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First published online 22 January 2008
doi: 10.1242/jcs.015826

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Tetrahymena IFT122A is not essential for cilia assembly but plays a role in returning IFT proteins from the ciliary tip to the cell body

Department of Biology, University of Rochester, Rochester, NY 14627, USA

View larger version (34K): [in this window] [in a new window]   Fig. 1. Sequence analysis of the Tetrahymena IFT122A gene. (A) An un-rooted neighbor-joining tree of IFT122A and other IFT and OSEG proteins, which share similar domain organization. Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster; Hs, Homo sapiens; Tt, Tetrahymena thermophila. The number at the branches indicates the value of 1000 bootstraps. (B) Primary and secondary structure analysis of Ift122Ap. Yellow boxes indicate six WD motifs, and green boxes show two degenerate TRP-like repeat units. Secondary structure prediction shows two distinct regions of Ift122Ap: an N-terminal β-strand-rich region (yellow) and an -helix-rich region (green). (C) Sequence alignment of the degenerate repeat units on Ift122Ap. Identical residues are shaded.

View larger version (62K): [in this window] [in a new window]   Fig. 2. IFT122A is a ciliary gene. (A) Northern analysis of IFT122A expression during cilia regeneration. CU428 wild-type cells were deciliated and allowed to regenerate cilia. Total RNAs extracted from log phase cells and from the cells collected at 0, 1 and 2 hours in the recovery buffer were probed with isotope-labeled IFT122A cDNA. The upper panel shows that IFT122A transcripts were highly induced during cilia regeneration in wild-type cells. The lower panel shows the ethidium bromide staining as loading control. (B) Detection of HA-tagged Ift122Ap in cilia by immunoblotting. Total proteins from cilia and cell body fractions were isolated from HA-tagged IFT122A, HA-tagged IFT172, and wild-type CU428 strains. In the upper panel, the blot was probed with anti-HA monoclonal antibody. A 140 kDa protein and 190 kDa protein were detected in the cilia fraction (Cil) from IFT122A-HA and IFT172-HA cells, respectively. In the lower panel, the same blot was re-probed with a control antibody against polyglycine. The cytoplasmic protein Pgp1p, shown as multiple high-molecular mass bands, was only present in the cell body fraction (CB).

View larger version (45K): [in this window] [in a new window]   Fig. 3. IFT122A knockout cells. (A) Strategy to generate knockout cells. The wild-type IFT122A locus was homologously targeted by the disruption construct, replacing most of the coding region with a neo3 cassette in the knockout locus. Arrowheads indicate the primers that were used in the PCR assay to determine the genotype. (B) PCR analysis of the genotype. Genomic DNAs extracted from CU428 strains and from IFT122A knockout cells were analyzed using a forward primer and two locus-specific primers. In the wild-type cells (WT), a 1.8 kb band was obtained, while in the IFT122A knockout (KO), only the 2.4 kb corresponding to the knockout locus could be amplified. (C) Ink uptake assay to test the function of oral cilia. IFT122A knockout cells can ingest ink particles to form black food vacuoles. Scale bar: 10 µm. (D) The proportion of cells that formed black food vacuoles at different time points. Open squares, CU428 cell; filled (red) circles, IFT122A knockout cells. (E-G). Immunostaining of IFT172 knockout (E), IFT122A knockout (F) and CU428 (G) cells using anti-tubulin antibody. The arrowhead indicates the anterior end of the cell. Scale bars, 10 µm.

View larger version (50K): [in this window] [in a new window]   Fig. 4. Distal tip localization of Ift88p-GFP in the IFT122A knockout cells. (A) Strategy to introduce IFT88-GFP constructs into the wild-type cells or IFT122A knockout cells. (B) Ift88p-GFP fluorescence in the wild-type cells. (C) Enlarged image from the boxed region in B. Ift88p-GFP localized to the cilia in oral apparatus (OA), along the somatic cilia (arrowheads) and to the basal body rows (arrows). (D) Ift88p-GFP fluorescence in the IFT122A knockout cells. (E) Ift88p-GFP fluorescence merged with DIC images. (F,G), Enlarged images from the boxed region in E. In the IFT122A knockout background, Ift88p-GFP became enriched at the distal tips of oral cilia (OA) and somatic cilia (arrowhead). Scale bars, 10 µm.

View larger version (57K): [in this window] [in a new window]   Fig. 5. Distal tip localization of Ift172p-HA in the IFT122A knockout cells. (A) Strategy to generate tagged strains with IFT172-HA knocked-in to the endogenous locus. (B) Growth curves of the CU428 wild-type cells and IFT knockout cells. (C-H) Immunofluorescence staining of the cells with wild-type IFT122A (C,D) or with IFT122A deleted (E-H). Cells were stained with anti-HA monoclonal antibody (red) and anti-tubulin antiserum (green), and D, F and H are merged images of Ift172p-HA and tubulin staining. Ift172p-HA became enriched at the distal region of some cilia (arrow). Scale bars, 10 µm.

View larger version (86K): [in this window] [in a new window]   Fig. 6. Localization of Ift172p-HA during cilia regeneration in the IFT122A knockout cells. IFT122A knockout cells with HA-tagged IFT172 were deciliated by pH shock and allowed to regenerate cilia. Cells collected after 20 minutes (A-C), 40 minutes (D-F) and 60 minutes (G-I) in the regeneration buffer were fixed and stained with anti-tubulin (green) and anti-HA antibody (red). The Ift172p-HA first appeared in nascent cilia and gradually became enriched at the tips of some cilia. Arrowhead, nascent cilia at 20 minutes. Arrow, the Ift172p-HA in the nascent cilia (C) and accumulation at the tips of cilia (F and I). Scale bar, 10 µm.

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