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Molecular Mechanism Underlying OS Vesicular Trafficking and Models for OS Disc Morphogenesis in Mammalian Rods (A) The ''vesicular targeting model'' suggests new discs are assembled and ''grow'' via SARA-, PI3P-, and SNARE-mediated vesicular trafficking and membrane fusion events. This set of events is likely to continue until a disc membrane reaches its mature size. The axonemal vesicles and the primitive disc sacs could be derived from internalized OS basal membranes, although it is also probable that some of the axonemal vesicles are directly shipped from the IS through the connecting cilium. (B) Our data suggest that at the basal OS, the axonemally localized vesicles recruit SARA via rhodopsin's cytoplasmic tail. SARA tethers axonemal vesicles onto nascent discs through the interaction between its FYVE domain and PI3P. In addition, SARA may regulate SNARE fusion complex assembly and/or activity via its direct interaction with syntaxin 3. (C) The ''evagination/disc rim formation'' model suggests that discs are formed by a series of evagination events of the basal OS plasma membrane, depicted in the diagram in a longitudinal view. In this model, the most proximal open disc and most basal OS plasma membrane are fused by bilaterally growing ciliary membranes (i.e., disc rims). The two growing ciliary membranes meet at the opposite side of axoneme, which results in a closed disc.
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The light-sensing organelle of the vertebrate rod photoreceptor, the outer segment (OS), is a modified cilium containing approximately 1,000 stacked disc membranes that are densely packed with visual pigment rhodopsin. The mammalian OS is renewed every ten days; new discs are assembled at the base of the OS by a poorly understood mechanism. Our res...
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Context 1
... this end, a plasmid (SARA-sh/RFP) containing both a short-hairpin RNA (sh) against SARA and an RFP ex- pression cassette was generated ( Figure 5A). The speci- ficity and effectiveness of SARA-sh/RFP in silencing SARA expression was validated by immunoblotting assays ( Figure 5A) and immunostaining in cell cultures ( Figure S7). ...
Context 2
... now, the single predominant hypothesis for disc biogenesis, the evagination/disc rim formation model proposed by Steinberg and his colleagues ) was based solely upon their morphological studies of adult monkey rods. These authors observed a few outfolded plasma membranes, which were referred to as open discs, at the base of OS and thus hypothesized that discs are formed by a series of evaginations of the basal OS plasma membranes followed by a ''disc rim for- mation'' process that pinches off discs ( Figure 7C). Basal open discs have also been described for rodent rods (Carter-Dawson and LaVail, 1979). ...
Context 3
... results presented in this report complementarily support a novel model that we refer to as the ''vesicular targeting model,'' in which SARA-, PI3P-, and SNARE- regulated vesicular trafficking underlies the disc incorpo- ration of rhodopsin as well as disc assembly (Figures 7A and 7B). First, our ultrastructural analysis of adult rodent rods revealed abundant tubulo-vesicles in the proximal OS axonemal cytoplasmic space. ...
Context 4
... the molecular details of syntaxin 3's action in the OS require future identification of its cognate part- ners, it is tempting to suggest that the strategic topologi- cal restriction of syntaxin 3 in the OS likely plays a central role in determining the sites where the membrane fusions occur. We further speculate that the syntaxin 3-mediated SNARE activity is employed for the ''heterotypic fusion'' between axonemal vesicles and nascent discs, the ''ho- motypic fusion'' of axonemal vesicles, or both ( Figure 7A). ...
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Citations
... We sought a reliable antibody marker of the IS plasma membrane so that single ISs could be identified in our localization experiments. Antibodies that target STX3, a t-SNARE protein previously localized at the IS plasma membrane in mouse retina [55,67,68], and the Na/ K-ATPase channel, which has also been used as a general IS marker [22,69], were tested. Using SIM and STORM super-resolution microscopy experiments, the STX3 antibody resulted in more densely labeled IS boundaries of individual rods (Fig 4A and 4B). ...
Photoreceptor cells in the vertebrate retina have a highly compartmentalized morphology for efficient phototransduction and vision. Rhodopsin, the visual pigment in rod photoreceptors, is densely packaged into the rod outer segment sensory cilium and continuously renewed through essential synthesis and trafficking pathways housed in the rod inner segment. Despite the importance of this region for rod health and maintenance, the subcellular organization of rhodopsin and its trafficking regulators in the mammalian rod inner segment remain undefined. We used super-resolution fluorescence microscopy with optimized retinal immunolabeling techniques to perform a single molecule localization analysis of rhodopsin in the inner segments of mouse rods. We found that a significant fraction of rhodopsin molecules was localized at the plasma membrane, at the surface, in an even distribution along the entire length of the inner segment, where markers of transport vesicles also colocalized. Thus, our results collectively establish a model of rhodopsin trafficking through the inner segment plasma membrane as an essential subcellular pathway in mouse rod photoreceptors.
... Rod outer segment rhodopsin is synthesized in the endoplasmic reticulum and further modified in the Golgi, both organelles located in the IS, far from the OS. Rhodopsin is delivered to the OS by vesicular transport within the connecting cilium, and these intracellular vesicles then directly coalesce into the disk endomembranes [41][42][43][44][45][46][47][48]. Rhodopsin's terminal carboxyl sequence plays a fundamental role in this transport, the V(valine)XP(proline)X-COOH sequence interacting with the light chain of the molecular motor cytoplasmic dynein [49][50][51]. ...
An increasing number of articles have been published in recent years on the role of the endocannabinoid system (ECS) in different cellular processes. Here we review and discuss findings on the ECS in visual processing and present the structure of the retina. We focus on the photoreceptor cell and the events that occur in the phototransduction process, considering the conformational light-induced changes in rhodopsin and in particular its chromophore (11-cis retinal). Advances in the distribution and function of the endocannabinoid system in the retina with special reference to its function in the physiological light process are also addressed, as is the relationship between rhodopsin, retinal pathologies and the ECS.
... 160977 We previously reported that Smad anchor for receptor activation (SARA) is essential for maintaining the epithelial cell phenotype (26). Despite SARA being originally reported as an adaptor for the transforming growth factor-β (TGF-β) receptor (27,28), we and others subsequently reported that SARA is dispensable for TGF-β signaling and exerts functions that are not directly associated with TGF-β pathway, such as in intracellular molecular trafficking, cellular phenotype maintenance, and neuronal development (29)(30)(31)(32)(33). Interestingly, reanalysis of publicly available National Center for Biotechnology Information Gene Expression Omnibus (NCBI GEO) database data sets showed that SARA is detected in normal skin tissue and decreased in skin samples from patients with SSc (GSE9285 reported in ref. 34) and in cultured fibroblasts treated with fibrotic stimuli (GSE27165 reported in ref. 35). Our in vitro studies suggested that low SARA levels lead to spontaneous acquisition of mesenchymal phenotype in epithelial cells, implicating loss of SARA as one of the initial events leading to myofibroblast precursor transdifferentiation (26). ...
... SARA is a highly conserved protein, originally reported as a modulator of the TGF-β pathway. However, subsequent works by us and others showed that SARA is indeed dispensable for TGF-β signaling (30,31,33) and defined a role of SARA independent of the TGF-β pathway, including in intracellular molecular trafficking (27) and neuronal development (29,32). We have previously demonstrated that SARA is critical in maintaining epithelial cell phenotype in culture. ...
We previously reported that Smad Anchor for Receptor Activation (SARA) plays a critical role in maintaining epithelial cell phenotype. Here, we show that SARA suppresses myofibroblast precursor transdifferentiation in a mouse model of scleroderma. Mice overexpressing SARA specifically in PDGFRβ+ pericytes and pan-leukocytes (SARATg) developed significantly less skin fibrosis in response to bleomycin injection compared to wild-type littermates (SARAWT).Single cell RNASeq analysis of skin PDGFRβ+ cells implicated pericyte subsets assuming myofibroblast characteristics under fibrotic stimuli, and SARA overexpression blocked the transition. In addition, a cluster that expresses molecules associated with Th2 cells and macrophage activation was enriched in SARAWT, but not in SARATg mouse, after bleomycin treatment. Th2- specific Il-31 expression was increased in skin of the bleomycin-treated SARAWT mice, and scleroderma patients. Receptor-ligand analyses indicated that lymphocytes mediate pericyte transdifferentiation in SARAWT mice, while with SARA overexpression the myofibroblast activity of pericytes was suppressed. Together, these data suggest a novel crosstalk between myofibroblast precursors and immune cells in the pathogenesis of SSc, for which SARA plays a critical role.
... Thus, the photoreceptor synaptic ribbon appears to have several components in common with the primary cilium, raising the possibility that common functional mechanisms could also prevail at these two compartments. In agreement with this proposal, the t-SNARE protein Syntaxin-3 is essential for vesicle fusion both at the photoreceptor cilium as well as at the synaptic ribbon [61][62][63][64]. Future analyses might reveal further molecular and functional similarities between the synaptic ribbon and primary cilia. ...
The Unc119 protein mediates transport of myristoylated proteins to the photoreceptor outer segment, a specialized primary cilium. This transport activity is regulated by the GTPase Arl3 as well as by Arl13b and Rp2 that control Arl3 activation/inactivation. Interestingly, Unc119 is also enriched in photoreceptor synapses and can bind to RIBEYE, the main component of synaptic ribbons. In the present study, we analyzed whether the known regulatory proteins, that control the Unc119-dependent myristoylated protein transport at the primary cilium, are also present at the photoreceptor synaptic ribbon complex by using high-resolution immunofluorescence and immunogold electron microscopy. We found Arl3 and Arl13b to be enriched at the synaptic ribbon whereas Rp2 was predominantly found on vesicles distributed within the entire terminal. These findings indicate that the synaptic ribbon could be involved in the discharge of Unc119-bound lipid-modified proteins. In agreement with this hypothesis, we found Nphp3 (Nephrocystin-3), a myristoylated, Unc119-dependent cargo protein enriched at the basal portion of the ribbon in close vicinity to the active zone. Mutations in Nphp3 are known to be associated with Senior–Løken Syndrome 3 (SLS3). Visual impairment and blindness in SLS3 might thus not only result from ciliary dysfunctions but also from malfunctions of the photoreceptor synapse.
... Thus, the photoreceptor synaptic ribbon appears to have several components in common with the primary cilium, raising the possibility that common functional mechanisms could also prevail at these two compartments. In agreement with this proposal, the t-SNARE protein Syntaxin-3 is essential for vesicle fusion both at the photoreceptor cilium as well as at the synaptic ribbon [61][62][63][64]. Future analyses might reveal further molecular and functional similarities between the synaptic ribbon and primary cilia. ...
The Unc119 protein mediates transport of myristoylated proteins to the photoreceptor outer segment, a specialized primary cilium. This transport activity is regulated by the GTPase Arl3 as well as by Arl13b and Rp2 that control Arl3 activation/inactivation. Interestingly, Unc119 is also enriched in photoreceptor synapses and can bind to RIBEYE, the main component of synaptic ribbons. In the present study, we analyzed whether the known regulatory proteins, that control the Unc119-dependent myristoylated protein transport at the primary cilium, are also present at the photoreceptor synaptic ribbon complex by using high-resolution immunofluorescence and immunogold electron microscopy. We found Arl3 and Arl13b to be enriched at the synaptic ribbon whereas Rp2 was predominantly found on vesicles distributed within the entire terminal. These findings indicate that the synaptic ribbon could be involved in the discharge of Unc119-bound lipid-modified proteins. In agreement with this hypothesis, we found Nphp3 (Nephrocystin-3), a myristoylated, Unc119-dependent cargo protein enriched at the basal portion of the ribbon in close vicinity to the active zone. Mutations in Nphp3 are known to be associated with Senior–Løken Syndrome 3 (SLS3). Visual impairment and blindness in SLS3 might thus not only result from ciliary dysfunctions but also from malfunctions of the photoreceptor synapse.This article is protected by copyright. All rights preserved.
... Disruption of this CC complex leads to rhodopsin IS mislocalization and defective OS membranes, releasing extracellular vesicles. This model is drawn compatible with the prevailing "evagination model" for OS biogenesis (53), in which rhodopsin moves within the CC membrane, while it does not refute the "endosome" model (54)(55)(56)(57), in which intraciliary traffic of rhodopsin vesicles is suggested. In the latter case, rhodopsin might still interact with the CC membrane complex to facilitate IFT. ...
Significance
The connecting cilium (CC) of the photoreceptor provides the only route for the trafficking of the outer segment (OS) proteins. Failure of OS protein transport causes degenerative photoreceptor diseases, including retinitis pigmentosa. We demonstrate that Tmem138, a protein linked to ciliopathy, is localized to the photoreceptor CC. Germline deletion of Tmem138 abolished OS morphogenesis, followed by rapid photoreceptor degeneration. Tmem138 interacts with rhodopsin and two additional CC compartment proteins, Ahi1 and Tmem231, likely forming a membrane complex to facilitate trafficking of rhodopsin and other OS-bound proteins across the CC. The study thus implicates a new line of regulation on the delivery of OS proteins through interactions with CC membrane complex(es) and provides insights into photoreceptor ciliopathy diseases.
... In addition, iron is an essential cofactor of fatty acid desaturases enzymes which play an important role in photoreceptor outer segment disc biogenesis (Figure 1C; Shichi, 1969;Song and Dunaief, 2013); whereby old photoreceptor discs containing phototransduction proteins are completely replaced by new ones over the course of about 10 days (Young, 1967;Young and Bok, 1969;Chuang et al., 2007). In addition to the above retina specific role for iron, like the CNS, the retina expresses several irondependent enzymes including tyrosine hydroxylase (Ramsey et al., 1996), phenylalanine hydroxylase (Gottschall et al., 1982), and tryptophan hydroxylase (Kuhn et al., 1980) that are involved in the biosynthesis and regulation of neurotransmitters such as dopamine, serotonin, and melatonin (Kaushik et al., 2007). ...
Iron plays an important role in a wide range of metabolic pathways that are important for neuronal health. Excessive levels of iron, however, can promote toxicity and cell death. An example of an iron overload disorder is hemochromatosis (HH) which is a genetic disorder of iron metabolism in which the body’s ability to regulate iron absorption is altered, resulting in iron build-up and injury in several organs. The retina was traditionally assumed to be protected from high levels of systemic iron overload by the blood-retina barrier. However, recent data shows that expression of genes that are associated with HH can disrupt retinal iron metabolism. Thus, the effects of iron overload on the retina have become an area of research interest, as excessively high levels of iron are implicated in several retinal disorders, most notably age–related macular degeneration. This review is an effort to highlight risk factors for excessive levels of systemic iron build-up in the retina and its potential impact on the eye health. Information is integrated across clinical and preclinical animal studies to provide insights into the effects of systemic iron loading on the retina.
... Contrairement aux cônes, les segments externes des bâtonnets sont constitués d'un empilement de disques membranaires individualisés non reliés à la membrane plasmique du segment interne. Par conséquent, les lipides sont transférés de la membrane plasmique vers les disques alors que pour les cônes, la membrane plasmide est en continuité avec celles de leurs segments externes (Burgoyne et al., 2015;Chuang et al., 2007;Volland et al., 2015) Le renouvellement des segments externes des bâtonnets a été découvert par Richard W. ...
Chez les patients souffrant de rétinopathie pigmentaire (RP), les cônes dégénèrent après les bâtonnets, indépendamment de la mutation de l'un des 69 gènes connus pour causer cette maladie héréditaire. Rod-derived Cone Viability Factor (RdCVF) qui est produit par épissage alternatif du gène nucleoredoxin-like 1 (NXNL1) est sécrété par les bâtonnets et exerce un effet protecteur sur les cônes en se liant à son récepteur à la surface des cônes, un complexe composé de basigine-1 (BSG1) et du transporteur de glucose, GLUT1. RdCVF stimule l’entrée du glucose qui est métabolisé par les cônes via la glycolyse aérobie pour permettre le renouvellement quotidien de leurs segments externes. Ce renouvellement est essentiel à la vision centrale chez l’homme et à la vision des cônes chez tous les mammifères. RdCVF est une future thérapie pour la RP. Dans le cadre d’un programme pharmacologique sur la dégénérescence maculaire lié à l’âge, j’ai identifié avec ma collègue une nouvelle acétogénine qui stimule elle aussi la survie des cônes via la glycolyse aérobie. C’est par la recherche du mécanisme d’action de cette molécule que nous avons identifié PFKFB2, une enzyme bifonctionelle, comme un membre de la signalisation métabolique d RdCVF. En effet, l’expression du messager de PFKFB2 est régulée par RdCVF dans les cônes via l’action du glucose intracellulaire. J’ai pu identifier et valider dans les cônes le rôle d’un élément de réponse, liant le facteur de transcription MONDOA dans le promoteur de PFKFB2. L’inactivation du gène Pfkfb2 dans les cônes entraine une perte de la vision chez la souris. Mes résultats montrent que la protéine PFKFB2 est exprimé spécifiquement par les cônes. Paradoxalement, le messager du gène Pfkfb2 est exprimé dans les bâtonnets et les cônes. Nous avons mis en évidence que le gène Pfkfb2 produisait plusieurs ARN messagers différents, avec ou sans élément ciblé par le microARN (miR182). Une étude in silico montre que l’ARN messager contenant l’élément ciblé par miR182 est majoritaire dans la rétine. L’étude du profil d’expression chez la souris rd1 montre que l’isoforme avec l’élément ciblé par miR182 est exprimée par les bâtonnets et un second ne possédant cet élément est exprimé par les cônes. L'enzyme bi-fonctionnelle PFKFB2 régule la production ou la destruction du fructose 2,6 biphosphate (F26BP), le principal activateur allostérique de la phosphofructokinase, l'enzyme limitante de la glycolyse, par l’action différentielle de ces deux domaines, kinase ou phosphatase. La signalisation de l’insuline inactive le domaine phosphatase de PFKFB2 et en favorisant la production de F26BP, stimule la glycolyse aérobie en l’absence de RdCVF. J’ai montré que l'expression ectopique du domaine kinase PFKFB2 (PFKFB2KD) protège les cônes in vitro. La modélisation mathématique du système montre que la présence de PFKFB2 dans les cônes dirige le flux métabolique de RdCVF des bâtonnets vers les cônes. PFKFB2 est un transistor biologique qui amplifie de signal RdCVF en accélérant le flux glycolytique dans les cônes.Mon travail montre que le flux glycolytique des cônes repose sur RdCVF, via PFKFB2 et de l’action de l’insuline, via une kinase qui n’est pas clairement identifiée. Il est ainsi concevable que la combinaison RdCVF et PFKFB2KD puisse nous permettre de progresser du concept de protection de la vision centrale des patients souffrant de RP vers une restauration de la vision centrale par stimulation de la reconstruction des segments externes des cônes chez la plupart des patients pour lesquels la cécité ne résulte pas de la mort des cônes mais de la perte de leur segment externe.
... rodent retina (Robichaux et al. 2019). It has been implicated in the trafficking of rhodopsin to the outer segments of rod photoreceptors (Chuang et al. 2007;Mazelova et al. 2009), and in the trafficking of peripherin-2 (RDS, PRPH2, OMIM 179605) and rod outer segment protein 1 (ROM1, OMIM 180721) (Zulliger et al. 2015). Finally, a role of STX3 in the release of neurotransmitter at photoreceptor and bipolar cell synaptic terminals was revealed in non-mammalian vertebrate retina (Curtis et al. 2010;Datta et al. 2017;Hays et al. 2020). ...
... Given the high level of STX3 expression in the murine and non-mammalian retina and roles for Stx3 in protein trafficking in photoreceptors (Chuang et al. 2007;Kakakhel et al. 2020;Mazelova et al. 2009;Zulliger et al. 2015), and in neurotransmitter release at the first two synapses in the visual throughput pathway (Curtis et al. 2008(Curtis et al. , 2010Datta et al. 2017;Liu et al. 2014) we asked whether MVID subjects with STX3 variants might have visual impairment. To address this question, we studied five newly identified MVID subjects in which we found homozygous STX3 variants; in addition, we investigated five previously described MVID subjects with STX3 variants (Alsaleem et al. 2017;Julia et al. 2019;Maddirevula et al. 2019;Wiegerinck et al. 2014) for the presence or absence of symptoms of marked visual impairment. ...
... Ribeye shares a domain with the nuclear protein CtBP2, and thus CtBP2 antibodies label synaptic ribbons in addition to some nuclei, such as those in the inner nuclear layer (INL). Strong STX3 immunolabeling of photoreceptor inner and rhodopsin-labeled outer segments was observed in the human retina ( Fig. 2e-h), contrasting with the labeling of only inner segments in mouse photoreceptors (Fig. 3) (Chuang et al. 2007;Zulliger et al. 2015). In addition, the inner and outer segments of cone photoreceptors also exhibited immunolabeling for STX3 in the human retina ( Fig. 2i-l). ...
Biallelic STX3 variants were previously reported in five individuals with the severe congenital enteropathy, microvillus inclusion disease (MVID). Here, we provide a significant extension of the phenotypic spectrum caused by STX3 variants. We report ten individuals of diverse geographic origin with biallelic STX3 loss-of-function variants, identified through exome sequencing, single-nucleotide polymorphism array-based homozygosity mapping, and international collaboration. The evaluated individuals all presented with MVID. Eight individuals also displayed early-onset severe retinal dystrophy, i.e., syndromic—intestinal and retinal—disease. These individuals harbored STX3 variants that affected both the retinal and intestinal STX3 transcripts, whereas STX3 variants affected only the intestinal transcript in individuals with solitary MVID. That STX3 is essential for retinal photoreceptor survival was confirmed by the creation of a rod photoreceptor-specific STX3 knockout mouse model which revealed a time-dependent reduction in the number of rod photoreceptors, thinning of the outer nuclear layer, and the eventual loss of both rod and cone photoreceptors. Together, our results provide a link between STX3 loss-of-function variants and a human retinal dystrophy. Depending on the genomic site of a human loss-of-function STX3 variant, it can cause MVID, the novel intestinal-retinal syndrome reported here or, hypothetically, an isolated retinal dystrophy.
... There are two main competing theories: The first is that RTCs themselves are transported through the connecting cilium via motor driven transport and fuse with nascent discs within the enclosed outer segment plasma membrane. This idea is supported by some electron microscopy-based studies that appear to show vesicular structures within the lumen of the connecting cilium [35,65]. However, it is known that harsh treatment of retinal tissue, fixation, and processing can induce artifacts. ...
Retinal photoreceptors are neurons that convert dynamically changing patterns of light into electrical signals that are processed by retinal interneurons and ultimately transmitted to vision centers in the brain. They represent the essential first step in seeing without which the remainder of the visual system is rendered moot. To support this role, the major functions of photoreceptors are segregated into three main specialized compartments—the outer segment, the inner segment, and the pre-synaptic terminal. This compartmentalization is crucial for photoreceptor function—disruption leads to devastating blinding diseases for which therapies remain elusive. In this review, we examine the current understanding of the molecular and physical mechanisms underlying photoreceptor functional compartmentalization and highlight areas where significant knowledge gaps remain.
