Preparation of Doublet Microtubule Fraction for Single Particle Cryo-electron Microscopy

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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