Centriole and transition zone structures in photoreceptor cilia revealed by cryo-electron tomography.

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.

Mutations in SAMD7 cause autosomal-recessive macular dystrophy with or without cone dysfunction.

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.

Expansion Microscopy of Mouse Photoreceptor Cilia.

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.

Super-resolution microscopy reveals photoreceptor-specific subciliary location and function of ciliopathy-associated protein CEP290.

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.

Knockout of mouse receptor accessory protein 6 leads to sperm function and morphology defects†.

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.

Mutations in ARL2BP, a protein required for ciliary microtubule structure, cause syndromic male infertility in humans and mice.

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.

ARL13B, a Joubert Syndrome-Associated Protein, Is Critical for Retinogenesis and Elaboration of Mouse Photoreceptor Outer Segments.

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.

ARL2BP, a protein linked to retinitis pigmentosa, is needed for normal photoreceptor cilia doublets and outer segment structure.

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.