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The outer segments (OS) of rod and cone photoreceptor cells are specialized sensory cilia that contain hundreds of opsin-loaded stacked membrane disks that enable phototransduction. The biogenesis of these disks is initiated at the OS base, but the driving force has been debated. Here, we studied the function of the protein encoded by the photoreceptor-specific gene C2orf71, which is mutated in inherited retinal dystrophy (RP54). We demonstrate that C2orf71/PCARE (photoreceptor cilium actin regulator) can interact with the Arp2/3 complex activator WASF3, and efficiently recruits it to the primary cilium. Ectopic coexpression of PCARE and WASF3 in ciliated cells results in the remarkable expansion of the ciliary tip. This process was disrupted by small interfering RNA (siRNA)-based down-regulation of an actin regulator, by pharmacological inhibition of actin polymerization, and by the expression of PCARE harboring a retinal dystrophy-associated missense mutation. Using human retinal organoids and mouse retina, we observed that a similar actin dynamics-driven process is operational at the base of the photoreceptor OS where the PCARE module and actin colocalize, but which is abrogated in Pcare-/- mice. The observation that several proteins involved in retinal ciliopathies are translocated to these expansions renders it a potential common denominator in the pathomechanisms of these hereditary disorders. Together, our work suggests that PCARE is an actin-associated protein that interacts with WASF3 to regulate the actin-driven expansion of the ciliary membrane at the initiation of new outer segment disk formation.

The adhesion class of G protein-coupled receptors (GPCRs) is the second largest family of GPCRs (33 members in humans). Adhesion GPCRs (aGPCRs) are defined by a large extracellular N-terminal region that is linked to a C-terminal seven transmembrane (7TM) domain via a GPCR-autoproteolysis inducing (GAIN) domain containing a GPCR proteolytic site (GPS). Most aGPCRs undergo autoproteolysis at the GPS motif, but the cleaved fragments stay closely associated, with the N-terminal fragment (NTF) bound to the 7TM of the C-terminal fragment (CTF). The NTFs of most aGPCRs contain domains known to be involved in cell-cell adhesion, while the CTFs are involved in classical G protein signaling, as well as other intracellular signaling. In this workshop report, we review the most recent findings on the biology, signaling mechanisms, and physiological functions of aGPCRs.

The USH1G protein SANS is a small multifunctional scaffold protein. It is involved in several different cellular processes, such as intracellular transport, in the cytoplasm, or splicing of pre-mRNA, in the cell nucleus. Here, we aimed to gain insight into the regulation of the subcellular localization and the nuclear–cytoplasmic shuttling of SANS and its paralog ANKS4B, not yet reported in the nucleus. We identified karyopherins mediating the nuclear import and export by screening the nuclear interactome of SANS. Sequence analyses predicted in silico evolutionarily conserved nuclear localization sequences (NLSs) and nuclear export sequences (NESs) in SANS, but only NESs in ANKS4B, which are suitable for karyopherin binding. Quantifying the nuclear–cytoplasmic localization of wild-type SANS and NLS/NES mutants, we experimentally confirmed in silico predicted NLS and NES functioning in the nuclear–cytoplasmic shuttling in situ in cells. The comparison of SANS and its paralog ANKS4B revealed substantial differences in the interaction with the nuclear splicing protein PRPF31 and in their nuclear localization. Finally, our results on pathogenic USH1G/SANS mutants suggest that the loss of NLSs and NESs and thereby the ability to control nuclear–cytoplasmic shuttling is disease-relevant. [ABSTRACT FROM AUTHOR]

In a subset of inherited retinal degenerations (including cone, cone-rod, and macular dystrophies), cone photoreceptors are more severely affected than rods; ABCA4 mutations are the most common cause of this heterogeneous class of disorders. To identify retinal-disease-associated genes, we performed exome sequencing in 28 individuals with "cone-first" retinal disease and clinical features atypical for ABCA4 retinopathy. We then conducted a gene-based case-control association study with an internal exome data set as the control group. TTLL5, encoding a tubulin glutamylase, was highlighted as the most likely disease-associated gene; 2 of 28 affected subjects harbored presumed loss-of-function variants: c.[1586_1589delAGAG];[1586_1589delAGAG], p.[Glu529Valfs(∗)2];[Glu529Valfs(∗)2], and c.[401delT(;)3354G>A], p.[Leu134Argfs(∗)45(;)Trp1118(∗)]. We then inspected previously collected exome sequence data from individuals with related phenotypes and found two siblings with homozygous nonsense variant c.1627G>T (p.Glu543(∗)) in TTLL5. Subsequently, we tested a panel of 55 probands with retinal dystrophy for TTLL5 mutations; one proband had a homozygous missense change (c.1627G>A [p.Glu543Lys]). The retinal phenotype was highly similar in three of four families; the sibling pair had a more severe, early-onset disease. In human and murine retinae, TTLL5 localized to the centrioles at the base of the connecting cilium. TTLL5 has been previously reported to be essential for the correct function of sperm flagella in mice and play a role in polyglutamylation of primary cilia in vitro. Notably, genes involved in the polyglutamylation and deglutamylation of tubulin have been associated with photoreceptor degeneration in mice. The electrophysiological and fundus autofluorescence imaging presented here should facilitate the molecular diagnosis in further families.

Pre-mRNA splicing is an essential process orchestrated by the spliceosome, a dynamic complex assembled stepwise on pre-mRNA. We have previously identified that USH1G protein SANS regulates pre-mRNA splicing by mediating the intranuclear transfer of the spliceosomal U4/U6.U5 tri-snRNP complex. During this process, SANS interacts with the U4/U6 and U5 snRNP-specific proteins PRPF31 and PRPF6 and regulates splicing, which is disturbed by variants of USH1G/SANS causative for human Usher syndrome (USH), the most common form of hereditary deaf–blindness. Here, we aim to gain further insights into the molecular interaction of the splicing molecules PRPF31 and PRPF6 to the CENTn domain of SANS using fluorescence resonance energy transfer assays in cells and in silico deep learning-based protein structure predictions. This demonstrates that SANS directly binds via two distinct conserved regions of its CENTn to the two PRPFs. In addition, we provide evidence that these interactions occur sequentially and a conformational change of an intrinsically disordered region to a short α-helix of SANS CENTn2 is triggered by the binding of PRPF6. Furthermore, we find that pathogenic variants of USH1G/SANS perturb the binding of SANS to both PRPFs, implying a significance for the USH1G pathophysiology. [ABSTRACT FROM AUTHOR]

VLGR1/ADGRV1 (very large G protein‐coupled receptor‐1) is the largest adhesion G protein‐coupled receptor (aGPCR). Mutations in VLGR1/ADGRV1 are associated with human Usher syndrome, the most common form of deaf‐blindness, and also with epilepsy in humans and mice. VLGR1 is expressed almost ubiquitously but is mainly found in the CNS and in the sensory cells of the eye and inner ear. Little is known about the pathogenesis of the diseases related to VLGR1. We previously identified VLGR1 as a vital component of focal adhesions (FAs) serving as a metabotropic mechanoreceptor controls cell spreading and migration. FAs are highly dynamic and turnover in response to internal and external signals. Here, we aimed to elucidate how VLGR1 participates in FA turnover. Nocodazole washouts and live cell imaging of paxillin‐DsRed2 consistently showed that FA disassembly was not altered, but de novo assembly of FA was significantly delayed in Vlgr1‐deficient astrocytes, indicating that VLGR1 is enrolled in FA assembly. In FRAP experiments, recovery rates were significantly reduced in Vlgr1‐deficient FAs, indicating reduced turnover kinetics in VLGR1‐deficient FAs. We showed that VLGR1 regulates cell migration by controlling the FA turnover during their assembly and expect novel insights into pathomechanisms related to pathogenic dysfunctions of VLGR1. [ABSTRACT FROM AUTHOR]

(f) The percentages of GPR115+ and KRT1/10+ cells were determined in these constructs built with HaCaT WT cells (n = 10 optical fields, n = 20-27 cells/field, means ± SEM). (c) Western blot analysis of lysates of HA-GPR115 Cos-7 cells treated with PNGase F (left) or of HA-GPR115 Cos-7 cells cultured with tunicamycin (right); the Abs used are indicated. (b) HaCaT WT cells and Cos-7 cells, transfected with GPR115 pcDNA3.1, were cell-surface-stained with the GPR115ECD Ab and analyzed by flow cytometry. [Extracted from the article]