Ktu/PF13 is required for cytoplasmic pre-assembly of axonemal dyneins

Abstract

Cilia and flagella are highly conserved organelles that have diverse roles in cell motility and sensing extracellular signals. Motility defects in cilia and flagella often result in primary ciliary dyskinesia. However, the mechanisms underlying cilia formation and function, and in particular the cytoplasmic assembly of dyneins that power ciliary motility, are only poorly understood. Here we report a new gene, kintoun (ktu), involved in this cytoplasmic process. This gene was first identified in a medaka mutant, and found to be mutated in primary ciliary dyskinesia patients from two affected families as well as in the pf13 mutant of Chlamydomonas. In the absence of Ktu/PF13, both outer and inner dynein arms are missing or defective in the axoneme, leading to a loss of motility. Biochemical and immunohistochemical studies show that Ktu/PF13 is one of the long-sought proteins involved in pre-assembly of dynein arm complexes in the cytoplasm before intraflagellar transport loads them for the ciliary compartment.

Figure 1. Medaka ktu mutant

a, b, Heart looping (dashed line) at 3 days postfertilization (dpf). c, d, Medaka Kupffer’s vesicle (KV, arrow in e) at 1.5 dpf and its schematic structure. e, f, Scanning EM of cilia in KV. g–j, Transmission EM of KV cilia (g, h; 9+0) and sperm flagella (i, j; 9+2), showing a partial or complete loss of ODA (arrowheads) and IDA (arrows) in ktu. k, i, Swimming sperm. m, The belly of the mutant adult (3-month-old) becomes expanded (arrowhead). n, o, PKD in ktu mutant kidney. p, The exon-intron structure of ktu, encoding a protein (solid box) with the conserved N-terminal domain (black bracket) and a domain weekly homologous to yeast PIH1/NOP17 (red bracket). A premature stop codon introduced in ktu is indicated by an asterisk. q, r, Overlay and high-power images of a nephric tubule (dotted lines) double-stained with anti-Ktu (green) and γ-tubulin (red) antibodies. Nuclei are in blue.

Figure 2. Recessive loss-of-function KTU mutations

a, b. Homozygous mutations (c.C23A [pS8X]; c.1214^1215insACGATACCTGCGTGGC [p.G406Rfs89X]) in two PCD patients. c, KTU is detectable in control respiratory cells but not in KTU mutant cells. The double band is probably explained by distinct alternative splicing products. GAPDH (loading control). d, In control respiratory ciliary extracts KTU is absent, whereas DNAI2 is abundant. KTU is only present in the cytoplasm. (e–h), Consistent with randomization of left/right body asymmetry OP-146-II-1 exhibits situs solitus (e, f) and her brother (OP-146-II-3) situs inversus totalis (g, h). Both have chronic lung disease. HT; heart, LV; liver, KD; kidney.

Figure 3. PF13 is the Chlamydomonas homolog of Ktu

a, micrographs of axonemes from wild type (wt), pf13-3, and pf13 transformed with λ1A (pf13-R). b, averages of 108 wild type and 118 pf13 doublets, and a difference image (wt-13). c–f, Western blots of C. reinhardtii proteins. ODA proteins (HCα, HCβ, IC2, LC1, LC2 and LC3), IDA proteins (IC140, IC97, and p28) and dynein c heavy chain HC9 in pf13 in flagella (c) and cytoplasm (d). e, PF13 occurs in whole cells (WC) and cell bodies (CB) but not in flagella. Absence of cytochrome (Cyt) f demonstrates lack of cell body contamination in flagellar fractions. FL 50:1, a 50-fold excess of flagella. f, IPs of ODA dyneins from wild-type and pf13 cell extracts probed with antibodies to ODA subunits HCβ, IC2 and LC6. g, A hypothesized role of PF13 in ODA assembly.

Figure 4. Ktu binds to dyneins and Hsp70

a, The anti-mouse (m) Ktu antibody detects a single band of 110 –120 kDa, slightly larger size than expected (814 amino acids, 88.3 kDa), which is undetectable in mutant mice. b, Immunoprecipitates with anti-mKtu or rabbit (r) IgG (control) from wild-type mouse testis extracts probed with anti-DNAI2, anti-Hsp70, anti-Hsp90 and anti-Actin. c, Full and truncated constructs of mKtu used in d. d, GST pull-down assay. Immunoblots with the indicated antibodies after incubation of GST-recombinant proteins with testis extracts. WCE, whole-cell extracts used. The amount of GST-recombinant proteins was confirmed by Coomassie staining (Arrowheads indicate GST-proteins used for the assay).