Features of the Tetrahymena IFT172 gene sequence. (A) Secondary structure prediction and domain organization of Ift172p. Black boxes indicate 7 WD motifs. Light gray boxes show 13 degenerate repeat units. The dark gray box indicates the repeat unit which is aligned and homologous to the LIM-transcription factor interaction domain (LIM-ID) of rat SLB/IFT172. (B) Comparison of Tetrahymena Ift172p and orthologues from different species. The overall sequence identity and similarity were compared with Tetrahymena Ift172p. Tt, Tetrahymena thermophila, EF495115; Cr, Chlamydomonas reinhardtii, AAT99263.1; Ce, Caenorhabditis elegans, NP_510681.2; Dm, Drosophila melanogaster, NP_647700.1; Rn, Rattus norvegicus, NP_446244.1; Hs, Homo sapiens, NP_056477.1. (C) Sequence alignment of Tetrahymena Ift172p repeat units. Black and gray shadings show conserved residues with 60 or 35% identity, respectively. The predicted alpha helices are shown as cylinders above the alignment.

Construction and characterization of IFT172 knockout cells. (A) Strategy to generate knockout cells. The wild-type locus of the IFT172 gene was targeted with the disruption construct by homologous recombination, replacing most of the coding region with a neo3 cassette in the knockout locus. Short bar, the probe for Southern analysis. N, NsiI site. (B). Southern blot analysis of the genotype. Genomic DNA extracted from CU428 wild-type strain and IFT172 knockout strains was digested with NsiI and resolved on an agarose gel. The blot was probed with an isotope labeled IFT172 cDNA fragment as indicated in A. The absence of the 2.2-kb band from the knockout cells (KO) shows that the wild-type IFT172 gene (WT) is replaced by a knockout allele. (C) Northern analysis of IFT172 expression during cilia regeneration. CU428 wild-type cells and IFT172 knockout cells were treated with a pH shock and allowed to regenerate cilia. Total RNAs were extracted from log phase cells and from the cells collected at 0, 60, and 120 min after deciliation and probed with the IFT172 cDNA probe. IFT172 transcripts were highly induced during cilia regeneration in wild-type cells. No IFT172 messages could be detected in knockout cells. The bottom panel shows the ethidium bromide staining to control for loading. (D–F). Morphology of wild-type and IFT172 knockout cells. The cells were stained with anti-tubulin antibodies. Wild-type cells contain hundreds of cilia (D), but IFT172 knockout cells cannot assemble normal length cilia (E and F). With the loss of ciliary beating, IFT172 knockout cells fail to finish division, fuse, and form large, multinucleated monster cells (F). Bar, 10 μm. (G) Percentage of the chain cells and monster cells cultured with or without vigorous shaking. Culturing cells with shaking can partially rescue the cytokinesis defect of IFT172 knockout cells. (H–J) Ink uptake assay to test oral cilia function. Black ink was added into the medium, and the numbers of cells that contained ink particles were measured after 4 h. Although all the wild-type cells contain large black food vacuoles (H), <5% of IFT172 knockout cells form black vacuoles (I). Bar, 10 μm.

Rescue experiment. (A) Strategy to map the functional domains and generate “knock-in” mutant cells by rescue of motility using IFT172 knockout cells. (B) Constructs for rescue experiments and for expressing Ift172p in the wild-type cells.

Localization of the Ift172p-HA. Confocal images of nondividing (A–C) and dividing (D–F) cells expressing IFT172-HA. The cells were stained with both anti-glutamylated and anti-glycylated tubulin antiserum (A and D, green) to visualize cilia and basal bodies and with anti-HA mAb (B and E, red) to detect Ift172p-HA. The overlay images (C and F, yellow) indicate that Ift172p localizes along the cilia and to the basal bodies. The oral apparatus (Oa), which has a high density of oral cilia, is stained strongly by anti-HA antibodies in both nondividing and dividing cells. The single arrowhead indicates the old oral apparatus, and the double arrowhead marks the newly formed oral apparatus in the dividing cell. Bar, 10 μm. (G–I) High-magnification images to show that Ift172p-HA (red) localized to the cilia (arrow) and is closely associated with the basal body (arrowhead). Bar, 5 μm. (J–O) Merged confocal images of glutamylated/glycylated tubulin and Ift172p- HA staining on regenerating cilia. (M–O), enlarged images of the boxed regions shown in J–L, respectively. (J and M) Deciliated cells; (K and N) 30 min in the recovery buffer; (L and O) 60 min in the recovery buffer. Bar, 10 μm.

Phenotypic analyses of cells rescued with RPD deletion constructs. (A) Deletion constructs and rescue results. Black regions represent the WDD, gray regions represent the RPD, and the dark gray box indicates the repeat unit corresponding to LIM-ID. The amino acid residue numbers are labeled according to the full-length protein. +, the construct rescued the knockout cells; −, the construct failed to rescue. N.D., not determined. (B) Immunolocalization of the FLAG-tagged Ift172p WDD or PRD expressed in the wild-type CU428 cells. Untransformed CU428 was used as negative control for staining. Both truncated proteins showed punctate localization in the cytosol and did not appear in the cilia or basal body. Bar, 10 μm. (C). Growth curve of different cell lines rescued by deletion constructs. Solid lines show the cells rescued with constructs targeting to the IFT172 endogenous locus. Dotted lines show cells in which the rescue constructs were targeted to the MTT1 locus and the cells were grown with 1 μg/ml cadmium chloride in SPP medium. The rescued results are consistent between the two targeted loci. Although cells rescued by full-length IFT172 had the same doubling time as CU428 wild-type cells, cells rescued by RPD truncated forms divided slower. (D) Negative images of the swimming paths of the cells rescued with four different truncation constructs. The paths were recorded by 2-s exposures of the swimming cells under low magnification with a dark-field microscope. The constructs used for rescue are shown above each image. (E) Frequency distribution of the path lengths measured from C.

Morphology of the rescued cells and the localization of RPD-truncated Ift172p. (A–E) Merged confocal images of tubulin (green) and Ift172p-FLAG (red) staining on different rescued cells. The anterior end of the cell containing an oral apparatus (Oa) was positioned to the top. The diagrams below the cell images represents the truncated forms of IFT172 used to generate the rescued knock-in cells. These FLAG-tagged truncation constructs were targeted to the MTT1 locus. (A) Cells rescued with full-length IFT172 showed that most of these proteins localized to the basal bodies in the cell cortex and oral apparatus, and some Ift172p-FLAG localized along the cilia (arrow). (B–E) In the cells rescued with truncated forms of IFT172, deletion of part of the RPD led to accumulation of the Ift172p at the distal tip of the cilia (arrowhead). (E) Higher magnification of the box region in D. Also note that cells with truncated IFT172 assembled fewer cilia that showed increased length heterogeneity. (F–K) Fluorescent microscope images of tubulin (green) and Ift172p-FLAG (red) staining during cilia regeneration in the knocked-in cells rescued with truncated form IFT172(RPDΔ3). (F) Deciliated cell at zero time of regeneration; (G) 20 min in the recovery buffer; (H and I) 40 min in the recovery buffer; (J and K) 60 min in the recovery buffer. Bar, 100 μm.

Localization of Ift88p-GFP in cells rescued with either full-length or RPD-truncated IFT172. (A) In the cells with full-length IFT172, Ift88p-GFP localized to oral cilia (Oa), basal body rows (arrow) and somatic cilia along the axoneme (arrowhead). (B–E) In the cells with a knocked in RPD-truncated construct IFT172(RPDΔ3), brightly fluorescent Ift88p-GFP accumulated in the cilia. The signals appeared to be enriched at the distal regions of ciliary tips, which could be observed more clearly in somatic cilia pointing out parallel to the focal plain (arrowhead) and in oral cilia (double arrowhead). (A, B, and D) Fluorescent microscope images. (C and E) Phase-contrast image merged with the fluorescent image. Bar, 10 μm.

Summary of Ift172p functional domains involved in the IFT process.