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Jan 2020

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Preparation of Doublet Microtubule Fraction for Single Particle Cryo-electron Microscopy
单粒子低温电子显微镜双态微管馏分的制备   

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Abstract

Over the years, studying the ultrastructure of the eukaryotic cilia/flagella using electron microscopy (EM) has contributed significantly toward our understanding of ciliary function. Major complexes in the cilia, such as inner and outer dynein arms, radial spokes, and dynein regulatory complexes, were originally discovered by EM. Classical resin-embedding EM or cryo-electron tomography can be performed directly on the isolated cilia or in some cases, cilia directly attached to the cell body. Recently, single particle cryo-EM has emerged as a powerful structural technique to elucidate high-resolution structures of macromolecular complexes; however, single particle cryo-EM requires non-overlapping complexes, i.e., the doublet microtubule of the cilia. Here, we present a protocol to separate the doublet microtubule from the isolated cilia bundle of two species, Tetrahymena thermophila and Chlamydomonas reinhardtii, using ATP reactivation and sonication. Our approach produces good distribution and random orientation of the doublet microtubule fragments, which is suitable for single particle cryo-EM analysis.

Keywords: Tetrahymena thermophila (嗜热四膜虫), Chlamydomonas reinhardtii (莱茵衣藻), Cilia (纤毛), Flagella (鞭毛), Doublet microtubule (偶极微管), Cryo-EM (冷冻电镜), Mass spectrometry (质谱分析法)

Background

Cilia are hairlike organelles that exist on the surface of cells and are responsible for motility and sensory functions. Cilia consist of a bundle of nine doublet microtubules surrounding the two central singlet microtubules in the case of motile cilia. Cilia comprise hundreds of unique protein molecules. Due to this complexity, the structural biology of cilia mainly relies on isolation of the intact cilia (Craige et al., 2013; Gaertig et al., 2013). In the last 15 years, cryo-electron tomography aided by subtomogram averaging has been a major force in pushing the molecular architecture of the intact eukaryotic cilia (Bui et al., 2008; Heuser et al., 2009; Imhof et al., 2019). However, the resolution of cryo-electron tomography is typically in the range of 20-40 Å, which limits the ability to create precise atomic models of the proteins in the cilia or interactions between different proteins for the mechanistic understanding of its function. Recently, advances in the single particle cryo-electron microscopy (cryo-EM) technique have allowed high-resolution structures of macromolecular complexes typically in the range of 3-4 Å; however, single particle cryo-EM does not work with the entire cilia due to the overlapping of the doublet microtubules. Our method of doublet microtubule purification and fractionation for cryo-EM overcomes this limitation by splitting the doublet microtubules out of the cilia. This is facilitated by reactivation of dyneins using ATP, sonication, and salt extraction (Figure 1), which produces well-separated fragments of the doublet microtubules suitable for single-particle cryo-EM (Ichikawa et al., 2017) (Figure 2). In fact, this allows the reconstruction of the doublet microtubule structures of both Chlamydomonas and Tetrahymena to 3-4 Å resolution and the visualization of the microtubule proteins inside the doublet (Ichikawa et al., 2019; Khalifa et al., 2020). In addition, these doublet microtubule purification protocols are suitable for proteomics analyses of the central pairs and microtubule inner proteins because they enrich those proteins during the salt wash of all the outer and inner dynein arm components (Dai et al., 2020).



Figure 1. The workflow of the sample preparation for cryo-EM



Figure 2. Cryo-EM images of the Tetrahymena (A) and Chlamydomonas (B) doublet fragments. Scale bar: 50 nm.

Materials and Reagents

  1. Tetrahymena doublet purification

    1. 24-well cell culture plate (Millipore Sigma, catalog number: CLS3527)

    2. SnakeSkin Dialysis Tubing, 10K MWCO, 22 mm (Thermo Fisher Scientific, catalog number: 68100)

    3. Tetrahymena thermophila cells (Tetrahymena Stock Center, SB255 mucocyst-free strain)

      Note: Our protocol works best with the SB255 strain rather than strains with mucocyst.

    4. Proteose Peptone No. 3 (Thermo Fisher Scientific, catalog number: 211693)

    5. Glucose (Research Products International, catalog number: G32040)

    6. Yeast Extract (Thermo Fisher Scientific, catalog number: 211929)

    7. Ethylenediaminetetraacetic acid iron (III) sodium salt, Fe-EDTA (Millipore Sigma, catalog number: EDFS)

    8. Dibucaine Hydrochloride (Millipore Sigma, catalog number: D0638)

    9. 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES]

    10. MgSO4 (Thermo Fisher Scientific, catalog number: AC447165000)

    11. Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid, EGTA (Millipore Sigma, catalog number: E4378)

    12. Dithiothreitol, DTT (Millipore Sigma, catalog number: 11583786001)

    13. Sucrose (Research Products International, catalog number: S24060)

    14. Trehalose (Research Products International, catalog number: T82000)

    15. Phenylmethylsulfonyl fluoride [PMSF] (Millipore Sigma, catalog number: 52332)

    16. 10% NP-40 Alternative (Millipore Sigma, catalog number: 492016)

    17. 40 mM Adenosine triphosphate [ATP] (Millipore Sigma, catalog number: A2383)

    18. SPP Liquid Media (see Recipes)

    19. 25 mg/ml Dibucaine (see Recipes)

    20. Cilia Wash Buffer (see Recipes)

    21. Cilia Final Buffer (see Recipes)

    22. Dialysis Buffer (see Recipes)


  2. Chlamydomonas doublet purification

    1. SnakeSkin Dialysis Tubing, 10K MWCO, 22 mm (Thermo Fisher Scientific, catalog number: 68100)

    2. Wild type Chlamydomonas Cells (Chlamydomonas resource center, CC-124 wild type mt-[137c])

      Note: Our protocol also works with other Chlamydomonas strains with flagella or some strains that do not grow flagella under normal culture conditions.

    3. Tris base (BioShop, catalog number: TRS001.5)

    4. NH4Cl (BioShop, catalog number: AMC303.1)

    5. MgSO4 (BioShop, catalog number: MAG522.1)

    6. CaCl2·2H2O (BioShop, catalog number: CCL302.1)

    7. K2HPO4 (BioShop, catalog number: PPD303.1)

    8. KH2PO4 (BioShop, catalog number: PPM666.1)

    9. Potassium Hydroxide, KOH (Thermo Fisher Scientific, catalog number: P2501)

    10. Acetic Acid, CH3COOH (Thermo Fisher Scientific, catalog number: FLA38212)

    11. Sodium Chloride, NaCl (BioShop, catalog number: SOD001.5)

    12. Aprotinin (Millipore Sigma, catalog number: A1153)

    13. Leupeptin (Millipore Sigma, catalog number: L2884)

    14. Potassium acetate (BioShop, catalog number: POA303.5)

    15. Polyethylene glycol, MW 20,000 (Millipore Sigma, catalog number: 817018)

    16. Paclitaxel (Millipore Sigma, catalog number: T7402)

    17. PMSF (Millipore Sigma, catalog number: 52332)

    18. 10% NP-40 Alternative (Millipore Sigma, catalog number: 492016)

    19. 100 mM Adenosine di-phosphate [ADP] (Millipore Sigma, catalog number: A2754)

    20. 10 mM ATP (Millipore Sigma, catalog number: A2383)

    21. Tris-acetatephophate (TAP) salt solution (see Recipes)

    22. Phosphate solution (see Recipes)

    23. TAP liquid media (see Recipes)

    24. 500 mM Potassium Hydroxide (KOH) (see Recipes)

    25. 500 mM Acetic Acid (CH3COOH) (see Recipes)

    26. 3 M Sodium Chloride (3 M NaCl) (see Recipes)

    27. HMDS solution (see Recipes)

    28. HMDEKP solution (see Recipes)

Equipment

  1. Tetrahymena doublet purification

    1. 250-ml Erlenmeyer flask (Millipore Sigma, catalog number: CLS4980250)

    2. 500-ml Erlenmeyer flask

    3. Floor shaker (Thermo Scientific, model: MAXQ8000)

    4. Spectrophotometer (Thermo Scientific, model: 840-208100 UV/Vis)

    5. Floor centrifuge (Beckman Coulter, model: Avanti J-20 XP, Rotors JLA-8.1 and JA25.5)

    6. Tabletop centrifuge (Thermo Scientific, model: Sorvall ST 16R, Rotor 75003181)

    7. Microfuge (Eppendorf, model: Centrifuge 5415 D, Rotor F45-24-11)

    8. pH meter (Hanna HI 2210 Benchtop pH/Temperature Meter)

    9. Sonicator (Fisher Scientific Sonic Dismembrator Model 100)


  2. Chlamydomonas doublet purification

    1. Floor shaker (Thermo Scientific, model: MAXQ8000)

    2. Spectrophotometer (Thermo Scientific, model: 840-208100 UV/Vis)

    3. Floor centrifuge (Beckman Coulter, model: Avanti J-20 XP, Rotor JLA-8.1, Rotor JA25.5)

    4. Tabletop centrifuge (Thermo Scientific, model: Sorvall ST 16R, Rotor 75003181)

    5. Microcentrifuge (Eppendorf, model: Centrifuge 5415 D, Rotor F45-24-11)

    6. pH meter (Hanna HI 2210 Benchtop pH/Temperature Meter)

    7. Sonicator (Fisher Scientific Sonic Dismembrator Model 100)

Procedure

  1. Tetrahymena doublet purification

    1. Growth of Tetrahymena cells for isolation

      1. Tetrahymena cells (SB255 or CU-428) are stored in bean media (Williams et al., 1980).

      2. Transfer 50 μl bean media into 1 ml SPP media in a 24-well cell culture plate. Culture at room temperature (RT) for 4-5 days until the cells reach a density of 1.6 × 106 cells/ml.

        Note: Observe the health of the cells under a light microscope before Step A1c.

      3. Transfer 40 μl saturated Tetrahymena cells to 40 ml liquid SPP media in a 250-ml Erlenmeyer flask and grow for approximately one week at RT.

        Note: 40 μl saturated Tetrahymena cells can be passaged to another 40 ml liquid SPP media in a 500-ml Erlenmeyer flask and kept for about one week at RT or for several weeks at 15-16°C.

      4. Transfer 2 ml saturated cells from the 40 ml RT culture into 100 ml liquid SPP media and grow overnight with shaking at 150 rpm and 30°C in the MAXQ8000 shaker incubator (Figure 3A).

      5. Add the entire 100 ml overnight culture to 1 L liquid SPP media and grow for approximately 2 days with shaking at 150 rpm and 30°C (MAXQ8000). The optimal OD600 is 0.7.

      6. Incubate cells at 15°C with shaking for 30 min to 1 h (Figure 3B).

    2. Cilia isolation by dibucaine treatment

      1. Divide the entire cell culture into 2 or 4 equal volumes and pour into appropriate centrifuge tubes. Centrifuge at 700 × g for 10 min at 4°C with slow deceleration using the Avanti, Rotor JLA-8.1.

        Note: After this step, keep the cells at 4°C or on ice to minimize the secretion of mucus.

      2. Resuspend pellet containing cells with 10 ml ice-cold SPP media and then adjust the total volume of solution to 24 ml.

      3. Transfer the 24 ml resuspended cells into a 250-ml Erlenmeyer flask on ice (Figure 3C).

      4. Have the 1 ml dibucaine (25 mg/ml) and 75 ml ice-cold liquid SPP media ready.

      5. Quickly add the 1 ml dibucaine to the 24 ml resuspended cells (final 1 mg/ml dibucaine) and swirl the flask for exactly 1 min.

      6. Quickly add 75 ml ice-cold liquid SPP media to stop the reaction and transfer the whole cilia solution into two centrifuge tubes for the Beckman Coulter JA25.5 rotor.

      7. Centrifuge the entire cilia suspension at 2,000 × g for 10 min at 4°C with slow deceleration (Avanti, Rotor JA25.5) (Figure 3D).

      8. Using a pipet gun, carefully aspirate the cilia-containing supernatant without disturbing the cellular debris and mucus. Divide the supernatant into 4 equal volumes in centrifuge tubes for the Beckman Coulter JA25.5 rotor, approximately 20 ml per centrifuge tube.

      9. Centrifuge the cilia suspension at 17,000 × g for 40 min at 4°C with slow deceleration using the Avanti, Rotor JA25.5 (Figure 3E).

      10. Remove the supernatant and gently wash away the transparent layer of mucus around the cilia pellet using a pipet and 100 μl volumes of ice-cold Cilia Wash Buffer. When the cilia pellet is clean, resuspend it with 250 μl ice-cold Cilia Wash Buffer for each centrifuge tube and resuspend the cilia pellet (total 1 ml).

      11. Transfer the cilia solution into two 1.5-ml microcentrifuge tubes (~500 μl each) and centrifuge the cilia suspension at 7,800 × g for 10 min at 4°C in a microfuge (Eppendorf, Centrifuge 5415 D).

      12. Remove the supernatant and resuspend the cilia pellet in 250 μl cilia wash buffer (Figure 3F).

        Note: The cilia pellet can be snap frozen here with liquid nitrogen and stored at -80°C, but the splitting works best with cilia without freezing.



      Figure 3. Purification of Tetrahymena cilia with dibucaine. Images depict Tetrahymena cell cultures (A-B), cell suspension before dibucaine treatment (C), pellet and supernatant after dibucaine treatment (D), and cilia pellet before (E) and after final washing (F).


    3. Purification of doublet microtubule fraction

      1. Resuspend the cilia pellet to 250 μl with Cilia Final Buffer for each tube.

        Note: From here, the amounts are for each tube.

      2. Add 44.1 μl 10% NP-40 alternative (final concentration of 1.5% NP-40) and resuspend the total solution. Incubate on ice for 30 min to de-membrane the cilia.

      3. Centrifuge at 7,800 × g for 10 min at 4°C in a microfuge (Eppendorf, Centrifuge 5415 D).

      4. Remove the supernatant and resuspend the pellet to 247 μl with Cilia Final Buffer.

      5. Add 2.5 μl 40 mM ATP and incubate for 10 min at RT for the axoneme to split apart.

      6. Centrifuge at 16,000 × g for 10 min at 4°C in a microcentrifuge (Eppendorf, Centrifuge 5415 D).

      7. Remove the supernatant and resuspend the doublet microtubule pellet to 250 μl with Cilia Final Buffer.

      8. Add 62.5 μl 3 M NaCl to a final concentration of 0.6 M NaCl and incubate on ice for 30 min to remove dyneins.

      9. Centrifuge at 16,000 × g for 10 min at 4°C in the microcentrifuge (Eppendorf, Centrifuge 5415 D).

      10. Remove the supernatant and resuspend the pellet to 250 μl with Cilia Final Buffer.

      11. Repeat Steps h-j.

      12. 250 μl doublet fractions are applied to the dialysis membrane and dialyzed against 200 ml Dialysis buffer overnight at 4°C with stirring to deplete radial spokes.

      13. Collect sample from dialysis tube and centrifuge at 16,000 × g for 10 min at 4°C in the microcentrifuge (Eppendorf, Centrifuge 5415 D).

      14. Remove the supernatant and resuspend the pellet to 250 μl with Cilia Final Buffer.

        Note: After dialysis, the purified doublet microtubule fraction with all the outside proteins is removed.

    4. Sonication of the doublet microtubule for cryo-EM

      1. Sonication conditions: power 4, 10 s (sample in 1.5-ml microtube on ice).

        Note: Slightly move the probe inside the solution while sonicating.

      2. Centrifuge at 2,000 × g for 10 min at 4°C in the microcentrifuge (Eppendorf, Centrifuge 5415 D)

        Note: This step only precipitates aggregated doublet fragments and depolymerized tubulin will stay in the supernatant.

      3. Resuspend the doublet microtubule pellet to 25 μl with Cilia Final Buffer (supplemented with 0.6 M NaCl).

        Note: 0.6 M NaCl helps the dissociation of aggregated doublet fragments.

      4. Quantitate protein concentration (i.e., Bradford assay with spectrophotometer), then adjust the volume to the required concentration for application. For cryo-EM, 4 mg/ml sonicated Tetrahymena doublet fragments is appropriate. For mass spectrometry, 1 mg/ml sonicated doublet fragment is appropriate.


  2. Chlamydomonas doublet purification

    1. Growth of Chlamydomonas cells for isolation

      1. Chlamydomonas cells are struck onto TAP solid plates containing 1.5% agar for storage.

      2. Grow cells on TAP solid plates on a 12 h alternating light and dark cycle for approximately two weeks at RT.

      3. Scrape off approximately 3-5 mm of cells and transfer to 50 ml liquid TAP media then grow for one week with shaking or stirring conditions on 12 h alternating light and dark cycles at RT (Figure 4A).

        Note: After one week, observe the health of the cells under a light microscope before Step B1d.

      4. Remove 10 ml of the 50 ml liquid culture, transfer it to 1 L TAP media, and grow with shaking or stirring conditions and 12 h alternating light and dark cycle at RT for approximately 4-6 days until the OD600 reaches 0.5-0.6 (Figure 4B).

    2. Flagella growth

      Note: This step is optional and useful for Chlamydomonas mutant strains that do not grow flagella under normal culture conditions.

      1. Centrifuge the 1 L Chlamydomonas culture at 700 × g for 7 min at 4°C using the Avanti, Rotor JLA-8.1.

      2. Resuspend the pellet in 50 ml deionized water vigorously using a pipette gun and transfer into a 50-ml conical tube.

        Note: Pipet up and down 10 times to separate the cells.

      3. Transfer 50 ml resuspended cells into 1 L deionized water (with a stir bar) and wrap the entire flask with aluminum to prevent contact with light.

      4. Place on a stirring or shaking platform and leave to incubate for 1-2 h.

      5. Observe the cells under a light microscope after the incubation period to see if Chlamydomonas cells are swimming.

    3. Flagella isolation by pH shock

      Note: We use pH shock because it gives us a cleaner doublet fraction with less protein contaminants, making it more suitable for mass spectrometry. The dibucaine method described for Tetrahymena can also be used.

      1. Centrifuge the 1 L cell culture at 700 × g for 7 min at 4°C using the Avanti, Rotor JLA-8.1 (Figure 4C).

        Note: From here, keep the samples on ice or 4°C.

      2. Resuspend the pellet containing cells in 3-5 ml HMDS solution and transfer into a 50-ml conical tube.

      3. Adjust the total volume of solution to 15 ml using HMDS solution (with a small stir bar).

      4. Adjust the pH of the solution to 4.5 with acetic acid and wait for 1 min using a pH probe with stirring.

      5. Quickly adjust the pH of the solution to 7.5 with 0.5 M KOH and remove the stir bar.

      6. Centrifuge the pH-shocked solution at 1,800 × g at 5 min at 4°C (Sorvall ST 16R, Rotor 75003181) with a deceleration power of 4 to remove the cell bodies (Figure 4D).

      7. After centrifugation, the isolated flagella will be in the supernatant. Carefully transfer the supernatant to a new 50-ml conical tube.

        Note: Avoid any debris or pellet.

      8. Centrifuge the supernatant at 4,700 × g for 40 min at 4°C (Sorvall ST 16R, Rotor 75003181) with a deceleration power of 7 (Figure 4E).

      9. The flagella are now pelleted. Remove the supernatant and resuspend the pellet in 500 μl HMDEKP solution.

      10. Transfer the flagella solution into a 1.5-ml microcentrifuge tube and centrifuge at 7,800 × g for 10 min at 4°C in the Eppendorf Centrifuge 5415 D (Figure 4F).

        Note: The green color of the pellet is from leakage of cell debris due to harsh dibucaine treatment or when working with some cell wall-less CLiP mutants (Chlamydomonas resource center). After the flagella are treated with NP-40 alternative and subsequently pelleted, the green color will not be present.

      11. Remove the supernatant.

        Note: Whole flagella pellet can be snap frozen here with liquid nitrogen and stored in the -80°C; however, splitting of doublet works better with flagella without freezing and storage.



      Figure 4. Purification of Chlamydomonas cilia with pH shock. Images depict Chlamydomonas cell cultures (A-B), centrifugation of cell culture (C), centrifugation after pH shock (D), and flagella pellet before (E) and after washing (F).


    4. Purification of doublet microtubule fraction

      1. Resuspend the flagella pellet to 250 μl with HMDEKP.

      2. Add 44.1 μl 10% NP-40 alternative (final concentration of 1.5% NP-40) and resuspend the total solution. Incubate on ice for 30 min to get rid of the flagella membrane.

      3. Centrifuge at 7,800 × g for 10 min at 4°C in a microcentrifuge (Eppendorf, Centrifuge 5415 D).

      4. Remove the supernatant and resuspend the pellet to 250 μl with HMDEKP.

      5. For cryo-EM, sonicate the flagella solution. Conditions specific to the Fisher Scientific Sonic Dismembrator Model 100: Small probe (1 ml volume), power 4, 10 s on ice.

        Note: Slightly move the probe inside the solution while sonicating.

        For Chlamydomonas doublet, sonication was done at an earlier step since it was harder to split.

      6. Centrifuge at 2,000 × g for 1 min at 4°C in the microcentrifuge (Eppendorf, Centrifuge 5415 D).

        Note: This step only precipitates aggregated doublets but leaves tubulins in the supernatant.

      7. Resuspend the doublet microtubule pellet to 250 μl with HMDEKP solution.

      8. Add 2.5 μl 100 mM ADP to a final concentration of 1 mM ADP and incubate for 10 min at RT.

        Note: This step is done only for Chlamydomonas doublet splitting to activate dyneins since it was harder to split.

      9. Add 2.5 μl 10 mM ATP to a final concentration of 0.1 mM ATP and incubate for another 10 min at RT for the doublet to split away from the flagella.

        Note: Concentration of ATP is lower in the Chlamydomonas protocol to get higher activity of dyneins. We did not add protease for splitting since we found that the addition of elastase affects the structure of MIPs.

      10. Centrifuge at 16,000 × g for 10 min at 4°C in a microcentrifuge (Eppendorf, Centrifuge 5415 D).

      11. Remove the supernatant and resuspend the pellet to 250 μl with HMDEKP.

      12. Add 62.5 μl 3 M NaCl (final concentration of 0.6 M NaCl) and incubate on ice for 30 min.

      13. Centrifuge at 16,000 × g for 10 min at 4°C in the microcentrifuge (Eppendorf, Centrifuge 5415 D).

      14. Remove the supernatant and resuspend the pellet to 250 μl with HMDEKP buffer.

      15. Repeat Steps B4l to B4n once more.

      16. After the second salt wash, the final pellet will contain the purified doublet microtubule fraction with most of the outside proteins removed except for radial spokes.

        Note: We tried several different conditions to remove radial spokes from Chlamydomonas doublets, but we were unable to remove radial spokes and keep Chlamydomonas doublet microtubules intact.

      17. Quantitate protein concentration (i.e., Bradford assay with spectrophotometer), then adjust the volume to the required concentration for application. For cryo-EM, 4 mg/ml sonicated doublet fragments is appropriate. For mass spectrometry, 1 mg/ml sonicated doublet fragment is appropriate.

Notes

In cryo-EM, obtained doublet microtubule fragments take random orientations in vitrifies ice compared with non-sonicated doublet microtubules (Figure 2). Tetrahymena doublet microtubule structure obtained with this procedure retained most of the MIPs except for inner junction (IJ) filament (FAP20/PACRG). In contrast, Chlamydomonas doublet structure retained IJ filament but some of the MIPs inside the A-tubule were lost (Ichikawa et al., 2017 and 2019; Khalifa et al., 2020).

Recipes

  1. SPP Liquid Media (Gorovsky et al., 1975)

    For 1 L media:

    1% Proteose Peptone No. 3, 10 g

    0.2% Glucose, 2 g

    0.1% Yeast Extract, 1 g

    0.003% Ethylenediaminetetraacetic acid iron (III) sodium salt [Fe-EDTA], 0.03 g

    Make up to 1 L with Milli-Q water and autoclave

  2. 25 mg/ml Dibucaine

    For 1 ml:

    Dibucaine Hydrochloride 0.025 g

    Make up to 1 ml with SPP Liquid Media

  3. Cilia Wash Buffer

    50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES], pH 7.4

    3 mM MgSO4

    0.1 mM Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid [EGTA]

    1 mM dithiothreitol (DTT) (add fresh)

    250 mM Sucrose

  4. Cilia Final Buffer

    50 mM HEPES, pH 7.4

    3 mM MgSO4

    0.1 mM EGTA

    1 mM DTT (add fresh)

    0.5% Trehalose

    1 mM PMSF (add fresh)

  5. Dialysis Buffer

    5 mM HEPES, pH 7.4

    1 mM DTT (add fresh)

    0.5 mM EDTA

  6. Tris-acetatephophate (TAP) salt solution

    For 1 L:

    NH4Cl, 15 g

    MgSO4, 1.95 g

    CaCl2·2H2O, 2 g

    Make up to 1 L with Milli-Q water

  7. Phosphate solution

    For 100 ml:

    K2HPO4, 28.8 g

    KH2PO4,14.4 g

    Make up to 100 ml with Milli-Q water

  8. TAP liquid media (Gorman and Levine, 1965)

    For 1 L media:

    Tris base, 2.42 g

    TAP salt solution, 25 ml

    Phosphate solution, 0.375 ml

    Hutner's trace elements (Chlamydomonas resource center),1.0 ml

    Glacial acetic acid, 1.0 ml

    Make up to 1 L with Milli-Q water and autoclave

    Note: For TAP solid media, add 20 g Agar to this recipe.

  9. 500 mM Potassium Hydroxide (KOH)

  10. 500 mM Acetic Acid (CH3COOH)

  11. 3 M Sodium Chloride (3 M NaCl)

  12. HMDS solution

    10 mM HEPES, pH 7.4

    5 mM MgSO4

    1 mM DTT (add fresh)

    4% sucrose

    10 μg/ml aprotinin (add fresh)

    5 μg/ml leupeptin (add fresh)

  13. HMDEKP solution

    30 mM HEPES, pH 7.4

    5 mM MgSO4

    1 mM DTT (add fresh)

    0.5 mM EGTA

    25 mM potassium acetate

    0.5% polyethylene glycol (MW 20,000)

    10 μM paclitaxel (add fresh)

    1 mM PMSF (add fresh)

    10 μg/ml aprotinin (add fresh)

    5 μg/ml leupeptin (add fresh)

    Note: Paclitaxel was added to the HMDEKP buffer for Chlamydomonas doublet preparation since it was less stable than the Tetrahymena doublet.

Acknowledgments

This research was financially supported by the Natural Sciences and Engineering Research Council of Canada (RGPIN-2016-04954), Canada Institute of Health Research (CIHR PJT-156354), and the Canada Institute for Advanced Research Arzieli Global Scholars Program to K.H.B. MI was supported by JST, PRESTO Grant Number JPMJPR20E1, JSPS KAKENHI Grant Numbers JP19K23726 and JP20K15733, and the Foundation for Nara Institute of Science and Technology (R2290001). This protocol was adapted with minor modification from previous study published by Khalifa et al. (2020).

Competing interests

The authors declare no conflicts of interests.

References

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  8. Ichikawa, M., Khalifa, A. A. Z., Kubo, S., Dai, D., Basu, K., Maghrebi, M. A. F., Vargas, J. and Bui, K. H. (2019). Tubulin lattice in cilia is in a stressed form regulated by microtubule inner proteins. Proc Natl Acad Sci U S A 116(40):19930-19938.
  9. Ichikawa, M., Liu, D., Kastritis, P. L., Basu, K., Hsu, T. C., Yang, S. and Bui, K. H. (2017). Subnanometre-resolution structure of the doublet microtubule reveals new classes of microtubule-associated proteins. Nat Commun 8: 15035. 
  10. Imhof, S., Zhang, J., Wang, H., Bui, K. H., Nguyen, H., Atanasov, I., Hui, W. H., Yang, S. K., Zhou, Z. H. and Hill, K. L. (2019). Cryo electron tomography with volta phase plate reveals novel structural foundations of the 96-nm axonemal repeat in the pathogen Trypanosoma brucei. Elife 8: e52058. 
  11. Khalifa, A. A. Z., Ichikawa, M., Dai, D., Kubo, S., Black, C. S., Peri, K., McAlear, T. S., Veyron, S., Yang, S. K., Vargas, J., Bechstedt, S., Trempe, J. F. and Bui, K. H. (2020). The inner junction complex of the cilia is an interaction hub that involves tubulin post-translational modifications. Elife 9: e52760. 
  12. Williams, N. E., Wolfe, J. and Bleyman, L. K. (1980). Long-term maintenance of Tetrahymena spp. J Protozool 27(3): 327.

简介

[摘要]多年来,使用电子显微镜(EM)研究真核纤毛/鞭毛的超微结构为我们对睫毛功能的理解做出了重要贡献。在纤毛主要络合物,如内,外动力蛋白臂,径向轮辐,而动力蛋白调控复合物,是最初由EM发现。可以在分离的纤毛上直接进行经典的树脂包埋EM或冷冻电子断层扫描,或者在某些情况下,可以直接将纤毛直接附着在细胞体上。最近,单粒子冷冻电镜已成为一个结构的强有力的技术,以阐明高分辨率结构小号大分子复合物; ħ H但是,单粒子冷冻电镜需要非重叠的复合物,即,纤毛的双峰微管。在这里,我们提出了一个协议到双峰微管从两个物种的分离的纤毛束分离,四膜虫嗜热和衣藻,使用ATP激活和超声处理。我们的方法可以产生良好的双峰微管片段分布和随机取向,适用于单颗粒冷冻-EM分析。


[背景]纤毛是存在的细胞的表面上的毛发状细胞器,并且负责运动和感觉功能。纤毛由一束9个双态微管组成,在活动性纤毛的情况下,纤毛围绕着两个中央单态微管。纤毛包含数百种独特的蛋白质分子。由于这种复杂性,纤毛的结构生物学主要依赖于完整纤毛的分离(Craige等,2013; Gaertig等,2013)。在过去的15年中,借助子图平均技术进行的冷冻电子断层扫描一直是推动完整的真核纤毛分子结构的主要力量(Bui等人,2008; Heuser等人,2009; Imhof等人,2019 )。然而,低温电子断层扫描的分辨率通常被设定在范围内的20 -40埃,这限制的能力创建的纤毛蛋白的精确原子模型或相互作用小号对于其功能的机制理解不同蛋白质之间。最近,在前进的单粒子低温电子显微镜(低温EM)技术已允许编高-分辨率结构小号大分子复合物典型地在3-4埃范围内的; ħ H但是,单粒子冷冻电镜不与整个工作纤毛由于重叠的二重峰的微管。我们针对冷冻EM的双峰微管纯化和分级分离方法通过将双峰微管从纤毛中分离出来,克服了这一局限性。这促进了通过再活化动力蛋白利用ATP,超声处理,和盐提取(图1) ,这产生良好-适用于单粒子冷冻电镜的双重微管的分离的片段(市川等人,2017)(图2) 。实际上,这允许将衣藻和四膜虫的双胞胎微管结构重建到3-4Å分辨率,并使双胞胎内的微管蛋白可视化(Ichikawa等人,2019年; Khalifa等人,2020年)。此外,这些双峰微管纯化方案适合于蛋白质组学analys Ë中心对和微管内的蛋白质是因为它们所有的外表面和内动力蛋白臂组件的盐洗涤过程中富集的那些蛋白质(戴等人。,20 20)。


图1.cryo-EM样品制备的工作流程


图2.四膜虫(A)和衣藻(B)双峰片段的Cryo-EM图像。比例尺:50 nm。

关键字:嗜热四膜虫, 莱茵衣藻, 纤毛, 鞭毛, 偶极微管, 冷冻电镜, 质谱分析法



材料和试剂


四膜虫双重净化
24孔细胞培养板(Millipore Sigma,目录号:CLS3527)
SnakeSkin透析管,10K MWCO,22毫米(Thermo Fisher Scientific ,目录号:68100)
四膜虫嗜热细胞(四膜虫保藏中心,SB255无粘液囊菌株)
注:我们的协议效果最好的SB255应变而不是株mucocyst 。


3号蛋白P(Thermo Fisher Scientific,目录号211693)
葡萄糖(国际研究产品,目录号:G32040)
酵母提取物(赛默飞世尔科技,目录号:211929)             
乙二胺四乙酸铁(III)钠盐,Fe-EDTA(Millipore Sigma,目录号:EDFS)
盐酸地布卡因(Millipore Sigma,目录号:D0638 )
4-(2-羟乙基)-1-哌嗪乙烷磺酸[HEPES]
MgSO 4 (Thermo Fisher Scientific,目录号:AC447165000)
乙二醇-双(2-氨基乙基醚)-N,N,N',N'-四乙酸,EGTA (Millipore Sigma,目录号:E4378)
二硫苏糖醇,DTT(Millipore Sigma,目录号:11583786001)
蔗糖(国际研究产品,目录号:S24060)
海藻糖(国际研究产品,目录号:T82000)
苯甲基磺酰氟[PMSF](Millipore Sigma,目录号:52332)
              10%NP-40替代品(Millipore Sigma,目录号:492016)
40 mM三磷酸腺苷[ATP](Millipore Sigma,目录号:A2383)
SPP液体介质(请参阅配方)
25毫克/毫升地布卡因(请参阅食谱)
纤毛洗涤缓冲液(请参阅食谱)
              纤毛最终缓冲液(请参见食谱)
透析缓冲液(请参见配方)


衣藻双胞胎纯化
SnakeSkin透析管,10K MWCO,22毫米(Thermo Fisher Scientific ,目录号:68100)
野生型衣藻细胞(衣藻资源中心,CC-124野生型mt- [137c])
注:我们的协议也可以与其他具有鞭毛衣菌株或某些菌株是不长鞭毛下正常培养条件小号。


Tris base (BioShop ,目录号:TRS001.5 )                           
NH 4 Cl(BioShop ,目录号:AMC303.1 )
MgSO 4 (BioShop ,目录号:MAG522.1 )
CaCl 2 · 2H 2 O(BioShop ,目录号:CCL302.1 )
K 2 HPO 4 (BioShop ,目录号:PPD303.1 )
KH 2 PO 4 (BioShop ,目录号:PPM666.1 )
氢氧化钾,KOH (Thermo Fisher Scientific,目录号:P2501)
乙酸,CH 3 COOH (Thermo Fisher Scientific,目录号:FLA38212)
氯化钠NaCl (BioShop ,目录号:SOD001.5 )
抑肽酶(Millipore Sigma,目录号:A1153)
Leupeptin(Millipore Sigma,目录号:L2884)
醋酸钾(BioShop ,目录号:POA303.5 )
聚乙二醇,分子量20,000(Millipore Sigma,目录号:817018)
紫杉醇(Millipore Sigma,目录号:T7402)
PMSF(Millipore Sigma,目录号:52332)
10%NP-40替代品(Millipore Sigma,目录号:492016)
100 mM二磷酸腺苷[ADP](Millipore Sigma,目录号:A2754)
10 mM ATP(Millipore Sigma,目录号:A2383)
磷酸三乙酸盐(TAP)盐溶液(请参阅食谱)
磷酸盐溶液(请参阅食谱)
TAP液体介质(请参阅配方)
500 mM氢氧化钾(KOH)(请参阅食谱)
500 mM乙酸(CH 3 COOH)(请参阅食谱)
3 M氯化钠(3 M NaCl)(请参阅食谱)
HMDS解决方案(请参阅食谱)
HMDEKP解决方案(请参阅食谱)


设备


四膜虫双重净化
250 ml锥形瓶(Millipore Sigma,目录号:CLS4980250)
500 -毫升三角瓶
振动筛(Thermo Scientific,型号:MAXQ8000)
分光光度计(Thermo Scientific,型号:840-208100 UV / Vis)
落地式离心机(贝克曼库尔特(Beckman Coulter),型号:Avanti J-20 XP,转子JLA-8.1和JA25.5)
台式离心机(Thermo Scientific,型号:Sorvall ST 16R,转子75003181)
微量离心机(Eppendorf,型号:Centrifuge 5415 D,转子F45-24-11)
pH计(Hanna HI 2210台式pH /温度计)
声波发生器(Fisher Scientific声波分解器100型)


衣藻双t纯化
振动筛(Thermo Scientific,型号:MAXQ8000)
分光光度计(Thermo Scientific,型号:840-208100 UV / Vis)
落地式离心机(贝克曼库尔特(Beckman Coulter),型号:Avanti J-20 XP,JLA-8.1转子,JA25.5转子)
台式离心机(Thermo Scientific,型号:Sorvall ST 16R,转子75003181)
微量离心机(Eppendorf,型号:5415 D离心机,F45-24-11转子)
pH计(Hanna HI 2210台式pH /温度计)
声波发生器(Fisher Scientific声波分解器100型)


程序


四膜虫双重净化
四膜虫细胞生长以进行分离
四膜虫细胞(SB255或CU-428)存储在豆类培养基中(Williams等,1980 )。
将50μl豆培养基转移至24孔细胞培养板中的1 ml SPP培养基中。在室温(RT)下培养4-5天,直到细胞密度达到1.6 × 10 6细胞/ ml。
注意:在步骤A1c之前,请在光学显微镜下观察细胞的健康状况。


转移40微升饱和四膜虫的细胞至40ml液体培养基SPP在一个250毫升的锥形烧瓶中并在生长大约一个星期RT 。
              注:40个微升饱和四膜虫细胞可传代到另一个40毫升的液体介质SPP在一个500 -毫升锥形瓶中,并保持约一周在RT或几个星期在15-16℃。


将40 ml RT培养物中的2 ml饱和细胞转移到100 ml液体SPP培养基中,并在MAXQ8000振荡器培养箱中以150 rpm和30°C摇动过夜生长(图3A)。
将整个100 ml过夜培养物添加到1 L液体SPP培养基中,并在150 rpm和30°C(MAXQ8000)摇动下生长约2天。最佳OD 600为0.7。
将细胞在15°C摇动孵育30分钟至1小时(图3B)。
用地布卡因治疗隔离纤毛
将整个细胞培养物分成2或4个相等体积,然后倒入适当的离心管中。使用Avanti转子JLA-8.1,在4 °C下以700 × g的速度离心10分钟,然后缓慢减速。
注意:完成此步骤后,请将细胞保持在4 °C或冰上,以最大程度地减少粘液的分泌。


用10 ml冰冷的SPP培养基重悬含有细胞的沉淀,然后将溶液的总体积调整为24 ml。
将24 ml重悬的细胞转移到冰上的250 ml锥形瓶中(图3C)。
准备好1毫升地布卡因(25毫克/毫升)和75毫升冰冷的液体SPP介质。
将1 ml的dibucaine快速添加到24 ml的重悬细胞中(最终1 mg / ml的dibucaine),并摇匀烧瓶1分钟。
快速添加75 ml冰冷的液态SPP介质以终止反应,并将整个纤毛溶液转移到Beckman Coulter JA25.5转子的两个离心管中。
将整个纤毛悬浮液在4°C下以2,000 × g的速度缓慢离心10分钟(Avanti,转子JA25.5)(图3D)。
使用移液枪小心地吸出含纤毛的上清液,而不会干扰细胞碎片和粘液。对于Beckman Coulter JA25.5转子,将上清液分成4个等体积的离心管,每个离心管约20 ml。
使用Avanti转子JA25.5在4°C下以17,000 × g的速度将纤毛悬浮液离心40分钟,并进行缓慢减速(图3E)。
除去上清液,并用移液器和100μl冰冷的Cilia Wash Buffer轻轻洗掉纤毛沉淀物周围的透明粘液层。当纤毛沉淀干净时,将250μl冰冷的纤毛洗涤缓冲液重悬于每个离心管中,并重悬纤毛沉淀(总计1 ml)。
转移纤毛溶液分成两个1.5毫升微量离心管(〜500微升每),并在7800离心纤毛悬浮×克在微量仪(Eppendorf,离心机5415 d)处理10分钟,在4℃。
除去上清液并将纤毛沉淀重悬于250μl纤毛洗涤缓冲液中(图3F)。
注意:纤毛沉淀可以在此处用液氮速冻并保存在-80°C,但对于纤毛最好的分离方法是不冷冻。




图3.用地布卡因纯化纤毛四膜虫。图像描绘四膜虫的细胞培养物(AB),细胞悬液地布卡因治疗前(C),p ellet和地布卡因后处理(d)的上清液,和纤毛沉淀之前(E)和最终洗涤(F)之后。


双峰微管级分的纯化
重悬沉淀纤毛到250微升具有纤毛最终缓冲液对每个管中。
注意:从这里开始,每个管的数量。


添加44.1微升10%NP-40的替代(1.5%终浓度的NP-40)和重悬总溶液。在冰上孵育30分钟,以去除纤毛的膜。
在微量离心机(Eppendorf,Centrifuge 5415 D)中于4°C以7,800 × g离心10分钟。
去除上清,重悬沉淀到247微升具有纤毛最终缓冲液。
添加2.5微升40毫摩尔在ATP和孵育10分钟RT的轴丝分裂开。
离心16 ,000 ×克10分钟,在4℃在微量仪(Eppendorf,离心机5415 d)。
去除上清,重悬双峰微管球团到250微升具有纤毛最终缓冲液。
添加62.5微升3M的NaCl的至0.6M NaCl对冰的终浓度和温育30分钟,以除去动力蛋白。
在微量离心机(Eppendorf,Centrifuge 5415 D)中于4°C以16,000 × g离心10分钟。
去除上清,重悬沉淀到250微升具有纤毛最终缓冲液。
重复步骤hj。
将250μl双重峰级分应用于透析膜,并在200 °C的透析缓冲液中于4 °C搅拌过夜,以耗尽radial骨辐条。
从透析管中收集样品,并在微量离心机(Eppendorf,Centrifuge 5415 D)中于4°C以16,000 × g离心10分钟。
去除上清,重悬沉淀到250微升具有纤毛最终缓冲液。
注:透析后,将所有的外蛋白纯化双微管部分被删除。


低温EM的双峰微管的超声处理
超声处理条件:p奥尔4,10秒(在冰上的1.5ml微管中样品)。
注意:在进行超声处理时,将探针轻轻移入溶液中。


在微量离心机(Eppendorf,Centrifuge 5415 D)中于4°C以2,000 × g离心10分钟
注意:此步骤仅沉淀聚集的双峰片段,解聚的微管蛋白将保留在上清液中。


用Cilia Final Buffer(补充0.6 M NaCl)将双峰微管沉淀重悬至25μl 。
注意:0.6 M NaCl有助于解离聚集的双峰片段。


孔定量泰特蛋白浓度(即,Bradford测定用分光光度计),然后调节该体积的对应用程序所需的浓度。对于cryo-EM,超声处理的四膜虫四联体片段为4 mg / ml是合适的。对于质谱分析,合适的是1 mg / ml超声双重峰片段。              


衣藻双胞胎纯化
衣藻细胞的生长用于分离
将衣藻细胞敲击到含有1.5%琼脂的TAP固体平板上进行储存。
在RT下,在12h的光照和黑暗交替循环下,在TAP固体板上生长细胞大约两周。
刮下约3-5 mm的细胞,转移到50 ml液体TAP培养基中,然后在摇动或搅拌条件下于室温下交替进行明暗循环12 h,生长一周(图4A)。
注意:一周后,在步骤B1d之前,在光学显微镜下观察细胞的健康状况。


除去50 ml液体培养物中的10 ml ,将其转移至1 L TAP培养基中,并在摇动或搅拌条件下生长,并在室温下交替进行12 h的明暗循环约4-6天,直到OD 600达到0.5-0.6 (图4B)。
鞭毛生长
注意:这一步是可选的,并且有用的衣突变株是不长鞭毛下正常文化条件。


使用Avanti转子JLA-8.1将1 L衣藻培养物以700 × g于4°C离心7分钟。
重悬的沉淀物在50毫升大力去离子水用移液枪和转移到一个50ml的锥形管中。
注意:上下吸移10次以分离单元格。


转移50毫升重悬浮的细胞到1L的去离子水(用一搅拌棒)和包住整个烧瓶与铝,以防止接触的光。
搅拌或摇晃的平台和离开孵化的地方1 - 2小时。
潜伏期后,在光学显微镜下观察细胞,以了解衣藻细胞是否在游动。
通过pH震荡分离鞭毛
注:我们使用pH休克,因为它给了我们一个更清洁的双重分数较少的蛋白质contamina NTS ,使之更适合于质谱分析。也可以使用针对四膜虫所述的地布卡因方法。


使用Avanti转子JLA-8.1在4°C下以700 × g离心1 L细胞培养物7分钟(图4C)。
注意:从这里开始,将样品放在冰上或4°C下。


重悬在含有沉淀的细胞在3-5毫升HMDS溶液并转移到50毫升锥形管中。
使用HMDS溶液(带小搅拌棒)将溶液的总体积调节至15 ml。
用乙酸将溶液的pH调节至4.5,然后在搅拌下使用pH探针等待1分钟。
用0.5 M KOH快速将溶液的pH调节至7.5,然后移开搅拌棒。
离心机pH值-在1800震惊溶液×克在5分钟,4°C(SORVALL为4减速功率以除去细胞体ST 16R,转子75003181) (图4D)。
离心后,分离出的鞭毛将在上清液中。小心地将上清液转移到新的50 ml锥形管中。
注意:避免任何杂物或颗粒。


在4°C(Sorvall ST 16R,Rotor 75003181)下以4,700 × g的速度离心上清液40分钟,减速力为7 (图4E)。
鞭毛现已成团。除去上清,重悬在500沉淀微升HMDEKP溶液。
将鞭毛溶液转移至1.5 ml微量离心管中,并在Eppendorf离心机5415 D中于4°C在7,800 × g下离心10分钟(图4F)。
注意:沉淀的绿色是由于严格的地布卡因处理或与某些无细胞壁的CLiP突变体(衣原体资源中心)一起工作造成的细胞碎片渗漏引起的。将鞭毛用NP-40替代物处理并随后制成颗粒后,将不会出现绿色。


除去上清液。
注意:整个鞭毛沉淀物可在此处用液氮速冻并保存在-80°C ;^ h H但是,双峰分裂作品带鞭毛更好的无严寒和存储。




图4.纯化的衣藻纤毛与pH休克。图像描绘了衣藻细胞培养物(AB),细胞培养物的离心作用(C),pH冲击后的离心作用(D)和(E)之前和洗涤后(F)的鞭毛沉淀物。


双峰微管级分的纯化
重悬沉淀鞭毛到250微升具有HMDEKP。
添加44.1微升10%NP-40的替代(1.5%终浓度的NP-40)和重悬总溶液。在冰上孵育30分钟以除去鞭毛膜。
在微量离心机(Eppendorf,Centrifuge 5415 D)中于4°C以7,800 × g离心10分钟。
去除上清,重悬沉淀到250微升具有HMDEKP。
对于冷冻EM,超声处理鞭毛溶液。条件的特定的Fisher Scientific公司声波粉碎仪型号100:小探针(1ml体积)中,p奥尔4,10秒,在冰上。
注意:在进行超声处理时,将探针轻轻移入溶液中。


对于衣藻(Chlamydomonas doublet),由于难以分裂,因此在较早的步骤中进行了超声处理。


在微量离心机(Eppendorf,Centrifuge 5415 D)中于4°C以2,000 × g离心1分钟。
注意:此步骤仅沉淀聚集的双峰,但将微管蛋白留在上清液中。


重悬双峰微管球团以250微升与HMDEKP溶液。
添加2.5微升100毫ADP至1mM ADP的终浓度,孵育在10分钟RT 。
注意:此步骤仅适用于衣藻双合子分裂以激活动力蛋白,因为它更难分裂。


添加2.5微升10毫在ATP至0.1毫摩尔ATP的终浓度并孵育另外10min RT为双峰从鞭毛分裂了。
注:ATP浓度在较低的衣协议以获得更高的活性动力蛋白。我们没有添加蛋白酶用于分裂,因为我们发现添加弹性蛋白酶会影响MIP的结构。


在微量离心机(Eppendorf,Centrifuge 5415 D)中于4°C以16,000 × g离心10分钟。
去除上清,重悬沉淀到250微升具有HMDEKP。
添加62.5微升3M的NaCl的(0.6摩尔NaCl的最终浓度)和在冰上孵育30分钟。
在微量离心机(Eppendorf,Centrifuge 5415 D)中于4°C以16,000 × g离心10分钟。
去除上清,重悬沉淀到250微升具有HMDEKP缓冲器。
重复步骤B4L到B4N上ç E多。
在第二次盐洗之后,最终的沉淀将包含纯化的双峰微管级分,除放射状辐条外,大部分外部蛋白质被去除。
注:我们尝试了几种不同的条件,从衣双峰去除放射状的轮辐,但我们未能够消除径向轮辐和保持衣双峰微管完好无损。


孔定量泰特蛋白浓度(即,Bradford测定用分光光度计),然后调节该体积的对应用程序所需的浓度。对于cryo-EM,4 mg / ml超声处理的双峰片段是合适的。对于质谱分析,合适的是1 mg / ml超声双重峰片段。


笔记


在cryo-EM中,与未超声处理的双峰微管相比,获得的双峰微管碎片在玻璃化冰中具有随机取向(图2)。通过此程序获得的四膜虫双合子微管结构保留了除内连接(IJ)细丝(FAP20 / PACRG)之外的大多数MIP 。相比之下,衣藻衣原体的双重结构保留了IJ细丝,但A管内的一些MIP丢失了(Ichikawa等人,2017年和2019年; Khalifa等人,2020年)。


菜谱


SPP液体介质(Gorovsky等,1975 )
对于1 L介质:


1%3号蛋白P ,10克           

0.2%葡萄糖,2克             

0.1%酵母提取物,1克             

0.003%乙二胺四乙酸铁(III)钠盐[Fe-EDTA] ,0.03 g


用Milli-Q水和高压灭菌器补足1 L


25毫克/毫升地布卡因
对于1毫升:


盐酸地布卡因0.025克


使用SPP液体培养基补足1毫升


纤毛洗涤缓冲液
50 mM 4-(2-羟乙基)-1-哌嗪乙烷磺酸[HEPES] ,pH 7.4


3毫米MgSO 4


0.1 mM乙二醇-双(2-氨基乙基醚)-N,N,N',N'-四乙酸[EGTA]


1 mM二硫苏糖醇(DTT)(新鲜添加)


250毫米蔗糖


              纤毛最终缓冲液
50 mM HEPES ,pH 7.4


3毫米MgSO 4


0.1毫米EGTA


1 mM DTT(新鲜添加)


0.5%海藻糖


1 mM PMSF(新鲜添加)


透析缓冲液
5 mM HEPES ,pH 7.4


1 mM DTT(新鲜添加)


0.5毫米EDTA


醋酸三乙酸盐(TAP)盐溶液
对于1 L :


NH 4 Cl ,15克


用MgSO 4 ,1 0.95克


氯化钙2· 2H 2 O ,2克


用Milli-Q水补足1 L


磷酸盐溶液
对于100毫升:


ķ 2 HPO 4 ,28.8克


KH 2 PO 4 ,14.4克


用Milli-Q水补足100毫升


TAP液体介质(Gorman和Levine,1965年)
对于1 L介质:


特里斯碱,2.42克


TAP盐溶液,25毫升


磷酸盐溶液,0.375毫升


Hutner的微量元素(衣藻资源中心),1.0毫升


冰醋酸,1.0毫升


用Milli-Q水和高压灭菌器补足1 L


注意:对于TAP固体培养基,在此配方中添加20克琼脂。


500 mM氢氧化钾(KOH)
500 mM乙酸(CH 3 COOH)
3 M氯化钠(3 M NaCl)
HMDS解决方案
10 mM HEPES ,pH 7.4


5毫米MgSO 4


1 mM DTT(新鲜添加)


4%蔗糖


10微克/ ml的抑肽酶(补充新鲜)


5微克/毫升亮抑酶肽(补充新鲜)


HMDEKP解决方案
30 mM HEPES ,pH 7.4


5毫米MgSO 4


1 mM DTT(新鲜添加)


0.5毫米EGTA


25 mM乙酸钾


0.5%聚乙二醇(MW 20,000)


10 μM紫杉醇(补充新鲜)


1 mM PMSF(新鲜添加)


10微克/ ml的抑肽酶(补充新鲜)


5微克/毫升亮抑酶肽(补充新鲜)


注意:将紫杉醇添加到HMDEKP缓冲液中用于衣原体双联体的制备,因为它比四膜虫双联体的稳定性差。


致谢


这项研究在财政支持的加拿大自然科学和工程研究理事会(RGPIN-2016-04954),健康研究的加拿大研究院(CIHR PJT-156354) ,和加拿大高级研究所Arzieli全球学者计划到KHB心肌梗塞由JST,PRESTO授予号JPMJPR20E1,JSPS KAKENHI授予号JP19K23726和JP20K15733以及奈良科学技术学院基金会(R2290001)支持。该协议是根据Khalifa等人先前发表的研究进行了少量修改而改编的。(2020年)


利益争夺


作者声明没有利益冲突。


参考


1 Bui,KH,Sakakibara,H.,Movassagh,T.,Oiwa,K. and Ishikawa,T.(2008)。              莱茵衣藻鞭毛内部原动力的分子结构。J Cell Biol 183(5):923-932。             

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Copyright Black et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Black, C., Dai, D. C., Peri, K., Ichikawa, M. and Bui, K. H. (2021). Preparation of Doublet Microtubule Fraction for Single Particle Cryo-electron Microscopy. Bio-protocol 11(11): e4041. DOI: 10.21769/BioProtoc.4041.
  2. Khalifa, A. A. Z., Ichikawa, M., Dai, D., Kubo, S., Black, C. S., Peri, K., McAlear, T. S., Veyron, S., Yang, S. K., Vargas, J., Bechstedt, S., Trempe, J. F. and Bui, K. H. (2020). The inner junction complex of the cilia is an interaction hub that involves tubulin post-translational modifications. Elife 9: e52760. 
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