SummaryThe ciliary tip has been implicated in ciliary assembly and disassembly, and signaling, yet information on its protein composition is limited. Using comparative, quantitative proteomics based on the fact that tip proteins will be approximately twice as concentrated in half-length compared with full-length flagella, we have identified FAP256 as a tip protein in Chlamydomonas. FAP256 localizes to the tips of both central pair and outer doublet microtubules (MTs) and it remains at the tip during flagellar assembly and disassembly. Similarly, its vertebrate counterpart, CEP104, localizes on the distal ends of both centrioles of nondividing cells until the mother centriole forms a cilium and then localizes at the tip of the elongating cilium. A null mutant of FAP256 in Chlamydomonas and RNAi in vertebrate cells showed that FAP256/CEP104 is required for ciliogenesis in a high percentage of cells. In those cells that could form cilia, there were structural deformities at the ciliary tips.

Tubulin polymerization promoting proteins (TPPPs) belong to a family of neomorphic moon lighting proteins, involved in various physiological and pathological conditions. In physiological conditions, TPPPs play an important role in microtubule dynamics regulating mitotic spindle assembly and in turn cell proliferation. In pathological situations, TPPPs interact with α-synuclein and β-amyloid and promote their aggregation leading to Parkinson’s disease and multiple system atrophy. Orthologs of TPPP family proteins were identified in ciliary proteomes from various organisms including Chlamydomonas but their role in ciliogenesis was not known. Here we showed that Flagellar Associated Protein, FAP265, a Chlamydomonas homologue of TPPP family proteins, localizes in the cytosol, at the basal bodies and in the flagella of vegetative Chlamydomonas cells. During cell division, the protein was found as a distinct spot in the nucleus and at the cleavage furrow which forms between the daughter cells. Further null mutants of Chlamydomonas FAP265 protein, fap265, showed severe defects in hatching from the mother sporangium. Daughter cells of fap265 were significantly larger in size compared with wild type cells. Moreover, the daughter cells present within the mother sporangium failed to form flagella before hatching. They reassembled their flagella only after hatching from the sporangium suggesting that FAP265 plays an important role in flagellar reassembly after cell division.

Several nuclear and nucleic acid-binding proteins were detected in the proteomic analyses of ciliary fractions from various organisms. Yet very little is known about the role of these proteins in ciliogenesis and ciliary signaling. In an attempt to characterize the role of these nuclear proteins, we identified a hypothetical protein from Chlamydomonas reinhardtii, CrRuvBL1, which is homologous to human DNA helicase, HsRuvBL1. CrRuvBL1 localizes to flagella and nucleus in vegetative Chlamydomonas cells. It accumulates in the nucleus specifically during initial stages of flagellar assembly and cell division indicating its role in these processes. Mammalian counterpart of this protein, HsRuvBL1, was found to be present at the basal bodies and in the primary cilium of quiescent Retinal Pigment Epithelial (RPE1) cells. In interphase cells, HsRuvBL1 is present at centrioles while the protein localizes on spindle fibers, spindle poles and midbodies, which are important structures formed during different phases of cell division. Depletion of HsRuvBL1 by using siRNAs leads to complete loss of primary cilia in RPE1 cells. Together these results suggest that nuclear proteins play an important role in ciliogenesis.

The cover image, by Damayanti Tammana and Trinadh Venkata Satish Tammana, is based on the Short Report Human DNA helicase, RuvBL1 and its Chlamydomonas homologue, CrRuvBL1 plays an important role in ciliogenesis, DOI: .