| The axonemes of motile flagella typically contain nine outer doublet microtubules and two central pair (CP) microtubules, termed C1 and C2. Doublet microtubules are continuous with the triplet microtubules of the basal body. In contrast, the CP microtubules are restricted to the axonemal lumen and their nucleation and assembly are poorly understood. Hydin, a component of a projection of the C2 microtubule, is present at the earliest times in short regenerating flagella but largely absent from flagella of the CP-deficient mutants pf15, pf18, and pf19; therefore it is a useful CP marker. To analyze CP assembly independently from outer doublet assembly, pf15 was mated with a wild-type strain expressing HA-tagged alpha-tubulin. Triple immuofluorescence microscopy of quadriflagellates with anti-HA, anti-hydin, and anti-acetylated tubulin showed HA-tubulin first in the middle segments of the formerly mutant flagella. The CP marker hydin was present along the entire length of the HA-positive structure, suggesting that it is the developing CP. In later stages, the position of the elongating CP varied between the proximal and distal flagellar regions, indicating that the developing CP can slide up and down inside the axonemal cylinder. In pf18 x PF18 HA-alpha-tubulin quadriflagellates, CP assembly was not apparent. Nevertheless, hydin accumulated in the mid region of the formerly mutant flagella, suggesting that some CP proteins are stockpiled in that location ahead of CP assembly. In contrast to hydin, PF6, a component of a projection of the C1 microtubule, was present in the proximal region of CP-deficient flagella of pf15 gametes. After formation of quadriflagellates, PF6 redistributed and associated with the developing CP, progressing from proximal to distal. The distinct patterns observed for PF6 and hydin during CP repair could be indicative of different transport routes. The data show that CP assembly during repair of CP-deficient flagella
is a self-templated process that does not require the transition zone as a nucleating structure. |