S和CO 2模块S。
软件
基于ImageJ的斐济语(1.52p版)(https://imagej.net/Fiji)(Schindelin等人,2012 )
FRET和共定位分析仪
(https://imagej.nih.gov/ij/plugins/fret-analyzer/fret-analyzer.htm)(Hachet-哈斯等人。,2006 )
程序
对于此协议中,我们使用人P-gp的FRET探针小号(二俣等人,2020 )。带有荧光蛋白标签的P-gps的示意图如图1所示。人P-gp FRET探针可从通讯作者上田一和(ueda.kazumitsu.7w@kyoto-u.ac.jp)获得。
图1.荧光蛋白标记的P-gps的示意图。我们生成了一个FRET构建体,即人P-gp-FRET,其中在NBD1之后插入了mCerulean(供体),在NBD2之后融合了mVenus(受体)。预测另一种FRET构建体P-gp-VsCn,其中mVenus和mCerulean在NBD2之后串联融合,尽管其构象仍显示出高水平的FRET。构建了在NBD1之后插入mCerulean的P-gp–mCerulean和在NBD2之后融合mVenus的P-gp–mCerulean作为阴性对照。TMD,跨膜结构域。NBD,核苷酸结合结构域,aa,氨基酸。
注意:HEK293细胞在37 °C,5%CO 2下的100 mm细胞培养皿中保存在补充有10%FBS(10%FBS-DMEM)的DMEM中。为了进行培养,使用了CO 2培养箱。
完整细胞
荧光蛋白标记的P-gps的瞬时表达
天1个添加100μ升PLL溶液至35毫米的玻璃基盘,孵育在室温下30分钟。
注意:PLL溶液用于促进细胞粘附在培养皿上。
用1.5 ml 1 × PBS(-)洗涤两次。
准备在100 mm细胞培养皿中培养的H EK293细胞,并弃去培养基。
用1.5 ml 1 × PBS(-)洗涤两次
添加1米升0.05%EDTA-T rypsin和孵育2分钟,在37℃,5%CO下2 。
加入9 ml 10%FBS-DMEM,然后转移到15 ml管中。
在室温下以1100 × g离心2分钟。
弃去上清液并加入10 ml 10%FBS-DMEM。
按照制造商的协议使用CellDrop计数细胞数。
将细胞稀释至1.5 × 10 5细胞/ ml 。
将HEK293细胞以3.0 × 10 5个细胞/孔接种到玻璃皿中,并在5%CO 2下于37°C孵育24小时。
第2天准备DNA-脂质复合物。
注意:最小质粒DNA集是P-gp-FRET,两个阴性对照(P-gp-mCerulean和P-gp-mVenus)和一个阳性对照(P-gp-VsCn)。
拌2.5微克质粒DNA和2.5μ升PLUS试剂在500μ升的Opti-MEM和与混合涡流混合器。
在室温下孵育5分钟。
添加6.25 μ升脂质体LTX并用旋涡混合器混合。
在室温下孵育30分钟。
替换500的介质μ升DNA-脂质复合物和1.5M升在10%FBS-DMEM 23小时,孵育37℃,5%CO 2 。
图像采集
第3天用2 ml的FluoroBrite DMEM洗涤两次。
将培养基替换为添加了10%FBS,1 mM丙酮酸钠和1 × GlutaMAX的1 m l FluoroBrite DMEM,并孵育1 h。
将培养皿转移到配备有显微镜的培养箱中。
转移50 0 μ升米edium到1.5米升管中并用2混合μ升维拉帕米氯碱IDE储备溶液(最终100μM)。
ñ OTE小号:
不要将维拉帕米氯化物原液直接添加到玻璃皿中,因为可以通过移液将HEK293细胞剥离。
维拉帕米是P-gp底物。
将培养基放回玻璃皿中,再孵育5分钟。
根据以下参数获取mVenus,mCerulean和FRET信号的图像,并保存为.lsm或.czi格式。
mVenus是在488nm处激发,在荧光发射使用带通收集过滤器(521 - 600纳米)(图2) 。mCerulean在445 nm处激发,并使用短通滤光片(490 nm)收集荧光发射。对于FRET信号图像时,激光被设置为445 nm,并且敏化发射使用带通滤波器(521收集- 600纳米)(图3) 。
注意:所有四个质粒的每个通道的代表性原始图像如图4所示。
图2.用于mVenus成像的Zen软件设置
图3.用于mCerulean成像和FRET信号成像的Zen软件设置
图4.所有四个质粒每个通道的代表性原始图像。比例尺= 10μm。
半完整细胞
注意:膜通透性适合控制小分子(如核苷酸)的浓度。
荧光蛋白标记的P-gp构建体的瞬时表达。
天1个种子的HEK293细胞作为描述在步骤d小号A1A-A1K 。
第2天如下制备DNA-脂质复合物。
注意:进行膜通透时,请勿使用Lipofectamine LTX,因为它会抑制SLO形成孔。
混合2微克质粒DNA和98μ升的Opti-MEM中为1.5米的升管中,用涡旋混合器混合。
混合10 μ升1毫克/米升PEI-MAX和90 μ升的Opti-MEM中另外的1.5米升管中,用涡旋混合器混合。
混合DNA和PEI-MAX,并与涡旋混合器混合。
在室温下孵育30分钟。
替换200的介质μ升DNA-PEI复合物和1.8米升在10%FBS-DMEM 24小时,孵育37℃,5%CO下2 。
膜通透性和图像采集
注意:我们建议一次使用一个或两个样本执行此步骤,以避免细胞长时间渗透。
第3天用2 ml冰冷的1 × PBS(-)洗涤。
添加1米升含有50ng /无血清DMEM μ升SLO并孵育在冰上5分钟。
注意小号:
在使用前稀释e SLO。
SLO是形成25个链球菌毒素-质膜,其允许离子和小分子的自由通道内30nm的水性孔。
用2 ml冰冷的1 × PBS(-)洗涤3次。
加入1米升1 ×转运缓冲液在37预热℃,补充有2mM的MgCl 2 ,5毫摩尔ATP二钠盐,和2微克/立方米升DAPI。为ATP耗竭,添加1 ×转运缓冲液补充有2mM的MgCl 2 ,2微克/立方米升DAPI,和10mM的NaN 3 。
在5%CO 2下于37°C孵育10分钟。
用预加热至37°C的2 ml 1 ×运输缓冲液洗涤两次。
添加1米升预加热1 ×补充有2mM的MgCl转运缓冲液2 ,5毫摩尔ATP二钠盐,和100 μM维拉帕米酰氯。
将培养皿转移至配备有显微镜的培养箱中,并在5%CO 2下于37°C孵育5分钟。
根据在步骤A2f中描述的参数,获取DAPI阳性细胞的mVenus,mCerulean和FRET信号的图像。
数据分析
使用公式1计算FRET效率(接近率)。
 (式1),
其中是FRET信号或致敏发射受体供体激发(激发445纳米/发射期间521 - 600 nm)时,被供体时的供体荧光激发(激发445纳米/发射490纳米),并且是在受体荧光受体激发(激发488纳米/发射521 - 600纳米)。和分别是受体和供体的渗漏。     
质膜的分割和邻近比的计算:
用ImageJ打开.lsm或.czi格式的图像,然后转换为.tiff格式。
将图像分割到每个通道(图像菜单&gt;颜色&gt;分割通道)。
使用“ FRET和共定位分析仪”分别计算表达P-gp-mCerulean和P-gp-mVenus的图像的供体和受体渗出。
从https://imagej.nih.gov/ij/plugins/fret-analyzer/fret-analyzer.htm下载“ FRET和Colocalization分析器”到plugins文件夹。
重新启动ImageJ以将“ Fret Analyzer”命令添加到“插件”菜单。
从“插件”菜单中打开“ FRET分析器”。
对于供体渗出评估,请输入对照数量(最多10个对照)。
选择第一个控件的施主通道图像和FRET信号图像。
点击“获取”按钮。
对所有控件执行(c)和(d),并获取供体渗漏的平均值。
计算供体渗漏的方式与供体渗漏的方式相同。
如有必要,裁剪图像,以便仅可见单个单元格。
将mCerulean,mVenus,FRET信号的图像保存到单独的文件夹中。
从FRET信号图像中减去供体和受体的渗漏,并创建校正后的FRET(cFRET)图像。该过程由下面描述的自制ImageJ宏自动执行。
注意小号:
若要使用下面的ImageJ宏,请将宏文本以.txt格式保存在ImageJ目录的Macro子文件夹中。安装宏(插件&gt;宏&gt;安装)。运行宏(插件&gt;宏&gt;宏名称)。
在执行此ImageJ宏之前,请取消选中“转换时缩放”(“编辑”菜单&gt;“选项”&gt;“转换...”)。
setBatchMode(true); //批量模式开启
// setBatchMode(false); //关闭批处理模式
dir1 = getDirectory(“选择mCerulean文件夹”);
//选择保存mCerulean图像的文件夹
dir2 = getDirectory(“选择mVenus文件夹”);
//选择保存mVenus图像的文件夹
dir3 = getDirectory(“选择FRET信号文件夹”);
//选择保存FRET图像的文件夹
dir4 = getDirectory(“选择cFRET文件夹”);
//选择要保存cFRET图像的文件夹
list1 = getFileList(dir1);
list2 = getFileList(dir2);
list3 = getFileList(dir3);
A = getNumber(“输入施主BT”,0);
B = getNumber(“输入接受者BT”,0);
对于(i = 0; i 调整&gt;自动局部阈值&gt;方法“全部尝试”) ***
              run(“ Options ...”,“ iterations = 1 count = 3 black do = Open”);
//通过“打开(腐蚀后稀释)”使质膜光滑
              run(“ Analyze Particles ...”,“ size = 1-100 pixel include add”);
              setForegroundColor(0,0,0);
              roiManager(“ deselect”);
              roiManager(“ Fill”);
//删除小于100像素的对象
              setOption(“ BlackBackground”,true);
              run(“ Erode”);
roiManager(“ Delete”);
运行(“分析粒子...”,“大小= 50-无穷大像素圆度= 0.00-0.3添加”);
//选择尺寸大于50像素且圆度小于0.3的对象
run(“全选”);
//选择所有投资回报率
              roiManager(“ Combine”);
//合并所有ROI
              setBackgroundColor(0,0,0);
              运行(“清除外部”);
//在mVenus图片中的ROI外部删除
              Save_name = dir5 +“ mem_” + Ori_name;
              saveAs(“ Tiff”,Save_name);
              关闭();
              selectImage(1);
setOption(“ Show All”,true);
roiManager(“ Measure”);
              Save_name = dir4 +“ ROI_” + Ori_name +“ .zip”;
roiManager(“ Save”,Save_name); //保存ROI信息
关闭();
roiManager(“ Delete”);
}
list4 = getFileList(dir4);
//从mCerulean和cFRET图像评估接近率
对于(i = 0; i 0){
                                          setSlice(2);
                                                        val2 = getPixel(ii,iii);
                                                        比率= val2 / val;
//计算接近率(cFRET图像的像素值/ mCerulean图像的像素值) 
                                                        和+ =比率;
                                                        计数+ = 1;
                                                        setSlice(3);
                                                        ratiop =比率* 100;
                                                        setPixel(ii,iii,ratiop); 
                                                        setSlice(1);
                                          }
                            }
              }
              updateDisplay();
              Save_name = dir6 +“ ratio_” + Ori_name;
              saveAs(“ Tiff”,Save_name);
              ratiom =总和/计数;
//将总接近率除以计数的像素数
              ar = newArray(sum,count,ratiom);
              Array.print(ar);
//在Log窗口中显示接近率的总和,质膜的像素数以及接近率的平均值
              总和= 0;
              计数= 0;
              run(“全部关闭”);
              roiManager(“ Delete”);
}
菜谱
PLL解决方案             
              0.01%PLL解决方案1 m l                                                          
1 × PBS( - )29米升               
储存在4 °C
10 × PBS(-)
注意:(-)表示不含镁或钙。
氯化钠80克                                         
Na 2 HPO 4 · 12H 2 O 29克             
氯化钾2克                                         
KH 2 PO 4 2克             
用milliQ填充至1L。室温保存
1 × PBS(-)
10 × PBS( - )百米升             
MilliQ 900百万升             
高压灭菌
储存在4 °C
10 ×传输缓冲区
1 M Hepes-KOH(pH 7.4)125 m l              
CH 3煮饭56.44克             
氯化镁2 · 6H 2 O 2.54 g             
用MilliQ填充至500 m l ,并用0.22μm过滤器灭菌
存放在室温下。
1 ×传输缓冲区
10 ×传输缓冲区100 m l             
MilliQ 900百万升                                                                                                                                                                                                   
储存在4 °C
0.05%胰蛋白酶-EDTA
0.5%胰蛋白酶-EDTA(10 × )10 m l                                         
1 × PBS( - )90米升             
储存在4°C
100 mM ATP储备液
ATP二钠盐551.1毫克
用MilliQ填充多达1000万升
用NaOH将pH调节至7
用0.22μm过滤器灭菌
储存在-30 °C
50 mM维拉帕米原液
注意:使用前请做好准备。
维拉帕米氯化物24.6毫克             
DMSO 1米升             
用0.22μm过滤器灭菌
储存在-30 °C
1 mg / m l DAPI储备溶液
DAPI 1毫克                           
DMSO 1米升             
用0.22μm过滤器灭菌
储存在-30 °C
100 mM NaN 3储备溶液
NaN 3 6.501毫克             
的MilliQ 1米升             
用0.22μm过滤器灭菌
储存在-30 °C
1 M MgCl 2储备溶液
MgCl 2 ·6H 2 O 20.33 g  
用MilliQ填充多达1亿升
高压灭菌
储存在4 °C
1 mg / m l PEI-最大
PEI-MAX 10毫克    
1 × PBS( - )10米升    
用0.22μm过滤器灭菌
储存在4 °C
致谢
这项工作得到了JSPS KAKENHI资助号18H05269的支持。
利益争夺
作者宣称没有任何竞争性的经济利益。
参考
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Copyright: © 2021 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. Futamata, R., Kioka, N. and Ueda, K. (2021). Live Cell FRET Analysis of the Conformational Changes of Human P-glycoprotein. Bio-protocol 11(4): e3930. DOI: 10.21769/BioProtoc.3930.
  2. Futamata, R., Ogasawara, F., Ichikawa, T., Kodan, A., Kimura, Y., Kioka, N. and Ueda, K. (2020). In vivo FRET analyses reveal a role of ATP hydrolysis-associated conformational changes in human P-glycoprotein. J Biol Chem 295(15): 5002-5011.
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