Ciliary proteins Fap43 and Fap44 interact with each other and are essential for proper cilia and flagella beating

Abstract

Cilia beating is powered by the inner and outer dynein arms (IDAs and ODAs). These multi-subunit macrocomplexes are arranged in two rows on each outer doublet along the entire cilium length, except its distal end. To generate cilia beating, the activity of ODAs and IDAs must be strictly regulated locally by interactions with the dynein arm-associated structures within each ciliary unit and coordinated globally in time and space between doublets and along the axoneme. Here, we provide evidence of a novel ciliary complex composed of two conserved WD-repeat proteins, Fap43p and Fap44p. This complex is adjacent to another WD-repeat protein, Fap57p, and most likely the two-headed inner dynein arm, IDA I1. Loss of either protein results in altered waveform, beat stroke and reduced swimming speed. The ciliary localization of Fap43p and Fap44p is interdependent in the ciliate Tetrahymena thermophila.Electronic supplementary materialThe online version of this article (10.1007/s00018-018-2819-7) contains supplementary material, which is available to authorized users.

FAP57/WDR65 targets assembly of a subset of inner arm dyneins and connects to regulatory hubs in cilia

“…We found that FAP57 co-elutes with the I1 tether head subunits during FPLC fractionation (Figure 4, Supplemental Table 4). Although none of these proteins require FAP57 for assembly into the axoneme (Table 2) nor vice versa (Supplemental Figure 3; see also Urbanska et al, 2018), collectively the data suggest that FAP57 is located in close proximity to both I1 dynein and the MIA complex.…”

“…Five other polypeptides co-eluted with FAP57; these included FAP44, FAP43, FAP244, FAP159, and FAP75, in order of peptide abundance ( Supplemental Table 4). Little is known about FAP159 and FAP75, but FAP43, FAP44, and FAP244 have been identified as subunits of a tether-tether head (T/TH) complex linking the I1 dynein motor domains to the DMT and also interacting with the base of dynein d Kubo et al, 2018;Urbanska et al, 2018). FAP43, FAP44, and FAP244 share structural similarity with FAP57 with respect to the arrangement of their WD repeat and coiled coil domains Kubo et al, 2018), and FAP43 and FAP44 have been proposed to interact with FAP57 in Tetrahymena cilia based on proximity labeling .…”

Rare Human Diseases: Model Organisms in Deciphering the Molecular Basis of Primary Ciliary Dyskinesia

“…As was shown in studies using model organisms, some mutations can cause small changes in cilia beating [36]. Thus, it is possible that individuals harboring similar mutations have no symptoms or manifest only mild symptoms and are never diagnosed for PCD.…”

The LisH Domain-Containing N-Terminal Fragment is Important for the Localization, Dimerization, and Stability of Katnal2 in Tetrahymena

“…To overexpress Kat2-2V5 in the GRL3/GRL4 region, we amplified a 0.8 kb fragment of the GRL4 gene, adding SacII and BglII restriction sites at 5 and 3 ends, respectively (primers in Table S1), and cloned it into a p4T2 vector carrying a neo2 cassette digested with SacII and BamHI. In parallel, the HA coding region and BTU1 3 UTR (untranslated region) were removed from the Kat2-HA-overexpressing plasmid using BamHI and XhoI sites, and were replaced by the 2V5 coding region, followed by 0.6 kb of 3 UTR of BTU1 (transcription terminator) obtained from the native locus expression plasmid [26] using BamHI and EcoRV restriction enzymes and 1.1 kb of the GRL3 gene amplified from the genomic DNA with the addition of EcoRV and XhoI sites at 5 and 3 ends, respectively (Table S1). Next, the GRL4-neo2 fragment was cloned into the pKat2-2V5-GRL3 vector using SacI and Cla I restriction sites.…”