Mapping the ciliary axoneme by proteomic analysis

Eukaryotic cilia and flagella play key roles in cell motility, sensory reception and development. Though a large number of ciliary proteins have been identified, the distribution of many of these proteins within the axoneme, the ciliary scaffold, is unknown. To address this issue, we used a biochemical assay to differentially solubilize the A- and B-tubules of the doublets and the central-pair microtubules within the 9+2 structure of Chlamydomonas reinhardtii axonemes. We identified a set of proteins that are found almost exclusively in the A-tubule fraction and define the A-tubule protein. We find that many proteins in the central-apparatus (CA) fraction (which are extracted with the central-pair microtubules) are also found in the B- and A-fractions, suggesting that they are promiscuous microtubule binders. Furthermore, many of the proteins absent in the CA-deficient paralyzed filament mutant pf18 are also found in the A-tubule fraction, some exclusively so. This indicates that the pf18 gene may be required for assembly of doublet-associated structures in addition to the CA, and raises the possibility that defects in the A-tubule proteins give rise to the paralyzed filament phenotype. By identifying the A-tubule protein and providing evidence that CA and doublet assembly are not independent, this study advances our understanding of the ciliary protein organization and ciliopathies caused by axonemal assembly mutants.