Figure 1.
Figure 1. Localization of full-length and truncated NPHP4 in multiciliated cells of the Xenopus epidermis. At the four-cell stage, Xenopus embryos were injected with mRNA for GFP-NPHP4 (green in merge), together with mRNAs for RFP-Centrin or RFP-Clamp (red in merge) to label basal bodies and ciliary rootlets, respectively. At stage 32, confocal microscopy was performed to study the localization of full-length and truncated NPHP4 in the cells with multiple motile cilia of epidermal skin. Maximum intensity projection of obtained confocal datasets is shown, unless noted otherwise. (A) Colocalization of GFP-NPHP4 with RFP-Centrin. Area enclosed by the white box is magnified in the right panel (5.3× magnification; see Fig. S1 A for separate GFP and RFP channels). (B) 3D reconstruction of confocal datasets is shown. Injection of mRNA encoding for GFP-NPHP4 (200 pg) revealed the broad localization of GFP-NPHP4 extending from the basal body to the presumptive transition zone distal to the basal body, whereas lower-dose injection of mRNA (70 pg) revealed a more confined localization of GFP-NPHP4 to the presumptive transition zone. The insets are magnified on the right panels. (C–F) Localization of NPHP4 truncations is studied. Numbers on the left of the panels indicate the first and last amino acid of the NPHP4 truncations. Expression of all constructs was confirmed by Western blot. (C) The C-terminal half of NPHP4 (amino acids 863–1,426) is sufficient for localization to the basal body. (D–F) The C-terminal half of NPHP4 contains two distinct subdomains targeting NPHP4 to the basal bodies: amino acids 863–1,250 and amino acids 1,251–1,426. (E) The first subdomain (amino acids 863–1,250) showed additional localization to the ciliary rootlet, colocalizing with RFP-Clamp. The inset is magnified by 2.4× on the right panels. (F) The C-terminal fragment spanning amino acids 1,225–1,426 showed enhanced localization to the plasma membrane (arrowhead). Area enclosed by the white box is magnified in the right panel. Bars: (main) 5 µm; (B, magnified insets) 500 nm.

Localization of full-length and truncated NPHP4 in multiciliated cells of the Xenopus epidermis. At the four-cell stage, Xenopus embryos were injected with mRNA for GFP-NPHP4 (green in merge), together with mRNAs for RFP-Centrin or RFP-Clamp (red in merge) to label basal bodies and ciliary rootlets, respectively. At stage 32, confocal microscopy was performed to study the localization of full-length and truncated NPHP4 in the cells with multiple motile cilia of epidermal skin. Maximum intensity projection of obtained confocal datasets is shown, unless noted otherwise. (A) Colocalization of GFP-NPHP4 with RFP-Centrin. Area enclosed by the white box is magnified in the right panel (5.3× magnification; see Fig. S1 A for separate GFP and RFP channels). (B) 3D reconstruction of confocal datasets is shown. Injection of mRNA encoding for GFP-NPHP4 (200 pg) revealed the broad localization of GFP-NPHP4 extending from the basal body to the presumptive transition zone distal to the basal body, whereas lower-dose injection of mRNA (70 pg) revealed a more confined localization of GFP-NPHP4 to the presumptive transition zone. The insets are magnified on the right panels. (C–F) Localization of NPHP4 truncations is studied. Numbers on the left of the panels indicate the first and last amino acid of the NPHP4 truncations. Expression of all constructs was confirmed by Western blot. (C) The C-terminal half of NPHP4 (amino acids 863–1,426) is sufficient for localization to the basal body. (D–F) The C-terminal half of NPHP4 contains two distinct subdomains targeting NPHP4 to the basal bodies: amino acids 863–1,250 and amino acids 1,251–1,426. (E) The first subdomain (amino acids 863–1,250) showed additional localization to the ciliary rootlet, colocalizing with RFP-Clamp. The inset is magnified by 2.4× on the right panels. (F) The C-terminal fragment spanning amino acids 1,225–1,426 showed enhanced localization to the plasma membrane (arrowhead). Area enclosed by the white box is magnified in the right panel. Bars: (main) 5 µm; (B, magnified insets) 500 nm.

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