Isolation and Dissociation of Chlamydomonas Axonemes

Axonemes were isolated from wild type and fap236 mutant Chlamydomonas cells and dissociated into microtubules as previously described^{2 (link)}. Briefly, cells in HMDS buffer (10 mM HEPES, 5 mM MgSO₄, 1 mM DTT, 4% Sucrose, pH 7.4) were treated with 25 mM dibucaine (Sigma-Aldrich) to induce deflagellation. Cell bodies were removed by centrifugation at 1,800 x g for 5 min. The flagella-containing supernatant was collected and laid over a 25% sucrose suspension in HMDS buffer. After centrifugation at 2,400 x g for 10 min, the supernatant was collected down to the sucrose interface. NP-40 (USB Chemicals) was added to the flagella to a final concentration of 1% to remove membranes. Axonemes were collected by centrifugation at 30,000 x g for 20 min and then resuspended in HMDEKP buffer (30 mM HEPES, 5 mM MgSO₄, 1 mM DTT, 0.5 mM EGTA, 25 mM KCl, pH 7.4) containing 1x ProteaseArrest protease inhibitors (G-Biosciences). The volume and optical density (at 280 nm) of the purified axonemes was adjusted to 10 μL and 32 absorbance units. Then the sample was mildly digested in HMDEKP buffer containing 10 μg/mL subtilisin A (Sigma-Aldrich) and 2 mM ATP, in the presence of ProteaseArrest protease inhibitors. Protease digestion was performed on ice and stopped after 30 min by plunge freezing.