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First published online 2 December 2008
doi: 10.1242/jcs.037408

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Localization of Inv in a distinctive intraciliary compartment requires the C-terminal ninein-homolog-containing region

Dai Shiba¹, Yoshihisa Yamaoka^2,*, Haruo Hagiwara^3,*, Tetsuro Takamatsu², Hiroshi Hamada⁴ and Takahiko Yokoyama^1,

¹ Department of Anatomy and Developmental Biology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan
² Department of Pathology and Cell Regulation, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan
³ Department of Anatomy and Cell Biology, Gunma University Graduate School of Medicine, Gunma, Japan
⁴ Developmental Genetics Group, Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan

View larger version (78K): [in this window] [in a new window]   Fig. 1. GFP-tagged Inv is detected at the base of primary cilium. (A) GFP-tagged Inv localization in vivo. GFP-tagged Inv (Inv-GFP) signal is detected at the tubule lumen from isolated renal tubules of Inv-GFP mice. Primary cilia were visualized by anti-acetylated -tubulin antibody (ac-tubulin; red). Merged picture of the boxed area is also shown. Inv-GFP signal is not present along the entire length of the primary cilium (arrowheads). Scale bar: 5 µm. (B) Visualization of primary cilia and Inv-GFP fluorescence from the top view (right) and from the side view (left) in living cells. Primary cilia of mouse renal epithelial cells derived from Inv-GFP mice were observed by Nomarski differential interference contrast (DIC). Primary cilia in a living cell can be seen as a dot when viewed from the top (arrowheads). The Inv-GFP signal is predominantly detected at the base of the primary cilium. Corresponding successive z-axis confocal images of both primary cilia and Inv-GFP fluorescence are available in supplementary material Movie 1. From the side view the Inv-GFP signal is also observed at the base of the primary cilium (white arrowheads). Scale bars: 5 µm. (C) Immunocytochemistry further confirms Inv-GFP localization. The primary cilium was detected with anti-acetylated -tubulin antibody (ac-tubulin; red), and the basal body and/or centrosome was detected with the anti--tubulin antibody (red). The Inv-GFP signal is predominantly detected at the base of the primary cilium, excluding the basal-body area. Localization of polycystin-2 (red) only partially overlaps with the Inv-GFP signal. Scale bars: 10 µm. Line scans of the fluorescent signal from `*' to `**' of the inserted images are shown on the right. A.U., arbitrary units.

View larger version (59K): [in this window] [in a new window]   Fig. 2. Endogenous Inv is exclusively detected at the base of primary cilia (the Inv compartment) but not in the basal body or ciliary necklace segment. (A) Localization of endogenous Inv. Inv was detected with the anti-Inv antibody in wild-type and inv cells. Primary cilia were stained with the anti-acetylated -tubulin antibody (ac-tubulin; green), and the basal body or centrosome were stained with the anti--tubulin antibody (green). The Inv signal (red) is detected at the base of the primary cilium, but not in the basal body area. In inv cells, there is no Inv signal in primary cilia. Thus, the antibody recognizes true endogenous Inv. Scale bars: 10 µm. Line scans of the fluorescent signal from `*' to `**' of the inserted images are shown on the right. A.U., arbitrary units. (B) Immunoelectron microscopical images of wild-type cells probed with an anti-Inv antibody. Significant Inv staining can be seen at the proximal segment of the primary cilium (Inv compartment), but not at the distal segment. The Inv signal is only seen between the ciliary membrane and axoneme, and not in the ciliary necklace segment (CN), basal body (BB) or transition zone (TZ). (C) Immunoblotting with anti-Inv antibody. Cell lysates extracted from inv cells (lane –) and inv cells expressing Inv protein (lane +) were separated by 7.5% SDS-PAGE, transferred to PVDF membrane and stained with anti-Inv antibody. A single major band is detected at approximately the predicted Inv molecular weight [117 kDa in lane + (arrowhead)].

View larger version (48K): [in this window] [in a new window]   Fig. 3. C-terminal Inv regulates Inv protein localization to the Inv compartment in primary cilia. (A) A schema of the Inv protein. Numbers indicate the position of amino acids; white ovals represent ankyrin repeats; the blue and gray boxes represent the IQ1/IQ2 domains and the basic region (BR), respectively. (B) Localization of Inv (1-1062), Inv (1-743) and Inv (742-1062) in the primary cilium was observed by GFP fluorescence and DIC in living cells. Numbers indicate the position of amino acids. Inv-GFP constructs were introduced into the inv cell line. Arrowheads in the images indicate GFP signal and primary cilia. Primary cilia in living cells are seen as a dot. Results of the localization of GFP-tagged truncated Inv are summarized on the right (+, Inv present; +/–, part of the transfected cells shows ciliary localization of truncated Inv). Percentages of ciliated cells with ciliary GFP signal in transfected cells are also indicated. (C) Localization of Inv-GFP proteins in cilia was further confirmed by immunocytochemistry. Primary cilia were detected by anti-acetylated -tubulin antibody (ac-tubulin; red), and basal body or centrosome by anti--tubulin antibody (red). Inv (1-743) signal was localized throughout the entire length of the cilia with a punctate staining pattern. By contrast, the Inv (742-1062) signal was predominantly detected at the base of primary cilia excluding the basal body. Scale bars: 10 µm.

View larger version (46K): [in this window] [in a new window]   Fig. 4. Two indispensable domains for primary-cilium targeting exist in the C-terminus of Inv. (A) Primary-cilium localization of GFP-tagged truncated forms of C-terminal Inv was observed by GFP fluorescence and DIC in living cells. Truncated Inv constructs were introduced into the inv cell line. Numbers indicate the position of amino acids. The wedged marking indicates the deletion of the IQ2 domain. Arrowheads in the images indicate the GFP signal and primary cilia. Primary cilia in living cells are seen as a dot. Results of the localization of GFP-tagged truncated Inv are summarized on the right. –, Inv absent; +, Inv present; +/–, part of the transfected cells shows ciliary localization of truncated Inv. Percentages of ciliated cells with positive ciliary GFP signal in transfected cells are also indicated. At least two independent regions, amino acids 915-935 (blue box: the IQ2 domain) and amino acids 1030-1062 (red box), are necessary for cilium targeting. (B) Localization of Inv (842-1062) in the primary cilia was further confirmed by immunocytochemistry. The primary cilium was detected by anti-acetylated -tubulin antibody (ac-tubulin; red). Inv (842-1062) signal was predominantly detected at the base of the primary cilium. Scale bars: 10 µm.

View larger version (50K): [in this window] [in a new window]   Fig. 5. Ciliary targeting of the Inv C-terminus depends on the IQ2 domain. (A) Localization of calmodulin was observed by immunocytochemistry. Calmodulin was detected by the anti-calmodulin antibody (green) and the primary cilium was detected by anti-acetylated -tubulin antibody (ac-tubulin; red). The calmodulin signal is predominantly present at the peri-basal-body area. Calmodulin staining in primary cilia is faint and distributed over the entire length of the primary cilium. Calmodulin localization in primary cilia and the peri-basal body is identical in wild-type cells and inv cells. Scale bars: 10 µm. (B) The IQ2 domain of mouse Inv is aligned with human and rat Inv. The construct in which glutamic acid (E) is substituted for isoleucine (I) in Inv (742-1062: I921E) is indicated in red. (C) GFP-tagged Inv (742-1062) and Inv (742-1062: I921E) proteins were expressed and ciliary targeting was studied. Mutation of an amino acid in the IQ2 domain resulted in loss of ciliary targeting. Arrowheads indicate GFP signal and primary cilia. Percentages of ciliated cells with positive ciliary GFP signal in transfected cells are indicated on the right. (D) Detection of direct calmodulin binding to the IQ2 domain. GST-fused truncated-Inv proteins expressed in E. coli under IPTG induction are shown. Inv (742-1062) binds to calmodulin, but deletion or mutation of the IQ2 domain results in loss of calmodulin binding (upper panel; arrowhead). Expression of GST fusion proteins was confirmed by anti-GST antibody staining (lower panel).

View larger version (45K): [in this window] [in a new window]   Fig. 6. The Inv C-terminal region contains a ninein homologous region. (A) The C-terminal ninein homologous region is highly conserved. The mouse C-terminal ninein homologous region of Inv is aligned with Inv and ninein from rat and human. Similar characteristic amino acids are in the same color. Numbers indicate the position of the amino acids. (B) Inv (1001-1062) signal (green) is detected at the centrosome and this is confirmed by anti--tubulin staining (red). Line-scan data (right) also shows colocalization of Inv (1001-1062) and -tubulin. Scale bar: 10 µm. (C) GFP-tagged ninein (1875-2035) signal (green) is detected at the centrosome and confirmed by anti--tubulin staining (red). Line-scan data (right) also shows the colocalization of GFP-tagged ninein (1875-2035) and -tubulin. Scale bar: 10 µm.

View larger version (57K): [in this window] [in a new window]   Fig. 7. The 60 C-terminal amino acids of Inv are required for localization of the full-length Inv protein to the Inv compartment. Inv (1-1062: BR), Inv (1-1062: IQ2) and Inv (1-1002) were fused with GFP. The constructs were introduced into inv cells and GFP signals were examined. Numbers indicate the position of the amino acids. Gray, blue and red boxes represent the BR domain, IQ2 domain and C-terminal ninein-homology-containing region, respectively. Wedged markings indicate the deletion of the BR domain, IQ2 domain or C-terminal ninein-homology-containing region. Primary cilia are detected with the anti-acetylated -tubulin antibody (ac-tubulin; red). GFP signals of Inv (1-1062), Inv (1-1062: BR) and Inv (1-1062: IQ2) are detected at the base of the primary cilium. By contrast, GFP-tagged Inv (1-1002) signal is localized over the entire length of cilia with a punctate staining. Scale bars: 10 µm.

View larger version (43K): [in this window] [in a new window]   Fig. 8. The Inv protein is a dynamic component along the primary cilium. (A) Schematic representation of FRAP analysis in Inv-GFP cells. Formvar film on which ciliated cells were cultured was folded over onto itself with the cell side out. Inv-GFP was partially photobleached. (B) Representative data of side-view FRAP. Photobleaching was performed at time 0. Bleaching widths of each FRAP experiment were 0.31 µm (top), 0.52 µm (middle) and 0.26 µm (bottom). The bleaching point is indicated by a yellow arrowhead. Line scans of fluorescent signal from `*' to `**' are shown below. Inv-GFP fluorescence rapidly recovered after photobleaching in all experiments, and the bleaching gap almost completely recovered within 60 seconds. Scale bar: 5 µm.

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