Figure 5.

LC2 is required for assembly of outer dynein arms. (A) Electron micrographs of cross-sections of flagella of wild-type g1 cells show prominent outer dynein arms (arrows) on all of the doublet microtubules except for doublet one (arrowhead), which does not normally have an outer dynein arm (Hoops and Witman, 1983). In contrast, the outer dynein arms are completely missing in flagella of the oda12-1 mutant (F56). The number of outer dynein arms in flagella of oda12-2 cells (V3) is variable; in this section outer dynein arms (arrows) are seen on only three doublet microtubules. (B) Axonemes from wild-type and the two oda12 mutants were separated by SDS-PAGE, transferred to a PVDF membrane, and analyzed by Western blotting. The blots were probed with antisera to IC140 (an inner arm IC), IC1 (an outer arm IC), and LC2. The anti-IC140 antisera reacted equivalently with a 140-kDa band in all three lanes, indicating that each lane was loaded with an equal number of axonemes. Neither the anti-LC2 antibody nor the anti-IC1 antibody recognized bands of the appropriate size in the oda12-1 axonemes, confirming that this mutant is completely lacking the outer dynein arm. However, these two antibodies did reveal a small amount of IC1 and LC2 in the oda12-2 axonemes. The antibody to IC1 also reacts with a smaller band (*) (Fowkes and Mitchell, 1998) that is not part of the outer arm and is not affected by the oda12 mutation. The bottom panel shows a longer exposure of the blot probed with the LC2 antibody to demonstrate the small amount of LC2 in the oda12-2 axonemes.