The WAVE complex drives the morphogenesis of the photoreceptor outer segment cilium.

The photoreceptor outer segment is a modified cilium filled with hundreds of flattened "disc" membranes responsible for efficient light capture. To maintain photoreceptor health and functionality, outer segments are continuously renewed through the addition of new discs at their base. This process is driven by branched actin polymerization nucleated by the Arp2/3 complex. To induce actin polymerization, Arp2/3 requires a nucleation promoting factor. Here, we show that the nucleation promoting factor driving disc morphogenesis is the pentameric WAVE complex and identify all protein subunits of this complex. We further demonstrate that the knockout of one of them, WASF3, abolishes actin polymerization at the site of disc morphogenesis leading to formation of disorganized membrane lamellae emanating from the photoreceptor cilium instead of an outer segment. These data establish that, despite the intrinsic ability of photoreceptor ciliary membranes to form lamellar structures, WAVE-dependent actin polymerization is essential for organizing these membranes into a proper outer segment.

Absolute Quantification of Photoreceptor Outer Segment Proteins.

Photoreceptor cells generate neuronal signals in response to capturing light. This process, called phototransduction, takes place in a highly specialized outer segment organelle. There are significant discrepancies in the reported amounts of many proteins supporting this process, particularly those of low abundance, which limits our understanding of their molecular organization and function. In this study, we used quantitative mass spectrometry to simultaneously determine the abundances of 20 key structural and functional proteins residing in mouse rod outer segments. We computed the absolute number of molecules of each protein residing within an individual outer segment and the molar ratio among all 20 proteins. The molar ratios of proteins comprising three well-characterized constitutive complexes in outer segments differed from the established subunit stoichiometries of these complexes by less than 7%, highlighting the exceptional precision of our quantification. Overall, this study resolves multiple existing discrepancies regarding the outer segment abundances of these proteins, thereby advancing our understanding of how the phototransduction pathway functions as a single, well-coordinated molecular ensemble.

TMEM67, TMEM237, and Embigin in Complex With Monocarboxylate Transporter MCT1 Are Unique Components of the Photoreceptor Outer Segment Plasma Membrane.

The outer segment (OS) organelle of vertebrate photoreceptors is a highly specialized cilium evolved to capture light and initiate light response. The plasma membrane which envelopes the OS plays vital and diverse roles in supporting photoreceptor function and health. However, little is known about the identity of its protein constituents, as this membrane cannot be purified to homogeneity. In this study, we used the technique of protein correlation profiling to identify unique OS plasma membrane proteins. To achieve this, we used label-free quantitative MS to compare relative protein abundances in an enriched preparation of the OS plasma membrane with a preparation of total OS membranes. We have found that only five proteins were enriched at the same level as previously validated OS plasma membrane markers. Two of these proteins, TMEM67 and TMEM237, had not been previously assigned to this membrane, and one, embigin, had not been identified in photoreceptors. We further showed that embigin associates with monocarboxylate transporter MCT1 in the OS plasma membrane, facilitating lactate transport through this cellular compartment.

A Ciliary Branched Actin Network Drives Photoreceptor Disc Morphogenesis.

The light-detecting organelle of the photoreceptor cell is a modified primary cilium, called the outer segment. The outer segment houses hundreds of light-sensitive membrane, "discs," that are continuously renewed by the constant formation of new discs at the outer segment base and the phagocytosis of old ones from outer segment tips by the retinal pigment epithelium. In this chapter, we describe how an actin cytoskeleton network, residing precisely at the site of disc formation, provides the driving force that pushes out the ciliary plasma membrane to form each disc evagination that subsequently can mature into a bona fide disc. We highlight the functions of actin-binding proteins, particularly PCARE and Arp2/3, that are known to participate in disc formation. Finally, we describe a working model of disc formation built upon the many studies focusing on the role of actin during disc morphogenesis.

Photoreceptor disc membranes are formed through an Arp2/3-dependent lamellipodium-like mechanism.

The light-sensitive outer segment of the vertebrate photoreceptor is a highly modified primary cilium filled with disc-shaped membranes that provide a vast surface for efficient photon capture. The formation of each disc is initiated by a ciliary membrane evagination driven by an unknown molecular mechanism reportedly requiring actin polymerization. Since a distinct F-actin network resides precisely at the site of disc morphogenesis, we employed a unique proteomic approach to identify components of this network potentially driving disc morphogenesis. The only identified actin nucleator was the Arp2/3 complex, which induces the polymerization of branched actin networks. To investigate the potential involvement of Arp2/3 in the formation of new discs, we generated a conditional knockout mouse lacking its essential ArpC3 subunit in rod photoreceptors. This knockout resulted in the complete loss of the F-actin network specifically at the site of disc morphogenesis, with the time course of ArpC3 depletion correlating with the time course of F-actin loss. Without the actin network at this site, the initiation of new disc formation is completely halted, forcing all newly synthesized membrane material to be delivered to the several nascent discs whose morphogenesis had already been in progress. As a result, these discs undergo uncontrolled expansion instead of normal enclosure, which leads to formation of unusual, large membrane whorls. These data suggest a model of photoreceptor disc morphogenesis in which Arp2/3 initiates disc formation in a "lamellipodium-like" mechanism.

PRCD is essential for high-fidelity photoreceptor disc formation.

Progressive rod-cone degeneration (PRCD) is a small protein residing in the light-sensitive disc membranes of the photoreceptor outer segment. Until now, the function of PRCD has remained enigmatic despite multiple demonstrations that its mutations cause blindness in humans and dogs. Here, we generated a PRCD knockout mouse and observed a striking defect in disc morphogenesis, whereby newly forming discs do not properly flatten. This leads to the budding of disc-derived vesicles, specifically at the site of disc morphogenesis, which accumulate in the interphotoreceptor matrix. The defect in nascent disc flattening only minimally alters the photoreceptor outer segment architecture beyond the site of new disc formation and does not affect the abundance of outer segment proteins and the photoreceptor's ability to generate responses to light. Interestingly, the retinal pigment epithelium, responsible for normal phagocytosis of shed outer segment material, lacks the capacity to clear the disc-derived vesicles. This deficiency is partially compensated by a unique pattern of microglial migration to the site of disc formation where they actively phagocytize vesicles. However, the microglial response is insufficient to prevent vesicular accumulation and photoreceptors of PRCD knockout mice undergo slow, progressive degeneration. Taken together, these data show that the function of PRCD is to keep evaginating membranes of new discs tightly apposed to each other, which is essential for the high fidelity of photoreceptor disc morphogenesis and photoreceptor survival.

PRCD Is a Small Disc-Specific Rhodopsin-Binding Protein of Unknown Function.

PRCD (progressive rod-cone degeneration) is a small ~6 kDa protein with unknown function that specifically resides in photoreceptor discs and interacts with rhodopsin. PRCD's discovery resulted from decades-long study of a canine retinal disease called progressive rod-cone degeneration which is one of the most frequent causes of blindness in dogs characterized by the slow, progressive death of rod photoreceptors followed by cones. A series of genetic studies eventually mapped the disease to a single point mutation in a novel gene which was then named Prcd. Highlighting the importance of this gene, this and several other mutations have been identified in human patients suffering from retinitis pigmentosa. In this review, we highlight what is currently known about PRCD protein, including the etiology and pathology of the retinal disease caused by its mutation, the protein's trafficking, localization, and biochemical characterization.

Progressive Rod-Cone Degeneration (PRCD) Protein Requires N-Terminal S-Acylation and Rhodopsin Binding for Photoreceptor Outer Segment Localization and Maintaining Intracellular Stability.

The light-sensing outer segments of photoreceptor cells harbor hundreds of flattened membranous discs containing the visual pigment, rhodopsin, and all the proteins necessary for visual signal transduction. PRCD (progressive rod-cone degeneration) protein is one of a few proteins residing specifically in photoreceptor discs, and the only one with completely unknown function. The importance of PRCD is highlighted by its mutations that cause photoreceptor degeneration and blindness in canine and human patients. Here we report that PRCD is S-acylated at its N-terminal cysteine and anchored to the cytosolic surface of disc membranes. We also showed that mutating the S-acylated cysteine to tyrosine, a common cause of blindness in dogs and a mutation found in affected human families, causes PRCD to be completely mislocalized from the photoreceptor outer segment. We next undertook a proteomic search for PRCD-interacting partners in disc membranes and found that it binds rhodopsin. This interaction was confirmed by reciprocal precipitation and co-chromatography experiments. We further demonstrated this interaction to be critically important for supporting the intracellular stability of PRCD, as the knockout of rhodopsin caused a drastic reduction in the photoreceptor content of PRCD. These data reveal the cause of photoreceptor disease in PRCD mutant dogs and implicate rhodopsin to be involved in PRCD's unknown yet essential function in photoreceptors.

Ingested soluble CD14 from milk is transferred intact into the blood of newborn rats.

BACKGROUND: Milk acts as an edible immune system that is transferred from mother to newborn. Soluble Cluster of Differentiation 14 (sCD14) is a protein found in significant quantities in human milk (~8-29 µg/ml). At a 10-fold lower concentration in the blood (~3 µg/ml), the most notable role of sCD14 is to sequester lipopolysaccharides of Gram-negative bacteria from immune cells. METHODS: To explore the pharmacodynamics of this milk protein and its biological fate, the biodistribution of radiolabeled sCD14 ((14)C, (125)I) was monitored in 10-d-old rat pups. RESULTS: Up to 3.4 ± 2.2% of the radiolabeled sCD14 administered was observed, intact, in the pup blood for up to 8 h post-ingestion. Additionally, 30.3 ± 13.0% of the radiolabeled sCD14 administered was observed degraded in the stomach at 8 h post-ingestion. A reservoir of intact, administered sCD14 (3.2 ± 0.3%), however, remained in the stomach at 8 h post-ingestion. Intact sCD14 was observed in the small intestine at 5.5 ± 1.6% of the dose fed at 8 h post-ingestion. CONCLUSION: The presence of intact sCD14 in the blood and the gastrointestinal tract of newborns post-ingestion has implications in the development of allergies, obesity, and other inflammation-related pathogeneses later in life.

Proteomic identification of unique photoreceptor disc components reveals the presence of PRCD, a protein linked to retinal degeneration.

Visual signal transduction takes place on the surface of flat membrane vesicles called photoreceptor discs, which reside inside the light-sensitive outer segment organelle of vertebrate photoreceptor cells. Although biochemical studies have indicated that discs are built with a handful of highly specialized proteins, proteomic studies have yielded databases consisting of hundreds of entries. We addressed this controversy by employing protein correlation profiling, which allows identification of unique components of organelles that can be fractionated but not purified to absolute homogeneity. We subjected discs to sequential steps of fractionation and identified the relative amounts of proteins in each fraction by label-free quantitative mass spectrometry. This analysis demonstrated that the photoreceptor disc proteome contains only eleven components, which satisfy the hallmark criterion for being unique disc-resident components: the retention of a constant molar ratio among themselves across fractionation steps. Remarkably, one of them is PRCD, a protein whose mutations have been shown to cause blindness, yet cellular localization remained completely unknown. Identification of PRCD as a novel disc-specific protein facilitates understanding its functional role and the pathobiological significance of its mutations. Our study provides a striking example how protein correlation profiling allows a distinction between constitutive components of cellular organelles and their inevitable contaminants.

Extracellular vesicles highlight many cases of photoreceptor degeneration.

The release of extracellular vesicles is observed across numerous cell types and serves a range of biological functions including intercellular communication and waste disposal. One cell type which stands out for its robust capacity to release extracellular vesicles is the vertebrate photoreceptor cell. For decades, the release of extracellular vesicles by photoreceptors has been documented in many different animal models of photoreceptor degeneration and, more recently, in wild type photoreceptors. Here, I review all studies describing extracellular vesicle release by photoreceptors and discuss the most unifying theme among them-a photoreceptor cell fully, or partially, diverts its light sensitive membrane material to extracellular vesicles when it has defects in the delivery or morphing of this material into the photoreceptor's highly organized light sensing organelle. Because photoreceptors generate an enormous amount of light sensitive membrane every day, the diversion of this material to extracellular vesicles can cause a massive accumulation of these membranes within the retina. Little is known about the uptake of photoreceptor derived extracellular vesicles, although in some cases the retinal pigment epithelial cells, microglia, Müller glia, and/or photoreceptor cells themselves have been shown to phagocytize them.

Absolute quantification of photoreceptor outer segment proteins.

Photoreceptor cells generate neuronal signals in response to capturing light. This process, called phototransduction, takes place in a highly specialized outer segment organelle. There are significant discrepancies in the reported amounts of many proteins supporting this process, particularly those of low abundance, which limits our understanding of their molecular organization and function. In this study, we used quantitative mass spectrometry to simultaneously determine the abundances of twenty key structural and functional proteins residing in mouse rod outer segments. We computed the absolute number of molecules of each protein residing within an individual outer segment and the molar ratio amongst all twenty proteins. The molar ratios of proteins comprising three well-characterized constitutive complexes in outer segments differed from the established subunit stoichiometries of these complexes by less than 7%, highlighting the exceptional precision of our quantification. Overall, this study resolves multiple existing discrepancies regarding the outer segment abundances of these proteins, thereby advancing our understanding of how the phototransduction pathway functions as a single, well-coordinated molecular ensemble.

(14)C radiolabeling of proteins to monitor biodistribution of ingested proteins.

The economical preparation of microgram quantities of (14)C-labeled proteins by in vacuo methylation with methyl iodide is described. The (14)C radiolabeling was achieved by the covalent attachment of [(14)C]methyl groups onto amino and imidazole groups by reaction in vacuo with [(14)C]methyl iodide. The method was tested by investigating the biodistribution of (14)C in rats that were fed (14)C-labeled human soluble cluster of differentiation 14 (CD14) protein, a receptor for bacterial lipopolysaccharide. Two other control proteins, bovine serum albumin (BSA) and casein, were also labeled with (14)C and used for comparative analysis to determine the following: (i) the efficacy and cost efficiency of the in vacuo radiolabeling procedure and (ii) the extent of incorporation of the (14)C label into the organs of orogastrically fed 10-day-old Sprague-Dawley rats. [(14)C]BSA, [(14)C]casein, and [(14)C]CD14 were individually prepared with specific radioactivities of 34,400, 18,800, and 163,000 disintegrations per minute (dpm)/microg, respectively. It was found that the accumulation of (14)C label in the organs of [(14)C]CD14-fed rats, most notably the persistence of (14)C in the stomach 480 min postgavage, was temporally and spatially distinct from [(14)C]BSA and [(14)C]casein-fed rats.

Alpha-lactalbumin in human milk alters the proteolytic degradation of soluble CD14 by forming a complex.

Mother's milk represents a foundational step in the proper development of newborn immunity. This is achieved, in part, through the action of numerous regulatory proteins such as soluble cluster of differentiation 14 (sCD14) found in significant quantities in human milk (~25-50 µg/mL). In adults, CD14 stimulates cytokine production in response to lipopolysaccharide (LPS), the major lipid component found in the outer membrane of Gram-negative bacteria. However, the fate and function of sCD14 in the neonatal gastrointestinal (GI) tract are unknown and may function differently from adults. Therefore, we administered human sCD14 to experimental animals and observed that it persisted in the upper GI tract after feeding. In our search for potential proteolytic protectants, immunoprecipitation of sCD14 from human milk revealed a 15-kD novel protein that copurified with sCD14. Mass spectrometry analysis of the protein identified alpha-lactalbumin. CD14 was also identified by immunoblot after immunoprecipitation of alpha-lactalbumin from milk. In vitro digestion assays revealed that purified alpha-lactalbumin decreases the proteolytic degradation of human milk derived sCD14 in vitro, suggesting a mechanism by which this key LPS receptor may remain functional in the neonate gut.

Photoreceptor Discs: Built Like Ectosomes.

The light-sensitive outer segment organelle of the vertebrate photoreceptor cell is a modified cilium filled with hundreds of flattened 'disc' membranes that provide vast light-absorbing surfaces. The outer segment is constantly renewed with new discs added at its base every day. This continuous process is essential for photoreceptor viability. In this review, we describe recent breakthroughs in the understanding of disc morphogenesis, with a focus on the molecular mechanisms responsible for initiating disc formation from the ciliary membrane. We highlight the discoveries that this mechanism evolved from an innate ciliary process of releasing small extracellular vesicles, or ectosomes, and that both disc formation and ectosome release rely on the actin cytoskeleton.

Comprehensive identification of mRNA isoforms reveals the diversity of neural cell-surface molecules with roles in retinal development and disease.

Genes encoding cell-surface proteins control nervous system development and are implicated in neurological disorders. These genes produce alternative mRNA isoforms which remain poorly characterized, impeding understanding of how disease-associated mutations cause pathology. Here we introduce a strategy to define complete portfolios of full-length isoforms encoded by individual genes. Applying this approach to neural cell-surface molecules, we identify thousands of unannotated isoforms expressed in retina and brain. By mass spectrometry we confirm expression of newly-discovered proteins on the cell surface in vivo. Remarkably, we discover that the major isoform of a retinal degeneration gene, CRB1, was previously overlooked. This CRB1 isoform is the only one expressed by photoreceptors, the affected cells in CRB1 disease. Using mouse mutants, we identify a function for this isoform at photoreceptor-glial junctions and demonstrate that loss of this isoform accelerates photoreceptor death. Therefore, our isoform identification strategy enables discovery of new gene functions relevant to disease.

Transducin ß-Subunit Can Interact with Multiple G-Protein γ-Subunits to Enable Light Detection by Rod Photoreceptors.

The heterotrimeric G-protein transducin mediates visual signaling in vertebrate photoreceptor cells. Many aspects of the function of transducin were learned from knock-out mice lacking its individual subunits. Of particular interest is the knockout of its rod-specific γ-subunit (Gγ1). Two studies using independently generated mice documented that this knockout results in a considerable >60-fold reduction in the light sensitivity of affected rods, but provided different interpretations of how the remaining α-subunit (Gαt) mediates phototransduction without its cognate Gß1γ1-subunit partner. One study found that the light sensitivity reduction matched a corresponding reduction in Gαt content in the light-sensing rod outer segments and proposed that Gαt activation is supported by remaining Gß1 associating with other Gγ subunits naturally expressed in photoreceptors. In contrast, the second study reported the same light sensitivity loss but a much lower, only approximately sixfold, reduction of Gαt and proposed that the light responses of these rods do not require Gßγ at all. To resolve this controversy and elucidate the mechanism driving visual signaling in Gγ1 knock-out rods, we analyzed both mouse lines side by side. We first determined that the outer segments of both mice have identical Gαt content, which is reduced ∼65-fold from the wild-type (WT) level. We further demonstrated that the remaining Gß1 is present in a complex with endogenous Gγ2 and Gγ3 subunits and that these complexes exist in wild-type rods as well. Together, these results argue against the idea that Gαt alone supports light responses of Gγ1 knock-out rods and suggest that Gß1γ1 is not unique in its ability to mediate vertebrate phototransduction.

Photoreceptor discs form through peripherin-dependent suppression of ciliary ectosome release.

The primary cilium is a highly conserved organelle housing specialized molecules responsible for receiving and processing extracellular signals. A recently discovered property shared across many cilia is the ability to release small vesicles called ectosomes, which are used for exchanging protein and genetic material among cells. In this study, we report a novel role for ciliary ectosomes in building the elaborate photoreceptor outer segment filled with hundreds of tightly packed "disc" membranes. We demonstrate that the photoreceptor cilium has an innate ability to release massive amounts of ectosomes. However, this process is suppressed by the disc-specific protein peripherin, which enables retained ectosomes to be morphed into discs. This new function of peripherin is performed independently from its well-established role in maintaining the high curvature of disc edges, and each function is fulfilled by a separate part of peripherin's molecule. Our findings explain how the outer segment structure evolved from the primary cilium to provide photoreceptor cells with vast membrane surfaces for efficient light capture.

Guanylate cyclase 1 relies on rhodopsin for intracellular stability and ciliary trafficking.

Sensory cilia are populated by a select group of signaling proteins that detect environmental stimuli. How these molecules are delivered to the sensory cilium and whether they rely on one another for specific transport remains poorly understood. Here, we investigated whether the visual pigment, rhodopsin, is critical for delivering other signaling proteins to the sensory cilium of photoreceptor cells, the outer segment. Rhodopsin is the most abundant outer segment protein and its proper transport is essential for formation of this organelle, suggesting that such a dependency might exist. Indeed, we demonstrated that guanylate cyclase-1, producing the cGMP second messenger in photoreceptors, requires rhodopsin for intracellular stability and outer segment delivery. We elucidated this dependency by showing that guanylate cyclase-1 is a novel rhodopsin-binding protein. These findings expand rhodopsin's role in vision from being a visual pigment and major outer segment building block to directing trafficking of another key signaling protein.

Maternal separation and gastrointestinal transit time in neonate rats.

Gastrointestinal transit times (GItts) were compared in separate litters of 10- and 15-day-old Sprague Dawley rats using barium sulphate. By tracking the leading front of the bolus on radiographs, the gastrocaecal transit times in pups were estimated. To measure the total GItt, the duration from orogastric gavage until an observable defecation of barium sulphate was recorded. The gastrocaecal times for 10-day-old pups maintained with their dam (n = 5) ranged from 4-5 h and those removed from the dam ranged from 2.5-5 h. For 15-day-old pups with their dam (n = 6) and without dam (n = 5), gastrocaecal times ranged from 4-6 h and 3.5-5 h, respectively. Ten-day-old pups that remained with the dam had a GItt of 13.8 ± 0.9 h and those kept in the absence of the dam had a time of 9.3 ± 0.7 h. This decrease (P < 0.05) in GItt in the absence of the dam was age-dependent in 10-day-old pups, and was not observed (P > 0.05) in 15-day-old pups. The results provide a basis, for the design of future studies involving neonate rat metabolism, to include maternal presence.