The retinitis pigmentosa 28 protein FAM161A is a novel ciliary protein involved in intermolecular protein interaction and microtubule association

Loss-of-function mutations in the gene encoding FAM161A were recently discovered as the cause for RP28, an autosomal recessive form of retinitis pigmentosa. To initiate the characterization of the cellular role of FAM161A in the retina, we focused on its subcellular localization and conducted in vitro studies to identify FAM161A-interacting proteins and associated cellular structures. Immunohistochemistry revealed the presence of mouse FAM161A in the photoreceptor inner segments, the synaptic regions of the outer and inner plexiform layers and the ganglion cells. In mouse and human retinal sections from unfixed eyes, FAM161A localized to the ciliary region linking photoreceptor outer and inner segments. High-resolution immunofluorescence and immunoelectron microscopy mapped FAM161A to the connecting cilium, the basal body region and the adjacent centriole. Ectopic FAM161A was found in the centrosome and concentrated at the base of primary cilia in cultured cells. In addition, overexpressed FAM161A was clearly associated with microtubules during interphase and mitosis. The presence of FAM161A increased microtubule acetylation and stabilization. We further show that the evolutionarily conserved UPF0564 domain of FAM161A is crucial for its binding to microtubules and mediates homo- and heterotypic FAM161A and FAM161B interaction. In conclusion, our study shows that FAM161A is a microtubule-associated ciliary protein presumably involved in microtubule stabilization to maintain the microtubule tracks and/or in transport processes along microtubules in photoreceptors and other retinal cell types

The giant spectrin βV couples the molecular motors to phototransduction and Usher syndrome type I proteins along their trafficking route

International audienceMutations in the myosin VIIa gene cause Usher syndrome type IB (USH1B), characterized by deaf-blindness. A delay of opsin trafficking has been observed in the retinal photoreceptor cells of myosin VIIa-deficient mice. We identified spectrin bV, the mammalian b-heavy spectrin, as a myosin VIIa-and rhodopsin-interacting partner in photoreceptor cells. Spectrin bV displays a polarized distribution from the Golgi apparatus to the base of the outer segment, which, unlike that of other b spectrins, matches the trafficking route of opsin and other phototransduction proteins. Formation of spectrin bV-rhodopsin complex could be detected in the differentiating photoreceptors as soon as their outer segment emerges. A failure of the spectrin bV-mediated coupling between myosin VIIa and opsin molecules thus probably accounts for the opsin transport delay in myosin VIIa-deficient mice. We showed that spectrin bV also associates with two USH1 proteins, sans (USH1G) and harmonin (USH1C). Spectrins are supposed to function as heteromers of a and b subunits, but fluorescence resonance energy transfer and in vitro binding experiments indicated that spectrin bV can also form homodimers, which likely supports its aII-independent bV functions. Finally, consistent with its distribution along the connecting cilia axonemes, spectrin bV binds to several subunits of the microtubule-based motor proteins, kinesin II and the dynein complex. We therefore suggest that spectrin bV homomers couple some USH1 proteins, opsin and other phototransduction proteins to both actin-and microtubule-based motors, thereby contributing to their transport towards the photoreceptor outer disks

Disruption of the retinitis pigmentosa 28 gene Fam161a in mice affects photoreceptor ciliary structure and leads to progressive retinal degeneration

Mutations in the FAM161A gene were previously identified as the cause for autosomal-recessive retinitis pigmentosa 28. To study the effects of Fam161a dysfunction in vivo, we generated gene-trapped Fam161a(GT/GT) mice with a disruption of its C-terminal domain essential for protein protein interactions. We confirmed the absence of the fulllength Fam161a protein in the retina of Fam161a(GTIGT) mice using western blots and showed weak expression of a truncated Fam161a protein by immunohistochemistry. Histological analyses demonstrated that photoreceptor segments were disorganized in young Fam161a(GTIGT) mice and that the outer retina was completely lost at 6 months of age. Reactive microglia appeared in the outer retina and electroretinography showed an early loss of photoreceptor function in 4-month-old Fam161a(GT/GT) animals. Light and electron microscopy revealed a remarkable phenotype of a significantly shortened connecting cilium, spread ciliary microtubule doublets and disturbed disk organization in Fam161a(GT/GT) photoreceptor cells. Co-immunolabeling experiments demonstrated reduced expression and mislocalization of centrin 3 and disturbed targeting of the Fam161a interactors lebercilin and Cep290, which were restricted to the basal body and proximal connecting cilium in Fam161a(GT/GT) retinas. Moreover, we identified misrouting of the outer segment cargo proteins opsin and rds/peripherin 2 in Fam161a(GT/GT) mice. In conclusion, our results suggest a critical role for the C-terminal domain of Fam161a for molecular interactions and integrity of the connecting cilium. Fam 161a is required for the molecular delivery into the outer segment cilium, a function which is essential for outer segment disk formation and ultimately visual function

Usherin defects lead to early-onset retinal dysfunction in zebrafish

Mutations in USH2A are the most frequent cause of Usher syndrome and autosomal recessive nonsyndromic retinitis pigmentosa. To unravel the pathogenic mechanisms underlying USH2A-associated retinal degeneration and to evaluate future therapeutic strategies that could potentially halt the progression of this devastating disorder, an animal model is needed. The available Ush2a knock-out mouse model does not mimic the human phenotype, because it presents with only a mild and late-onset retinal degeneration. Using CRISPR/Cas9-technology, we introduced protein-truncating germline lesions into the zebrafish ush2a gene (ush2armc1: c.2337_2342delinsAC; p.Cys780GlnfsTer32 and ush2ab1245: c.15520_15523delinsTG; p.Ala5174fsTer). Homozygous mutants were viable and displayed no obvious morphological or developmental defects. Immunohistochemical analyses with antibodies recognizing the N- or C-terminal region of the ush2a-encoded protein, usherin, demonstrated complete absence of usherin in photoreceptors of ush2armc1, but presence of the ectodomain of usherin at the periciliary membrane of ush2ab1245-derived photoreceptors. Furthermore, defects of usherin led to a reduction in localization of USH2 complex members, whirlin and Adgrv1, at the photoreceptor periciliary membrane of both mutants. Significantly elevated levels of apoptotic photoreceptors could be observed in both mutants when kept under constant bright illumination for three days. Electroretinogram (ERG) recordings revealed a significant and similar decrease in both a- and b-wave amplitudes in ush2armc1 as well as ush2ab1245 larvae as compared to strain- and age-matched wild-type larvae. In conclusion, this study shows that mutant ush2a zebrafish models present with early-onset retinal dysfunction that is exacerbated by light exposure. These models provide a better understanding of the pathophysiology underlying USH2A-associated RP and a unique opportunity to evaluate future therapeutic strategies

Tecnologie digitali per la valorizzazione del patrimonio culturale. La rappresentazione di beni non accessibili

Il contributo rientra nel filone delle sperimentazioni di comunicazione multimediale e di ricostruzione virtuale di architetture e frammenti urbani. Il lavoro presenta i risultati di alcune applicazioni digitali che adoperano il linguaggio multimediale per consentire la fruizione virtuale di siti per diverse ragioni non accessibili, per raccontare un percorso reale o concettuale. I casi studio analizzati si configurano quali possibili prodotti strategici di divulgazione e comunicazione per la conoscenza e la comprensione del patrimonio culturale e per la veicolazione di contenuti scientifici.The contribution is part of the study and experimentation of multimedia communication, virtual analysis and reconstruction of architectures and urban fragments. This paper shows the results of some digital applications that use multimedia language for virtual fruition and enhancement of some sites, which for several reasons, are not accessible, to tell a real or conceptual path. The cases analyzed are considered as possible strategic products of dissemination and communication for the knowledge and understanding of cultural heritage and for the transmission of scientific content

Additional file 4: of KIAA0556 is a novel ciliary basal body component mutated in Joubert syndrome

IFT analysis in C. elegans K04F10.2( tm1830 ) mutants. a Intraflagellar transport rates in wild-type and K04F10.2(tm1830) mutant worms. Shown are the anterograde and retrograde velocities (μm.s-1/standard deviation (SD)) of GFP-tagged IFT proteins along amphid and phasmid channel cilia (combined; top rows), or phasmid cilia only (bottom rows). t-test pairwise comparison with wild-type controls, n number of particles, N measured number of amphids and phasmids. OSM-3 is the worm orthologue of KIF17; CHE-11 is the worm orthologue of IFT140; OSM-6 is the worm orthologue of IFT52. b Representative fluorescence images of phasmid cilia showing normal IFT protein localisations and distributions in tm1830 mutants. ds distal segment, ms middle segment, bb basal body region, den dendrite. All images are similarly scaled and orientated (arrow denotes basal body). Scale bar, 3 μm. c Representative kymographs (time (t) over distance (d) plots) used to generate IFT rate measurements. For each kymograph, the horizontal axis (distance) is 5 μm and the vertical axis (time) is 25 seconds. d Distribution plots of IFT protein velocities. (JPG 1951 kb

An siRNA-based functional genomics screen for the identification of regulators of ciliogenesis and ciliopathy genes

Defects in primary cilium biogenesis underlie the ciliopathies, a growing group of genetic disorders. We describe a whole-genome siRNA-based reverse genetics screen for defects in biogenesis and/or maintenance of the primary cilium, obtaining a global resource. We identify 112 candidate ciliogenesis and ciliopathy genes, including 44 components of the ubiquitin-proteasome system, 12 G-protein-coupled receptors, and 3 pre-mRNA processing factors (PRPF6, PRPF8 and PRPF31) mutated in autosomal dominant retinitis pigmentosa. The PRPFs localize to the connecting cilium, and PRPF8- and PRPF31-mutated cells have ciliary defects. Combining the screen with exome sequencing data identified recessive mutations in PIBF1, also known as CEP90, and C21orf2, also known as LRRC76, as causes of the ciliopathies Joubert and Jeune syndromes. Biochemical approaches place C21orf2 within key ciliopathy-associated protein modules, offering an explanation for the skeletal and retinal involvement observed in individuals with C21orf2 variants. Our global, unbiased approaches provide insights into ciliogenesis complexity and identify roles for unanticipated pathways in human genetic disease

An siRNA-based functional genomics screen for the identification of regulators of ciliogenesis and ciliopathy genes

© 2015 Macmillan Publishers Limited. Defects in primary cilium biogenesis underlie the ciliopathies, a growing group of genetic disorders. We describe a whole-genome siRNA-based reverse genetics screen for defects in biogenesis and/or maintenance of the primary cilium, obtaining a global resource. We identify 112 candidate ciliogenesis and ciliopathy genes, including 44 components of the ubiquitin-proteasome system, 12 G-protein-coupled receptors, and 3 pre-mRNA processing factors (PRPF6, PRPF8 and PRPF31) mutated in autosomal dominant retinitis pigmentosa. The PRPFs localize to the connecting cilium, and PRPF8- and PRPF31-mutated cells have ciliary defects. Combining the screen with exome sequencing data identified recessive mutations in PIBF1, also known as CEP90, and C21orf2, also known as LRRC76, as causes of the ciliopathies Joubert and Jeune syndromes. Biochemical approaches place C21orf2 within key ciliopathy-associated protein modules, offering an explanation for the skeletal and retinal involvement observed in individuals with C21orf2 variants. Our global, unbiased approaches provide insights into ciliogenesis complexity and identify roles for unanticipated pathways in human genetic disease