Negative regulation of ciliary length by ciliary male germ cell-associated kinase (Mak) is required for retinal photoreceptor survival

Abstract

Cilia function as cell sensors in many organs, and their disorders are referred to as "ciliopathies." Although ciliary components and transport machinery have been well studied, regulatory mechanisms of ciliary formation and maintenance are poorly understood. Here we show that male germ cell-associated kinase (Mak) regulates retinal photoreceptor ciliary length and subcompartmentalization. Mak was localized both in the connecting cilia and outer-segment axonemes of photoreceptor cells. In the Mak-null retina, photoreceptors exhibit elongated cilia and progressive degeneration. We observed accumulation of intraflagellar transport 88 (IFT88) and IFT57, expansion of kinesin family member 3A (Kif3a), and acetylated α-tubulin signals in the Mak-null photoreceptor cilia. We found abnormal rhodopsin accumulation in the Mak-null photoreceptor cell bodies at postnatal day 14. In addition, overexpression of retinitis pigmentosa 1 (RP1), a microtubule-associated protein localized in outer-segment axonemes, induced ciliary elongation, and Mak coexpression rescued excessive ciliary elongation by RP1. The RP1 N-terminal portion induces ciliary elongation and increased intensity of acetylated α-tubulin labeling in the cells and is phosphorylated by Mak. These results suggest that Mak is essential for the regulation of ciliary length and is required for the long-term survival of photoreceptors.

Fig. 1.

Expression and subcellular localization of Mak in the retina. (A and B) In situ hybridization analysis of retinal sections at E15.5 (A) and postnatal day 9 (P9) (B). Mak mRNA is expressed in both photoreceptor precursors and developing photoreceptors in the retina. (C–L) Subcellular localization of Mak in photoreceptors. Retinal sections at P14 (E) and 1 mo (C, D, F, and G) and dissociated photoreceptor cells at P14 (H–L) were stained with anti-Mak (red in C–L) and anti-acetylated α-tubulin (a marker for cilia; green in C, D, and I), anti-RPGR (a marker for connecting cilia; green in E and J), anti-RP1 (a marker for outer-segment axonemes; green in F and K), anti-ROM1 (a marker for outer-segment disks, green in H), or anti–γ-tubulin (a marker for basal bodies; green in G and L) antibodies. [Scale bars: 100 μm (A and B), 2 μm (H–L and D–G Center and Right), and 10 μm (C and D–G Left).] Arrowheads (D–L) indicate basal body-connecting cilium junctions. Brackets (E and J) indicate connecting cilia. CC, connecting cilia; GCL, ganglion cell layer; INL, inner nuclear layer; IS, inner segments; NBL, neuroblastic layer; ONL, outer nuclear layer; OPL, outer plexiform layer OS, outer segments; RPE, retinal pigment epithelium.

Fig. 2.

Loss of Mak leads to photoreceptor degeneration (A–H). Retinal sections from wild-type and Mak-KO mice at age P14 (A and B), 1 mo (C and D), 6 mo (E and F), and 12 mo (G and H) were stained with toluidine blue. Progressive degeneration of the ONL (a photoreceptor layer) occurs in the Mak-KO retina. (Scale bar: 100 μm.) IPL, inner plexiform layer. (I) Thickness of retinal layers was measured at age P14, 1 mo, 6 mo, and 12 mo. ONL thickness decreased progressively in the Mak-KO retina. Average of layer thickness in the wild-type retina at P14 was set to 100%. Error bars show SD. *P < 0.03. (J) ERGs recorded from Mak-KO mice. Scotopic ERGs elicited by four different stimulus intensities (−5.0 to 1.0 log cd-s/m²).

Fig. 3.

Ciliary defect in Mak-null photoreceptors. (A–H) Immunohistochemical analysis of the Mak-null photoreceptor cilia. Retinal sections from wild-type mice (A, A′, C, E, and G) and Mak-KO mice (B, B′, D, F, and H) at age P14 (A, A′, B, and B′) and 1 mo (C–H) were stained with anti-Mak (red in A, A′, B, and B′), anti-acetylated α-tubulin (a ciliary marker; green in A, B, G, and H; red in E and F), anti-RPGR (a connecting cilium marker; red in C and D), anti-RP1 (a marker for the outer-segment axonemes; green in E and F), anti-IFT88 (a component of IFT complex; red in G and H) or anti–γ-tubulin (a marker for the basal bodies; green in C and D) antibodies. Arrowheads in E and F indicate the distal tips of acetylated microtubules in the outer-segment axonemes. (I and J) Rhodopsin is mislocalized in the Mak-KO retina. Retinal sections from wild-type mice (I and I′) and Mak-KO mice (J and J′) at age P14 were stained with an anti-rhodopsin antibody. [Scale bars: 10 μm (A, C, E, and G, Upper), 100 μm (I and I′), and 2 μm (A′, C, E, and G, Lower).] (K and L) Length of the ciliary axonemes stained with the anti-acetylated α-tubulin antibody (K and L) and connecting cilia stained with the anti-RPGR (L) antibody in the wild-type photoreceptors (black bars) and Mak-KO photoreceptors (gray bars) were measured. Error bars show SE. *P < 0.03. (M and N) Longitudinal profiles of the connecting cilia in 1-mo-old wild-type photoreceptors (M) and Mak-KO photoreceptors (N) observed by electron microscopy. Arrowheads indicate the basal body-connecting cilium junctions in the photoreceptors. (Scale bar in M: 1 μm.)

Fig. 4.

RP1 controls ciliary length and is phosphorylated by Mak. (A–I) Overexpression of RP1 induces ciliary elongation. (A) Schematic diagrams of the RP1-FL, -N, -C1 and -C2 constructs. DC, doublecortin domain. (B–I) FLAG-tagged constructs expressing GFP (B and C), RP1-FL (D and E) or RP1 lacking the C-terminal portion (RP1-N) (F–I) were transfected into NIH 3T3 cells. Localization of FLAG-tagged proteins was observed using anti-FLAG (green) and anti-acetylated α-tubulin (red) antibodies and DAPI (blue). Arrows indicate transfected cells. Arrowheads indicate basal part of cilia. (J and K) RP1 and Mak antagonistically regulate ciliary length. FLAG-tagged constructs expressing GFP, RP1-FL, or RP1-N were transfected with or without a Mak expression plasmid into NIH 3T3 cells. (J) Cilia were observed using the anti-acetylated α-tubulin (red) antibody. (K) The length of the cilia stained with the anti-acetylated α-tubulin antibody (n > 30 for each construct). Error bars show SE. *P < 0.03. (L) Mak interacts with RP1. A Mak expression plasmid was transfected with or without FLAG-tagged RP1 expression plasmids (RP1-FL or RP1-N) into HEK293 cells. RP1 proteins were immunoprecipitated with the anti-FLAG antibody. Immunoprecipitated Mak was detected by Western blotting analysis using the anti-Mak antibody. (M) Mak phosphorylates RP1 in vitro. GST-RP1-N (residues 1–661), GST-RP1-C1 (residues 662–1,380), and GST-RP1-C2 (residues 1,381–2,095) were purified from bacterial extracts and stained with Coomassie brilliant blue (CBB) (Left). GST-RP1 deletion proteins were applied for the in vitro kinase assay using purified GST-Mak (Right). [Scale bars: 100 μm (B, D, and F), 10 μm (C, E, G, H, and I), and 2 μm (J).]