DYF-1 Is Required for Assembly of the Axoneme in Tetrahymena thermophila

In most cilia, the axoneme can be subdivided into three segments: proximal (the transition zone), middle (with outer doublet microtubules), and distal (with singlet extensions of outer doublet microtubules). How the functionally distinct segments of the axoneme are assembled and maintained is not well understood. DYF-1 is a highly conserved ciliary protein containing tetratricopeptide repeats. In Caenorhabditis elegans, DYF-1 is specifically needed for assembly of the distal segment (G. Ou, O. E. Blacque, J. J. Snow, M. R. Leroux, and J. M. Scholey. Nature. 436:583-587, 2005). We show that Tetrahymena cells lacking an ortholog of DYF-1, Dyf1p, can assemble only extremely short axoneme remnants that have structural defects of diverse natures, including the absence of central pair and outer doublet microtubules and incomplete or absent B tubules on the outer microtubules. Thus, in Tetrahymena, DYF-1 is needed for either assembly or stability of the entire axoneme. Our observations support the conserved function for DYF-1 in axoneme assembly or stability but also show that the consequences of loss of DYF-1 for axoneme segments are organism specific.Cilia are microtubule-rich cellular extensions that arise from basal bodies near the surfaces of most eukaryotic cell types. Defective cilia cause a wide variety of diseases, including polycystic kidney disease, primary ciliary dyskinesia, and retinal degeneration (3). A typical motile cilium has a microtubule-based framework, the axoneme, which contains nine outer (mostly doublet) microtubules and two central (singlet) microtubules. In most cilia, the axoneme can be subdivided into three segments: proximal (transition zone), middle (containing outer doublet microtubules), and distal (containing singlet extensions of peripheral microtubules). The outer doublet microtubules of the middle segment have a complete tubule A made of 13 protofilaments and an incomplete tubule B made of 11 protofilaments that is fused to the wall of the A tubule (36, 57). The outer microtubules in the distal segment lack the B tubule (32, 49). The distal segment also lacks dynein arms and radial spokes, and its microtubules are terminated by caps that are associated with the plasma membranes at the tips of cilia (11, 50). The distal segments are characterized by a high level of microtubule turnover, which could play a role in the regulation of the length of cilia (31).The mechanisms that establish the segmental subdivision of the axoneme are not well understood. Studies of Caenorhabditis elegans indicate that the distal segment is assembled using a mechanism that differs from the one utilized in the middle and proximal segments (54). In most cell types, ciliogenesis is dependent on the intraflagellar transport (IFT) pathway, a bidirectional motility of protein aggregates, known as IFT particles, that occurs along outer microtubules (10, 28, 29, 42). IFT particles are believed to provide platforms for transport of axonemal precursors (23, 44). The anterograde component of IFT that delivers cargo from the cell body to the tips of cilia is carried out by kinesin-2 motors (28, 63), whereas the cytoplasmic dynein DHC1b is responsible for the retrograde IFT (41, 43, 53). Importantly, in the well-studied amphid cilia of C. elegans, two distinct kinesin-2 complexes are involved in the anterograde IFT and differ in movement velocity: the “slow” heterotrimeric kinesin-II and the “fast” homodimeric OSM-3 kinesin (54). While kinesin-II and OSM-3 work redundantly to assemble the middle segment, OSM-3 alone functions in the assembly of the distal segment (39, 56).In C. elegans, DYF-1 is specifically required for assembly of the distal segment (39). In the DYF-1 mutant, the rate of IFT in the remaining middle segment is reduced to the level of the slow kinesin-II, suggesting that the Osm3 complex is nonfunctional and that kinesin-II functions alone in the middle segment. Thus, DYF-1 could either activate OSM-3 kinesin or dock OSM-3 to IFT particles (14, 39).However, a recent study of zebrafish has led to a different model for DYF-1 function. Zebrafish embryos that are homozygous for a loss of function of fleer, an ortholog of DYF-1, have shortened olfactory and pronephric cilia and ultrastructural defects in the axonemes. In the middle segment, the fleer axonemes have B tubules that are disconnected from the A tubule, indicating that DYF-1 functions in the middle segment and could play a role in the stability of doublet microtubules (40). Earlier, a similar mutant phenotype was reported in Tetrahymena for a mutation in the C-terminal tail domain of β-tubulin, at the glutamic acid residues that are used by posttranslational polymodifications (glycylation and glutamylation) (47). Glycylation (46) and glutamylation (12) are conserved polymeric posttranslational modifications that affect tubulin and are highly enriched on microtubules of axonemes and centrioles (reviewed in reference 20). Other studies have indicated that tubulin glutamylation contributes to the assembly and stability of axonemes and centrioles (4, 8). The fleer mutant zebrafish cilia have reduced levels of glutamylated tubulin (40). Pathak and colleagues proposed that the primary role of DYF-1/fleer is to serve as an IFT cargo adapter for a tubulin glutamic acid ligase (25) and that the effects of lack of function of DYF-1/fleer could be caused by deficiency in tubulin glutamylation in the axoneme (40). As an alternative hypothesis, the same authors proposed that DYF-1 is a structural component that stabilizes the doublet microtubules in the axoneme (40).Here, we evaluate the significance of a DYF-1 ortholog, Dyf1p, in Tetrahymena thermophila. Unexpectedly, we found that Tetrahymena cells lacking Dyf1p either fail to assemble an axoneme or can assemble an axoneme remnant. While our observations revealed major differences in the significance of DYF-1 for segmental differentiation in diverse models, it is clear that DYF-1 is a conserved and critical component that is required for assembly of the axoneme.