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Tmem138 is localized to the connecting cilium essential for rhodopsin localization and outer segment biogenesis.

ABSTRACT: Photoreceptor connecting cilium (CC) is structurally analogous to the transition zone (TZ) of primary cilia and gates the molecular trafficking between the inner and the outer segment (OS). Retinal dystrophies with underlying CC defects are manifested in a broad array of syndromic conditions known as ciliopathies as well as nonsyndromic retinal degenerations. Despite extensive studies, many questions remain in the mechanism of protein trafficking across the photoreceptor CC. Here, we genetically inactivated mouse Tmem138, a gene encoding a putative transmembrane protein localized to the ciliary TZ and linked to ciliopathies. Germline deletion of Tmem138 abolished OS morphogenesis, followed by rapid photoreceptor degeneration. Tmem138 was found localized to the photoreceptor CC and was required for localization of Ahi1 to the distal subdomain of the CC. Among the examined set of OS proteins, rhodopsin was mislocalized throughout the mutant cell body prior to OS morphogenesis. Ablation of Tmem138 in mature rods recapitulated the molecular changes in the germline mutants, causing failure of disc renewal and disintegration of the OS. Furthermore, Tmem138 interacts reciprocally with rhodopsin and a related protein Tmem231, and the ciliary localization of the latter was also altered in the mutant photoreceptors. Taken together, these results suggest a crucial role of Tmem138 in the functional organization of the CC, which is essential for rhodopsin localization and OS biogenesis.

SUBMITTER: Guo D

PROVIDER: S-EPMC9169668 | biostudies-literature | 2022 Apr

REPOSITORIES: biostudies-literature

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Tmem138 is localized to the connecting cilium essential for rhodopsin localization and outer segment biogenesis.

Guo Dianlei D Ru Jiali J Xie Lijing L Wu Mingjuan M Su Yingchun Y Zhu Shiyong S Xu Shujuan S Zou Bin B Wei Yanhong Y Liu Xialin X Liu Yizhi Y Liu Chunqiao C

Proceedings of the National Academy of Sciences of the United States of America 20220408 15

Photoreceptor connecting cilium (CC) is structurally analogous to the transition zone (TZ) of primary cilia and gates the molecular trafficking between the inner and the outer segment (OS). Retinal dystrophies with underlying CC defects are manifested in a broad array of syndromic conditions known as ciliopathies as well as nonsyndromic retinal degenerations. Despite extensive studies, many questions remain in the mechanism of protein trafficking across the photoreceptor CC. Here, we genetically ...[more]

PMID: 35394880

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Project description:BackgroundThe retinal rod outer segment is a sensory cilium that is specialized for the conversion of light into an electrical signal. Within the cilium, up to several thousand membranous disks contain as many as a billion copies of rhodopsin for efficient photon capture. Disks are continually turned over, requiring the daily synthesis of a prodigious amount of rhodopsin. To promote axial diffusion in the aqueous cytoplasm, the disks have one or more incisures. Across vertebrates, the range of disk diameters spans an order of magnitude, and the number and length of the incisures vary considerably, but the mechanisms controlling disk architecture are not well understood. The finding that transgenic mice overexpressing rhodopsin have enlarged disks lacking an incisure prompted us to test whether lowered rhodopsin levels constrain disk assembly.Methodology/principal findingsThe structure and function of rods from hemizygous rhodopsin knockout (R+/-) mice with decreased rhodopsin expression were analyzed by transmission electron microscopy and single cell recording. R+/- rods were structurally altered in three ways: disk shape changed from circular to elliptical, disk surface area decreased, and the single incisure lengthened to divide the disk into two sections. Photocurrent responses to flashes recovered more rapidly than normal. A spatially resolved model of phototransduction indicated that changes in the packing densities of rhodopsin and other transduction proteins were responsible. The decrease in aqueous outer segment volume and the lengthened incisure had only minor effects on photon response amplitude and kinetics.Conclusions/significanceRhodopsin availability limits disk assembly and outer segment girth in normal rods. The incisure may buffer the supply of structural proteins needed to form larger disks. Decreased rhodopsin level accelerated photoresponse kinetics by increasing the rates of molecular collisions on the membrane. Faster responses, together with fewer rhodopsins, combine to lower overall sensitivity of R+/- rods to light.

Project description:Primary cilia exhibit a distinct complement of proteins, including G-protein-coupled receptors (GPCRs) that mediate sensory and developmental signals. The localization of GPCRs to the ciliary membrane involves ciliary localization sequences (CLSs), but it is not known how CLSs might relate to cilium type. Here, we studied the localization of two rhodopsin (RHO)-like GPCRs, somatostatin receptor (SSTR3) and RHO, in three types of cilia, from inner medullary collecting duct (IMCD3) cells, hTERT-RPE1 cells (possessing pocket cilia), and rod photoreceptors (whose cilia grow into elaborate phototransductive outer segments). SSTR3 was localized specifically to all three types of cilia, whereas RHO showed more selectivity for the photoreceptor cilium. Focusing on C-terminal CLSs, we characterized a novel CLS in the SSTR3 C terminus, which was required for the robust ciliary localization of SSTR3. Replacing the C terminus of RHO with this SSTR3 CLS-enhanced ciliary localization, compared with full-length RHO in IMCD3 and hTERT-RPE1 cells. Addition of the SSTR3 CLS to the single transmembrane protein CD8A enabled ciliary localization. In hTERT-RPE1 cells, a partial SSTR3 CLS added to CD8A effected specific localization to the periciliary (pocket) membrane, demonstrating C-terminal localization sequence targeting to this domain. Using retinas from mice, including both sexes, we show that deletion of the C terminus of RHO reduced the rod outer segment localization and that addition of the SSTR3 C-terminal CLS to the truncated RHO partly rescued this mislocalization. Overall, the study details elements of the different C termini of SSTR3 and RHO that are major effectors in determining specificity of cilium (or pericilium) localization among different types of cilia.SIGNIFICANCE STATEMENT The localization of G-protein-coupled receptors to primary cilia is key to many types of signal transduction. After characterizing a novel C-terminal CLS in SSTR3, we investigated how SSTR3 and RHO localization to the cilium relates to C-terminal CLSs and to cilium type. We found that the SSTR3 C-terminal CLS was effective in three different types of cilia, but the RHO C terminus showed a clear localization preference for the highly elaborate photoreceptor cilium. When added to CD8A, part of the SSTR3 CLS promoted specific periciliary membrane localization in hTERT-RPE1 cells, demonstrating an effective CLS for this domain. Thus, we demonstrate that elements of the C termini of SSTR3 and RHO determine different localization patterns among different types of cilia.

Dataset Information

Tmem138 is localized to the connecting cilium essential for rhodopsin localization and outer segment biogenesis.

Publications

Tmem138 is localized to the connecting cilium essential for rhodopsin localization and outer segment biogenesis.

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