Figure 8. dyf13 mutant reduces the levels of inner dynein arm components in its flagella.

(A) Elution patterns of inner-arm dyneins of oda1 single and oda1dyf13 double mutant C. reinhardtii cells. Inner-arm dyneins were extracted with high salt from outer-armless axonemes of oda1 and oda1dyf13 cells and fractionated by ion-exchange chromatography on a Mono Q column. The elution positions of each dyneins are indicated by a–g. (B) SDS-PAGE of peak fractions showing the dynein heavy chain bands (circles). Peak fractions were subjected to SDS-PAGE with a 3–5% acrylamide gradient and a 3–8 M urea gradient. Dynein a and f were reduced in oda1dyf13 compared with oda1. (C) 2D-DIGE proteomic analysis of wild-type and dyf13 mutant flagella. Fluorescence image of the 2D-DIGE analytical gel, using isoelectric focusing (IEF) in the first dimension and SDS polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension. Wild-type is shown in green and dyf13 mutant is shown in red.

DOI: http://dx.doi.org/10.7554/eLife.01566.023

Figure 8—figure supplement 1. dyf13 mutant does not change the levels of outer dynein arm components in its flagella.

(A) Elution patterns of outer-arm and inner-arm dyneins of wild-type and dyf13 mutant C. reinhardtii cells. The elution positions of each dyneins are indicated by αβ, γ (outer-arm), and a–g (inner-arm). (B) SDS-PAGE of peak fractions showing the dynein heavy chain bands (circles). Peak fractions were subjected to SDS-PAGE with a 3–5% acrylamide gradient and a 3–8 M urea gradient. Contaminations of membrane fraction and degradation products are indicated by white arrowhead and asterisk, respectively. Dynein a and f were reduced in dyf13 mutant but αβ, γ were not changed compared with wild-type flagella. FPLC and SDS-PAGE were separately performed in the figure. (C) Western blotting images of isolated flagella fractions from wild-type cc125 and dyf13 mutant cells probed with antibodies centrin and α-tubulin. The ratio column shows the relative intensity ratio of dyf13 mutant band normalized with wild-type band intensity, showing that the relative amount of centrin was reduced in dyf13 mutant flagella by approximately 50%, comparable to the abundance change measured by 2D-DIGE.

DOI: http://dx.doi.org/10.7554/eLife.01566.024