RESUMO
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.
Assuntos
Actinas , Cílios , Actinas/metabolismo , Cílios/química , Células Fotorreceptoras/metabolismo , Citoplasma , MorfogêneseRESUMO
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.
Assuntos
Proteínas , Segmento Externo da Célula Bastonete , Animais , Camundongos , Proteínas/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Transdução de Sinal Luminoso , Retina/metabolismoRESUMO
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.
Assuntos
Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Segmento Externo das Células Fotorreceptoras da Retina/metabolismo , Simportadores/metabolismo , Animais , Bovinos , Camundongos Endogâmicos C57BLRESUMO
Glaucoma, one of the leading causes of irreversible blindness, is commonly associated with elevated intraocular pressure due to impaired aqueous humour (AH) drainage through the trabecular meshwork. The aetiological mechanisms contributing to impaired AH outflow, however, are poorly understood. Here, we identified the secreted form of vasorin, a transmembrane glycoprotein, as a common constituent of human AH by mass spectrometry and immunoblotting analysis. ELISA assay revealed a significant but marginal decrease in vasorin levels in the AH of primary open-angle glaucoma patients compared to non-glaucoma cataract patients. Human trabecular meshwork (HTM) cells were confirmed to express vasorin, which has been shown to possess anti-apoptotic and anti-TGF-ß activities. Treatment of HTM cells with vasorin induced actin stress fibres and focal adhesions and suppressed TGF-ß2-induced SMAD2/3 activation in HTM cells. Additionally, cobalt chloride-induced hypoxia stimulated a robust elevation in vasorin expression, and vasorin suppressed TNF-α-induced cell death in HTM cells. Taken together, these findings reveal the importance of vasorin in maintenance of cell survival, inhibition of TGF-ß induced biological responses in TM cells, and the decreasing trend in vasorin levels in the AH of glaucoma patients suggests a plausible role for vasorin in the pathobiology of ocular hypertension and glaucoma.
Assuntos
Proteínas de Transporte , Glaucoma de Ângulo Aberto , Glaucoma , Proteínas de Membrana , Malha Trabecular , Proteínas de Transporte/metabolismo , Células Cultivadas , Glaucoma/metabolismo , Glaucoma de Ângulo Aberto/metabolismo , Glicoproteínas/metabolismo , Humanos , Hipóxia/metabolismo , Proteínas de Membrana/metabolismo , Malha Trabecular/metabolismo , Fator de Crescimento Transformador beta2/metabolismoRESUMO
Individuals with pseudoexfoliation (PEX) syndrome exhibit various connective tissue pathologies associated with dysregulated extracellular matrix homeostasis. PEX glaucoma is a common, aggressive form of open-angle glaucoma resulting from the deposition of fibrillary material in the conventional outflow pathway. However, the molecular mechanisms that drive pathogenesis and genetic risk remain poorly understood. PEX glaucoma-associated single-nucleotide polymorphisms are located in and affect activity of the promoter of LOXL1-AS1, a long non-coding RNA (lncRNA). Nuclear and non-nuclear lncRNAs regulate a host of biological processes, and when dysregulated, contribute to disease. Here we report that LOXL1-AS1 localizes to the nucleus where it selectively binds to the mRNA processing protein, heterogeneous nuclear ribonucleoprotein-L (hnRNPL). Both components of this complex are critical for the regulation of global gene expression in ocular cells, making LOXL1-AS1 a prime target for investigation in PEX syndrome and glaucoma.
Assuntos
Síndrome de Exfoliação/genética , Glaucoma de Ângulo Aberto/genética , RNA Longo não Codificante/genética , Ribonucleoproteínas/genética , Aminoácido Oxirredutases/genética , Síndrome de Exfoliação/patologia , Regulação da Expressão Gênica/genética , Predisposição Genética para Doença , Glaucoma de Ângulo Aberto/patologia , Humanos , Complexos Multiproteicos/genética , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas/genéticaRESUMO
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.
RESUMO
Strong evidence suggests that dysregulated lipid metabolism involving dysfunction of the retinal pigmented epithelium (RPE) underlies the pathogenesis of age-related macular degeneration (AMD), the leading cause of irreversible blindness in the elderly. A hallmark of AMD is the overproduction of lipid- and protein-rich extracellular deposits that accumulate in the extracellular matrix (Bruch's membrane (BrM)) adjacent to the RPE. We analyzed apolipoprotein A-1 (ApoA-1)-containing lipoproteins isolated from BrM of elderly human donor eyes and found a unique proteome, distinct from high-density lipoprotein (HDL) isolated from donor plasma of the same individuals. The most striking difference is higher concentrations of ApoB and ApoE, which bind to glycosaminoglycans. We hypothesize that this interaction promotes lipoprotein deposition onto BrM glycosaminoglycans, initiating downstream effects that contribute to RPE dysfunction/death. We tested this hypothesis using two potential therapeutic strategies to alter the lipoprotein/protein profile of these extracellular deposits. First, we used short heparan sulfate oligosaccharides to remove lipoproteins already deposited in both the extracellular matrix of RPE cells and aged donor BrM tissue. Second, an ApoA-1 mimetic, 5A peptide, was demonstrated to modulate the composition and concentration of apolipoproteins secreted from primary porcine RPE cells. Significantly, in a mouse model of AMD, this 5A peptide altered the proteomic profile of circulating HDL and ameliorated some of the potentially harmful changes to the protein composition resulting from the high-fat, high-cholesterol diet in this model. Together, these results suggest that targeting HDL interactions with BrM represents a new strategy to slow AMD progression in humans.
Assuntos
Lipoproteínas HDL/metabolismo , Degeneração Macular/metabolismo , Animais , Apolipoproteína A-I/análise , Apolipoproteína A-I/metabolismo , Lâmina Basilar da Corioide/metabolismo , Células Cultivadas , Humanos , Lipoproteínas HDL/sangue , Lipoproteínas HDL/isolamento & purificação , Camundongos , Epitélio Pigmentado da Retina/metabolismo , SuínosRESUMO
The retinal pigmented epithelium (RPE) forms the outer blood-retinal barrier, provides nutrients, recycles visual pigment, and removes spent discs from the photoreceptors, among many other functions. Because of these critical roles in visual homeostasis, the RPE is a principal location of disease-associated changes in age-related macular degeneration (AMD), emphasizing its importance for study in both visual health and disease. Unfortunately, there are no early indicators of AMD or disease progression, a void that could be filled by the development of early AMD biomarkers. Exosomes are lipid bilayer membrane vesicles of nanoscale sizes that are released in a controlled fashion by cells and carry out a number of extra- and intercellular activities. In the RPE they are released from both the apical and basal sides, and each source has a unique signature/content. Exosomes released from the basolateral side of RPE cells enter the systemic circulation via the choroid and thus represent a potential source of retinal disease biomarkers in blood. Here we discuss the potential of targeted immunocapture of eye-derived exosomes and other small extracellular vesicles from blood for eye disease biomarker discovery.
Assuntos
Biomarcadores/sangue , Exossomos/metabolismo , Retina/citologia , Epitélio Pigmentado da Retina/metabolismo , Corioide , Humanos , Degeneração Macular/patologiaRESUMO
Glaucoma, a leading cause of irreversible blindness, is commonly associated with elevated intraocular pressure (IOP) due to impaired aqueous humor (AH) drainage through the trabecular meshwork (TM). Although dysregulated production and organization of extracellular matrix (ECM) is presumed to increase resistance to AH outflow and elevate IOP by altering TM cell contractile and adhesive properties, it is not known whether regulation of ECM protein phosphorylation via the secretory vertebrate lonesome kinase (VLK) influences TM cellular characteristics. Here, we tested this possibility. Experiments carried out in this study reveal that the 32 kDa protein is a prominent VLK isoform detectable in lysates and conditioned media (CM) of human TM cells. Increased levels of VLK were observed in CM of TM cells subjected to cyclic mechanical stretch, or treated with dexamethasone, TGF-ß2, and TM cells expressing constitutively active RhoA GTPase. Downregulation of VLK expression in TM cells using siRNA decreased tyrosine phosphorylation (TyrP) of ECM proteins and focal adhesions, and induced changes in cell shape in association with reduced levels of actin stress fibers and phospho-paxillin. VLK was also demonstrated to regulate TGF-ß2-induced TyrP of ECM proteins. Taken together, these results suggest that VLK secretion can be regulated by external cues, intracellular signal proteins, and mechanical stretch, and VLK can in turn regulate TyrP of ECM proteins secreted by TM cells and control cell shape, actin stress fibers, and focal adhesions. These observations indicate a potential role for VLK in homeostasis of AH outflow and IOP, and in the pathobiology of glaucoma. J. Cell. Physiol. 232: 2447-2460, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Adesão Celular , Forma Celular , Proteínas da Matriz Extracelular/metabolismo , Proteínas Tirosina Quinases/metabolismo , Malha Trabecular/enzimologia , Adulto , Idoso , Humor Aquoso/metabolismo , Adesão Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados/metabolismo , Dexametasona/farmacologia , Adesões Focais/enzimologia , Glaucoma/enzimologia , Glaucoma/patologia , Glaucoma/fisiopatologia , Humanos , Pressão Intraocular , Mecanotransdução Celular , Pessoa de Meia-Idade , Mutação , Paxilina/metabolismo , Fosforilação , Proteínas Tirosina Quinases/genética , Interferência de RNA , Fibras de Estresse/enzimologia , Fatores de Tempo , Malha Trabecular/efeitos dos fármacos , Transfecção , Fator de Crescimento Transformador beta2/farmacologia , Tirosina , Adulto Jovem , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
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.
Assuntos
Proteínas do Olho/química , Proteínas de Membrana/química , Células Fotorreceptoras de Vertebrados/metabolismo , Rodopsina/metabolismo , Acilação , Animais , Cromatografia em Gel , Eletroporação , Proteínas do Olho/metabolismo , Espectrometria de Massas , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Inherited retinal degenerations, caused by mutations in over 100 individual genes, affect approximately 2 million people worldwide. Many of the underlying mutations cause protein misfolding or mistargeting in affected photoreceptors. This places an increased burden on the protein folding and degradation machinery, which may trigger cell death. We analyzed how these cellular functions are affected in degenerating rods of the transducin γ-subunit (Gγ1) knockout mouse. These rods produce large amounts of transducin ß-subunit (Gß1), which cannot fold without Gγ1 and undergoes intracellular proteolysis instead of forming a transducin ßγ-subunit complex. Our data revealed that the most critical pathobiological factor leading to photoreceptor cell death in these animals is insufficient capacity of proteasomes to process abnormally large amounts of misfolded protein. A decrease in the Gß1 production in Gγ1 knockout rods resulted in a significant reduction in proteasomal overload and caused a striking reversal of photoreceptor degeneration. We further demonstrated that a similar proteasomal overload takes place in photoreceptors of other mutant mice where retinal degeneration has been ascribed to protein mistargeting or misfolding, but not in mice whose photoreceptor degenerate as a result of abnormal phototransduction. These results establish the prominence of proteasomal insufficiency across multiple degenerative diseases of the retina, thereby positioning proteasomes as a promising therapeutic target for treating these debilitating conditions.
Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Retina/metabolismo , Degeneração Retiniana/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Animais , Western Blotting , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Retina/patologia , Degeneração Retiniana/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estresse Fisiológico , Transducina/genética , Transducina/metabolismoRESUMO
This study illustrates a vital role for ankyrin-B in lens architecture, growth and function through its involvement in membrane protein and spectrin-actin cytoskeletal organization and stability The transparent ocular lens is essential for vision by focusing light onto the retina. Despite recognizing the importance of its unique cellular architecture and mechanical properties, the molecular mechanisms governing these attributes remain elusive. This study aims to elucidate the role of ankyrin-B (AnkB), a membrane scaffolding protein, in lens cytoarchitecture, growth and function using a conditional knockout (cKO) mouse model. AnkB cKO mouse has no defects in lens morphogenesis, but exhibited changes that supported a global role for AnkB in maintenance of lens clarity, size, cytoarchitecture, and stiffness. Notably, absence of AnkB led to nuclear cataract formation, evident from P16. AnkB cKO lens fibers exhibit progressive disruption in membrane organization of the spectrin-actin cytoskeleton, channel proteins, cell-cell adhesion, shape change, loss and degradation of several membrane proteins (e.g., NrCAM. N-cadherin and aquaporin-0) along with a disorganized plasma membrane and impaired ball-and-socket membrane interdigitations. Furthermore, absence of AnkB led to decreased lens stiffness. Collectively, these results illustrate the essential role for AnkB in lens architecture, growth and function through its involvement in membrane protein and cytoskeletal organization.
RESUMO
Among neurons, retinal ganglion cells (RGCs) are uniquely sensitive to mitochondrial dysfunction. The RGC is highly polarized, with a somatodendritic compartment in the inner retina and an axonal compartment projecting to targets in the brain. The drastically dissimilar functions of these compartments implies that mitochondria face different bioenergetic and other physiological demands. We hypothesized that compartmental differences in mitochondrial biology would be reflected by disparities in mitochondrial protein composition. Here, we describe a protocol to isolate intact mitochondria separately from mouse RGC somatodendritic and axonal compartments by immunoprecipitating labeled mitochondria from RGC MitoTag mice. Using mass spectrometry, 471 and 357 proteins were identified in RGC somatodendritic and axonal mitochondrial immunoprecipitates, respectively. We identified 10 mitochondrial proteins exclusively in the somatodendritic compartment and 19 enriched ≥2-fold there, while 3 proteins were exclusively identified and 18 enriched in the axonal compartment. Our observation of compartment-specific enrichment of mitochondrial proteins was validated through immunofluorescence analysis of the localization and relative abundance of superoxide dismutase ( SOD2 ), sideroflexin-3 ( SFXN3 ) and trifunctional enzyme subunit alpha ( HADHA ) in retina and optic nerve specimens. The identified compartmental differences in RGC mitochondrial composition may provide promising leads for uncovering physiologically relevant pathways amenable to therapeutic intervention for optic neuropathies.
RESUMO
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.
Assuntos
Proteínas do Olho/genética , Degeneração Retiniana/genética , Segmento Externo das Células Fotorreceptoras da Retina/química , Sequência de Aminoácidos , Animais , Bovinos , Fracionamento Celular , Proteínas do Olho/química , Proteínas do Olho/isolamento & purificação , Proteínas do Olho/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Espectrometria de Massas , Dados de Sequência Molecular , Mutação , Proteômica , Degeneração Retiniana/patologia , Segmento Externo das Células Fotorreceptoras da Retina/metabolismoRESUMO
The retina is a highly ordered tissue whose outermost layers are formed by subcellular compartments of photoreceptors generating light-evoked electrical responses. We studied protein distributions among individual photoreceptor compartments by separating the entire photoreceptor layer of a flat-mounted frozen retina into a series of thin tangential cryosections and analyzing protein compositions of each section by label-free quantitative mass spectrometry. Based on 5038 confidently identified peptides assigned to 896 protein database entries, we generated a quantitative proteomic database (a "map") correlating the distribution profiles of identified proteins with the profiles of marker proteins representing individual compartments of photoreceptors and adjacent cells. We evaluated the applicability of several common peptide-to-protein quantification algorithms in the context of our database and found that the highest reliability was obtained by summing the intensities of all peptides representing a given protein, using at least the 5-6 most intense peptides when applicable. We used this proteome map to investigate the distribution of glycolytic enzymes, critical in fulfilling the extremely high metabolic demands of photoreceptor cells, and obtained two major findings. First, unlike the majority of neurons rich in hexokinase I, but similar to other highly metabolically active cells, photoreceptors express hexokinase II. Hexokinase II has a very high catalytic activity when associated with mitochondria, and indeed we found it colocalized with mitochondria in photoreceptors. Second, photoreceptors contain very little triosephosphate isomerase, an enzyme converting dihydroxyacetone phosphate into glyceraldehyde-3-phosphate. This may serve as a functional adaptation because dihydroxyacetone phosphate is a major precursor in phospholipid biosynthesis, a process particularly active in photoreceptors because of the constant renewal of their light-sensitive membrane disc stacks. Overall, our approach for proteomic profiling of very small tissue amounts at a resolution of a few microns, combining cryosectioning and liquid chromatography-tandem MS, can be applied for quantitative investigation of proteomes where spatial resolution is paramount.
Assuntos
Glicólise , Células Fotorreceptoras de Vertebrados/metabolismo , Proteômica/métodos , Algoritmos , Animais , Bases de Dados de Proteínas , Proteínas do Olho/metabolismo , Hexoquinase/metabolismo , Isoenzimas/metabolismo , Marcação por Isótopo , Proteínas Mitocondriais/metabolismo , Células Fotorreceptoras de Vertebrados/citologia , Células Fotorreceptoras de Vertebrados/enzimologia , Transporte Proteico , Ratos , Ratos Long-Evans , Epitélio Pigmentado da Retina/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Frações Subcelulares/metabolismo , Triose-Fosfato Isomerase/metabolismoRESUMO
The small GTPase Arl3 is important for the enrichment of lipidated proteins to primary cilia, including the outer segment of photoreceptors. Human mutations in the small GTPase Arl3 cause both autosomal recessive and dominant inherited retinal dystrophies. We discovered that dominant mutations result in increased active G-protein-Arl3-D67V has constitutive activity and Arl3-Y90C is fast cycling-and their expression in mouse rods resulted in a displaced nuclear phenotype due to an aberrant Arl3-GTP gradient. Using multiple strategies, we go on to show that removing or restoring the Arl3-GTP gradient within the cilium is sufficient to rescue the nuclear migration defect. Together, our results reveal that an Arl3 ciliary gradient is involved in proper positioning of photoreceptor nuclei during retinal development.
Assuntos
Fatores de Ribosilação do ADP , Proteínas de Membrana , Células Fotorreceptoras Retinianas Bastonetes , Animais , Humanos , Camundongos , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Cílios/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas de Membrana/metabolismo , Transporte Proteico , Células Fotorreceptoras Retinianas Bastonetes/metabolismoRESUMO
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.
RESUMO
The first steps of vision take place within a stack of tightly packed disc-shaped membranes, or "discs", located in the outer segment compartment of photoreceptor cells. In rod photoreceptors, discs are enclosed inside the outer segment and contain deep indentations in their rims called "incisures". The presence of incisures has been documented in a variety of species, yet their role remains elusive. In this study, we combined traditional electron microscopy with three-dimensional electron tomography to demonstrate that incisures are formed only after discs become completely enclosed. We also observed that, at the earliest stage of their formation, discs are not round as typically depicted but rather are highly irregular in shape and resemble expanding lamellipodia. Using genetically manipulated mice and frogs and measuring outer segment protein abundances by quantitative mass spectrometry, we further found that incisure size is determined by the molar ratio between peripherin-2, a disc rim protein critical for the process of disc enclosure, and rhodopsin, the major structural component of disc membranes. While a high perpherin-2 to rhodopsin ratio causes an increase in incisure size and structural complexity, a low ratio precludes incisure formation. Based on these data, we propose a model whereby normal rods express a modest excess of peripherin-2 over the amount required for complete disc enclosure in order to ensure that this important step of disc formation is accomplished. Once the disc is enclosed, the excess peripherin-2 incorporates into the rim to form an incisure.
RESUMO
Visual signal transduction takes place within a stack of flattened membranous "discs" enclosed within the light-sensitive photoreceptor outer segment. The highly curved rims of these discs, formed in the process of disc enclosure, are fortified by large hetero-oligomeric complexes of two homologous tetraspanin proteins, PRPH2 (a.k.a. peripherin-2 or rds) and ROM1. While mutations in PRPH2 affect the formation of disc rims, the role of ROM1 remains poorly understood. In this study, we found that the knockout of ROM1 causes a compensatory increase in the disc content of PRPH2. Despite this increase, discs of ROM1 knockout mice displayed a delay in disc enclosure associated with a large diameter and lack of incisures in mature discs. Strikingly, further increasing the level of PRPH2 rescued these morphological defects. We next showed that disc rims are still formed in a knockin mouse in which the tetraspanin body of PRPH2 was replaced with that of ROM1. Together, these results demonstrate that, despite its contribution to the formation of disc rims, ROM1 can be replaced by an excess of PRPH2 for timely enclosure of newly forming discs and establishing normal outer segment structure.
RESUMO
Visual signal transduction takes place within a stack of flattened membranous 'discs' enclosed within the light-sensitive photoreceptor outer segment. The highly curved rims of these discs, formed in the process of disc enclosure, are fortified by large hetero-oligomeric complexes of two homologous tetraspanin proteins, PRPH2 (a.k.a. peripherin-2 or rds) and ROM1. While mutations in PRPH2 affect the formation of disc rims, the role of ROM1 remains poorly understood. In this study, we found that the knockout of ROM1 causes a compensatory increase in the disc content of PRPH2. Despite this increase, discs of ROM1 knockout mice displayed a delay in disc enclosure associated with a large diameter and lack of incisures in mature discs. Strikingly, further increasing the level of PRPH2 rescued these morphological defects. We next showed that disc rims are still formed in a knockin mouse in which the tetraspanin body of PRPH2 was replaced with that of ROM1. Together, these results demonstrate that, despite its contribution to the formation of disc rims, ROM1 can be replaced by an excess of PRPH2 for timely enclosure of newly forming discs and establishing normal outer segment structure.