Disruption of the basal body protein POC1B results in autosomal-recessive cone-rod dystrophy

Exome sequencing revealed a homozygous missense mutation (c.317C>G [p.Arg106Pro]) in POC1B, encoding POC1 centriolar protein B, in three siblings with autosomal-recessive cone dystrophy or cone-rod dystrophy and compound-heterozygous POC1B mutations (c.199_201del [p.G1n67del] and c.810+1G>T) in an unrelated person with cone-rod dystrophy. Upon overexpression of POC1B in human TERT-immortalized retinal pigment epithelium 1 cells, the encoded wild-type protein localized to the basal body of the primary cilium, whereas this localization was lost for p.Arg106Pro and p.G1n67del variant forms of POC1B. Morpholino-oligonucleotide-induced knockdown of poc1b translation in zebrafish resulted in a dose-dependent small-eye phenotype, impaired optokinetic responses, and decreased length of photoreceptor outer segments. These ocular phenotypes could partially be rescued by wild-type human POC1B mRNA, but not by c.199_201del and c.317C>G mutant human POC1B mRNAs. Yeast two-hybrid screening of a human retinal cDNA library revealed FAM161A as a binary interaction partner of POC1B. This was confirmed in coimmunoprecipitation and colocalization assays, which both showed loss of FAM161A interaction with p.Arg106Pro and p.G1n67del variant forms of POC1B. FAM161A was previously implicated in autosomal-recessive retinitis pigmentosa and shown to be located at the base of the photoreceptor connecting cilium, where it interacts with several other ciliopathy-associated proteins. Altogether, this study demonstrates that POC1B mutations result in a defect of the photoreceptor sensory cilium and thus affect cone and rod photoreceptors

Role of Noncovalent Binding of 11-cis-Retinal to Opsin in Dark Adaptation of Rod and Cone Photoreceptors

AbstractRegeneration of visual pigments of vertebrate rod and cone photoreceptors occurs by the initial noncovalent binding of 11-cis-retinal to opsin, followed by the formation of a covalent bond between the ligand and the protein. Here, we show that the noncovalent interaction between 11-cis-retinal and opsin affects the rate of dark adaptation. In rods, 11-cis-retinal produces a transient activation of the phototransduction cascade that precedes sensitivity recovery, thus slowing dark adaptation. In cones, 11-cis-retinal immediately deactivates phototransduction. Thus, the initial binding of the same ligand to two very similar G protein receptors, the rod and cone opsins, activates one and deactivates the other, contributing to the remarkable difference in the rates of rod and cone dark adaptation

Molecular characterization of the interaction between peripherin-2 and opsins in rod and cone photoreceptors

The tetraspanin peripherin-2 is a glyco-membrane protein exclusively expressed in the outer segments of rod and cone photoreceptors. Mutations in peripherin-2 are associated with retinal disorders characterized by Degeneration of rod or cone cells. Previous unpublished work identified peripherin-2 as a potential novel part of the protein complex comprising the B-subunit of the cyclic nucleotide-gated channel (CNGB1a and the light detector rhodopsin. In the first part of this study, using a combination of protein biochemical and FRET approaches in transfected HEK293 cells and in virally transduced murine rod outer segments, it could be demonstrated that peripherin-2 simultaneously binds to both, CNGB1a and rhodopsin. The interaction between peripherin-2 and rhodopsin was not described in previous studies. The binding domain mediating the peripherin-2/rhodopsin interaction could be narrowed down to the fourth transmembrane domain (TM4) of peripherin-2. Finally, the data revealed that the G266D point mutation in TM4 of peripherin-2 that is linked to a rod degenerative disease selectively disrupts the peripherin-2/rhodopsin interaction. To analyze if peripherin-2 also binds to cone opsins in the second part of this study, a similar experimental approach was conducted as used for the investigation of the peripherin-2/rhodopsin interaction. In this context, it was unveiled that peripherin-2 binds to both, short wavelength-and medium wavelength-sensitive cone opsin (S-opsin and M-opsin, respectively) in transfected HEK293 cells and in outer segments of transduced murine cones. Co-immunoprecipitation and quantitative FRET analysis revealed that binding of peripherin-2 to M-opsin was stronger than the peripherin-2/S-opsin interaction. This result was supported by transmission electron microscopy studies using gold particles coupled to opsin- and peripherin-2-specific antibodies. Finally, quantitative FRET analysis in transfected HEK293 cells and in transduced cone outer segments demonstrated that the V268I Point mutation in TM4 of peripherin-2 associated with a degenerative cone disease significantly attenuates the peripherin-2/M-opsin interaction. Taken together, this study provides a proof-of-principle for FRET-based analysis of protein-protein interactions in the outer segments of rod and cone photoreceptors. This approach led to the identification of hitherto unknown Protein complexes between peripherin-2 and opsins suggesting a novel physiological role of peripherin-2 in rods and cones. Finally, Analysis of disease-linked point mutations unveiled the molecular determinants of the peripherin-2/opsin interaction. These results might contribute to understanding the differential penetrance of certain point mutations in rods and cones

Cone-specific mediation of rod sensitivity in trichromatic observers

PURPOSE. The slope of the rod threshold versus the illuminance (TVI) function changes with the wavelength of the background light. This study was conducted to determine whether the changes in slope are due to the stimulation of specific cone classes. METHODS. An eight-channel optical system was used to generate lights that differed in cone and rod photoreceptor illuminance. Rod flicker TVI functions were measured in normal trichromatic observers at mesopic light levels. The independent variables were (1) the relative contribution of the short (S)-and long (L)-wavelength cones to the background light (i.e., the background lights varied along S-only and L-only lines), and (2) the temporal frequency of the flickering lights (4, 7.5, and 15 Hz). RESULTS. The 4-Hz rod flicker TVI function had a slope of 0.87 when measured near W (MacLeod-Boynton chromaticity of 0.66, 1.0). At 4 and 7.5 Hz, an increase in the relative L-cone illuminance steepened the slope of the rod-only TVI curve, but an increase in the relative S-cone illuminance had no effect. The slope of the 7.5-Hz TVI function decreased at higher illuminance levels. At 15 Hz, the thresholds could be measured over only a limited range. CONCLUSIONS. The L-cone system contributes to the desensitization of the rod system at mesopic light levels, whereas, in the range of lights used in these experiments, the S-cone system apparently does not. The possibility that S-cone stimulation desensitizes the response to rod signals at higher levels of S-cone illumination cannot be eliminated. (Invest Ophthalmol Vis Sci. 2002;43:898 -905) T he primate visual system operates over a range of 10 log units. This ability is due in part to the duplex retina in which scotopic (i.e., rod-dominated) vision operates at low light levels and photopic (i.e., cone-dominated) vision operates at high light levels. In several early studies, researchers proposed that these two systems behave independently of each other under many conditions, 1-4 but there is now clear evidence of the rods' influence on the cone systems and the cones' influence on the rod system. Visual signals originating in the rod photoreceptors do not have their own pathway to the brain but instead combine with neural signals originating in the cone photoreceptors. Signals originating with the rod photoreceptors are transmitted to the retinal ganglion cells through at least two anatomic pathways. One pathway combines through second-order cells. Rod photoreceptors connect to rod bipolar cells, which in turn connect to rod (AII) amacrine cells. The rod amacrine cells have gap junction connections with on-center ganglion cells in sublamina b of the inner plexiform layer, and have inhibitory synapses with off-center ganglion cells in sublamina a. Rod signals may also enter the cone circuit through gap junctions between rod spherules and cone pedicles (see Refs. 5-7). There is also recent evidence in rodents of a third pathway connecting the rod photoreceptors directly to OFF cone bipolar cells. 8,9 The general perceptual consequences of interaction between rods and cones have been documented extensively. We know, for instance, that the rod photoreceptor system influences cone-mediated sensitivity 10 -13 and vice versa 14 -18 ; that interaction between the rod and cone systems is more evident with flashed lights than with steady lights 19 ; and that location, spatial extent, and temporal frequency play an important role in determining the magnitude of rod and cone interaction. 17,20 -24 Rod-cone interaction (how rods influence cones) and cone-rod interaction (how cones influence rods) have become umbrella terms that characterize many classes of visual processing. One historical difficulty with experiments that investigate rod-cone (and cone-rod) interaction is that the narrowbandwidth lights (i.e., lights of a few spectral wavelengths) used as experimental stimuli often stimulate more than one class of photoreceptor. These experiments therefore do not lend themselves as easily to physiological interpretation. Many previous researchers have addressed such topics by measuring rod sensitivity to lights to which the rod system is much more sensitive than the cone systems (e.g., Ref. 25) or by investigating the responses of monochromatic and dichromatic observers. 26 -28 To investigate questions concerned with cone-rod interaction, I used an approach based on the cone-rod photoreceptor space defined by Shapiro et al. For this article, I examined rod TVI functions for 4-Hz flickering lights. Aguilar and Stiles 25 measured a rod TVI function by optimizing experimental parameters to isolate the rod system. One of these optimizations was to desensitize the cone systems with a long-wavelength adaptation light. They found that the slope of a major portion of the curve (i.e., when the adaptation light is between Ϫ2 and 2.2 log scotopic trolands [td]) is approximately 1.0. However, Sharpe et al

Cone-specific mediation of rod sensitivity in trichromatic observers

PURPOSE. The slope of the rod threshold versus the illuminance (TVI) function changes with the wavelength of the background light. This study was conducted to determine whether the changes in slope are due to the stimulation of specific cone classes. METHODS. An eight-channel optical system was used to generate lights that differed in cone and rod photoreceptor illuminance. Rod flicker TVI functions were measured in normal trichromatic observers at mesopic light levels. The independent variables were (1) the relative contribution of the short (S)-and long (L)-wavelength cones to the background light (i.e., the background lights varied along S-only and L-only lines), and (2) the temporal frequency of the flickering lights (4, 7.5, and 15 Hz). RESULTS. The 4-Hz rod flicker TVI function had a slope of 0.87 when measured near W (MacLeod-Boynton chromaticity of 0.66, 1.0). At 4 and 7.5 Hz, an increase in the relative L-cone illuminance steepened the slope of the rod-only TVI curve, but an increase in the relative S-cone illuminance had no effect. The slope of the 7.5-Hz TVI function decreased at higher illuminance levels. At 15 Hz, the thresholds could be measured over only a limited range. CONCLUSIONS. The L-cone system contributes to the desensitization of the rod system at mesopic light levels, whereas, in the range of lights used in these experiments, the S-cone system apparently does not. The possibility that S-cone stimulation desensitizes the response to rod signals at higher levels of S-cone illumination cannot be eliminated. (Invest Ophthalmol Vis Sci. 2002;43:898 -905) T he primate visual system operates over a range of 10 log units. This ability is due in part to the duplex retina in which scotopic (i.e., rod-dominated) vision operates at low light levels and photopic (i.e., cone-dominated) vision operates at high light levels. In several early studies, researchers proposed that these two systems behave independently of each other under many conditions, 1-4 but there is now clear evidence of the rods' influence on the cone systems and the cones' influence on the rod system. Visual signals originating in the rod photoreceptors do not have their own pathway to the brain but instead combine with neural signals originating in the cone photoreceptors. Signals originating with the rod photoreceptors are transmitted to the retinal ganglion cells through at least two anatomic pathways. One pathway combines through second-order cells. Rod photoreceptors connect to rod bipolar cells, which in turn connect to rod (AII) amacrine cells. The rod amacrine cells have gap junction connections with on-center ganglion cells in sublamina b of the inner plexiform layer, and have inhibitory synapses with off-center ganglion cells in sublamina a. Rod signals may also enter the cone circuit through gap junctions between rod spherules and cone pedicles (see Refs. 5-7). There is also recent evidence in rodents of a third pathway connecting the rod photoreceptors directly to OFF cone bipolar cells. 8,9 The general perceptual consequences of interaction between rods and cones have been documented extensively. We know, for instance, that the rod photoreceptor system influences cone-mediated sensitivity 10 -13 and vice versa 14 -18 ; that interaction between the rod and cone systems is more evident with flashed lights than with steady lights 19 ; and that location, spatial extent, and temporal frequency play an important role in determining the magnitude of rod and cone interaction. 17,20 -24 Rod-cone interaction (how rods influence cones) and cone-rod interaction (how cones influence rods) have become umbrella terms that characterize many classes of visual processing. One historical difficulty with experiments that investigate rod-cone (and cone-rod) interaction is that the narrowbandwidth lights (i.e., lights of a few spectral wavelengths) used as experimental stimuli often stimulate more than one class of photoreceptor. These experiments therefore do not lend themselves as easily to physiological interpretation. Many previous researchers have addressed such topics by measuring rod sensitivity to lights to which the rod system is much more sensitive than the cone systems (e.g., Ref. 25) or by investigating the responses of monochromatic and dichromatic observers. 26 -28 To investigate questions concerned with cone-rod interaction, I used an approach based on the cone-rod photoreceptor space defined by Shapiro et al. For this article, I examined rod TVI functions for 4-Hz flickering lights. Aguilar and Stiles 25 measured a rod TVI function by optimizing experimental parameters to isolate the rod system. One of these optimizations was to desensitize the cone systems with a long-wavelength adaptation light. They found that the slope of a major portion of the curve (i.e., when the adaptation light is between Ϫ2 and 2.2 log scotopic trolands [td]) is approximately 1.0. However, Sharpe et al

A model for transport of membrane-associated phototransduction polypeptides in rod and cone photoreceptor inner segments

AbstractWe discuss putative mechanisms of membrane protein transport in photoreceptors based on Pde6d and Gucy2e/Gucy2f knockout mice. Knockout of the Pde6d gene encoding PrBP/δ, a prenyl binding protein present in the retina at relatively high levels, was shown to impair transport of G-protein coupled receptor kinase 1 (GRK1) and cone phosphodiesterase α′ subunit (PDE6α′) to the rod and cone outer segments. Other prenylated proteins are minimally affected, suggesting some specificity of interaction. Knockout of the Gucy2e gene encoding guanylate cyclase 1 (GC1) disrupted transport of G-protein coupled receptor kinase 1 (GRK1), cone PDE6α′, cone transducin α and γ subunits (cTα and cTγ) to the cone outer segments, while a GC1/GC2 double knockout prevented transport of rod PDE6, but not transducin, GRK1, or rhodopsin, to the rod outer segments. These knockout phenotypes suggest that PrBP/δ functions in extracting prenylated proteins from the endoplasmic reticulum (ER) where they dock after prenylation, and that GC-bearing membranes may co-transport peripheral membrane proteins in vesicles. We conclude that distinct pathways have evolved in rods and cones for transport of integral and peripherally membrane-associated proteins

The transcription factor GTF2IRD1 regulates the topology and function of photoreceptors by modulating photoreceptor gene expression across the retina

The mechanisms that specify photoreceptor cell-fate determination, especially as regards to short-wave-sensitive (S) versus medium-wave-sensitive (M) cone identity, and maintain their nature and function, are not fully understood. Here we report the importance of general transcription factor II-I repeat domain-containing protein 1 (GTF2IRD1) in maintaining M cone cell identity and function as well as rod function. In the mouse, GTF2IRD1 is expressed in cell-fate determined photoreceptors at postnatal day 10. GTF2IRD1 binds to enhancer and promoter regions in the mouse rhodopsin, M- and S-opsin genes, but regulates their expression differentially. Through interaction with the transcription factors CRX and thyroid hormone receptor β 2, it enhances M-opsin expression, whereas it suppresses S-opsin expression; and with CRX and NRL, it enhances rhodopsin expression. In an apparent paradox, although GTF2IRD1 is widely expressed in multiple cell types across the retina, knock-out of GTF2IRD1 alters the retinal expression of only a limited number of annotated genes. Interestingly, however, the null mutation leads to altered topology of cone opsin expression in the retina, with aberrant S-opsin overexpression and M-opsin underexpression in M cones. Gtf2ird1-null mice also demonstrate abnormal M cone and rod electrophysiological responses. These findings suggest an important role for GTF2IRD1 in regulating the level and topology of rod and cone gene expression, and in maintaining normal retinal function

Rod-cone interaction in monocular but not binocular pathways

Photopic background stimulation elevates scotopic increment thresholds (rod-cone interaction) at moderate background levels when both test and concentric disk-background stimuli enter the same eye (monocular condition) but not when they enter different eyes (dichoptic condition). Only when background levels are made extremely high is there any measurable dichoptic interaction, and this interaction does not resemble that observed monocularly. Rod-cone interaction, as usually studied, is a property of monocular pathways in human vision.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26966/1/0000533.pd

Molecular Determinants of Photoreceptor Presynaptic Terminal Morphology

Rod and cone photoreceptors are light-receptive cells in the visual system that convert photons into an electrochemical signal to be processed through the retina and transmitted into the brain. From the first visual synapse, from photoreceptor to interneurons, rod spherules and cone pedicles diverge in morphology and connectivity patterns. It is known that the transcription factor Nrl is sufficient to drive the cone-to-rod cell fate conversion and morphological change. To dissect the source of the spherule versus pedicle differences, we performed a directed RNAi screen using in vivo electroporation to knock down a select portion of the Nrl regulome to identify genes associated with morphological features. We systematically characterized four distinct features of rod spherules and S-cone pedicles: spherule width, terminal position in the outer plexiform layer, ribbon number, and presence or absence of telodendrites. Using previously published next-generation sequencing data of the transcriptome of developing rod and cone-like photoreceptors as well as key transcription factor binding profiles, we defined a set of genes potentially associated with restricting spherule morphology from that of the default pedicle state. By knocking down genes individually, we were able to dissect the effects each gene has to restrict spherules. Our screen identified twenty-seven genes that control one or two independent features of rod photoreceptor spherule morphology, terminal width or outer plexiform layer position. Many of these were confirmed either through rescue experiments or examination of loss of function of mouse strains. Lastly, we generated a protein interaction network to connect the seemingly random sets of genes that controlled spherule morphology. Clustering of genes in this network did not show enrichment of our positive screen targets into communities. When we created shortest network pathways between all pairs of positive targets, we discovered that there was an enrichment of pathways that utilized Ncoa2, and this gene has a direct path to Nrl. We hypothesize that we have discovered a more directly involved gene regulatory network associated with the restriction of rod photoreceptor spherules. This knowledge should help in blinding disease treatment strategies to improve proper integration into the native retinal circuitry after loss of photoreceptors

Disruption of the Basal Body Protein POC1B Results in Autosomal-Recessive Cone-Rod Dystrophy

Exome sequencing revealed a homozygous missense mutation (c.317C>G [p.Arg106Pro]) in POC1B, encoding POC1 centriolar protein B, in three siblings with autosomal-recessive cone dystrophy or cone-rod dystrophy and compound-heterozygous POC1B mutations (c.199_201del [p.Gln67del] and c.810+1G>T) in an unrelated person with cone-rod dystrophy. Upon overexpression of POC1B in human TERT-immortalized retinal pigment epithelium 1 cells, the encoded wild-type protein localized to the basal body of the primary cilium, whereas this localization was lost for p.Arg106Pro and p.Gln67del variant forms of POC1B. Morpholino-oligonucleotide-induced knockdown of poc1b translation in zebrafish resulted in a dose-dependent small-eye phenotype, impaired optokinetic responses, and decreased length of photoreceptor outer segments. These ocular phenotypes could partially be rescued by wild-type human POC1B mRNA, but not by c.199_201del and c.317C>G mutant human POC1B mRNAs. Yeast two-hybrid screening of a human retinal cDNA library revealed FAM161A as a binary interaction partner of POC1B. This was confirmed in coimmunoprecipitation and colocalization assays, which both showed loss of FAM161A interaction with p.Arg106Pro and p.Gln67del variant forms of POC1B. FAM161A was previously implicated in autosomal-recessive retinitis pigmentosa and shown to be located at the base of the photoreceptor connecting cilium, where it interacts with several other ciliopathy-associated proteins. Altogether, this study demonstrates that POC1B mutations result in a defect of the photoreceptor sensory cilium and thus affect cone and rod photoreceptors