RESUMO
Sterile alpha motif domain containing 7 (SAMD7) is a component of the Polycomb repressive complex 1, which inhibits transcription of many genes, including those activated by the transcription factor Cone-Rod Homeobox (CRX). Here we report bi-allelic mutations in SAMD7 as a cause of autosomal-recessive macular dystrophy with or without cone dysfunction. Four of these mutations affect splicing, while another mutation is a missense variant that alters the repressive effect of SAMD7 on CRX-dependent promoter activity, as shown by in vitro assays. Immunostaining of human retinal sections revealed that SAMD7 is localized in the nuclei of both rods and cones, as well as in those of cells belonging to the inner nuclear layer. These results place SAMD7 as a gene crucial for human retinal function and demonstrate a significant difference in the role of SAMD7 between the human and the mouse retina.
Assuntos
Anormalidades do Olho , Degeneração Macular , Camundongos , Animais , Humanos , Transativadores/genética , Proteínas de Homeodomínio/genética , Retina , Mutação/genética , Degeneração Macular/genéticaRESUMO
The small size of ciliary structures that underlies photoreceptor function and inherited ciliopathies requires imaging techniques adapted to visualizing them at the highest possible resolution. In addition to powerful super-resolution imaging modalities, emerging approaches to sample preparation, including expansion microscopy (ExM), can provide a robust route to imaging specific molecules at the nanoscale level in the retina. We describe a protocol for applying ExM to whole retinas in order to achieve nanoscale fluorescence imaging of ciliary markers, including tubulin, CEP290, centrin, and CEP164. The results are consistent with those from other super-resolution fluorescence techniques and reveal new insights into their arrangements with respect to the subcompartments of photoreceptor cilia. This technique is complimentary to other imaging modalities used in retinal imaging, and can be carried out in virtually any laboratory, without the need for expensive specialized equipment.
Assuntos
Cílios , Microscopia , Camundongos , Animais , Retina/diagnóstico por imagem , Células FotorreceptorasRESUMO
Cilia are evolutionarily conserved hair-like structures with a wide spectrum of key biological roles, and their dysfunction has been linked to a growing class of genetic disorders, known collectively as ciliopathies. Many strides have been made towards deciphering the molecular causes for these diseases, which have in turn expanded the understanding of cilia and their functional roles. One recently-identified ciliary gene is ARL2BP, encoding the ADP-Ribosylation Factor Like 2 Binding Protein. In this study, we have identified multiple ciliopathy phenotypes associated with mutations in ARL2BP in human patients and in a mouse knockout model. Our research demonstrates that spermiogenesis is impaired, resulting in abnormally shaped heads, shortened and mis-assembled sperm tails, as well as in loss of axonemal doublets. Additional phenotypes in the mouse included enlarged ventricles of the brain and situs inversus. Mouse embryonic fibroblasts derived from knockout animals revealed delayed depolymerization of primary cilia. Our results suggest that ARL2BP is required for the structural maintenance of cilia as well as of the sperm flagellum, and that its deficiency leads to syndromic ciliopathy.
Assuntos
Proteínas de Transporte/genética , Ciliopatias/genética , Infertilidade Masculina/genética , Proteínas de Membrana Transportadoras/genética , Fotofobia/genética , Adulto , Animais , Cílios/patologia , Ciliopatias/patologia , Modelos Animais de Doenças , Feminino , Humanos , Infertilidade Masculina/patologia , Masculino , Camundongos , Camundongos Knockout , Microtúbulos/metabolismo , Pessoa de Meia-Idade , Linhagem , Fotofobia/patologia , Motilidade dos Espermatozoides/genética , Cauda do Espermatozoide/patologia , Espermatogênese/genética , Síndrome , Fatores de TranscriçãoRESUMO
The rod and cone photoreceptor cells of the vertebrate retina have highly specialized structures that enable them to carry out their function of light detection over a broad range of illumination intensities with optimized spatial and temporal resolution. Most prominent are their unusually large sensory cilia, consisting of outer segments packed with photosensitive disc membranes, a connecting cilium with many features reminiscent of the primary cilium transition zone, and a pair of centrioles forming a basal body which serves as the platform upon which the ciliary axoneme is assembled. These structures form a highway through which an enormous flux of material moves on a daily basis to sustain the continual turnover of outer segment discs and the energetic demands of phototransduction. After decades of study, the details of the fine structure and distribution of molecular components of these structures are still incompletely understood, but recent advances in cellular imaging techniques and animal models of inherited ciliary defects are yielding important new insights. This knowledge informs our understanding both of the mechanisms of trafficking and assembly and of the pathophysiological mechanisms of human blinding ciliopathies.
Assuntos
Cílios/ultraestrutura , Células Fotorreceptoras Retinianas Cones/ultraestrutura , Segmento Interno das Células Fotorreceptoras da Retina/ultraestrutura , Segmento Externo das Células Fotorreceptoras da Retina/ultraestrutura , Animais , Cílios/fisiologia , Humanos , Células Fotorreceptoras Retinianas Cones/fisiologia , Segmento Interno das Células Fotorreceptoras da Retina/fisiologia , Segmento Externo das Células Fotorreceptoras da Retina/fisiologiaRESUMO
Mutations in the Joubert syndrome-associated small GTPase ARL13B are linked to photoreceptor impairment and vision loss. To determine the role of ARL13B in the development, function, and maintenance of ciliated photoreceptors, we generated a pan-retina knock-out (Six3-Cre) and a rod photoreceptor-specific inducible conditional knock-out (Pde6g-CreERT2) of ARL13B using murine models. Embryonic deletion of ARL13B led to defects in retinal development with reduced cell proliferation. In the absence of ARL13B, photoreceptors failed to develop outer segment (OS) membranous discs and axonemes, resulting in loss of function and rapid degeneration. Additionally, the majority of photoreceptor basal bodies did not dock properly at the apical edge of the inner segments. The removal of ARL13B in adult rod photoreceptor cells after maturation of OS resulted in loss of photoresponse and vesiculation in the OS. Before changes in photoresponse, removal of ARL13B led to mislocalization of rhodopsin, prenylated phosphodiesterase-6 (PDE6), and intraflagellar transport protein-88 (IFT88). Our findings show that ARL13B is required at multiple stages of retinogenesis, including early postnatal proliferation of retinal progenitor cells, development of photoreceptor cilia, and morphogenesis of photoreceptor OS discs regardless of sex. Last, our results establish a need for ARL13B in photoreceptor maintenance and protein trafficking.SIGNIFICANCE STATEMENT The normal development of photoreceptor cilia is essential to create functional, organized outer segments with stacked membrane discs that house the phototransduction proteins necessary for sight. Our study identifies a complex role for ARL13B, a small GTPase linked to Joubert syndrome and visual impairment, at various stages of photoreceptor development. Loss of ARL13B led to defects in retinal proliferation, altered placement of basal bodies crucial for components of the cilium (transition zone) to emanate, and absence of photoreceptor-stacked discs. These defects led to extinguished visual response and dysregulated protein trafficking. Our findings show the complex role ARL13B plays in photoreceptor development, viability, and function. Our study accounts for the severe retinal impairment observed in ARL13B-linked Joubert syndrome patients.
Assuntos
Fatores de Ribosilação do ADP/fisiologia , Retina/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Fatores de Ribosilação do ADP/deficiência , Fatores de Ribosilação do ADP/genética , Envelhecimento/metabolismo , Animais , Axonema/metabolismo , Axonema/ultraestrutura , Cílios/metabolismo , Cílios/ultraestrutura , Proteínas do Olho/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Biogênese de Organelas , Transporte Proteico/fisiologia , Retina/anormalidades , Retina/embriologia , Retina/crescimento & desenvolvimento , Segmento Externo da Célula Bastonete/efeitos da radiação , Rodopsinas Sensoriais/metabolismoRESUMO
Bardet-Biedl syndrome (BBS) is an autosomal recessive ciliopathy characterized by developmental abnormalities and vision loss. To date, mutations in 21 genes have been linked to BBS. The products of eight of these BBS genes form a stable octameric complex termed the BBSome. Mutations in BBS8, a component of the BBSome, cause early vision loss, but the role of BBS8 in supporting vision is not known. To understand the mechanisms by which BBS8 supports rod and cone photoreceptor function, we generated animal models lacking BBS8. The loss of BBS8 protein led to concomitant decrease in the levels of BBSome subunits, BBS2 and BBS5 and increase in the levels of the BBS1 and BBS4 subunits. BBS8 ablation was associated with severe reduction of rod and cone photoreceptor function and progressive degeneration of each photoreceptor subtype. We observed disorganized and shortened photoreceptor outer segments (OS) at post-natal day 10 as the OS elaborates. Interestingly, loss of BBS8 led to changes in the distribution of photoreceptor axonemal proteins and hyper-acetylation of ciliary microtubules. In contrast to properly localized phototransduction machinery, we observed OS accumulation of syntaxin3, a protein normally found in the cytoplasm and the synaptic termini. In conclusion, our studies demonstrate the requirement for BBS8 in early development and elaboration of ciliated photoreceptor OS, explaining the need for BBS8 in normal vision. The findings from our study also imply that early targeting of both rods and cones in BBS8 patients is crucial for successful restoration of vision.
Assuntos
Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Células Fotorreceptoras/metabolismo , Animais , Síndrome de Bardet-Biedl/genética , Síndrome de Bardet-Biedl/metabolismo , Síndrome de Bardet-Biedl/patologia , Cílios/metabolismo , Proteínas do Citoesqueleto , Modelos Animais de Doenças , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Neurônios/patologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Cones/patologia , Células Fotorreceptoras Retinianas Bastonetes/metabolismoRESUMO
Receptor accessory protein 6 (REEP6) is a member of the REEP/Ypt-interacting protein family that we recently identified as essential for normal endoplasmic reticulum homeostasis and protein trafficking in the retina of mice and humans. Interestingly, in addition to the loss of REEP6 in our knockout (KO) mouse model recapitulating the retinal degeneration of humans with REEP6 mutations causing retinitis pigmentosa (RP), we also found that male mice are sterile. Herein, we characterize the infertility caused by loss of Reep6. Expression of both Reep6 mRNA transcripts is present in the testis; however, isoform 1 becomes overexpressed during spermiogenesis. In vitro fertilization assays reveal that Reep6 KO spermatozoa are able to bind the zona pellucida but are only able to fertilize oocytes lacking the zona pellucida. Although spermatogenesis appears normal in KO mice, cauda epididymal spermatozoa have severe motility defects and variable morphological abnormalities, including bent or absent tails. Immunofluorescent staining reveals that REEP6 expression first appears in stage IV tubules within step 15 spermatids, and REEP6 localizes to the connecting piece, midpiece, and annulus of mature spermatozoa. These data reveal an important role for REEP6 in sperm motility and morphology and is the first reported function for a REEP protein in reproductive processes. Additionally, this work identifies a new gene potentially responsible for human infertility and has implications for patients with RP harboring mutations in REEP6.
Assuntos
Proteínas do Olho/metabolismo , Proteínas de Membrana/metabolismo , Espermatozoides/citologia , Espermatozoides/fisiologia , Animais , Proteínas do Olho/genética , Regulação da Expressão Gênica , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Mutação , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Primary cilia mediate sensory signaling in multiple organisms and cell types but have structures adapted for specific roles. Structural defects in them lead to devastating diseases known as ciliopathies in humans. Key to their functions are structures at their base: the basal body, the transition zone, the "Y-shaped links," and the "ciliary necklace." We have used cryo-electron tomography with subtomogram averaging and conventional transmission electron microscopy to elucidate the structures associated with the basal region of the "connecting cilia" of rod outer segments in mouse retina. The longitudinal variations in microtubule (MT) structures and the lumenal scaffold complexes connecting them have been determined, as well as membrane-associated transition zone structures: Y-shaped links connecting MT to the membrane, and ciliary beads connected to them that protrude from the cell surface and form a necklace-like structure. These results represent a clearer structural scaffold onto which molecules identified by genetics, proteomics, and superresolution fluorescence can be placed in our emerging model of photoreceptor sensory cilia.
Assuntos
Centríolos , Cílios , Humanos , Animais , Camundongos , Tomografia com Microscopia Eletrônica , Microscopia Eletrônica de Transmissão , Corpos BasaisRESUMO
As signalling organelles, cilia regulate their G protein-coupled receptor content by ectocytosis, a process requiring localised actin dynamics to alter membrane shape. Photoreceptor outer segments comprise an expanse of folded membranes (discs) at the tip of highly-specialised connecting cilia, into which photosensitive GPCRs are concentrated. Discs are shed and remade daily. Defects in this process, due to mutations, cause retinitis pigmentosa (RP). Whilst fundamental for vision, the mechanism of photoreceptor disc generation is poorly understood. Here, we show membrane deformation required for disc genesis is driven by dynamic actin changes in a process akin to ectocytosis. We show RPGR, a leading RP gene, regulates actin-binding protein activity central to this process. Actin dynamics, required for disc formation, are perturbed in Rpgr mouse models, leading to aborted membrane shedding as ectosome-like vesicles, photoreceptor death and visual loss. Actin manipulation partially rescues this, suggesting the pathway could be targeted therapeutically. These findings help define how actin-mediated dynamics control outer segment turnover.
Assuntos
Actinas , Proteínas do Olho , Retinose Pigmentar , Animais , Actinas/metabolismo , Camundongos , Retinose Pigmentar/metabolismo , Retinose Pigmentar/genética , Proteínas do Olho/metabolismo , Proteínas do Olho/genética , Cílios/metabolismo , Humanos , Segmento Externo das Células Fotorreceptoras da Retina/metabolismo , Camundongos Knockout , Camundongos Endogâmicos C57BL , Membrana Celular/metabolismoRESUMO
Mutations in the cilium-associated protein CEP290 cause retinal degeneration as part of multiorgan ciliopathies or as retina-specific diseases. The precise location and the functional roles of CEP290 within cilia and, specifically, the connecting cilia (CC) of photoreceptors, remain unclear. We used super-resolution fluorescence microscopy and electron microscopy to localize CEP290 in the CC and in the primary cilia of cultured cells with subdiffraction resolution and to determine effects of CEP290 deficiency in 3 mutant models. Radially, CEP290 localizes in close proximity to the microtubule doublets in the region between the doublets and the ciliary membrane. Longitudinally, it is distributed throughout the length of the CC whereas it is confined to the very base of primary cilia in human retinal pigment epithelium-1 cells. We found Y-shaped links, ciliary substructures between microtubules and membrane, throughout the length of the CC. Severe CEP290 deficiencies in mouse models did not prevent assembly of cilia or cause obvious mislocalization of ciliary components in early stages of degeneration. There were fewer cilia and no normal outer segments in the mutants, but the Y-shaped links were clearly present. These results point to photoreceptor-specific functions of CEP290 essential for CC maturation and stability following the earliest stages of ciliogenesis.
Assuntos
Antígenos de Neoplasias/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cílios/metabolismo , Ciliopatias/genética , Proteínas do Citoesqueleto/metabolismo , Microscopia/métodos , Animais , Modelos Animais de Doenças , Humanos , CamundongosRESUMO
The outer segment (OS) of photoreceptor cells is an elaboration of a primary cilium with organized stacks of membranous disks that contain the proteins needed for phototransduction and vision. Though ciliary formation and function has been well characterized, little is known about the role of cilia in the development of photoreceptor OS. Nevertheless, progress has been made by studying mutations in ciliary proteins, which often result in malformed OSs and lead to blinding diseases. To investigate how ciliary proteins contribute to OS formation, we generated a knockout (KO) mouse model for ARL2BP, a ciliary protein linked to retinitis pigmentosa. The KO mice display an early and progressive reduction in visual response. Before photoreceptor degeneration, we observed disorganization of the photoreceptor OS, with vertically aligned disks and shortened axonemes. Interestingly, ciliary doublet microtubule (MT) structure was also impaired, displaying open B-tubule doublets, paired with loss of singlet MTs. On the basis of results from this study, we conclude that ARL2BP is necessary for photoreceptor ciliary doublet formation and axoneme elongation, which is required for OS morphogenesis and vision.