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Apr 2020

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Visualization of Host Cell Kinase Activation by Viral Proteins Using GFP Fluorescence Complementation and Immunofluorescence Microscopy
利用GFP荧光互补和免疫荧光显微镜观察病毒蛋白对宿主细胞激酶的激活   

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Abstract

Non-receptor protein-tyrosine kinases regulate cellular responses to many external signals and are important drug discovery targets for cancer and infectious diseases. While many assays exist for the assessment of kinase activity in vitro, methods that report changes in tyrosine kinase activity in single cells have the potential to provide information about kinase responses at the cell population level. In this protocol, we combined bimolecular fluorescence complementation (BiFC), an established method for the assessment of protein-protein interactions, and immunofluorescence staining with phosphospecific antibodies to characterize changes in host cell tyrosine kinase activity in the presence of an HIV-1 virulence factor, Nef. Specifically, two Tec family kinases (Itk and Btk) as well as Nef were fused to complementary, non-fluorescent fragments of the Venus variant of YFP. Each kinase was expressed in 293T cells in the presence or absence of Nef and immunostained for protein expression and activity with anti-phosphotyrosine (pTyr) antibodies. Multi-color confocal microscopy revealed the interaction of Nef with each kinase (BiFC), kinase activity, and kinase protein expression. Strong BiFC signals were observed when Nef was co-expressed with both Itk and Btk, indicative of interaction, and a strong anti-pTyr immunoreactivity was also seen. The BiFC, pTyr, and kinase expression signals co-localized to the plasma membrane, consistent with Nef-mediated kinase activation in this subcellular compartment. Image analysis allowed calculation of pTyr-to-kinase protein ratios, which showed a range of responses in individual cells across the population that shifted upward in the presence of Nef and back down in the presence of a kinase inhibitor. This method has the potential to reveal changes in steady-state non-receptor tyrosine kinase activity and subcellular localization in a cell population in response to other protein-kinase interactions, information that is not attainable from immunoblotting or other in vitro methods.

Keywords: Protein-tyrosine kinase (蛋白酪氨酸激酶), Tec family kinases (Tec家族激酶), Interleukin-2-inducible T cell kinase (Itk) (白细胞介素2诱导的T细胞激酶(Itk)), Bruton’s tyrosine kinase (Btk) (布鲁顿酪氨酸激酶(Btk)), HIV-1 Nef (HIV-1 Nef), Bimolecular fluorescence complementation (BiFC) (双分子荧光互补(BiFC)), Confocal microscopy (共聚焦显微镜), Signal transduction (信号传导), Protein-protein interaction (蛋白互作分析)

Background

Non-receptor protein-tyrosine kinases, exemplified by members of the Src and Tec kinase families, regulate many aspects of cell biology including growth, differentiation, and motility in response to diverse stimuli (Amatya et al., 2019). Methods that assess the spatial and temporal aspects of tyrosine kinase signaling at the single cell and population levels are essential to better understanding their function. In this protocol, we provide details of a cell-based method to evaluate protein-protein interaction, kinase activity, and subcellular localization of Tec family kinases in response to the interaction with the HIV-1 accessory protein, Nef. This approach is potentially applicable to many other kinase systems in which protein-protein interactions impact kinase activity.


Nef is a small (27-34 kDa, depending on the subtype) membrane-associated protein unique to the primate lentiviruses HIV-1, HIV-2, and SIV (Foster and Garcia, 2008). HIV-1 Nef enhances viral infectivity, supports high-titer replication in vivo, and promotes immune escape of HIV-infected cells (Basmaciogullari and Pizzato, 2014; Pawlak and Dikeakos, 2015). Rhesus macaques infected with nef-defective SIV exhibit very low viral loads and do not progress to simian AIDS (Kestler et al., 1991), illustrating an essential role for Nef in viral pathogenesis. Along the same lines, individuals infected with nef-defective HIV-1 can remain AIDS-free in the absence of antiretroviral therapy for many years (Deacon et al., 1995; Kirchhoff et al., 1995).


Nef lacks intrinsic biochemical activities, functioning instead through interactions with host cell proteins related primarily to endocytic trafficking and kinase signaling pathways (Staudt et al., 2020).Nef hijacks non-receptor tyrosine kinases of the Src and Tec families normally linked to immune receptor activation to enhance HIV-1 replication. Nef directly activates the Src family members Hck and Lyn by binding to their SH3 domains (Briggs et al., 1997; Trible et al., 2006). Selective inhibition of Nef-mediated Src family kinase activation blocks Nef-dependent enhancement of HIV-1 infectivity and replication (Emert-Sedlak et al., 2009 and 2013). Tec family kinases play essential roles in B- and T-cell receptor signaling (Andreotti et al., 2010), with the interleukin-2 inducible T-cell kinase (Itk) and Bruton's tyrosine kinase (Btk) expressed in primary HIV-1 target cells (CD4+ T cells and macrophages, respectively). Readinger et al. provided the first evidence linking Itk to HIV-1 entry, viral transcription, assembly, and release (Readinger et al., 2008). A subsequent study showed that Nef provides a link between HIV-1 infection and Tec family kinase signaling by demonstrating direct interaction between Nef and both Itk and Btk at the cell membrane (Tarafdar et al., 2014). Treatment of HIV-infected T cells with a selective Itk inhibitor blocked Nef-dependent enhancement of viral infectivity and replication. Importantly, activation of both Src and Tec family kinases is highly conserved across all M group HIV-1 subtypes, consistent with an important function in the HIV-1 life cycle and viral pathogenesis (Narute and Smithgall, 2012; Emert-Sedlak et al., 2013; Tarafdar et al., 2014). For an in-depth review of Nef interactions with host cell tyrosine kinases, see Staudt et al. (2020).


In a recent study, we explored the molecular mechanisms of Tec family kinase activation by the Nef proteins of HIV-1 and SIV (Li et al., 2020). By combining cell-based bimolecular fluorescence complementation (BiFC) (Romei and Boxer, 2019) and anti-phosphotyrosine immunofluorescence microscopy, we found that HIV-1 Nef interacts with Itk and Btk at the cell membrane and results in constitutive kinase activity. For the BiFC assay, Itk or Btk and Nef were fused to complementary, non-fluorescent fragments of the Venus variant (Nagai et al., 2002) of YFP. The kinases were then expressed in 293T cells either alone or together with Nef, followed by immunostaining for Nef and kinase protein expression as well as protein-tyrosine phosphorylation using anti-phosphotyrosine (pTyr) antibodies. Multi-color confocal microscopy enabled simultaneous assessment of Nef-kinase complex formation (BiFC), kinase activity (anti-pTyr immunofluorescence), and kinase protein expression (anti-kinase immunofluorescence). When expressed alone, both Itk and Btk showed a diffuse subcellular staining pattern with the anti-kinase antibody and weak reactivity with the anti-pTyr antibody. In contrast, co-expression with Nef induced a strong BiFC signal with both Itk and Btk, indicative of interaction, and a strong anti-pTyr immunoreactivity was seen. The BiFC, pTyr, and kinase expression signals co-localized to the cell membrane, consistent with Nef-mediated kinase activation in this subcellular compartment. As a control, cells were treated with Tec family kinase inhibitors (Lin et al., 2004; Roskoski, 2016) that suppressed the pTyr signal but did not affect BiFC, demonstrating that interaction of Nef with Tec family kinases at the membrane does not require kinase activity.


Results were quantitated at the single-cell level using the NIH ImageJ image analysis software (Schneider et al., 2012). Kinase expression and tyrosine phosphorylation immunofluorescence signal intensities for at least 100 cells for each condition were expressed as the mean pTyr-to-kinase protein fluorescence intensity ratios. This analysis enabled statistical comparisons of cell populations and showed that cells co-expressing Itk or Btk and Nef had significantly higher fluorescence ratios as compared with those expressing the kinase alone or the inhibitor-treated cells expressing the Nef-kinase complex.


Using the same approach, we also investigated Nef-stimulated Itk and Btk autophosphorylation on their respective activation loop tyrosine residues (pTyr511 and pTyr551, respectively), a step required for Tec family kinase activation (Joseph et al., 2013). Cells were transfected with the Nef and Itk/Btk Venus fusion constructs for BiFC as before, but in this case the cells were stained with phosphospecific antibodies for the activation loop phosphotyrosines in place of the general anti-pTyr antibody. Co-expression with Nef led to significant increases in Itk and Btk activation loop autophosphorylation, which also localized almost exclusively to the cell membrane. SIV Nef (mac239 allele) was also found to strongly induce membrane-associated autophosphorylation of both kinases, demonstrating that Tec family kinase activation is conserved across Nef proteins from diverse primate lentiviruses. A representative confocal image from this study is shown in Figure 1.


While the experimental approach described above was developed to explore kinase interaction with, and activation by, a viral protein in a cell-based setting, the overall concept should be readily adaptable to any combination of kinases and interacting partners. The cell-based approach has important advantages over older in vitro methods such as immune-complex kinase assays, which do not provide information about the subcellular localization of the active kinase complex or the range of responses across a population of individual cells. However, one important caveat includes consideration of where to add the fragments of Venus for the BiFC assay. For example, both Nef and Tec family kinases localize to the cell membrane by virtue of N-terminal signals. Nef is myristoylated on its N-terminus, while Tec kinases have an N-terminal Pleckstrin homology (PH) domain that binds to membrane phosphoinositides. To avoid interference with these membrane-targeting signals, we were careful to fuse the Venus fragments to the C-terminus of each protein. Control experiments are also essential to verify that Venus fragment fusion does not influence basal kinase activity or localization, which is readily accomplished by comparing unfused with fused versions of each kinase in transfected cells and staining with kinase and phosphospecific antibodies. Finally, it should be noted that the Venus fluorophore, once reconstituted via BiFC, is irreversible. While this feature of BiFC may help to stabilize transient interactions for endpoint assessment by microscopy as described here, other techniques are more appropriate to assess the kinetics of interaction, such as the split-FAST reversible complementation system (Tebo and Gautier, 2019).



Figure 1. HIV-1 and SIV Nef proteins induce constitutive Btk activation loop autophosphorylation at the cell membrane. Btk was expressed in 293T cells either alone or together with HIV-1 Nef (SF2 isolate) or SIV Nef (mac239) as BiFC pairs in the absence or presence of the Btk/Itk inhibitor ibrutinib (1 μM). Cells were fixed and stained for confocal microscopy with phosphospecific antibodies against the Btk activation loop phosphotyrosine (pY551; red) and the Btk protein (V5 epitope; cyan). Nef interaction with Btk is observed as fluorescence complementation of the YFP variant, Venus (BiFC; green). Note that interaction and kinase activation occur at the plasma membrane.

Materials and Reagents

Molecular biology reagents

  1. Phusion high-fidelity DNA polymerase (New England Biolabs, catalog number: M0530S)

  2. Venus template (gift from Dr. Atsushi Miyawaki, RIKEN Brain Science Institute, Saitama, Japan)

  3. HIV-1 (SF2 allele) and SIV (mac239) Nef clones (NIH AIDS Reagent Program, HIV #11431; SIV #2476)

  4. Full-length human Tec family kinase cDNA clones (Dana-Farber/Harvard Cancer Center PlasmID DNA Resource Core, Btk # HsCD00346954; Itk # HsCD00021352)

  5. Mammalian expression vector, pCDNA3.1(−) (Thermo Fisher, catalog number: V79520)


Antibodies
  1. Anti-V5 tag mouse monoclonal antibody (Thermo Fisher, catalog number: R960-25)

  2. Anti-V5 tag rabbit polyclonal antibody (Sigma, catalog number: AB3792)

  3. BTK anti-pY551 rabbit monoclonal antibody (Abcam, catalog number: ab40770)

  4. Anti-pTyr antibody pY99 (Santa Cruz, catalog number: sc-7020)

  5. Anti-HIV-1 Nef monoclonal antibody 6.2 (NIH AIDS Reagent Program, catalog number: 1539)

  6. Goat anti-rabbit IgG (H+L), mouse/human ads-TXRD (Texas Red conjugate; cross-adsorbed to mouse and human immunoglobulins; Southern Biotech, catalog number: 4050-07)

  7. Goat anti-mouse IgG (H+L), human ads-TXRD (Texas Red conjugate; cross-adsorbed to human immunoglobulins; Southern Biotech, catalog number: 1031-07)

  8. Pacific Blue goat anti-mouse IgG antibody (Thermo Fisher/Molecular Probes, catalog number: P31582)

  9. Pacific Blue goat anti-rabbit IgG antibody (Thermo Fisher/Molecular Probes, catalog number: P10994)


Cell culture reagents and kinase inhibitors
  1. 35 mm microwell dishes (MatTek, catalog number: P35G-1.5-14-C)

  2. Human embryonic kidney 293T cells (American Type Culture Collection, catalog number: CRL-11268)

  3. Dulbecco’s modified Eagle’s medium (DMEM; ThermoFisher/Invitrogen, catalog number: 11965-118)

  4. Fetal bovine serum (FBS; Gemini Bio-Products, catalog number: 900-108)

  5. Trypsin-EDTA, 0.05% (ThermoFisher/Invitrogen catalog number: 25300054)

  6. X-tremeGENE 9 DNA transfection reagent (Sigma-Aldrich, catalog number: 06365787001)

  7. Paraformaldehyde, 16% aqueous solution (Fisher, catalog number: 50980487)

  8. Triton X-100 (Sigma, catalog number: X100-1L)

  9. Bovine serum albumin (BSA, Sigma, catalog number: A3059-500G)

  10. Itk inhibitor, BMS-509744 (Calbiochem, catalog number: 41-982-05MG)

  11. Itk/Btk inhibitor, ibrutinib (SelleckChem, catalog number: S2680)

Equipment

  1. Olympus FluoView FV1000 Confocal Microscope

Software

  1. Prism v. 8.0 (GraphPad Software, Inc.; www.graphpad.com)

  2. ImageJ (National Institutes of Health; https://imagej.net/Welcome)

  3. Olympus FluoView Software (https://www.olympus-lifescience.com/en/)

Procedure

  1. Construction of expression vectors for BiFC based on Venus. This procedure is based on our published work with BiFC vectors for lentiviral Nef alleles and the Tec family kinases Btk and Itk (Tarafdar et al., 2014; Li et al., 2020) but can be easily adapted to virtually any combination of interacting protein partners. While our approach uses pcDNA3-based expression vectors that drive strong protein expression in 293T cells, other vectors can be substituted depending on the target cell type and application. Additional details related to primer design, subcloning strategy, and sequences of the final fusion constructs are provided in the Appendix.

    1. PCR-amplify the coding sequence for the Venus N-terminal (VN: residues Val2 to Asp173) and C-terminal (VC: residues Ala154 to Lys238) fragments containing the appropriate restriction sites and a stop codon, and subclone the PCR product into the mammalian expression vector, pcDNA3.1(–).

    2. PCR-amplify the coding sequence for HIV-1 Nef (SF2) and SIV Nef (mac239) in the same manner and subclone the product into the pcDNA3.1(–)/VN construct.

    3. PCR-amplify the full-length human Tec family kinase cDNA clones containing the appropriate restriction sites and a C-terminal V5 epitope tag for ligation in-frame into the pcDNA3.1(–)/VC construct.


  2. 293T cell culture and transfection

    1. Culture 293T cells in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum in a 37°C humidified incubator with a 5% CO2 atmosphere. Use 10 ml DMEM per 10-cm culture dish and maintain cells at a density of 2-5 × 104 cells/cm2. When cultures reach 80% confluence, remove the spent medium and split 1:10 by incubating with 1.0 ml 0.05% trypsin-EDTA solution for 1 min at 37°C to generate a single cell suspension. Add 10 ml fresh medium and spin at 500 × g for 5 min. Resuspend the cell pellet in 10 ml fresh medium and transfer 1.0 ml cell suspension to new 10-cm dishes with an additional 10 ml fresh medium. For transfection, seed 2.5 × 105 cells per MatTek plate and culture overnight.

    2. Transfect cells with BiFC expression vectors using X-tremeGene 9 DNA transfection reagent according to the manufacturer’s protocol. Briefly, mix 3 µl X-tremeGene 9 with 97 µl DMEM, add each BiFC expression plasmid (0.5 µg each VN and VC construct), and incubate the mixture at room temperature for 15 min. After incubation, add the transfection complex dropwise to the MatTek culture plate. Treat the cells with inhibitors or the DMSO carrier solvent (0.1% final concentration) 4 h after transfection as needed.


  3. Immunofluorescence staining

    1. Forty hours post-transfection, fix the cells by replacing the medium with 2 ml 4% paraformaldehyde and incubate for 10 min at room temperature. Wash cells with 2 ml PBS (pH 7.4) for 5 min with occasional gentle shaking by hand.

    2. Permeabilize the cells with 2 ml 0.2% Triton X-100 in PBS for 15 min. Wash cells twice with 2.0 ml PBS for 5 min each.

    3. Block the cells with 2 ml 2% BSA in PBS for 1 h at room temperature or overnight at 4°C.

    4. Incubate the cells with anti-V5 (kinase tag) and anti-Nef or anti-pTyr (or anti-Btk pY551/anti-Itk pY511) antibodies diluted 1:1,000 in 250 µl PBS with 2% BSA for 1 h at room temperature. Wash cells three times with 2 ml PBS for 5 min each.

    5. Incubate the cells with the appropriate secondary antibodies conjugated to Texas Red or Pacific Blue at a dilution of 1:500 or 1:1,000, respectively. Wash the cells three times with 2 ml PBS for 5 min each. Keep cells in 2 ml PBS for imaging.

      Note: This protocol was developed for 3-color imaging to allow simultaneous detection of interaction (Venus complementation) and protein expression of each interacting partner (kinase and Nef), or interaction, kinase expression, and kinase activity (overall cellular phosphotyrosine content or activation loop autophosphorylation).


  4. Fluorescence imaging and analysis

    1. Acquire images using multi-color confocal microscopy with a 60× objective using x-y scan mode. On the Olympus FluoView1000, we used the violet laser to detect Pacific Blue (405 nm), the green laser to detect fluorescence complementation (Venus; 543 nm), and the red laser to detect Texas Red (633 nm).

    2. Perform single-cell image analysis using the Java-based image processing program, ImageJ, as described under Data analysis.

Data analysis

As an example, the following protocol uses ImageJ to quantitate the mean fluorescence intensities (MFI) of cells stained with anti-phosphotyrosine (anti-pTyr) and anti-Btk antibodies. The ratio of the pTyr to Btk MFIs is then calculated for a minimum of 100 cells as a measure of the kinase activity within the cell population. The same approach can be used to quantitate other MFI ratios such as interaction (BiFC fluorescence) normalized to partner protein expression.


  1. File/Open: Open the Btk protein expression and confocal image pair (Figure 2). Minimize the pTyr image and do not click on it again. Click on the Btk protein image window.



    Figure 2. Confocal images of 293T cells stained for Btk protein expression and pTyr opened in ImageJ. Cells stained for Btk protein are shown on the left (cyan), with pTyr shown on the right (red). These images are from cells co-expressing Btk and HIV-1 Nef.


  2. Image/Type: Change to 8-bit, which will convert the image to black and white (Figure 3).



    Figure 3. Conversion of Btk protein image to an 8-bit greyscale. In the control bar, click on Image and then 8-bit, which will convert the image to an 8-bit grey scale image (right).


  3. Image/Adjust/Threshold: Set levels by moving the two sliders to set the min/max to include staining but minimize the background and overexposure. Check the “Dark Background” box. Click “Set” and “OK.” Close the Threshold box. The red highlighted areas show the pixels that will be included in the analysis (Figure 4).



    Figure 4. Setting the threshold. Click on Image in the control bar, select Adjust, and then Threshold. The box on the upper left will appear, and the pixels that will be included in the analysis will be shown in red on the image (right). Click Set; the lower box shown will appear. Click OK to finish.


  4. Analyze/Set Measurements: Select these options (Figure 5):

    1. Area, Min & Max Gray Value, and Mean Gray Value.

    2. Set the “Redirect to” box to “None.”



      Figure 5. Setting the analysis parameters. From the control bar, click on Analyze and then Set Measurements; the box shown on the left will appear. Select the parameters shown.


  5. Analyze/Analyze Particles (Figure 6):

    1. Size: Determines the cell area that will be analyzed. Typically, [20-infinity] and [50-infinity] works best for 400× and 100× images of 293T cells, respectively.

    2. Check the “pixel units” box.

    3. Circularity: Start with [0.0-1.0] to exclude background specks and noise.

    4. Show: Chose the “Outlines” option to reveal the location and size of the areas selected for measurement (controlled by adjusting the size/circularity values above).

    5. Check the “Display Results” box and uncheck the “Clear results” box.

    6. Click “OK.” Two additional windows will be generated: One (Figure 7) shows outlines of the cell areas used in the analysis of the thresholded image (left), and the second (right) is the results table window containing MFI data (‘Mean’ column).

    7. Copy the data in the Results window into an Excel file and close the Results window only.



    Figure 6. Particle (pixel) analysis settings. From the control bar, select Analyze and then analyze particle, which will open this box. Select the parameters shown.



    Figure 7. Results of particle analysis. The center image shows outlines of the areas where pixel intensities have been analyzed and numbered. The table on the right shows the resulting data including the number of the analyzed feature and its area, followed by the mean and minimum and maximum fluorescence intensities. Note that cells will be analyzed individually, as groups, or as fragments, with several fragments from the same cell.


    The next step will use the same masks to analyze the anti-pTyr image. Be sure the “Results” box from the protein analysis is closed. Click on the ‘Nef + Btk’ protein box to activate it.


  6. Analyze/Set Measurements: Set the “Redirect to” box to the corresponding pTyr image file name (previously minimized and not modified) while keeping the other parameters the same. Click “OK.” The selected (outlined) fields established for the Btk protein image are now directly transposed onto the pTyr image and will analyze the pTyr levels relative to the identical cell locations (pixels) provided by the Btk immuno-stain.

  7. Analyze/Analyze Particles: Use the previous settings and click “OK.” Select all and copy the data in the Results window into the Excel file. The Results windows from the Btk protein and pTyr analyses are shown side-by-side below (Figure 8). Note that the number of rows in each are the same (54; not shown) and the areas used for analysis of each region are also identical. However, the MFI values differ (Mean columns) as expected, since the intensities are different.



    Figure 8. Results of particle analysis of the Btk pTyr image. The Results tables from the Btk protein (from Figure 7, left) and pTyr (right) analyses are shown side-by-side for comparison. Note that areas of the features in each image are identical, as are the number of features (54 in each case, not shown). Both conditions must be true for the subsequent calculations to be valid.


  8. Using Excel, calculate the ratio of pTyr to Btk mean pixel densities (“Mean” columns).

  9. Analyze at least 100 cells per condition, which may require several fields of cells depending on the cell density, size, and magnification used. Significant differences between groups can be analyzed using an unpaired Student’s t-test (GraphPad Prism v.8.0). For added rigor, perform three biological replicates of each experiment.

  10. The data can be presented in several different ways using Prism. We prefer showing the results for individual “cells” (really from masks of individual cells or cell fragments as identified by ImageJ) as a series of bars, which gives a view of the distribution of ratios across the cell population. Alternatively, box-and-whisker plots or violin plots can be used. Figure 9 shows the distribution of ratios obtained using the above approach following ImageJ analysis of the results shown in Figure 1.



    Figure 9. Image analysis of Btk autophosphorylation in the presence and absence of lentiviral Nef proteins. Mean fluorescence intensities for Btk activation loop autophosphoryla-tion (pY551) and Btk protein expression signals were determined for ≥100 cells from each condition using ImageJ and the data shown in Figure 1. The fluorescence intensity ratio (pY551: Btk expression) for each cell (or cell fragment as determined by ImageJ) is shown as a horizontal bar, with the median value indicated by the red bar. Student’s t-test shows significant increases in Btk activation loop phosphorylation in the presence of both HIV-1 and SIV Nef (P < 0.0001 in each case). When Btk is expressed alone, a wide range of pY551:Btk ratios are observed because the extent of Btk activity increases in a non-linear fashion as the amount of protein increases. When expressed with HIV-1 Nef, note that most of the black bars shift to the top of the stack, consistent with maximal activation of Btk by Nef in almost all the cells imaged. Co-expression with SIV Nef produces a more subtle, albeit statistically significant, shift in the pY551:Btk ratio. Ratios from all three populations shift downward in the presence of the Btk kinase inhibitor, ibrutinib.

Notes

  1. Do not allow the dishes to dry out prior to imaging. Drying will seriously affect staining and create false positive artifacts.

  2. Protect fluorophores from light to avoid bleaching by wrapping dishes in aluminum foil.

  3. Always include positive and negative BiFC pairs as controls, especially when developing the assay for a new protein-protein interaction.

  4. 293T cells should be maintained at a low passage number to ensure cell integrity (i.e., less than 10 passages).

  5. Keep all confocal image acquisition and ImageJ analysis settings the same throughout an experiment. Results should be reproducible when all conditions are kept constant.

  6. When testing a new phosphospecific antibody, titrate a range of antibody concentrations during the staining procedure to identify the optimal concentration.

  7. After staining, the cells can be imaged up to 2 days later, and as long as one week in some cases. When storing plates, protect from light and keep at 4°C.

  8. During imaging, adjust the laser power to avoid oversaturating the signal in the image, as this may affect subsequent image analysis.

Acknowledgments

This work was funded by the National Institutes of Health grants AI152677 and AI057083 to T.E.S. These protocols were originally reported by Li et al. (2020) (doi: 10.1074/jbc.RA120.012536). The following reagent was obtained through the NIH HIV Reagent Program, Division of AIDS, NIAID, NIH: Anti-Human Immunodeficiency Virus 1 (HIV-1) Nef Monoclonal (6.2), ARP-1539, contributed by Drs. Kai Krohn and Vladimir Ovod.

Competing interests

The authors have no competing interests.

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简介

[摘要]非受体蛋白酪氨酸激酶调节细胞对许多外部信号的反应,是癌症和传染病的重要药物发现靶点。尽管存在许多测定的激酶活性的评估体外,方法,在酪氨酸激酶活性变动报告书单细胞中有提供有关的细胞群水平激酶反应信息的潜力。在这个协议中,我们结合双分子荧光互补(附设),用于建立的方法的蛋白质评估-蛋白质相互作用小号,和immunofluorescen CE染色 使用磷酸特异性抗体来表征在 HIV-1 毒力因子Nef存在下宿主细胞酪氨酸激酶活性的变化。具体而言,两种 Tec家族激酶(Itk和Btk )以及Nef融合到 YFP 金星变体的互补非荧光片段上。每种激酶表达在293T细胞中的存在或不存在的Nef和免疫染色的蛋白质的表达和活性用抗磷酸酪氨酸(的pTyr )抗体。多色共聚焦显微镜显示的相互作用的Nef与每个激酶(附设),激酶活性,和激酶蛋白的表达。强附设观察信号时,Nef的是与两个共表达中Itk和Btk的,指示交互的,一个第二一强反的pTyr免疫反应也被看作。的附设,的pTyr ,和激酶表达信号共定位于质膜,具有一致的Nef在该亚细胞区室介导的激酶活化。图像分析允许的计算的pTyr -到-激酶蛋白比,这表现出了一系列的反应中跨在存在向上移动人口单个细胞的Nef和激酶抑制剂的存在下退缩。这种方法有可能揭示细胞群中稳态非受体酪氨酸激酶活性和亚细胞定位的变化,以响应其他蛋白激酶相互作用,这是免疫印迹或其他体外方法无法获得的信息。

背景]非受体蛋白酪氨酸激酶,由成员例举的Src的和Tec的激酶家族,调节细胞生物学的许多方面,包括生长,分化,和运动响应于各种刺激(Amatya等人,2019)。在单细胞和种群水平上评估酪氨酸激酶信号的空间和时间方面的方法对于更好地理解它们的功能至关重要。在该协议中,我们提供了一种基于细胞的方法的详细信息,以评估蛋白质-蛋白质相互作用、激酶活性和 Tec家族激酶的亚细胞定位,以响应与 HIV-1 辅助蛋白Nef的相互作用。这种方法可能适用于许多其他激酶系统,其中蛋白质 - 蛋白质相互作用影响激酶活性。

Nef的是一个小的(27-34 kDa的,这取决于亚型)膜相关蛋白独有的灵长类动物慢病毒HIV-1,HIV-2 ,和SIV (福斯特和加西亚,2008年)。HIV-1 Nef增强病毒感染性,支持体内高滴度复制,并促进 HIV 感染细胞的免疫逃逸(Basmaciogullari 和 Pizzato,2014 年;Pawlak 和 Dikeakos,2015 年)。感染了Nef缺陷型 SIV 的恒河猴表现出非常低的病毒载量,并且不会发展为猿猴 AIDS (Kestler等,1991),说明了Nef在病毒发病机制中的重要作用。同样,感染了nef缺陷型 HIV-1 的个体可以在多年没有抗逆转录病毒治疗的情况下保持无艾滋病(Deacon等人,1995 年;Kirchhoff等人,1995 年)。

Nef缺乏内在的生化活性,而是通过与主要与内吞运输和激酶信号通路相关的宿主细胞蛋白的相互作用来发挥作用(Staudt等,2020)。Nef劫持通常与免疫受体激活相关的Src和 Tec 家族的非受体酪氨酸激酶,以增强 HIV-1 复制。Nef通过与Src家族成员Hck和 Lyn 的 SH3 结构域结合直接激活它们(Briggs等人,1997 年;Trible等人,2006 年)。选择性抑制Nef介导的Src家族激酶激活可阻断Nef依赖性 HIV-1 感染性和复制的增强(Emert-Sedlak等人,2009 年和 2013 年)。Tec家族激酶发挥在乙重要作用-和T -细胞受体信号传导(安德烈奥蒂等人,2010),与白细胞介素-2诱导的T细胞激酶(中Itk )和布鲁顿酪氨酸激酶(Btk的)在初级HIV-1表达靶细胞(分别为 CD4 + T 细胞和巨噬细胞)。雷丁等人。提供的第一证据连接中Itk到HIV-1进入,病毒转录,组件,和释放(Readinger等人,2008)。随后的一项研究表明,Nef通过证明Nef与细胞膜上的Itk和Btk之间的直接相互作用,在 HIV-1 感染和 Tec家族激酶信号之间建立了联系(Tarafdar等人,2014 年)。用选择性Itk抑制剂处理 HIV 感染的 T 细胞阻止了病毒感染性和复制的Nef依赖性增强。重要的是,Src和 Tec家族激酶的激活在所有 M组 HIV-1 亚型中都是高度保守的,这与 HIV-1 生命周期和病毒发病机制中的重要功能一致(Narute 和 Smithgall,2012 年;Emert-Sedlak等人。,2013 年;Tarafdar等人,2014 年)。有关Nef与宿主细胞酪氨酸激酶相互作用的深入评论,请参阅 Staudt等人。(2020 年)。

在最近的一项研究中,我们探索了HIV-1 和 SIV的Nef蛋白激活Tec家族激酶的分子机制(Li等,2020)。通过结合基于细胞的双分子荧光互补 ( BiFC ) (Romei 和 Boxer,2019 年)和抗磷酸酪氨酸免疫荧光显微镜,我们发现 HIV-1 Nef与细胞膜上的Itk和Btk相互作用并导致组成型激酶活性。对于BiFC测定,Itk或Btk和Nef与 YFP的 Venus 变体(Nagai等,2002)的互补非荧光片段融合。然后将激酶单独或与Nef一起在 293T 细胞中表达,然后使用抗磷酸酪氨酸( pTyr ) 抗体对Nef和激酶蛋白表达以及蛋白酪氨酸磷酸化进行免疫染色。多色共聚焦显微镜能够同时评估Nef-激酶复合物形成 ( BiFC )、激酶活性(抗pTyr免疫荧光)和激酶蛋白表达(抗激酶免疫荧光)。当单独表达时,Itk和Btk均显示出与抗激酶抗体的弥漫性亚细胞染色模式和与抗pTyr抗体的弱反应性。相比之下,共表达与Nef的诱导强烈的附设信号两者中Itk和Btk的,指示交互的,一个第二一强反的pTyr免疫反应被看见。的附设,的pTyr ,和激酶表达信号共同定位于细胞膜上,具有一致的Nef在该亚细胞区室介导的激酶活化。作为对照,细胞用Tec的治疗家族激酶抑制剂(林等人,2004 ; Roskoski,2016)该抑制的pTyr信号,但并不影响附设,展示了该相互作用的Nef与Tec家族激酶在膜不要求激酶活性。

结果是孔定量达在使用NIH ImageJ的图像分析软件的单细胞水平版(施耐德等人,2012)。对于至少100个单元F激酶表达和酪氨酸磷酸化的免疫荧光信号强度或每个条件表示为平均值的pTyr -到-激酶蛋白的荧光强度比。此分析使能细胞群体的统计比较,并表明,细胞共表达中Itk或Btk的和Nef的有显著更高的荧光的比率为相比与那些表达单独的激酶或表达所述抑制剂处理的细胞的Nef -激酶复合物。

使用相同的方法,我们还研究了Nef刺激的Itk和Btk各自激活环酪氨酸残基(分别为 pTyr511 和 pTyr551)上的自磷酸化,这是 Tec家族激酶激活所需的步骤(Joseph等,2013)。如前所述,用用于BiFC的Nef和Itk / Btk Venus 融合构建体转染细胞,但在这种情况下,细胞用激活环磷酸酪氨酸的磷酸特异性抗体代替一般抗pTyr抗体染色。与Nef 的共表达导致Itk和Btk激活环自磷酸化显着增加,这也几乎完全定位于细胞膜。还发现SIV Nef (mac239 等位基因)强烈诱导两种激酶的膜相关自磷酸化,表明 Tec家族激酶激活在来自不同灵长类动物慢病毒的Nef蛋白中是保守的。本研究的代表性共焦图像如图 1所示。

虽然实验方法上面描述的开发是为了探索与激酶相互作用,并通过激活,在基于细胞的设置病毒蛋白,整体概念应该是容易适应激酶和相互作用配偶体的任意组合。基于细胞的方法比旧的体外方法具有重要的优势,例如免疫复合物激酶测定法,后者不提供有关活性激酶复合物的亚细胞定位或单个细胞群的反应范围的信息。然而,一个重要的警告包括考虑在何处添加金星碎片以进行BiFC检测。例如,Nef和 Tec家族激酶都通过 N 端信号定位于细胞膜。NEF被肉豆蔻酰化它的N-末端,而Tec的激酶具有N-末端普列克底物蛋白同源性(PH)结构域结合至膜磷酸肌醇。为了避免干扰这些膜靶向信号,我们小心地将 Venus 片段融合到每个蛋白质的 C 端。对照实验也是必不可少的,以验证金星片段融合不影响基础激酶活性或定位,这是容易地通过比较未融合来实现与在转染细胞每种激酶的融合版本和与激酶和染色的磷酸化的抗体。最后,应该指出的是,一旦通过BiFC重组,金星荧光团是不可逆的。虽然BiFC 的这一特性可能有助于稳定瞬态相互作用,以便通过显微镜进行终点评估,如此处所述,但其他技术更适合评估相互作用的动力学,例如 split-FAST 可逆互补系统(Tebo 和 Gautier,2019 年)。





图 1. HIV-1 和 SIV Nef蛋白在细胞膜上诱导组成型Btk激活环自磷酸化。BTK表达在293T细胞中单独或一起用HIV-1 Nef的(SF2分离物)或SIV的Nef (mac239)作为附设在不存在或存在对BTK /中Itk抑制剂依罗替尼(1 μM )。将细胞固定并染色以共聚焦显微镜的磷酸化的抗体对所述的Btk活化环磷酸酪氨酸(pY551;红色)和Btk的蛋白质(V5表位;青色)。Nef与Btk 的相互作用被观察为 YFP 变体金星(BiFC ;绿色)的荧光互补。请注意,相互作用和激酶激活发生在质膜上。

关键字:蛋白酪氨酸激酶, Tec家族激酶, 白细胞介素2诱导的T细胞激酶(Itk), 布鲁顿酪氨酸激酶(Btk), HIV-1 Nef, 双分子荧光互补(BiFC), 共聚焦显微镜, 信号传导, 蛋白互作分析



材料和试剂


分子生物学试剂

的Phusion ħ igh- ˚F idelity DNA聚合酶(New England Biolabs公司,目录号:M0530S)
金星模板(礼物从淳宫胁博士,RIKEN脑科学研究所,日本琦玉)
HIV-1(SF2 等位基因)和 SIV(mac239)Nef克隆(NIH AIDS Reagent Program,HIV #11431;SIV #2476)
全长人类 Tec家族激酶 cDNA 克隆(Dana-Farber/Harvard Cancer Center PlasmID DNA Resource Core,Btk # HsCD00346954;Itk # HsCD00021352)
哺乳动物表达载体,pCDNA3.1(-)(Thermo Fisher,目录号:V79520)


抗体

抗V5 t ag 小鼠单克隆抗体(Thermo Fisher ,目录号:R960-25)
抗V5 t ag 兔多克隆抗体(Sigma,目录号:AB3792)
BTK 抗 pY551 兔单克隆抗体(Abcam,目录号:ab40770)
抗的pTyr抗体PY99(Santa Cruz公司,目录号:SC-7020)
抗HIV-1 Nef的中号onoclonal抗体6.2(NIH AIDS试剂方案,目录号:1539)
山羊一个nti- ř abbit的IgG(H + L),米乌斯/ ħ UMAN广告-TXRD(德克萨斯红偶联物;交吸附于小鼠和人免疫球蛋白; Southern Biotech公司,目录号:4050-07)
山羊一个nti-米乌斯的IgG(H + L),ħ UMAN广告-TXRD(德克萨斯红偶联物;交吸附到人类免疫球蛋白; Southern Biotech公司,目录号:一〇三一年至1007年)
太平洋乙略的山羊抗小鼠IgG抗体(赛默飞世/ Molecular Probes公司,目录号:P31582)
太平洋乙略的山羊抗兔IgG抗体(赛默飞世/ Molecular Probes公司,目录号:P10994)


细胞培养试剂和激酶抑制剂

35 mm 微孔培养皿(MatTek ,目录号:P35G-1.5-14-C)
人胚肾293T细胞(美国典型培养物保藏中心,目录号:CRL-11268)
Dulbecco 改良的 Eagle 培养基(DMEM;ThermoFisher /Invitrogen,目录号:11965-118)
胎牛血清(FBS;Gemini Bio-Products,目录号:900-108)
胰蛋白酶-EDTA,0.05%(ThermoFisher /Invitrogen 目录号:25300054)
X- tremeGENE 9 DNA 转染试剂(Sigma - Aldrich,目录号:06365787001)
多聚甲醛,16% 水溶液(Fisher,目录号:50980487)
Triton X-100(Sigma,目录号:X100-1L)
牛血清白蛋白(BSA,Sigma,目录号:A3059-500G)
Itk抑制剂,BMS-509744(Calbiochem ,目录号:41-982-05MG)
Itk / Btk抑制剂,依鲁替尼(SelleckChem ,目录号:S2680)


设备


奥林巴斯FluoView FV1000 共聚焦显微镜


软件


Prism v. 8.0(GraphPad Software, Inc.;www.graphpad.com)
ImageJ(美国国立卫生研究院;https://imagej.net/Welcome)
奥林巴斯 FluoView 软件 ( https://www.olympus-lifescience.com/en/ )


程序


基于Venus的BiFC表达载体构建。该程序基于我们发表的针对慢病毒Nef等位基因以及 Tec家族激酶Btk和Itk 的BiFC载体工作(Tarafdar等人,2014 年;Li等人,2020 年),但可以轻松适应几乎任何相互作用的蛋白质组合伙伴。虽然我们的方法使用基于p c DNA3 的表达载体来驱动 293T 细胞中的强蛋白质表达,但也可以根据目标细胞类型和应用替换其他载体。其他细节与引物设计,亚克隆方案,并最终融合构建的序列中所提供的附录。
PCR 扩增 Venus N 端(VN:残基 Val 2至 Asp 173 )和 C 端(VC:残基Ala 154至 Lys 238 )的编码序列,其中包含适当的限制性位点和终止密码子,并亚克隆PCR 产物进入哺乳动物表达载体 pcDNA3.1( – )。
以相同的方式PCR 扩增HIV-1 Nef (SF2) 和 SIV Nef (mac239)的编码序列,并将产物亚克隆到pcDNA3.1( – )/VN 构建体中。
PCR-扩增全长人Tec的家族激酶的cDNA克隆含有为适当ligati限制性位点和一个C末端V5表位标签上在帧到所述的pcDNA3.1(- )/ VC构建体。


293T细胞培养和转染
在含有5% CO 2气氛的 37°C 加湿培养箱中,在补充有 10% 胎牛血清的 Dulbecco 改良 Eagle 培养基 (DMEM) 中培养 293T 细胞。使用10米升每10 DMEM -厘米培养皿和维持细胞以2-5的密度× 10 4细胞/ cm 2 。当培养物达到80%confluenc Ë ,取出的通过用1.0M孵育用过的培养基和分裂1:10升0.05%胰蛋白酶-EDTA溶液1在37℃分钟,以产生单细胞悬浮液。加入 10 ml新鲜培养基并以 500 × g旋转5分钟。重悬在10毫升的细胞沉淀升新鲜的培养基和转印1.0米升细胞悬浮液到新10 -厘米培养皿与一个附加10米升新鲜培养基中。对于转染,每个MatTek板接种 2.5 × 10 5 个细胞并培养过夜。
根据制造商的方案,使用 X- tremeGene 9 DNA 转染试剂用BiFC表达载体转染细胞。简而言之,将 3 µl X- tremeGene 9 与 97 µl DMEM 混合,加入每个BiFC表达质粒(每个 VN 和 VC 构建体 0.5 µg),并在室温下孵育混合物 15分钟。孵育后,将转染复合物逐滴添加到MatTek培养板中。根据需要在转染后4小时用抑制剂或 DMSO 载体溶剂(0.1% 终浓度)处理细胞。


免疫荧光染色
转染后40 小时,用 2 ml l 4% 多聚甲醛替换培养基固定细胞,并在室温下孵育 10分钟。洗涤细胞,用2M升PBS(pH为7.4)中5分钟,并偶尔轻轻摇动用手。
用 2 ml 0.2% Triton X-100 在 PBS 中渗透细胞15分钟。用 2.0 ml l PBS洗涤细胞两次,每次5分钟。
用 2 ml 2% BSA 的 PBS室温封闭细胞1小时或 4°C 过夜。
将细胞与抗 V5(激酶标签)和抗Nef或抗pTyr (或抗Btk pY551/抗Itk pY511)抗体在 250 µl PBS 和 2% BSA 中按 1 :1,000 稀释,室温孵育1 小时温度。用 2 ml PBS洗涤细胞 3 次,每次5分钟。
分别以 1:500 或 1:1,000的稀释度,用与德克萨斯红或太平洋蓝偶联的适当二级抗体孵育细胞。用 2 ml PBS洗涤细胞 3 次,每次5分钟。将细胞保存在 2 ml l PBS 中进行成像。
注意:该协议是为 3 色成像开发的,允许同时检测每个相互作用伙伴(激酶和Nef )的相互作用(金星互补)和蛋白质表达,或相互作用、激酶表达和激酶活性(整体细胞磷酸酪氨酸含量或激活环自磷酸化)。


荧光成像和分析
使用多采集图像-色共聚焦显微镜,具有60 ×物镜使用X-Y扫描模式。奥林巴斯FluoView1000,我们使用紫色激光来检测太平洋蓝(405纳米),绿色激光来检测荧光互补(金星; 543纳米),和红色激光来检测德克萨斯ř ED(633纳米)。
执行使用基于Java的图像处理程序,ImageJ的,数据下所述单细胞的图像分析一个nalysis 。


数据分析


作为一个例子,下面的协议使用的ImageJ到孔定量泰特用抗染色的细胞的平均荧光强度(MFI)磷酸酪氨酸(抗的pTyr )和抗Btk的抗体。然后计算最少 100 个细胞的pTyr与Btk MFI的比率,作为细胞群中激酶活性的量度。相同的方法可用于孔定量泰特其它MFI比率,如相互作用(附设标准化为伴侣蛋白表达荧光)。


文件/打开:Ó笔的Btk蛋白表达和共聚焦图像对(图2) 。最小化pTyr图像并且不要再次单击它。单击Btk蛋白质图像窗口。


图形用户界面描述已自动生成

˚F igure染色的293T细胞的2.共聚焦图像的Btk蛋白表达和的pTyr ImageJ中打开。染色细胞的Btk蛋白被示出在左侧(青色),用的pTyr右侧(红色)显示。这些图像来自共表达Btk和 HIV-1 Nef 的细胞。


图像/类型:更改为 8-bit ,这会将图像转换为黑白(图 3 )。


图形用户界面描述已自动生成

˚F igure 3.转化的Btk蛋白图像的8位灰度。在控制栏中,单击图像,然后单击 8 位,这会将图像转换为 8 位灰度图像(右图)。


图像/调整/阈值:通过移动两个滑块设置最小/最大以包括染色但最小化背景和过度曝光来设置级别。选中“深色背景”框。单击“设置”和“确定” 。” 关闭阈值框。红色突出显示的区域显示将包含在分析中的像素(图 4 )。


图形用户界面描述已自动生成

˚F igure 4.设置阈值。单击控制栏中的图像,选择调整,然后选择阈值。将出现左上角的框,将包含在分析中的像素将在图像上以红色显示(右)。点击设置;显示的下方框将出现。单击确定完成。


分析/设置测量:选择这些选项(图 5 ):
面积、最小和最大灰度值以及平均灰度值。
将“重定向到”框设置为“无”。


图形用户界面,应用程序描述已自动生成

˚F igure 5.设置分析参数。从控制栏中,点击分析,然后设置测量;所示的对话框上的左边会出现。选择显示的参数。


分析/分析粒子(图 6 ):
大小:确定将被分析的单元格区域。通常,[20-infinity] 和 [50-infinity] 分别适用于293T 细胞的400 ×和 100 ×图像。
选中“像素单位”框。
圆度:从 [0.0 - 1.0] 开始以排除背景斑点和噪音。
显示:选择“轮廓”选项以显示选定用于测量的区域的位置和大小(通过调整上面的大小/圆度值来控制)。
选中“显示结果”框并取消选中“清除结果”框。
单击“确定” 。”两个额外的窗口将被生成:一(图7 )中的的分析中使用的小区区域的显示轮廓阈值化的图像(左)和第二(右)是结果小号表窗口含MFI数据(‘平均数’列)。
将“结果”窗口中的数据复制到 Excel 文件中,然后仅关闭“结果”窗口。


图形用户界面,应用程序描述已自动生成

˚F igure 6.粒子(像素)分析设置。从控制栏中,选择分析,然后分析粒子,这将打开此框。选择显示的参数。


图形用户界面、应用程序、Word描述已自动生成

˚F igure 7.结果小号颗粒分析。中心图像显示了像素强度已被分析和编号的区域的轮廓。在吨能够在右侧示出了所得到的数据,其包括所分析的特征的数量和它的区域,其次是平均和最小和最大荧光强度。请注意,细胞将被单独、分组或作为片段进行分析,其中包含来自同一细胞的多个片段。


下一步将使用相同的掩码来分析反pTyr图像。确保蛋白质分析的“结果”框已关闭。单击“ Nef + Btk ”蛋白质框以激活它。


分析/设置测量:将“重定向到”框设置为相应的pTyr图像文件名(以前最小化且未修改),同时保持其他参数相同。单击“确定” 。” 为Btk蛋白质图像建立的选定(轮廓)字段现在直接转置到pTyr图像上,并将分析相对于Btk免疫染色提供的相同细胞位置(像素)的pTyr水平。
分析/分析粒子:使用之前的设置并单击“确定” 。” 全选并将结果窗口中的数据复制到 Excel 文件中。Btk蛋白和pTyr分析的结果窗口并排显示在下方(图 8 )。请注意,每个区域的行数相同(54;未显示),用于分析每个区域的区域也相同。然而,MFI 值与预期不同(平均列),因为强度不同。





˚F igure 8.结果小号的的颗粒分析的Btk的pTyr图像。从结果表格的Btk蛋白(从图7中,左)和的pTyr (右)的分析显示侧-通过-侧进行比较。请注意,每个图像中的特征区域是相同的,a s 是特征的数量(每种情况下为 54,未显示)。这两个条件都必须为真,后续计算才有效。


使用 Excel,计算pTyr与Btk平均像素密度(“平均”列)的比率。
每个条件至少分析 100 个细胞,这可能需要多个细胞区域,具体取决于使用的细胞密度、大小和放大倍数。组之间的差异显著可以使用分析的非配对吨-试验(的GraphPad Prism v.8.0)。为了更加严格,对每个实验进行三个生物学重复。
可以使用 Prism 以多种不同方式呈现数据。我们更喜欢将单个“细胞”的结果(实际上来自 ImageJ 识别的单个细胞或细胞片段的掩码)显示为一系列条形图,这给出了细胞群中比率分布的视图。或者,可以使用盒须图或小提琴图。FIGUR ë 9小号怎么样了使用上述方法按照图1所示的结果的ImageJ的分析而获得的比率的分布。 





图9.慢病毒Nef蛋白存在和不存在时Btk自磷酸化的图像分析。Btk激活环自磷酸化(pY551) 和Btk蛋白表达信号的平均荧光强度是使用 ImageJ 和图 1 中显示的数据确定每个条件下≥100 个细胞的。 每个细胞的荧光强度比(pY551:Btk表达) (或由 ImageJ 确定的细胞片段)显示为水平条,红色条表示中间值。学生吨-test显示小号显著增加的Btk的活化环磷酸化在两种HIV-1和SIV的存在的Nef (P <0.0001在每种情况下)。当Btk单独表达时,观察到广泛的pY 551 :Btk比率,因为随着蛋白质量的增加,Btk活性的程度以非线性方式增加。当用 HIV-1 Nef表达时,请注意大多数黑条移到堆栈的顶部,这与几乎所有成像细胞中Nef对Btk的最大激活一致。共用SIV表达Nef的产生更细微的,尽管统计学显著,在移位的pY 551:Btk的比率。在Btk激酶抑制剂依鲁替尼存在的情况下,所有三个群体的比率都会下降。


笔记


不要让菜肴在成像前变干。干燥会严重影响染色并产生假阳性伪影。
用铝箔包裹盘子,保护荧光团免受光照,以避免漂白。
始终包括阳性和阴性BiFC对作为对照,尤其是在开发新蛋白质-蛋白质相互作用的测定时。
293T细胞应保持在一个低的通道编号,以确保细胞的完整性(即,小于10代)。
在整个实验中保持所有共焦图像采集和 ImageJ 分析设置相同。当所有条件保持恒定时,结果应该是可重现的。
在测试新的磷酸化特异性抗体时,在染色过程中滴定一系列抗体浓度以确定最佳浓度。
染色后,细胞以后可以成像2天,并且只要一个星期某些情况下。储存板时,避光并保持在 4°C。
在成像过程中,调整激光功率以避免图像中的信号过饱和,因为这可能会影响后续的图像分析。


致谢


                                          这项工作由美国国立卫生研究院向 TES 提供 AI152677 和 AI057083 资助。这些协议最初由 Li等人报告。(2020) ( doi : 10.1074/jbc.RA120.012536 ) 。以下试剂通过 NIH HIV Reagent Program, Division of AIDS, NIAID, NIH: Anti-Human Immunodeficiency Virus 1 (HIV-1) Nef Monoclonal (6.2), ARP-1539,由 Drs. 凯·克罗恩和弗拉基米尔·奥沃德。


利益争夺


作者没有竞争利益。


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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. Shu, S. T., Li, W. F. and Smithgall, T. E. (2021). Visualization of Host Cell Kinase Activation by Viral Proteins Using GFP Fluorescence Complementation and Immunofluorescence Microscopy. Bio-protocol 11(13): e4068. DOI: 10.21769/BioProtoc.4068.
  2. Li, W. F., Aryal, M., Shu, S. T. and Smithgall, T. E. (2020). HIV-1 Nef dimers short-circuit immune receptor signaling by activating Tec-family kinases at the host cell membrane. J Biol Chem 295(15): 5163-5174.
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