FAP20 is an inner junction protein of doublet microtubules essential for both the planar asymmetrical waveform and stability of flagella in Chlamydomonas

The axoneme, the conserved core of eukaryotic cilia and flagella, contains highly specialized doublet microtubules (DMTs). A long-standing question had been what protein(s) compose the junctions between two tubules in DMT. Here we identified a highly conserved flagellar protein, FAP20 as an inner junction (IJ) component. The flagella of Chlamydomonas FAP20 mutants have normal length, but beat with an abnormal symmetrical three-dimensional pattern. In addition, the mutant axonemes are liable to disintegrate during beating, implying that interdoublet connections may be weakened. Conventional electron microscopy showed that the mutant axonemes lack the IJ and cryo-electron tomography combined with a structural labeling method revealed that the labeled FAP20 localizes at the IJ. The mutant axonemes also lack doublet-specific beak structures, which are localized in the proximal portion of axoneme and may be involved in the planar asymmetric flagellar bending. FAP20 itself, however, may not be a beak component because the uniform localization of FAP20 along the entire length of all nine DMTs is inconsistent with the beak's localization. FAP20 is the first confirmed component of the IJ. Our data also suggest that the IJ is important for both stabilizing the axoneme and scaffolding intra-B-tubular substructures required for a planar asymmetrical waveform.