RESUMO
Photoreceptors are highly polarized sensory neurons. Precise localization of signaling molecules within the ciliary outer segment is critical for photoreceptor function and viability. The small GTPase Arl3 plays a particularly important role in photoreceptors as it regulates outer segment enrichment of lipidated proteins essential for the visual response: transducin-α, transducin-γ, PDEα, PDE ß, and Grk1. Recently, mutations in Arl3 have been identified in human patients with nonsyndromic autosomal recessive and dominant inherited retinal degenerations as well as syndromic Joubert syndrome including retinal dystrophy.
Assuntos
Proteínas Monoméricas de Ligação ao GTP , Distrofias Retinianas , Humanos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Transducina/metabolismo , Cílios/genética , Cílios/metabolismo , Fatores de Ribosilação do ADP/genética , Distrofias Retinianas/genética , MutaçãoRESUMO
Vision begins when light is captured by the outer segment organelle of photoreceptor cells in the retina. Outer segments are modified cilia filled with hundreds of flattened disk-shaped membranes. Disk membranes are separated from the surrounding plasma membrane, and each membrane type has unique protein components. The mechanisms underlying this protein sorting remain entirely unknown. In this study, we investigated the outer segment delivery of the rod cyclic nucleotide-gated (CNG) channel, which is located in the outer segment plasma membrane, where it mediates the electrical response to light. Using Xenopus and mouse models of both sexes, we now show that the targeted delivery of the CNG channel to the outer segment uses the conventional secretory pathway, including protein processing in both ER and Golgi, and requires preassembly of its constituent α1 and ß1 subunits. We further demonstrate that the N-terminal glutamic acid-rich protein (GARP) domain of CNGß1 contains two distinct functional regions. The glutamic acid-rich region encodes specific information targeting the channel to rod outer segments. The adjacent proline-enriched region connects the CNG channel to photoreceptor disk rims, likely through an interaction with peripherin-2. These data reveal fine functional specializations within the structural domains of the CNG channel and suggest that its sequestration to the outer segment plasma membrane requires an interaction with peripherin-2.SIGNIFICANCE STATEMENT Neurons and other differentiated cells have a remarkable ability to deliver and organize signaling proteins at precise subcellular locations. We now report that the CNG channel, mediating the electrical response to light in rod photoreceptors, contains two specialized regions within the N terminus of its ß-subunit: one responsible for delivery of this channel to the ciliary outer segment organelle and another for subsequent channel sequestration into the outer segment plasma membrane. These findings expand our understanding of the molecular specializations used by neurons to populate their critical functional compartments.
Assuntos
Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Domínios Proteicos/fisiologia , Segmento Externo da Célula Bastonete/metabolismo , Animais , Animais Geneticamente Modificados , Animais Recém-Nascidos , Sítios de Ligação/fisiologia , Canais de Cátion Regulados por Nucleotídeos Cíclicos/química , Feminino , Masculino , Proteínas de Membrana/química , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas do Tecido Nervoso/química , Segmento Externo da Célula Bastonete/química , XenopusRESUMO
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.
Assuntos
Proteínas de Membrana/deficiência , Morfogênese/genética , Segmento Externo das Células Fotorreceptoras da Retina/patologia , Animais , Membrana Celular/metabolismo , Membrana Celular/patologia , Micropartículas Derivadas de Células/metabolismo , Micropartículas Derivadas de Células/ultraestrutura , Distrofias de Cones e Bastonetes/genética , Distrofias de Cones e Bastonetes/patologia , Distrofias de Cones e Bastonetes/veterinária , Modelos Animais de Doenças , Cães , Espaço Extracelular/metabolismo , Proteínas do Olho/genética , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Segmento Externo das Células Fotorreceptoras da Retina/metabolismo , Segmento Externo das Células Fotorreceptoras da Retina/ultraestrutura , Retinose Pigmentar/genética , Retinose Pigmentar/patologiaRESUMO
Arf4 is proposed to be a critical regulator of membrane protein trafficking in early secretory pathway. More recently, Arf4 was also implicated in regulating ciliary trafficking, however, this has not been comprehensively tested in vivo. To directly address Arf4's role in ciliary transport, we deleted Arf4 specifically in either rod photoreceptor cells, kidney, or globally during the early postnatal period. Arf4 deletion in photoreceptors did not cause protein mislocalization or retinal degeneration, as expected if Arf4 played a role in protein transport to the ciliary outer segment. Likewise, Arf4 deletion in kidney did not cause cystic disease, as expected if Arf4 were involved in general ciliary trafficking. In contrast, global Arf4 deletion in the early postnatal period resulted in growth restriction, severe pancreatic degeneration and early death. These findings are consistent with Arf4 playing a critical role in endomembrane trafficking, particularly in the pancreas, but not in ciliary function.
Assuntos
Fatores de Ribosilação do ADP/genética , Doenças Renais Císticas/genética , Pâncreas Exócrino/patologia , Degeneração Retiniana/genética , Animais , Cílios/genética , Cílios/patologia , Modelos Animais de Doenças , Humanos , Rim/metabolismo , Rim/patologia , Doenças Renais Císticas/patologia , Camundongos , Camundongos Knockout , Motivos de Nucleotídeos/genética , Pâncreas Exócrino/crescimento & desenvolvimento , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/patologia , Degeneração Retiniana/patologia , Deleção de SequênciaRESUMO
Meier-Gorlin syndrome (MGS) is a genetically heterogeneous primordial dwarfism syndrome known to be caused by biallelic loss-of-function mutations in one of five genes encoding pre-replication complex proteins: ORC1, ORC4, ORC6, CDT1, and CDC6. Mutations in these genes cause disruption of the origin of DNA replication initiation. To date, only an autosomal-recessive inheritance pattern has been described in individuals with this disorder, with a molecular etiology established in about three-fourths of cases. Here, we report three subjects with MGS and de novo heterozygous mutations in the 5' end of GMNN, encoding the DNA replication inhibitor geminin. We identified two truncating mutations in exon 2 (the 1(st) coding exon), c.16A>T (p.Lys6(∗)) and c.35_38delTCAA (p.Ile12Lysfs(∗)4), and one missense mutation, c.50A>G (p.Lys17Arg), affecting the second-to-last nucleotide of exon 2 and possibly RNA splicing. Geminin is present during the S, G2, and M phases of the cell cycle and is degraded during the metaphase-anaphase transition by the anaphase-promoting complex (APC), which recognizes the destruction box sequence near the 5' end of the geminin protein. All three GMNN mutations identified alter sites 5' to residue Met28 of the protein, which is located within the destruction box. We present data supporting a gain-of-function mechanism, in which the GMNN mutations result in proteins lacking the destruction box and hence increased protein stability and prolonged inhibition of replication leading to autosomal-dominant MGS.
Assuntos
Microtia Congênita/genética , Nanismo/genética , Geminina/genética , Transtornos do Crescimento/genética , Micrognatismo/genética , Mutação , Patela/anormalidades , Adolescente , Sequência de Aminoácidos , Sequência de Bases , Ciclo Celular/genética , Pré-Escolar , Microtia Congênita/metabolismo , Nanismo/metabolismo , Nanismo/patologia , Éxons , Feminino , Geminina/metabolismo , Expressão Gênica , Genes Dominantes , Transtornos do Crescimento/metabolismo , Heterozigoto , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Padrões de Herança , Masculino , Micrognatismo/metabolismo , Dados de Sequência Molecular , Patela/metabolismo , Linhagem , Estabilidade Proteica , Proteólise , Splicing de RNA , Alinhamento de SequênciaRESUMO
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
Complete congenital stationary night blindness (cCSNB) is a clinically and genetically heterogeneous group of retinal disorders characterized by nonprogressive impairment of night vision, absence of the electroretinogram (ERG) b-wave, and variable degrees of involvement of other visual functions. We report here that mutations in GPR179, encoding an orphan G protein receptor, underlie a form of autosomal-recessive cCSNB. The Gpr179(nob5/nob5) mouse model was initially discovered by the absence of the ERG b-wave, a component that reflects depolarizing bipolar cell (DBC) function. We performed genetic mapping, followed by next-generation sequencing of the critical region and detected a large transposon-like DNA insertion in Gpr179. The involvement of GPR179 in DBC function was confirmed in zebrafish and humans. Functional knockdown of gpr179 in zebrafish led to a marked reduction in the amplitude of the ERG b-wave. Candidate gene analysis of GPR179 in DNA extracted from patients with cCSNB identified GPR179-inactivating mutations in two patients. We developed an antibody against mouse GPR179, which robustly labeled DBC dendritic terminals in wild-type mice. This labeling colocalized with the expression of GRM6 and was absent in Gpr179(nob5/nob5) mutant mice. Our results demonstrate that GPR179 plays a critical role in DBC signal transduction and expands our understanding of the mechanisms that mediate normal rod vision.
Assuntos
Mutação , Miopia/genética , Miopia/fisiopatologia , Cegueira Noturna/genética , Cegueira Noturna/fisiopatologia , Receptores Acoplados a Proteínas G/genética , Células Bipolares da Retina/metabolismo , Células Bipolares da Retina/fisiologia , Animais , Mapeamento Cromossômico/métodos , Adaptação à Escuridão/genética , Eletrorretinografia/métodos , Oftalmopatias Hereditárias , Técnicas de Silenciamento de Genes/métodos , Doenças Genéticas Ligadas ao Cromossomo X , Heterozigoto , Humanos , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Miopia/metabolismo , Cegueira Noturna/metabolismo , Linhagem , Receptores de Glutamato Metabotrópico/genética , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Transdução de Sinais , Peixe-ZebraRESUMO
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
An important post-translational modification (PTM) of α-tubulin is the removal of amino acids from its C-terminus. Removal of the C-terminal tyrosine residue yields detyrosinated α-tubulin, and subsequent removal of the penultimate glutamate residue produces ΔC2-α-tubulin. These PTMs alter the ability of the α-tubulin C-terminal tail to interact with effector proteins and are thereby thought to change microtubule dynamics, stability, and organization. The peptidase(s) that produces ΔC2-α-tubulin in a physiological context remains unclear. Here, we take advantage of the observation that ΔC2-α-tubulin accumulates to high levels in cells lacking tubulin tyrosine ligase (TTL) to screen for cytosolic carboxypeptidases (CCPs) that generate ΔC2-α-tubulin. We identify CCP1 as the sole peptidase that produces ΔC2-α-tubulin in TTLΔ HeLa cells. Interestingly, we find that the levels of ΔC2-α-tubulin are only modestly reduced in photoreceptors of ccp1-/- mice, indicating that other peptidases act synergistically with CCP1 to produce ΔC2-α-tubulin in post-mitotic cells. Moreover, the production of ΔC2-α-tubulin appears to be under tight spatial control in the photoreceptor cilium: ΔC2-α-tubulin persists in the connecting cilium of ccp1-/- but is depleted in the distal portion of the photoreceptor. This work establishes the groundwork to pinpoint the function of ΔC2-α-tubulin in proliferating and post-mitotic mammalian cells.
Assuntos
Neurônios , Tubulina (Proteína) , Humanos , Camundongos , Animais , Tubulina (Proteína)/metabolismo , Células HeLa , Neurônios/metabolismo , Peptídeo Hidrolases/metabolismo , Tirosina/metabolismo , Microtúbulos/metabolismo , Processamento de Proteína Pós-Traducional , Mamíferos/metabolismoRESUMO
Photoreceptors are sensory neurons that capture light within their outer segment, a narrow cylindrical organelle stacked with disc-shaped membranes housing the visual pigment. Photoreceptors are the most abundant neurons in the retina and are tightly packed to maximize the capture of incoming light. As a result, it is challenging to visualize an individual cell within a crowded photoreceptor population. To address this limitation, we developed a rod-specific mouse model that expresses tamoxifen-inducible cre recombinase under the control of the Nrl promoter. We characterized this mouse using a farnyslated GFP (GFPf) reporter mouse and found mosaic rod expression throughout the retina. The number of GFPf-expressing rods stabilized within 3 days post tamoxifen injection. At that time, the GFPf reporter began to accumulate in basal disc membranes. Using this new reporter mouse, we attempted to quantify the time course of photoreceptor disc renewal in WT and Rd9 mice, a model of X-linked retinitis pigmentosa previously proposed to have a reduced disc renewal rate. We measured GFPf accumulation in individual outer segments at 3 and 6 days post-induction and found that basal accumulation of the GFPf reporter was unchanged between WT and Rd9 mice. However, rates of renewal based on the GFPf measurements were inconsistent with historical calculations from radiolabeled pulse-chase experiments. By extending GFPf reporter accumulation to 10 and 13 days we found that this reporter had an unexpected distribution pattern that preferentially labeled the basal region of the outer segment. For these reasons the GFPf reporter cannot be used for measuring rates of disc renewal. Therefore, we used an alternative method that labels newly forming discs with a fluorescent dye to measure disc renewal rates directly in the Rd9 model and found it was not significantly different from WT. Our study finds that the Rd9 mouse has normal rates of disc renewal and introduces a novel Nrl:CreERT2 mouse for gene manipulation of individual rods.
RESUMO
The primary cilium is a signaling organelle with a unique membrane composition maintained by a diffusional barrier residing at the transition zone. Many transition zone proteins, such as the tectonic complex, are linked to preserving ciliary composition but the mechanism remains unknown. To understand tectonic's role, we generate a photoreceptor-specific Tctn1 knockout mouse. Loss of Tctn1 results in the absence of the entire tectonic complex and associated MKS proteins yet has minimal effects on the transition zone structure of rod photoreceptors. We find that the protein composition of the photoreceptor cilium is disrupted as non-resident membrane proteins accumulate in the cilium over time, ultimately resulting in photoreceptor degeneration. We further show that fluorescent rhodopsin moves faster through the transition zone in photoreceptors lacking tectonic, which suggests that the tectonic complex acts as a physical barrier to slow down membrane protein diffusion in the photoreceptor transition zone to ensure proper removal of non-resident membrane proteins.
Assuntos
Cílios , Proteínas de Membrana , Animais , Camundongos , Proteínas de Membrana/genética , Rodopsina/genética , Neuritos , Corantes , Camundongos KnockoutRESUMO
Defects in primary or motile cilia result in a variety of human pathologies, and retinal degeneration is frequently associated with these so-called ciliopathies. We found that homozygosity for a truncating variant in CEP162, a centrosome and microtubule-associated protein required for transition zone assembly during ciliogenesis and neuronal differentiation in the retina, caused late-onset retinitis pigmentosa in 2 unrelated families. The mutant CEP162-E646R*5 protein was expressed and properly localized to the mitotic spindle, but it was missing from the basal body in primary and photoreceptor cilia. This impaired recruitment of transition zone components to the basal body and corresponded to complete loss of CEP162 function at the ciliary compartment, reflected by delayed formation of dysmorphic cilia. In contrast, shRNA knockdown of Cep162 in the developing mouse retina increased cell death, which was rescued by expression of CEP162-E646R*5, indicating that the mutant retains its role for retinal neurogenesis. Human retinal degeneration thus resulted from specific loss of the ciliary function of CEP162.
Assuntos
Degeneração Retiniana , Animais , Humanos , Camundongos , Centrossomo/metabolismo , Cílios/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Neurogênese/genética , Retina/metabolismo , Degeneração Retiniana/metabolismoRESUMO
MAD2L1BP-encoded p31comet mediates Trip13-dependent disassembly of Mad2- and Rev7-containing complexes and, through this antagonism, promotes timely spindle assembly checkpoint (SAC) silencing, faithful chromosome segregation, insulin signaling, and homology-directed repair (HDR) of DNA double-strand breaks. We identified a homozygous MAD2L1BP nonsense variant, R253*, in 2 siblings with microcephaly, epileptic encephalopathy, and juvenile granulosa cell tumors of ovary and testis. Patient-derived cells exhibited high-grade mosaic variegated aneuploidy, slowed-down proliferation, and instability of truncated p31comet mRNA and protein. Corresponding recombinant p31comet was defective in Trip13, Mad2, and Rev7 binding and unable to support SAC silencing or HDR. Furthermore, C-terminal truncation abrogated an identified interaction of p31comet with tp53. Another homozygous truncation, R227*, detected in an early-deceased patient with low-level aneuploidy, severe epileptic encephalopathy, and frequent blood glucose elevations, likely corresponds to complete loss of function, as in Mad2l1bp-/- mice. Thus, human mutations of p31comet are linked to aneuploidy and tumor predisposition.
Assuntos
Encefalopatias , Tumor de Células da Granulosa , Neoplasias Ovarianas , Feminino , Humanos , Animais , Camundongos , Proteínas Mad2/genética , Proteínas Mad2/metabolismo , Tumor de Células da Granulosa/genética , Mutação , AneuploidiaRESUMO
Expression of channels to specific neuronal sites can critically impact their function and regulation. Currently, the molecular mechanisms underlying this targeting and intracellular trafficking of transient receptor potential (TRP) channels remain poorly understood, and identifying proteins involved in these processes will provide insight into underlying mechanisms. Vision is dependent on the normal function of retinal depolarizing bipolar cells (DBCs), which couple a metabotropic glutamate receptor 6 to the TRP melastatin 1 (TRPM1) channel to transmit signals from photoreceptors. We report that the extracellular membrane-attached protein nyctalopin is required for the normal expression of TRPM1 on the dendrites of DBCs in mus musculus. Biochemical and genetic data indicate that nyctalopin and TRPM1 interact directly, suggesting that nyctalopin is acting as an accessory TRP channel subunit critical for proper channel localization to the synapse.
Assuntos
Dendritos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas/metabolismo , Retina/citologia , Células Bipolares da Retina/citologia , Canais de Cátion TRPM/metabolismo , Aminoácidos/farmacologia , Animais , Proteínas de Bactérias/genética , Biofísica , Linhagem Celular Transformada , Estimulação Elétrica , Antagonistas de Aminoácidos Excitatórios/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Técnicas In Vitro , Proteínas de Repetições Ricas em Leucina , Proteínas Luminescentes/genética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Biológicos , Técnicas de Patch-Clamp , Proteínas/genética , Proteoglicanas/genética , Proteoglicanas/metabolismo , Retina/crescimento & desenvolvimento , Canais de Cátion TRPM/deficiência , Transfecção/métodos , Xantenos/farmacologiaRESUMO
Mutations in TRPM1 are found in humans with an autosomal recessive form of complete congenital stationary night blindness (cCSNB). The Trpm1(-/-) mouse has been an important animal model for this condition. Here we report a new mouse mutant, tvrm27, identified in a chemical mutagenesis screen. Genetic mapping of the no b-wave electroretinogram (ERG) phenotype of tvrm27 localized the mutation to a chromosomal region that included Trpm1. Complementation testing with Trpm1(-/-) mice confirmed a mutation in Trpm1. Sequencing identified a nucleotide change in exon 23, converting a highly conserved alanine within the pore domain to threonine (p.A1068T). Consistent with prior studies of Trpm1(-/-) mice, no anatomical changes were noted in the Trpm1(tvrm27/tvrm27) retina. The Trpm1(tvrm27/tvrm27) phenotype is distinguished from that of Trpm1(-/-) by the retention of TRPM1 expression on the dendritic tips of depolarizing bipolar cells (DBCs). While ERG b-wave amplitudes of Trpm1(+/-) heterozygotes are comparable to wild type, those of Trpm1(+/tvrm27) mice are reduced by 32%. A similar reduction in the response of Trpm1(+/tvrm27) DBCs to LY341495 or capsaicin is evident in whole cell recordings. These data indicate that the p.A1068T mutant TRPM1 acts as a dominant negative with respect to TRPM1 channel function. Furthermore, these data indicate that the number of functional TRPM1 channels at the DBC dendritic tips is a key factor in defining DBC response amplitude. The Trpm1(tvrm27/tvrm27) mutant will be useful for elucidating the role of TRPM1 in DBC signal transduction, for determining how Trpm1 mutations impact central visual processing, and for evaluating experimental therapies for cCSNB.
Assuntos
Mutação Puntual , Células Bipolares da Retina/fisiologia , Canais de Cátion TRPM/genética , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Sequência de Aminoácidos , Aminoácidos/farmacologia , Animais , Capsaicina/farmacologia , Mapeamento Cromossômico , Cromossomos de Mamíferos/genética , Dendritos/fisiologia , Modelos Animais de Doenças , Éxons , Oftalmopatias Hereditárias/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Heterozigoto , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Miopia/genética , Cegueira Noturna/genética , Retina/patologia , Retina/fisiologia , Análise de Sequência de DNA , Canais de Cátion TRPM/metabolismo , Treonina/genética , Xantenos/farmacologiaRESUMO
Congenital stationary night blindness (CSNB) is a clinically and genetically heterogeneous group of retinal disorders characterized by nonprogressive impaired night vision and variable decreased visual acuity. We report here that six out of eight female probands with autosomal-recessive complete CSNB (cCSNB) had mutations in TRPM1, a retinal transient receptor potential (TRP) cation channel gene. These data suggest that TRMP1 mutations are a major cause of autosomal-recessive CSNB in individuals of European ancestry. We localized TRPM1 in human retina to the ON bipolar cell dendrites in the outer plexifom layer. Our results suggest that in humans, TRPM1 is the channel gated by the mGluR6 (GRM6) signaling cascade, which results in the light-evoked response of ON bipolar cells. Finally, we showed that detailed electroretinography is an effective way to discriminate among patients with mutations in either TRPM1 or GRM6, another autosomal-recessive cCSNB disease gene. These results add to the growing importance of the diverse group of TRP channels in human disease and also provide new insights into retinal circuitry.
Assuntos
Mutação , Cegueira Noturna/congênito , Cegueira Noturna/genética , Canais de Cátion TRPM/genética , Sequência de Aminoácidos , Estudos de Casos e Controles , Deleção Cromossômica , Estudos de Coortes , Eletrorretinografia/normas , Éxons , Feminino , Genes Recessivos , Heterozigoto , Homozigoto , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Cegueira Noturna/fisiopatologia , Núcleo Familiar , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Transdução de Sinais , População Branca/genéticaRESUMO
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.
Assuntos
Células Fotorreceptoras/metabolismo , Actinas/metabolismo , Animais , Micropartículas Derivadas de Células/metabolismo , Cílios/metabolismo , Humanos , Modelos Biológicos , PolimerizaçãoRESUMO
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.
Assuntos
Micropartículas Derivadas de Células/metabolismo , Cílios/metabolismo , Periferinas/metabolismo , Células Fotorreceptoras/metabolismo , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Retinitis pigmentosa (RP) is a blinding disease often associated with mutations in rhodopsin, a light-sensing G protein-coupled receptor and phospholipid scramblase. Most RP-associated mutations affect rhodopsin's activity or transport to disc membranes. Intriguingly, some mutations produce apparently normal rhodopsins that nevertheless cause disease. Here we show that three such enigmatic mutations-F45L, V209M and F220C-yield fully functional visual pigments that bind the 11-cis retinal chromophore, activate the G protein transducin, traffic to the light-sensitive photoreceptor compartment and scramble phospholipids. However, tests of scramblase activity show that unlike wild-type rhodopsin that functionally reconstitutes into liposomes as dimers or multimers, F45L, V209M and F220C rhodopsins behave as monomers. This result was confirmed in pull-down experiments. Our data suggest that the photoreceptor pathology associated with expression of these enigmatic RP-associated pigments arises from their unexpected inability to dimerize via transmembrane helices 1 and 5.
Assuntos
Mutação , Mutação Puntual , Retina/metabolismo , Retinose Pigmentar/genética , Rodopsina/química , Rodopsina/genética , Animais , Células COS , Bovinos , Chlorocebus aethiops , Proteínas de Ligação ao GTP/química , Células HEK293 , Humanos , Lipossomos/metabolismo , Camundongos Knockout , Proteínas de Transferência de Fosfolipídeos/metabolismo , Multimerização Proteica , Retina/química , Transducina/genéticaRESUMO
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.