Preparation of axonemes and dyneins from Chlamydomonas

Isolation of Chlamydomonas flagella was achieved through dibucaine treatment as described in a previous paper (48). After collection via centrifugation, flagella were demembraned with 0.2% Igepal in HMDS solution [10 mM Hepes (pH 7.4), 5 mM MgSO₄, 1 mM dithiothreitol, and 4% sucrose]. The axonemes were precipitated by centrifugation and washed with HMDEK solution [30 mM Hepes, 5 mM MgSO₄, 1 mM dithiothreitol, 1 mM EGTA, and 50 mM potassium acetate (pH 7.4)] to remove Igepal. Preparation of dyneins from Chlamydomonas was performed using an HPLC system with a Mono Q anion exchange column as described previously (49). The axonemes were treated with 0.6 M KCl in HMDEK on ice for 10 to 30 min to extract crude dyneins. The suspension was centrifuged, and the supernatant containing crude dyneins was pooled. The supernatant was diluted 10-fold by addition of HMDEK solution. The diluted supernatant was centrifuged to remove aggregates and loaded onto a Mono Q column. The Mono Q fractions were analyzed by SDS-PAGE using 3 to 5% acrylamide with a 3 to 8 M urea gradient to determine the positions of dynein species.