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Sep 2021
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APEX-mediated Proximity Labeling of Proteins in Cells Targeted by Extracellular Vesicles
APEX 介导的细胞外囊泡靶向细胞中蛋白质的邻近标记   

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Abstract

Extracellular vesicles (EVs) are thought to mediate intercellular communication through the delivery of cargo proteins and RNA to target cells. The uptake of EVs is often followed visually using lipophilic-dyes or fluorescently-tagged proteins to label membrane constituents that are then internalized into recipient cells (Christianson et al., 2013; De Jong et al., 2019). However, these methods do not probe the exposure of EV cargo to intracellular compartments, such as the cytoplasm and nucleus, where protein or RNA molecules could elicit functional changes in recipient cells. In this protocol, we employ an EV cargo protein-APEX fusion to detect proximity interactions with recipient cell cytoplasmic/nuclear targets. This approach results in the biotinylation of proteins in close contact with the reporter fusion and thus permits profiling of biotinylated proteins affinity purified on immobilized streptavidin beads.


Graphic abstract:


Schematic showing three steps of APEX-mediated proximity labeling of proteins in cells targeted by EVs.


Keywords: Extracellular vesicles (EVs) (细胞外囊泡(EVs)), APEX Proximity Labeling (APEX近似标记), mESC differentiation (微粒细胞分化), EV uptake ( EV的摄取), Mass Spectrometry (MS) (质谱分析(MS))

Background

Extracellular vesicles (EVs) are secreted by cells, circulate in body fluids, and ultimately generate functional changes through interaction with or uptake into recipient cells (Raposo and Stoorvogel, 2013). Protein, RNA, and possibly DNA are packaged by EVs and may be delivered into target cells to elicit changes in gene expression and cell behavior (Budnik et al., 2016; Van Niel et al., 2018; Shurtleff et al., 2016; Temoche-Diaz et al., 2019; Song et al., 2021). Most of the current methodologies used to visualize EV uptake, however, do not identify the molecular targets of molecules released into recipient cells. An approach to define such targets should be of broad utility.


APEX biotinylation labeling is a newly-developed technique that reveals the subcellular proteomes of many landmarks in the nucleus and cytoplasm (Chen and Perrimon, 2017). Using hydrogen peroxide (H2O2) as an electron donor, the enzyme APEX catalyzes the oxidation of the substrate biotin-phenol (BP) (Hung et al., 2016). The biotin-phenoxyl radical is a highly reactive, short-lived (<1 ms) species that conjugates to other proteins that are proximal to the APEX active site. Biotinylated protein products may then be isolated by streptavidin affinity purification and identified using conventional mass spectrometry (MS) techniques (Lobingier et al., 2017).


We used this approach to investigate the contacts made by the protein cyclinD1 when it is delivered to mouse embryonic cells (mESCs) from EVs produced by differentiating neural progenitor cells (Song et al., 2021). The use of a fusion of APEX to other EV cargo proteins should prove useful to identify molecular contacts within target cells.

Materials and Reagents

  1. Syringe, 10 ml (BD, catalog number: 302995)

  2. Syringe filter (0.45 µm; Thermo Scientific, catalog number: 44525-PP)

  3. Falcon tube (Fisher Scientific, catalog number: 08-771-23)

  4. 35 mm dishes (tissue culture dish) (Corning, catalog number: CLS430165)

  5. 10 cm dishes (tissue culture dish) (Corning, catalog number: CLS430167)

  6. 15 cm dishes (tissue culture dish) (Corning, catalog number: CLS430597)

  7. Open-Top Thinwall Ultra-Clear Tube (38.5 ml), 25 × 89 mm (Beckman Coulter, catalog number: 344058)

    Note: On text "38.5 ml ultra-clear tube".

  8. Open-Top Thinwall Ultra-Clear Tube (13.2 ml), 14 × 89 mm (Beckman Coulter, catalog number: 344059)

    Note: On text "13.2 ml ultra-clear tube".

  9. Open-Top Thinwall Ultra-Clear Tube (5 ml), 13 × 51 mm (Beckman Coulter, catalog number: 344057)

    Note: On text "5 ml ultra-clear tube".

  10. Transfer pipettes (Fisherbrand, catalog number: 13-711-7M)

  11. 293T cells (ATCC, catalog number: CRL-3216)

  12. N2A cells (ATCC, catalog number: CCL-131)

  13. mESCs (R1, ATCC, catalog number: SCRC-1011)

  14. Retinoic acid (RA) (Sigma, catalog number: R2625)

  15. Fetal bovine serum (FBS) (VWR, catalog number: 89510-194)

  16. Exosome-depleted FBS (System Biosciences (SBI), catalog number: EXO-FBS-250A-1)

  17. Puromycin (Sigma-Aldrich, catalog number: P8833-100MG)

  18. pcDNA3 APEX-nes (Addgene, catalog number: 49386)

  19. XPack CMV-XP-MCS-EF1α-Puro Cloning Lentivector (System Biosciences)

  20. psPAX (Addgene, catalog number: 12260)

  21. pMD2.G (Addgene, catalog number: 12259)

  22. Lipofectamine 2000 (Life Technologies, catalog number: 11668019)

  23. OPTI-MEM (Thermo Scientific, catalog number: 31985062)

  24. Sucrose (Fisher Chemical, catalog number: S5-3)

  25. Biotin-phenol (Sigma-Aldrich, catalog number: SML2135)

  26. Protease inhibitor cocktail (100×) (Sigma-Aldrich, catalog number: P8340)

  27. Streptavidin-HRP (Thermo Fisher Scientific; catalog number: 21130)

  28. H2O2 (Thermo Fisher, catalog number: 34062)

  29. Sodium ascorbate (Sigma-Aldrich, catalog number: A7631)

  30. Sodium azide (Sigma-Aldrich, catalog number: 26628-22-8)

  31. Trolox (Sigma-Aldrich, catalog number: 238813-5G)

  32. Biotin (Thermo Fisher Scientific, catalog number: 29129)

  33. DTT (Gold Biotechnology, catalog number: DTT25)

  34. DPBS (Dulbecco's phosphate-buffered saline; Thermo Fisher, catalog number: 14190144)

  35. Streptavidin-agarose beads (Sigma, catalog number: 16-126)

  36. NovexTM WedgeWellTM 10% Tris-glycine mini gels, 10-well (Thermo Fisher, catalog number: XP10200BOX)

  37. Ponceau S (Thermo Fisher Scientific, catalog number: XP00100PK2)

  38. Coomassie G250 (Sigma, catalog number: 1.15444)

  39. DMEM/F12 culture medium (Thermo Fisher Scientific, catalog number: 11320033)

  40. DMEM culture medium (Life Technologies, catalog number: 10566-024)

  41. Neurobasal culture medium (Thermo Fisher Scientific, catalog number: 21103049)

  42. B-27TM Supplement (50×) (Thermo Fisher Scientific, catalog number: 17504044)

  43. N-2 Supplement (100×) (Thermo Fisher Scientific, catalog number: 17502048)

  44. L-glutamine (Thermo Fisher Scientific, catalog number: 25030081)

  45. Non-essential amino acids (100×) (Thermo Fisher Scientific, catalog number: 11140050)

  46. β-mercaptoethanol (0.1 M) (Sigma, catalog number: M3148)

  47. DMEM + 10% FBS (see Recipes)

  48. RA (10 μM) + 1% exosome-depleted FBS in DMEM (see Recipes)

  49. Buffer C (see Recipes)

  50. Quencher solution (see Recipes)

  51. 4× Loading buffer (20 ml) (see Recipes)

  52. N2B27 medium (1 L) (see Recipes)

  53. RIPA (see Recipes)

  54. Buffer D (see Recipes)

  55. TBS-T (see Recipes)

Equipment

  1. Sorvall RC 6+ centrifuge (Thermo Scientific, model: 46910)

  2. Fixed angle rotor F14S-6X250y FiberLite (Thermo Scientific, catalog number: 78500)

  3. Ultracentrifuge (Beckman Coulter, model: Optima XE-90, catalog number: A94471)

  4. Swinging-bucket rotor SW 32 Ti and bucket set (Beckman Coulter, catalog number: 369694)

  5. Swinging-bucket rotor SW 28 Ti and bucket set (Beckman Coulter, catalog number: 342204)

  6. Swinging-bucket rotor SW 55 Ti and bucket set (Beckman Coulter, catalog number: 342194)

  7. Swinging-bucket rotor SW 41 Ti and bucket set (Beckman Coulter, catalog number: 331362)

  8. ChemiDoc MP Imaging System (Bio-Rad Laboratories, model: ChemiDoc MP 10)

  9. NanoSight NS300 instrument equipped with a 405-nm laser (Malvern Instruments, Malvern, United Kingdom)

  10. Refractometer (Fisher Scientific)

  11. Bath sonicator (Covaris, model: S220)

Software

  1. Nanosight NTA 3.1 software (Malvern Instruments)

  2. Excel (Microsoft, 2016)

  3. GraphPad Prism 7

  4. ImageLab software v4.0

  5. PEAKS Studio X+

  6. Vsn R package

Procedure

Part I: EV purification


  1. Plasmid construction

    1. Clone PCR fragments of cyclin D1 and APEX from pcDNA3 APEX-nes into the XPack CMV-XP-MCS-EF1α-Puro Cloning Lentivector.

      1. PCR cyclinD1 from the cDNA of mESCs by using the following primers:

        CyclinD1-F: acgGGATCCCATGGAACACCAGCTCCTG

        CyclinD1-R: acgGAATTCGATGTCCACATCTCGCACG

      2. Insert cyclinD1 PCR fragments into pcDNA3 APEX-nes through BamHI and EcoRI to get pcDNA3 cyclinD1-APEX-nes plasmid.

      3. PCR cyclinD1-Flag-APEX from the above vector by using the following primers:

        acgCTCGAGTATGGAACACCAGCTCCTG

        acgGGATCCtGATGTCCACATCTCGCACG

      4. Insert cyclinD1-Flag-APEX PCR fragments into XPack CMV-XP-MCS-EF1α-Puro through XhoI and BamHI to get XPack CMV-XP-cyclinD1-Flag-APEX -EF1α-Puro plasmid.

    2. Confirm vectors by using Sanger DNA sequencing (UC Berkeley DNA sequencing facility).


  2. Lentivirus package and transfection

    1. Vector transfection

      1. Seed 293T cells at early passage (P1 in our experiments) on 10 cm culture dish and culture to get 60-70% confluency.

      2. Mix 6 µg of transfer vector (XPack CMV-XP-cyclinD1-APEX-EF1α-Puro), 3.9 µg of psPAX, and 2.1 µg of pMD2.G in 0.5 ml of OPTI-MEM (total DNA 12 µg).

      3. In another tube, mix 36 µl of lipofectamine in 0.5 ml of OPTI-MEM.

      4. Mix the DNA and the lipofectamine, and incubate at room temperature for 20 min.

      5. Change the culture medium into 5 ml OPTI-MEM.

      6. Add the DNA and liposome mixture (1 ml) to the culture dish.

      7. After 8 h, remove OPTI-MEM media and discard into 100% bleach.

      8. Incubate cells in fresh growth medium (see Recipe 1) for 2 days.

    2. Harvest virus

      1. Centrifuge the medium at 1,000 × g for 5 min at 4°C.

      2. Filter the medium, which has the cyclinD1-APEX lentivirus, slowly through a 0.45 µm filter into a 15 ml falcon tube.

    3. Transduce 50% confluency N2A cells with lentivirus

      1. Add 1.5 ml cyclinD1-APEX lentivirus to N2A cells in 35 mm dish and culture for 24 h.

      2. Replace medium with growth medium (see Recipe 1) containing 5 µg/ml puromycin.

      3. Culture cells in 5 µg/ml puromycin for 4 days to select N2A cells stably expressing cyclinD1-APEX.


  3. Cell culture and differentiation

    1. Cell culture

      Culture cyclinD1-APEX stably expressing N2A cells in four 15 cm dishes in 30 ml cell culture growth medium (see Recipe 1) until cells reach 80% confluency (the cell number is ~3 × 107cells per dish).

      Note: The surface available for cell growth in these dishes is 151.9 sq. cm. The total volume of cell culture growth medium used for these dishes is 30 ml per plate.

    2. Cell differentiation

      1. Split cells in the four 15 cm dishes to a total of fourteen 15 cm dishes, each containing approximately 8 × 106 cells, and incubate in 30 ml of RA (10 µM) containing medium with 1% exosome-depleted FBS (see Recipe 2).

        Note: The maximum volume of medium that can be processed in the steps below is 420 ml at each time point.

      2. Incubate cells for 6 days. Feed the cells with RA-containing medium (see Recipe 2) every 3 days. Collect medium afterward at day 6.


  4. Collect conditioned medium and sediment EVs

    1. Collect the conditioned medium from the fourteen 15 cm plates into separate containers.

      Note: Approximately 420 ml of total condition medium should be collected.

    2. Centrifuge conditioned medium at 1,000 × g for 15 min at 4°C using Sorvall RC 6+ centrifuge with a fixed rotor of F14S-6X250y FiberLite (or equivalent centrifuge) in 250 ml tube to remove floating cells (Low Spin Speed).

    3. Decant the supernatant fraction into a new container immediately after the centrifuge finishes.

      Note: It is important to remember not to disturb the pellet.

    4. Use a Sorvall RC 6+ Centrifuge (with F14S-6X250y FiberLite fixed angle rotor) to centrifuge the supernatant at 10,000 × g for 15 min at 4°C and sediment large EVs and cellular debris (medium speed spin).

    5. After the centrifuge stops, immediately but carefully decant the supernatant fraction into a new container.

      Note: It is important to remember not to disturb the pellet.

    6. Transfer 32 ml of the collected supernatant into one 38.5 ml ultra-clear tube. Keep transferring until twelve 38.5 ml ultra-clear tubes are filled.

    7. Use SW-28 and SW32 Ti rotors at ~100,000 × g (28,000 RPM) for 1.5 h at 4°C at maximum acceleration/brake to centrifuge.

    8. Gently remove supernatant and resuspend the pellet fraction by pipetting two to three times in 200 µl of phosphate buffered saline (PBS, pH 7.4).

    9. Pipette the resuspended pellet fraction into a new 5 ml ultra-clear tube.

      Note: After combining the pellet fractions from 12 tubes, ~3 ml of resuspend pellet may be collected.

    10. Dilute resuspension from the above step with PBS to a total volume of ~4.8 ml.

    11. Use SW-55 rotor to centrifuge sample at approximately 150,000 × g (38,500 RPM) for 1 h at 4°C and with maximum acceleration and brake.

    12. Gently remove the supernatant. Add 100 µl of PBS into the tube and incubate at room temperature for 30 min.

    13. Resuspend pellet by gently pipetting.

    14. Add 900 µl of 60% sucrose. Mix thoroughly until a homogenous suspension is made.

      Note: Sucrose is dissolved in buffer C (see Recipe 3). Use the refractometer to measure the sucrose concentration. The read of the refractometer should be 60.

    15. First, carefully add 2 ml of 40% sucrose and then carefully place 1 ml of 20% sucrose on top. Add the sucrose medium very slowly and smoothly to avoid disturbing the layer between the cushion. Three separate layers of the different densities of sucrose should be apparent.

      Note: This step requires extreme caution. Dispense sucrose slowly to the side of the clear tube, and let it trickle down the wall of the tube.

    16. Use the SW-55 rotor to centrifuge sample at approximately 150,000 × g (38,500 RPM) for 16 h, at 4°C and with minimum acceleration with no brake.

      Note: It is important to have no brake during deceleration. Having the brake on can cause disruption of the gradient.

    17. Once the centrifugation stops, aspirate ~0.8 ml of supernatant from top to bottom. Collect the 20%/40% sucrose interface (Figure 1) (the total volume at this step is ~1 ml) into a new 5 ml ultra-clear tube.

      Note: Be careful not to disrupt the second gradient of the 40%/60% sucrose interface.



      Figure 1. EVs at 20%/40% interface after overnight centrifugation


    18. Dilute up to ~4.8 ml of PBS in the 5 ml ultra-clear tube.

    19. Centrifuge at ~150,000 × g (38,500 RPM) for 1 h in an SW-55 rotator at 4°C and with maximum acceleration and deceleration.

    20. Gently decant the supernatant fraction and add 100 µl of PBS into the tube to incubate at room temperature for 30 min.

    21. Resuspend the EV pellet by gently pipetting.


  5. EV measurement by NTA

    1. Estimate EV sizes and quantities using the NanoSight NS300 instrument equipped with a 405-nm laser; analyze the data in the scatter mode.

    2. Dilute collected vesicles as described above (D21) at 1,000× with filtered PBS.

    3. Introduce samples into the chamber automatically, at a constant flow rate during five repeats of 60-s captures, and at camera level 13 in scatter mode with Nanosight NTA 3.1 software.

    4. Estimate the EV size at the detection threshold using the Nanosight NTA 3.1 software, after which export “experiment summary” and “particle data” (Figure 2).



      Figure 2. Nanoparticle tracking analysis of the size distribution and the number of purified EVs from the 420-ml medium of N2A cells treated with RA for 6 days.


    5. Calculate the particle numbers in each size category from the particle data, and pool, bin, and count “true” particles with track length >3 with Excel.

      Note: ~5 × 108 to ~2.5 × 109 per 1 µl particle number is normally counted in step D21 EV sample.


    Part II: APEX biotinylated labeling in EV

    1. Incubate biotin-phenol (500 μM) with ~1 × 1010 purified EVs (see Step D21 in Part I) for 30 min at 37°C in a total mixture volume <50 μl in an Eppendorf tube. Prepare two samples at this step.

      Note: Keep in a small volume to ensure there is less of the biotin-phenol residual after the quencher solution wash.

    2. Transfer the mixture into two 13.2 ml ultra-clear tubes.

    3. Initiate APEX labeling by adding 1 mM (0.003%) H2O2 in one tube.

      Note: For the other tube, perform the same treatment as follows, but do not add H2O2. This serves as a negative control.

    4. After 1 min, add 12 ml quencher solution (see Recipe 4) immediately.

    5. Centrifuge at ~110,000 × g (31,500 RPM) for 1 h in an SW-41 rotor and 4°C with maximum acceleration and brake.

    6. Use quencher solution (see Recipe 4) to sediment and wash EVs once. Once stopped, aspirate the supernatant and add 12 ml quencher solution (see Recipe 4).

    7. Centrifuge at ~110,000 × g (31,500 RPM) for 1 h in an SW-41 rotor at 4°C and with maximum acceleration and deceleration.

    8. Gently decant the supernatant fraction. Add 40 µl of PBS into the tube and incubate at room temperature for 30 min.

    9. Resuspend the EV pellet fraction in 40 µl PBS by gently pipetting.

    10. Transfer the two EV samples into new Eppendorf tubes.

    11. Add 4× SDS-loading buffer (see Recipe 5) to prepare samples for SDS PAGE and streptavidin-HRP blotting.

      1. Incubate samples at 95°C for 10 min.

      2. Load 15 µl of samples per lane in a 10% Tris-glycine mini gels.

      3. Run gels at a constant voltage of 150 V for 1 h.

      4. Transfer proteins from gels onto a PVDF membrane with constant amps of 0.6 A for 1.5 h.

      5. Block membrane(s) with 5% BSA in TBST overnight in the cold room (4°C).

      6. Incubate membrane(s) with the Streptavidin-HRP (1:10,000) for 1 h at room temperature.

      7. Wash three times with TBST for 8 min of each wash.

      8. Add the HRP substrate following the manufacturer's specifications. Develop signal using Chemidoc Imaging System.

        Note: The remaining EVs can be used for the following steps after proving that the APEX biotinylated labeling works well in EVs.


Part III: APEX biotinylated labeling in cells

  1. APEX reaction in receipt mES cells

    1. Prepare two 35 mm dishes of ES cells at a density of 4 × 104-5 × 104/cm2.

    2. Incubate cylinD1-APEX EVs with mESCs in N2B27 medium (see Recipe 6) for 2 days at a concentration of ~5 × 109 EV/ml medium.

    3. Pre-warm N2B27 medium (see Recipe 6) at 37°C for 30 min and then add 2 μl of the biotin-phenol stock solution (500 mM) into 2 ml of N2B27 medium to make a 500 μM biotin-phenol solution.

    4. Incubate the above solution with cells for 30 min at 37°C.

    5. Immediately prior to use, dilute 1 mM (0.003%) H2O2 into the medium for each dish for a 1-min labeling reaction at room temperature. Use the other dish without H2O2 as a negative control.

    6. Quench the reaction by immediately washing cells thoroughly with room temperature quencher solution three times (see Recipe 4).

      Note: Ensure that the washes are performed using the quencher solution instead of purely DPB.

    7. Lyse cells in 1 ml RIPA (see Recipe 7) supplemented with 10 mM sodium ascorbate, 1 mM sodium azide, and 1× protease inhibitors.

    8. Sonicate cell lysate in a bath sonicator for 10 min on ice and then centrifuge at 10,000 × g for 10 min at 4°C.

    9. Add 40 μl of concentrated streptavidin-agarose beads to the supernatant fraction (800 μl), and then rotate the mixture overnight at 4°C.

      Note: Wash 40 μl of streptavidin-agarose beads once with 1 ml of RIPA.

    10. Centrifuge above sample at 3,000 × g for 10 min at 4°C. Discard the supernatant fraction.

      Note: Ensure all the following buffers and samples are kept on ice.

    11. Add 1 ml of RIPA buffer (see Recipe 7) to streptavidin-agarose beads and rotate the mixture at 4°C for 10 min.

    12. Centrifuge above sample at 3,000 × g for 10 min at 4°C. Discard the supernatant fraction.

      Note: The process from 10 to 11 is named the beads wash in the following steps.

    13. Wash beads once again by RIPA buffer (see Recipe 7).

    14. Wash beads once with buffer D (see Recipe 8).

    15. Elute biotinylated proteins from the beads by heating the sample in a 4× SDS-loading buffer (see Recipe 5) supplemented with 2 mM biotin and 20 mM DTT for 10 min at 95°C.

    16. Load 15 μl of sample per lane in 10% Tris-glycine mini gels. Run at a constant voltage of 150 V for 1 h.

    17. Ponceau S stain gels at room temperature for 3 min (Figure 3).



      Figure 3. Streptavidin-HRP blotting after cyclinD1-APEX EV incubation with mESC


      Note: Treat EV pre-incubation mESC with biotin-phenol with H2O2 (B+H) or not (B). Detect biotinylated protein by blotting with streptavidin-horseradish peroxidase (SA-HRP). Ponceau S staining (left of panel) of the same membrane serves as a loading control.

    18. Wash gel three times with ddH2O.

    19. Evaluate biotinylated proteins by blotting with streptavidin-HRP (Figure 3).

      Note: The same procedure in Part II, Step 11 from d to h.

  2. Mass Spectrometry (MS) analysis

    1. Electrophorese heated samples from Part III, Step A16 in a 10% Tris-glycine mini gel for ~3 min.

    2. Stain the proteins with Coomassie and excise stained bands from the gel with a fresh razor blade.

    3. Submit samples to a Spectrometry Facility (we used the Taplin Mass at Harvard Medical School) for in-gel tryptic digestion of proteins followed by liquid chromatography and mass spectrometry analysis according to their standards.

    4. MS data analysis

      1. After raw MS data is acquired, perform peptide identification, quantification, and filtering on the Peaks Studio X+ platform with the default settings via the Proteome Discoverer database.

      2. Implement Variance stabilization normalization (Vsn) with the justvsn function from the R/Bioconductor-package Vsn to normalize the quantified protein data.

      3. Perform T-test analyses to examine significant changes in protein abundances between two different groups.

Data analysis

  1. Export immunoblot images using ImageLab software v4.0 as .tif format.

  2. Use ImageJ to open and process immunoblot images, including rotating, cropping, adjusting brightness, and contrast when necessary.

  3. Prepare figures with GraphPad Prism or preferred program (Figure 2).

Recipes

  1. DMEM + 10% FBS (500 ml)

    50 ml FBS

    450 ml DMEM

  2. RA (10 μM) + 1% exosome-depleted FBS in DMEM (500 ml)

    5 ml exosome-depleted FBS

    RA (10 μM)

    Add DMEM to 500 ml

  3. Buffer C

    20 mM Tris-HCl pH 7.4

    137 mM NaCl

  4. Quencher solution

    10 mM sodium ascorbate

    5 mM Trolox

    10 mM sodium azide

    In DPBS (Dulbecco's phosphate-buffered saline)

  5. 4× Loading buffer (20 ml)

    Dissolve 1.6 g of SDS in 6 ml of ddH2O

    Add 5 ml of 1 M Tris-HCl pH 6.8

    Add 1.23 g of DTT. Dissolve

    Add 8 mg of bromophenol blue

    Add 8 ml of glycerol

    Add ddH2O to 20 ml

    Make 1 ml aliquots. Store at -20°C

  6. N2B27 medium (1 L)

    475 ml DMEM/F12

    475 ml Neurobasal

    20 ml B27

    10 ml N2

    10 ml L-glutamine (200mM)

    10 ml non-essential amino acids (100×)

    1 ml 0.1 M β-mercaptoethanol

  7. RIPA

    50 mM Tris, pH 7.4

    150 mM NaCl

    1% Triton X-100

    0.5% deoxycholate

    0.1% SDS

    1 mM Trolox

    1 mM DTT

  8. Buffer D

    1 M KCl

    0.1 M Na2CO3

    2 M urea

    10 mM Tris-HCl, (pH 7.4)

  9. TBS-T

    0.1% Tween 20 (v/v) in 1× TBS

Acknowledgments

We thank the technical suggestion from Prof. Alice Ting’s lab. We thank current and former Schekman lab members who provided technical advice in the development of this protocol. We acknowledge the Howard Hughes Medical Institute for funding. We also acknowledge UC Berkeley Tang family scholarship. This protocol has been adapted from Song et al. (2021; Doi: 10.1083/jcb.202101075).

Competing interests

The authors declare no competing interests.

References

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  11. Van Niel, G., D'Angelo, G. and Raposo, G. (2018). Shedding light on the cell biology of extracellular vesicles. Nat Rev Mol Cell Biol 19(4): 213-228.

简介

[摘要]细胞外囊泡 (EVs) 被认为通过将货物蛋白和 RNA 递送至靶细胞来介导细胞间通讯。通常使用亲脂性染料或荧光标记蛋白来标记膜成分,然后将膜成分内化到受体细胞中(Christianson等人,2013 年;De Jong等人,2013 年)。., 2019)。然而,这些方法并没有探测 EV 货物暴露于细胞内隔室,例如细胞质和细胞核,在那里蛋白质或 RNA 分子可以引起受体细胞的功能变化。在该协议中,我们采用 EV 货物蛋白-APEX 融合来检测与受体细胞质/核目标的近距离相互作用。这种方法导致与报告基因融合密切接触的蛋白质的生物素化,从而允许对在固定化链霉亲和素珠上纯化的生物素化蛋白质进行亲和分析。

图文摘要:


示意图显示了 APEX 介导的 EV 靶向细胞中蛋白质的邻近标记的三个步骤。



[背景]细胞外囊泡 (EV) 由细胞分泌,在体液中循环,并最终通过与受体细胞的相互作用或摄取产生功能变化(Raposo 和 Stoorvogel,2013)。蛋白质、RNA 和可能的 DNA 由 EV 包装,并可能被递送到靶细胞中以引起基因表达和细胞行为的变化(Budnik等人,2016 年;Van Niel等人,2018 年;Shurtleff等人,2016 年; Temoche-Diaz等人,2019 年;Song等人,2021 年)。然而,目前大多数用于可视化 EV 摄取的方法并没有识别释放到受体细胞中的分子的分子靶点。定义此类目标的方法应该具有广泛的用途。
APEX 生物素化标记是一项新开发的技术,可揭示细胞核和细胞质中许多标志物的亚细胞蛋白质组(Chen 和 Perrimon,2017)。使用过氧化氢 (H 2 O 2 ) 作为电子供体,APEX 酶催化底物生物素-苯酚 (BP) 的氧化(Hung等,2016)。生物素-苯氧基自由基是一种高活性、短寿命 (<1 ms) 的物质,可与靠近 APEX 活性位点的其他蛋白质结合。然后可以通过链霉亲和纯化分离生物素化蛋白质产物,并使用常规质谱 (MS) 技术进行鉴定(Lobingier等,2017)。
我们使用这种方法来研究蛋白质 cyclinD1 在从神经祖细胞分化产生的 EV 递送至小鼠胚胎细胞 (mESC) 时产生的接触(Song等人,2021)。使用 APEX 与其他 EV 货物蛋白的融合应该证明有助于识别靶细胞内的分子接触。

关键字:细胞外囊泡(EVs), APEX近似标记, 微粒细胞分化, EV的摄取, 质谱分析(MS)


材料和试剂

 
注射器,10 ml(BD,目录号:302995)
注射器过滤器 (0.45 µm; Thermo Scientific, 目录号:44525-PP)
猎鹰管(Fisher Scientific,目录号:08-771-23)
35 毫米培养皿(组织培养皿)(康宁,目录号:CLS430165)
10厘米培养皿(组织培养皿)(康宁,目录号:CLS430167)
15厘米培养皿(组织培养皿)(康宁,目录号:CLS430597)
开顶薄壁超透明管(38.5 ml),25 × 89 mm(Beckman Coulter,目录号:344058)
注意:在文字“38.5 ml 超清管”上。
开顶薄壁超透明管(13.2 ml),14 × 89 mm(Beckman Coulter,目录号:344059)
注意:在文字“13.2ml 超清管”上。
开顶薄壁超透明管(5 ml),13 × 51 mm(Beckman Coulter,目录号:344057)
注意:在文字“5 ml 超清管”上。
移液管(Fisherbrand,目录号:13-711-7M)
293T 细胞(ATCC,目录号:CRL-3216)
N2A 细胞(ATCC,目录号:CCL-131)
mESC(R1,ATCC,目录号:SCRC-1011)
视黄酸(RA)(Sigma,目录号:R2625)
胎牛血清(FBS)(VWR,目录号:89510-194)
外泌体耗尽的 FBS(System Biosciences(SBI),目录号:EXO-FBS-250A-1)
嘌呤霉素(Sigma-Aldrich,目录号:P8833-100MG)
pcDNA3 APEX-nes(Addgene,目录号:49386)
XPack CMV-XP-MCS-EF1α-Puro 克隆慢病毒载体(System Biosciences)
psPAX(Addgene,目录号:12260)
pMD2.G(Addgene,目录号:12259)
Lipofectamine 2000(Life Technologies,目录号:11668019)
OPTI-MEM(Thermo Scientific,目录号:31985062)
蔗糖(Fisher Chemical,目录号:S5-3)
生物素-苯酚(Sigma-Aldrich,目录号:SML2135)
蛋白酶抑制剂混合物(100×)(Sigma-Aldrich,目录号:P8340)
链霉亲和素-HRP(Thermo Fisher Scientific;目录号:21130)
H 2 O 2 (Thermo Fisher ,目录号:34062)
抗坏血酸钠(Sigma-Aldrich ,目录号:A7631)
叠氮化钠(Sigma-Aldrich ,目录号:26628-22-8)
Trolox(Sigma-Aldrich,目录号:238813-5G)
生物素(Thermo Fisher Scientific,目录号:29129)
DTT(Gold Biotechnology,目录号:DTT25)
DPBS(Dulbecco 磷酸盐缓冲盐水;Thermo Fisher,目录号:14190144)
链霉亲和素-琼脂糖珠(Sigma,目录号:16-126)
Novex TM WedgeWell TM 10% Tris-甘氨酸迷你凝胶,10 孔(Thermo Fisher,目录号:XP10200BOX)
Ponceau S(Thermo Fisher Scientific,目录号:XP00100PK2)
考马斯 G250(Sigma,目录号:1.15444)
DMEM/F12 培养基(Thermo Fisher Scientific,目录号:11320033)
DMEM培养基(Life Technologies,目录号:10566-024)
Neurobasal培养基(Thermo Fisher Scientific,目录号:21103049)
B-27 TM补充剂(50×)(Thermo Fisher Scientific,目录号:17504044) 
N-2补充剂(100×)(Thermo Fisher Scientific,目录号:17502048)
L-谷氨酰胺(Thermo Fisher Scientific,目录号:25030081)
非必需氨基酸(100×)(Thermo Fisher Scientific,目录号:11140050)
β-巯基乙醇(0.1 M)(Sigma,目录号:M3148)
DMEM + 10% FBS(见食谱)
RA (10 μM) + DMEM 中 1% 外泌体耗尽的 FBS(参见食谱)
缓冲液 C(见配方)
淬火溶液(见配方)
4 × 上样缓冲液(20 ml)(见配方)
N2B27 培养基(1 升)(见食谱)
RIPA(见食谱)
缓冲液 D(见配方)
TBS-T(见食谱)
 
设备
 
Sorvall RC 6+ 离心机(Thermo Scientific,型号:46910)
固定角转子 F14S-6X250y FiberLite(Thermo Scientific,目录号:78500)
超速离心机(Beckman Coulter,型号:Optima XE-90,目录号:A94471)
摆斗转子 SW 32 Ti 和铲斗套件(Beckman Coulter,目录号:369694)
摆斗转子 SW 28 Ti 和铲斗组(Beckman Coulter,目录号:342204)
摆斗转子 SW 55 Ti 和铲斗套件(Beckman Coulter,目录号:342194)
摆斗转子 SW 41 Ti 和铲斗组(Beckman Coulter,目录号:331362)
ChemiDoc MP 成像系统(Bio-Rad 实验室,型号:ChemiDoc MP 10)
NanoSight NS300 仪器配备 405-nm 激光器(Malvern Instruments,Malvern,英国)
折光仪(Fisher Scientific)
巴斯超声波仪(Covaris,型号:S220)
 
软件
 
Nanosight NTA 3.1 软件(马尔文仪器)
Excel(微软,2016 年)
GraphPad棱镜7
ImageLab软件 v4.0
PEAKS Studio X+
Vsn R 包
 
程序
 
第一部分:电动汽车净化
 
质粒构建
将 pcDNA3 APEX-nes 的细胞周期蛋白 D1 和 APEX 的 PCR 片段克隆到 XPack CMV-XP-MCS-EF1α-Puro Cloning Lentivector 中。
使用以下引物从 mESCs 的 cDNA 中 PCR cyclinD1 :
CyclinD1-F: acgGGATCCCATGGAACACCAGCTCCTG
CyclinD1-R: acgGAATTCGATGTCCACATCTCGCACG
通过BamHI和EcoRI将cyclinD1 PCR片段插入pcDNA3 APEX-nes,得到pcDNA3 cyclinD1-APEX-nes质粒。
使用以下引物从上述载体 PCR cyclinD1-Flag-APEX:
acgCTCGAGTATGGAACACCAGCTCCTG
acgGGATCCtGATGTCCACATCTCGCACG
将cyclinD1-Flag-APEX PCR片段通过XhoI和BamHI插入XPack CMV-XP-MCS-EF1α-Puro,得到XPack CMV-XP-cyclinD1-Flag-APEX-EF1α-Puro质粒。
使用 Sanger DNA 测序(加州大学伯克利分校 DNA 测序设施)确认载体。
 
慢病毒包装和转染
载体转染
将 293T 细胞在早期传代(我们实验中的 P1)上接种到 10 厘米培养皿并培养以获得 60-70% 的汇合度。 
混合 6 µg 转移载体(XPack CMV-XP-cyclinD1-APEX-EF1α-Puro)、3.9 µg psPAX 和 2.1 µg pMD2.G 在 0.5 ml OPTI-MEM 中(总 DNA 12 µg)。
在另一管中,将 36 µl lipofectamine 混合在 0.5 ml OPTI-MEM 中。
混合 DNA 和 lipofectamine,并在室温下孵育 20 分钟。
将培养基更改为 5 ml OPTI-MEM。
将 DNA 和脂质体混合物 (1 ml) 添加到培养皿中。
8 小时后,取出 OPTI-MEM 介质并丢弃到 100% 漂白剂中。
在新鲜生长培养基(见配方 1)中孵育细胞 2 天。
收获病毒
将培养基在4°C 下以 1,000 × g离心5 分钟。
将含有 cyclinD1-APEX 慢病毒的培养基缓慢通过 0.45 µm 过滤器过滤到 15 ml 猎鹰管中。
用慢病毒转导 50% 汇合 N2A 细胞
将 1.5 ml cyclinD1-APEX 慢病毒添加到 35 mm 培养皿中的 N2A 细胞中并培养 24 小时。
用含有 5 µg/ml 嘌呤霉素的生长培养基(见配方 1)替换培养基。
在 5 µg/ml 嘌呤霉素中培养细胞 4 天,以选择稳定表达 cyclinD1-APEX 的 N2A 细胞。
 
细胞培养和分化
细胞培养
文化 cyclinD1-APEX 在 30 ml 细胞培养生长培养基中的四个 15 cm 培养皿中稳定表达 N2A 细胞(参见配方 1),直到细胞达到 80% 汇合(细胞数为每培养皿约 3 × 10 7 个细胞)。
注意:这些培养皿中可用于细胞生长的表面为 151.9 平方厘米。用于这些培养皿的细胞培养基的总体积为每盘 30 毫升。
细胞分化
将四个 15 cm 培养皿中的细胞拆分为总共 14 个 15 cm 培养皿,每个培养皿包含大约 8 × 10 6 个细胞,并在含有 1% 外泌体耗尽 FBS 的 30 ml RA (10 µM) 培养基中孵育(参见配方 2 )。
注意:每个时间点可在以下步骤中处理的最大培养基体积为 420 毫升。
孵育细胞 6 天。每 3 天用含 RA 的培养基(参见配方 2)喂养细胞。然后在第 6 天收集培养基。
 
收集条件培养基和沉积物 EV
将 14 个 15 厘米板中的条件培养基收集到单独的容器中。
注意:应收集大约 420 毫升的总条件培养基。
使用带有 F14S-6X250y FiberLite 固定转子(或等效离心机)的 Sorvall RC 6+ 离心机在 250 ml 试管中以 1,000 × g在 4°C下以 1,000 × g离心条件培养基15 分钟,以去除漂浮细胞(低转速)。
离心机完成后立即将上清液部分倒入新容器中。
注意:重要的是要记住不要打扰颗粒。
使用 Sorvall RC 6+ 离心机(配备 F14S-6X250y FiberLite 固定角转子)在 4°C 下以 10,000 × g离心上清液15 分钟,并沉淀大 EV 和细胞碎片(中速旋转)。
离心机停止后,立即小心地将上清液倒入新容器中。
注意:重要的是要记住不要打扰颗粒。
将 32 毫升收集的上清液转移到一个 38.5 毫升的超清管中。继续转移,直到装满 12 个 38.5 毫升的超透明管。
使用 SW-28 和 SW32 Ti 转子,以 ~100,000 × g (28,000 RPM) 在 4°C 下以最大加速度/制动离心 1.5 小时。
通过在 200 µl 磷酸盐缓冲盐水(PBS,pH 7.4)中移液 2 到 3 次,轻轻去除上清液并重悬沉淀部分。
将重悬的沉淀部分移入新的 5 ml 超清管中。
注意:合并 12 个管中的颗粒部分后,可以收集约 3 ml 的重悬颗粒。
用 PBS 将上述步骤的重悬液稀释至总体积约为 4.8 ml。
使用 SW-55 转子以大约 150,000 × g (38,500 RPM) 的速度离心样品1 小时 4°C 和最大加速度和制动。
轻轻去除上清液。向管中加入 100 µl PBS,室温孵育 30 分钟。
通过轻轻移液重悬沉淀。
添加 900 µl 60% 蔗糖。彻底混合直到制成均匀的悬浮液。 
注意:蔗糖溶解在缓冲液 C 中(参见配方 3)。使用折光仪测量蔗糖浓度。折射计的读数应为 60。
首先,小心地加入 2 ml 40% 蔗糖,然后小心地将 1 ml 20% 蔗糖放在上面。非常缓慢和平稳地添加蔗糖培养基,以避免干扰垫子之间的层。三个不同密度的蔗糖应该是明显的。
注意:此步骤需要格外小心。将蔗糖慢慢分配到透明管的一侧,让它沿着管壁滴下。
使用 SW-55 转子以大约 150,000 × g (38,500 RPM) 的速度离心样品16 小时,温度为 4°C,且加速度最小,无制动。
注意:减速时不要刹车很重要。刹车可能会导致梯度中断。
一旦离心停止,从上到下吸出 ~0.8 ml 上清液。将 20%/40% 蔗糖界面(图 1)(此步骤的总体积约为 1 毫升)收集到新的 5 毫升超透明管中。
注意:注意不要破坏 40%/60% 蔗糖界面的第二个梯度。
 
 
图 1.过夜离心后 20%/40% 界面处的 EV
 
在 5 ml 超清管中稀释至 4.8 ml 的 PBS。
在SW-55 旋转器中以 ~150,000 × g (38,500 RPM)在 4°C 和最大加速和减速条件下离心1 小时。
轻轻倒出上清液,向管中加入100 µl PBS,室温孵育 30 分钟。
通过轻轻移液重悬 EV 颗粒。
 
NTA EV 测量
使用配备 405 nm 激光器的 NanoSight NS300 仪器估算 EV 尺寸和数量;以分散模式分析数据。
如上所述(D21)用过滤的 PBS 以 1,000 倍稀释收集的囊泡。
使用 Nanosight NTA 3.1 软件在 5 次重复 60 秒捕获期间以恒定流速自动将样品引入腔室,并在散射模式下在相机级别 13 下将样品引入腔室。
使用 Nanosight NTA 3.1 软件估计检测阈值处的 EV 大小,然后导出“实验摘要”和“粒子数据”(图 2)。
 
 
图 2. 用 RA 处理 6 天的 N2A 细胞的 420 毫升培养基的大小分布和纯化 EV 数量的纳米粒子跟踪分析。
 
从粒子数据中计算每个尺寸类别中的粒子数,并使用 Excel 合并、合并和计数轨道长度 >3 的“真实”粒子。
注意:~5 × 10 8到~2.5 × 10 9每 1 µl 粒子数通常在步骤 D21 EV 样本中计数。
 
第二部分:EV 中的 APEX 生物素化标记
 
孵化 生物素-苯酚 (500 μM) 和 ~1 × 10 10纯化的 EV(参见第 I 部分中的步骤 D21),在 Eppendorf 管中总体积 <50 μl 的混合物中,在 37°C 下持续 30 分钟。在这一步准备两个样品。
注意:保持小体积以确保淬灭剂溶液洗涤后生物素-苯酚残留较少。
将混合物转移到两个 13.2 毫升的超透明管中。
通过添加 1 mM 启动 APEX 标记 (0.003%) H 2 O 2在一管中。
注意:对于另一根管子,进行如下相同的处理,但不要添加H 2 O 2 。这用作阴性对照。 
1 分钟后,立即加入 12 毫升淬灭剂溶液(参见配方 4)。
在SW-41 转子和4°C 下以~110,000 × g (31,500 RPM)离心1 小时,最大加速度和制动。
使用猝灭剂溶液(见配方 4)沉淀和清洗 EV 一次。停止后,吸出上清液并加入 12 ml 猝灭剂溶液(参见配方 4)。
在SW-41 转子中以 ~110,000 × g (31,500 RPM) 在4°C 和最大加速和减速条件下离心1 小时。
轻轻倒出上清液部分。向管中加入40 µl PBS,室温孵育 30 分钟。
通过轻轻移液将 EV颗粒部分重悬在 40 µl PBS 中。
将两个 EV 样品转移到新的 Eppendorf 管中。
添加 4 × SDS 上样缓冲液(参见配方 5)以准备用于 SDS PAGE 和链霉亲和素-HRP 印迹的样品。
在 95°C 下孵育样品 10 分钟。
负载15 μ每泳道样品的升在10%Tris-甘氨酸凝胶的迷你。
在 150 V 的恒定电压下运行凝胶 1 小时。
将蛋白质从凝胶转移到 PVDF 膜上,恒定电流为 0.6 A,持续 1.5 小时。
在冷藏室 (4°C) 中,用 TBST 中的 5% BSA 封闭膜过夜。
将膜与链霉亲和素-HRP (1:10,000) 在室温下孵育 1 小时。
用 TBST 洗涤 3 次,每次洗涤 8 分钟。
按照制造商的规格添加 HRP 基材。使用 Chemidoc 成像系统开发信号。
注意:在证明 APEX 生物素化标记在 EV 中运行良好后,剩余的 EV 可用于以下步骤。
 
第三部分:细胞中的 APEX 生物素化标记
 
接收 mES 细胞中的 APEX 反应
准备两个 35 毫米的 ES 细胞培养皿,密度为 4 × 10 4 -5 × 10 4 /cm 2 。
将 cylinD1-APEX EV 与 MESC 在 N2B27 培养基(参见配方 6)中以 ~5 × 10 9 EV/ml 培养基的浓度孵育 2 天.
在 37°C 下预热 N2B27 培养基(参见配方 6)30 分钟,然后加入 2 将微升生物素-苯酚原液 (500 mM) 加入 2 ml N2B27 培养基中,制成 500 μM 生物素-苯酚溶液。
将上述溶液与细胞在 37°C 下孵育 30 分钟。
临用前,稀释 1 mM (0.003%) H 2 O 2进入每道菜的培养基中,在室温下进行 1 分钟的标记反应。使用没有 H 2 O 2的另一道菜作为阴性对照。
立即用室温淬灭剂溶液彻底清洗细胞 3 次以淬灭反应(参见配方 4)。
注意:确保使用淬灭剂溶液而不是纯 DPB 进行洗涤。
在补充有 10 mM 抗坏血酸钠、1 mM 叠氮化钠和 1× 蛋白酶抑制剂的 1 ml RIPA(参见配方 7)中裂解细胞。
在冰浴超声仪中对细胞裂解物进行超声处理 10 分钟,然后在 4°C 下以 10,000 × g离心10 分钟。
添加 40 微升 将浓缩的链霉亲和素琼脂糖珠加入上清液部分 (800 μl),然后将混合物在 4°C 下旋转过夜。
注意:用 1 ml RIPA清洗 40 μl链霉亲和素琼脂糖珠一次。
在 4°C 下以 3,000 × g离心上述样品10 分钟。丢弃上清液部分。
注意:确保以下所有缓冲液和样品都保存在冰上。
向链霉亲和素-琼脂糖珠中加入 1 ml RIPA 缓冲液(参见配方 7),并在 4°C 下旋转混合物 10 分钟。
在 4°C 下以 3,000 × g离心上述样品10 分钟。丢弃上清液部分。
注意:10 到 11 的过程在以下步骤中称为珠子洗涤。
用 RIPA 缓冲液再次清洗珠子(参见配方 7)。
用缓冲液 D 清洗珠子一次(参见配方 8)。
通过在补充有 2 mM 生物素和 20 mM DTT 的 4× SDS 加载缓冲液(参见配方 5)中在 95°C 下加热样品 10 分钟,从珠子上洗脱生物素化蛋白质。
负载15 μ每泳道样品的升在10%Tris-甘氨酸凝胶的迷你。在 150 V 的恒定电压下运行 1 小时。
Ponceau S 染色凝胶在室温下 3 分钟(图 3)。
 
 
图 3. cyclinD1-APEX EV 与 mESC 孵育后的链霉亲和素-HRP 印迹
 
注意:用 H 2 O 2 (B+H) 或不 (B)用生物素-苯酚处理 EV 预孵化 mESC 。通过使用链霉亲和素-辣根过氧化物酶 (SA-HRP) 进行印迹检测生物素化蛋白。同一膜的 Ponceau S 染色(面板左侧)用作加载控制。
 
用 ddH 2 O清洗凝胶 3 次。
通过使用链霉亲和素-HRP 进行印迹评估生物素化蛋白质(图 3)。
注意:与第二部分,步骤 11 从 d 到 h 中的程序相同。
 
质谱 (MS) 分析
将第 III 部分步骤 A16 中的样品在 10% Tris-甘氨酸微型凝胶中电泳加热约 3 分钟。
用考马斯染色蛋白质,用新鲜的刀片从凝胶上切下染色带。
将样品提交到光谱分析设施(我们使用哈佛医学院的 Taplin Mass)进行蛋白质的凝胶内胰蛋白酶消化,然后根据其标准进行液相色谱和质谱分析。
质谱数据分析
获取原始 MS 数据后,通过 Proteome Discoverer 数据库使用默认设置在 Peaks Studio X+ 平台上执行肽识别、定量和过滤。
使用 R/Bioconductor-package Vsn 中的 justvsn 函数实现方差稳定归一化 (Vsn),以归一化量化的蛋白质数据。
执行 T 检验分析以检查两个不同组之间蛋白质丰度的显着变化。
 
数据分析
 
使用 ImageLab 软件 v4.0 将免疫印迹图像导出为 .tif 格式。
使用 ImageJ 打开和处理免疫印迹图像,包括在必要时旋转、裁剪、调整亮度和对比度。
使用 GraphPad Prism 或首选程序准备数字(图 2)。
 
食谱
 
DMEM + 10% FBS(500 毫升)
50 毫升 FBS
450 毫升 DMEM
RA (10 μM) + DMEM 中的 1% 外泌体耗尽 FBS(500 ml)
5 ml 去除外泌体的 FBS
RA (10 μM)
添加DMEM至 500 毫升
缓冲液 C
20 mM Tris-HCl pH 7.4
137 毫米氯化钠
淬火液
10 mM 抗坏血酸钠
5 mM Trolox
10 mM 叠氮化钠
在 DPBS(Dulbecco 磷酸盐缓冲盐水)中
4 × 上样缓冲液(20 毫升)
将 1.6 g SDS 溶解在 6 ml ddH 2 O 中
添加 5 ml 1 M Tris-HCl pH 6.8
添加 1.23 克 DTT。溶解
加入8毫克溴酚蓝
加入8毫升甘油
添加 ddH 2 O 到 20 毫升
制作 1 ml 等分试样。储存在-20°C
N2B27 培养基(1 升)
475 毫升 DMEM/F12
475 毫升
20 毫升 B27
10毫升氮气
10 毫升 L-谷氨酰胺 (200 mM)
10毫升非必需氨基酸(100×)
1 ml 0.1 M β-巯基乙醇
RIPA
50 mM Tris,pH 7.4
150 毫米氯化钠
1% 海卫 X-100
0.5% 脱氧胆酸盐
0.1% SDS
1 mM Trolox
1 毫米 DTT
缓冲器 D
1 M氯化钾
0.1 M Na 2 CO 3
2 摩尔尿素
10 mM Tris-HCl,(pH 7.4)
TBS-T
0.1% 吐温 20 (v/v) 在 1× TBS 中
 
致谢
 
我们感谢 Alice Ting 教授实验室的技术建议。我们感谢现任和前任 Schekman 实验室成员,他们在本协议的开发过程中提供了技术建议。我们感谢霍华德休斯医学研究所的资助。我们也承认加州大学伯克利分校唐家的奖学金。该协议改编自宋等人。(2021 年;Doi:10.1083/jcb.202101075)。
 
利益争夺
 
作者声明没有竞争利益。
 
参考
 
Budnik, V.、Ruiz-Canada, C. 和 Wendler, F.(2016 年)。细胞外囊泡完善了神经系统中的交流。 Nat Rev Neurosci 17(3): 160-172。
Christianson, HC, Svensson, KJ, van Kuppevelt, TH, Li, JP 和 Belting, M. (2013)。癌细胞外泌体依赖于细胞表面硫酸乙酰肝素蛋白聚糖的内化和功能活性。 Proc Natl Acad Sci USA 110(43): 17380-17385。
Chen, CL 和 Perrimon, N. (2017)。用于活细胞蛋白质组学分析的邻近依赖性标记方法。 Wiley Interdiscip Rev Dev Biol 6(4)。             
De Jong, OG, Kooijmans, SAA, Murphy, DE, Jiang, L., Evers, MJW, Sluijter, JPG, Vader, P. 和 Schiffelers, RM (2019)。细胞外囊泡给药:从想象到创新。Acc Chem Res 52(7): 1761-1770。
Hung, V., Udeshi, ND, Lam, SS, Loh, KH, Cox, KJ, Pedram, K., Carr, SA 和 Ting, AY (2016)。使用工程化过氧化物酶 APEX2 在活细胞中空间解析蛋白质组图谱。国家议定书 11(3): 456-475。              
Lobingier, BT, Hüttenhain, R., Eichel, K., Miller, KB, Ting, AY, von Zastrow, M. 和 Krogan, NJ (2017)。时空解析活细胞中蛋白质相互作用网络的方法。单元格169(2):350-360.e312。              
Raposo, G. 和 Stoorvogel, W.(2013 年)。细胞外囊泡:外泌体、微囊泡和朋友。J Cell Biol 200(4): 373-383。
Shurtleff, MJ, Temoche-Diaz, MM, Karfilis, KV, Ri, S. 和 Schekman, R. (2016)。Y-box 蛋白 1 需要在细胞和无细胞反应中将 microRNA 分选为外泌体。Elife 5:e19276。
Song, L.、Tian, X. 和 Schekman, R. (2021)。来自神经元细胞的细胞外囊泡通过 cyclinD1 促进 mESCs 的神经诱导。J Cell Biol 220(9):e202101075。              
Temoche-Diaz, MM, Shurtleff, MJ, Nottingham, RM, Yao, J., Fadadu, RP, Lambowitz, AM 和 Schekman, R. (2019)。microRNA 分选到癌细胞衍生的细胞外囊泡亚型的不同机制。 Elife 8:e47544。
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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Song, L., Chen, J., Sun, A. and Schekman, R. (2021). APEX-mediated Proximity Labeling of Proteins in Cells Targeted by Extracellular Vesicles. Bio-protocol 11(21): e4213. DOI: 10.21769/BioProtoc.4213.
  2. Song, L., Tian, X. and Schekman, R. (2021). Extracellular vesicles from neuronal cells promote neural induction of mESCs through cyclinD1. J Cell Biol 220(9): e202101075.
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