Inv acts as a molecular anchor for Nphp3 and Nek8 in the proximal segment of primary cilia

Abstract

A primary cilium is an antenna-like structure extending from the surface of most vertebrate cells. It is structurally divided along its vertical axis into sub-compartments that include the ciliary tip, the shaft, the ciliary necklace segment, the transitional zone and the basal body. We recently discovered that the shaft of the primary cilia has a distinct molecular compartment, termed the "Inv compartment", which is characterized by the accumulation of Inv at the base of primary cilia. Inv was discovered as a causative gene in inv mutant mice. It was later found to be responsible for the infantile type of nephronophthisis (NPHP2). Nephronophthisis (NPHP) is an autosomal recessive kidney disease. Nine causative genes have been identified, with all examined products thought to function in cilia, basal body and/or centrioles. However, their exact intra-ciliary localization and relationship have not been clear. Here, we report that products of Nphp3 and Nek8 (the mouse orthologs of the causative genes for NPHP3 and NPHP9, respectively) localize to the Inv compartment. We also show that Inv is essential for the compartmental localization of Nphp3 and Nek8, whereas localization of Inv does not require Nphp3 or Nek8. Nphp1 and Nphp4 also localize at the proximal region of the cilium, but not in Inv compartment. Our results indicate that Inv acts as an anchor for Nphp3 and Nek8 in the Inv compartment, and suggest that Inv compartment is a candidate site for intra-ciliary interaction of Inv, Nphp3 and Nek8.

Fig. 1.. Inv, Nphp3, and Nek8 localization in the Inv compartment.

A: Localization of Inv, Nphp3, and Nek8 in wild-type mouse renal epithelial cells (Dai1 cells). Inv and Nek8 are detected with the anti-Inv antibody and the anti-Nek8 antibody, respectively. Localization of Nphp3 is detected by transfection with GFP-tagged full-length construct of Nphp3. The Inv signal (green) is detected at the base of the primary cilium (ac-tubulin; red), but not in the basal body area (γ-Tubulin; red), that is Inv compartment (a top panel). Nphp3 (a middle panel; green) and Nek8 (a bottom panel; green) are also detected at the base of primary cilia. Scale bars = 10 μm. B: Nphp3 localizes to the Inv compartment. Nphp3 signal (green) is completely overlapped with Inv signal (red). Primary cilia are stained with the anti-acetylated α-Tubulin antibody (ac-tubulin; blue). Scale bar = 10 μm. Line scan of the fluorescent signal from “*” to “**” is also shown (bottom). A.U., arbitrary units. C: Nek8 localizes to the Inv compartment. Nek8 signal (red) is completely overlapped with Inv signal (GFP-tagged full-length Inv). Primary cilia are stained with the anti-acetylated α-Tubulin antibody (ac-tubulin; blue). Scale bar = 10 μm. Line scan of the fluorescent signal from “*” to “**” is also shown (bottom). A.U., arbitrary units.

Fig. 2.. Localization of Nphp1 and Nphp4.

A: Localization of Nphp1 and Nphp4 are different from Inv compartment. Localization of Nphp1 and Nphp4 are observed using wild-type mouse renal epithelial cells (Dai1 cells). Cells were transfected with GFP-tagged full-length constructs of Nphp1 or Nphp4. Both Nphp1 and Nphp4 signals are detected at around the transition zone of primary cilia (ac-tubulin; red), and not overlapped with the basal body (γ-Tubulin; red). Localization of Nphp1 and Nphp4 (green) are different from Inv compartment (Inv; red) (bottom panels). Scale bars = 10 μm. B: Co-localization of Nphp1 and Nphp4 in wild-type mouse renal epithelial cells (Dai1 cells). Cells are transfected with GFP (green) or mKO2 (red)-tagged full-length constructs of Nphp1 or Nphp4. Both Nphp1 and Nphp4 signals overlap each other. Scale bars = 10 μm. C: Localization of Nphp1 and Nphp4 are not changed in Inv mutant cells. Localization of Nphp1 and Nphp4 are observed using Inv mutant cells (Dai2 cells). Cells were transfected with GFP-tagged full-length constructs of Nphp1 and Nphp4. Both Nphps are detected at the base of primary cilia (ac-tubulin; red), and these signals are consistent with the results obtained from wild type cells (Dai1 cells). Scale bars = 10 μm.

Fig. 3.. Nphp3 and Nek8 localization to the Inv compartment are dependent on Inv.

A: Localization of Nphp3 and Nek8 in Inv mutant cells (Dai2 cells) are different from those in wild-type cells (Dai1 cells). Nphp3-GFP transfected cells are identified with cytoplasmic GFP signal. Faint and patchy ciliary localization of Nphp3 is observed along entire length of primary cilia in Inv mutant cells (a top panel), and some cells have extremely faint or no detectable Nphp3 signals in the primary cilia (a middle panel). Nek8 localization to the primary cilia is not detected in Inv mutant cells (a bottom panel). Primary cilia are stained with the anti-acetylated α-Tubulin antibody (ac-tubulin; blue). Results of the localization of Nphp3 and Nek8 to the Inv compartment are summarized on the right (−, absent). Scale bars = 10 μm. B: Inv regulates localization of Nphp3 and Nek8 to the Inv compartment. Introduction of a full-length Inv construct, Inv (1–1062), into Inv mutant cells recovers Nphp3 and Nek8 localization to the Inv compartment. Localization of Nphp3 is detected by co-transfection with mKO2-tagged full-length construct of Nphp3. Nek8 is detected with the anti-Nek8 antibody. A C-terminal Inv construct, Inv (742–1062), localizes to the Inv compartment, but it does not rescue Nphp3 and Nek8 localization. Results of the localization of Nphp3 and Nek8 to the Inv compartment are summarized on the right (−, absent; +, present). Scale bars = 10 μm. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Fig. 4.. Classification of Nphps according to Inv compartment localization.

A schematic representation of Inv compartment and Nphps in the primary cilium. Inv, Nphp3, and Nek8 localize at the proximal segment of the primary cilia termed the “Inv compartment”. Inv compartment is a candidate site of intraciliary interaction of these molecules. Nphp1 and Nphp4 are not detected in the Inv compartment. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]