RESUMO
Despite lizards using a wide range of colour signals, the limited variation in photoreceptor spectral sensitivities across lizards suggests only weak selection for species-specific, spectral tuning of photoreceptors. Some species, however, have enhanced short-wavelength sensitivity, which probably helps with the detection of signals rich in ultraviolet and short wavelengths. In this study, we examined the visual system of Tiliqua rugosa, which has an ultraviolet/blue tongue, to gain insight into this species' visual ecology. We used electroretinograms, opsin sequencing and immunohistochemical labelling to characterize whole-eye spectral sensitivity and the elements that shape it. Our findings reveal that T. rugosa expresses all five opsins typically found in lizards (SWS1, SWS2, RH1, RH2 and LWS) but possesses greatly enhanced short-wavelength sensitivity compared with other diurnal lizards. This enhanced short-wavelength sensitivity is characterized by a broadening of the spectral sensitivity curve of the eye towards shorter wavelengths while the peak sensitivity of the eye at longer wavelengths (560â nm) remains similar to that of other diurnal lizards. While an increased abundance of SWS1 photoreceptors is thought to mediate elevated ultraviolet sensitivity in a couple of other lizard species, SWS1 photoreceptor abundance remains low in this species. Instead, our findings suggest that short-wavelength sensitivity is driven by multiple factors which include a potentially red-shifted SWS1 photoreceptor and the absence of short-wavelength-absorbing oil droplets. Examining the coincidence of enhanced short-wavelength sensitivity with blue tongues among lizards of this genus will provide further insight into the co-evolution of conspecific signals and whole-eye spectral sensitivity.
Assuntos
Lagartos , Animais , Eletrorretinografia , Olho , Opsinas/genética , FilogeniaRESUMO
Purpose: In Bornholm eye disease, a defect in the splicing of transcripts from a variant OPN1LW opsin gene leads to a depletion in spliced transcript levels and, consequently, a reduction in photopigment in photoreceptors expressing the variant gene. Methods: Myopic and age-matched control subjects were drawn from the Western Australian Pregnancy Cohort (Raine) Study and the Norfolk Island Eye Study groups. The OPN1LW opsin gene was amplified using long-range PCR methodology and was fully sequenced. Expression of variant opsins was evaluated using quantitative PCR (qPCR). RNA secondary structure changes arising from identified variants were predicted by modeling. Results: Forty-two nucleotide sites were found to vary across the 111 subjects studied. Of these, 15 had not been previously reported, with three present only in myopic individuals. Expression of these variants in transfected human embryonic kidney (HEK293T) cells demonstrated that splicing efficiencies were not affected. However, gene transcripts from two of the three variants were significantly depleted. RNA secondary structure modeling predicted that these single nucleotide changes could affect RNA stability. Conclusions: None of the variants identified in myopic individuals appeared to alter the efficiency of transcript splicing. However, two resulted in a significant reduction in the number of spliced and unspliced transcripts, indicating an overall reduction in steady-state transcript stability. Such a change would be expected to result in a reduced amount of photopigment, and this may be a contributing factor in the development of myopia.
Assuntos
Miopia/genética , Splicing de RNA , Estabilidade de RNA , RNA Mensageiro/genética , Opsinas de Bastonetes/genética , Adulto , Austrália , Estudos de Casos e Controles , Clonagem Molecular , Expressão Gênica , Variação Genética , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Ilhas , Masculino , Miopia/diagnóstico , Miopia/fisiopatologia , Conformação de Ácido Nucleico , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Opsinas de Bastonetes/deficiência , Análise de Sequência de DNARESUMO
A comprehensive description of the spectral characteristics of retinal photoreceptors in palaeognaths is lacking. Moreover, controversy exists with respect to the spectral sensitivity of the short-wavelength-sensitive-1 (SWS1) opsin-based visual pigment expressed in one type of single cone: previous microspectrophotometric (MSP) measurements in the ostrich (Struthio camelus) suggested a violet-sensitive (VS) SWS1 pigment, but all palaeognath SWS1 opsin sequences obtained to date (including the ostrich) imply that the visual pigment is ultraviolet-sensitive (UVS). In this study, MSP was used to measure the spectral properties of visual pigments and oil droplets in the retinal photoreceptors of the emu (Dromaius novaehollandiae). Results show that the emu resembles most other bird species in possessing four spectrally distinct single cones, as well as double cones and rods. Four cone and a single rod opsin are expressed, each an orthologue of a previously identified pigment. The SWS1 pigment is clearly UVS (wavelength of maximum absorbance [λmax] = 376 nm), with key tuning sites (Phe86 and Cys90) consistent with other vertebrate UVS SWS1 pigments. Palaeognaths would appear, therefore, to have UVS SWS1 pigments. As they are considered to be basal in avian evolution, this suggests that UVS is the most likely ancestral state for birds. The functional significance of a dedicated UVS cone type in the emu is discussed.
Assuntos
Dromaiidae/fisiologia , Opsinas/fisiologia , Células Fotorreceptoras Retinianas Cones/fisiologia , Pigmentos da Retina/fisiologia , Visão Ocular , Animais , Raios UltravioletaRESUMO
The physiological functions and cellular signaling of Class II phosphoinositide 3-kinases (PI3Ks) remain largely unknown. Platelets express two Class II PI3Ks: PI3KC2α and PI3KC2ß. PI3KC2α deficiency was recently reported to cause disruption of the internal membrane reserve structure of platelets (open canalicular system, OCS) that results in dysregulated platelet adhesion and impaired arterial thrombosis in vivo. Notably, these effects on platelets occurred despite normal agonist-induced 3-phosphorylated phosphoinositide (3-PPI) production and cellular activation in PI3KC2α-deficient platelets. However, the potential compensatory actions of PI3KC2ß in platelets have not yet been investigated. Here, we report the first mice deficient in both PI3KC2α and PI3KC2ß (no Class II PI3Ks in platelets) and reveal a nonredundant role for PI3KC2α in mouse platelet structure and function. Specifically, we show that the disrupted OCS and impaired thrombus stability observed in PI3KC2α-deficient platelets does not occur in PI3KC2ß-deficient platelets and is not exaggerated in platelets taken from mice deficient in both enzymes. Furthermore, detailed examination of 3-PPI production in platelets from this series of mice revealed no changes in either unactivated or activated platelets, including those with a complete lack of Class II PI3Ks. These findings indicate a nonredundant role for PI3KC2α in regulating platelet structure and function, and suggest that Class II PI3Ks do not significantly contribute to the acute agonist-induced production of 3-PPIs in these cells.
Assuntos
Plaquetas/metabolismo , Classe II de Fosfatidilinositol 3-Quinases/deficiência , Trombose/sangue , Trombose/genética , Animais , Plaquetas/ultraestrutura , Classe II de Fosfatidilinositol 3-Quinases/genética , Classe II de Fosfatidilinositol 3-Quinases/metabolismo , Classe III de Fosfatidilinositol 3-Quinases/genética , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica , Camundongos , Camundongos Knockout , Adesividade Plaquetária , Contagem de Plaquetas , Testes de Função PlaquetáriaRESUMO
Mutations in the KCNV2 gene, which encodes the voltage-gated K+ channel protein Kv8.2, cause a distinctive form of cone dystrophy with a supernormal rod response (CDSRR). Kv8.2 channel subunits only form functional channels when combined in a heterotetramer with Kv2.1 subunits encoded by the KCNB1 gene. The CDSRR disease phenotype indicates that photoreceptor adaptation is disrupted. The electroretinogram (ERG) response of affected individuals shows depressed rod and cone activity, but what distinguishes this disease is the supernormal rod response to a bright flash of light. Here, we have utilized knock-out mutations of both genes in the mouse to study the pathophysiology of CDSRR. The Kv8.2 knock-out (KO) mice show many similarities to the human disorder, including a depressed a-wave and an elevated b-wave response with bright light stimulation. Optical coherence tomography (OCT) imaging and immunohistochemistry indicate that the changes in six-month-old Kv8.2 KO retinae are largely limited to the outer nuclear layer (ONL), while outer segments appear intact. In addition, there is a significant increase in TUNEL-positive cells throughout the retina. The Kv2.1 KO and double KO mice also show a severely depressed a-wave, but the elevated b-wave response is absent. Interestingly, in all three KO genotypes, the c-wave is totally absent. The differential response shown here of these KO lines, that either possess homomeric channels or lack channels completely, has provided further insights into the role of K+ channels in the generation of the a-, b-, and c-wave components of the ERG.
Assuntos
Distrofia de Cones/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Retina/metabolismo , Canais de Potássio Shab/metabolismo , Animais , Distrofia de Cones/diagnóstico por imagem , Distrofia de Cones/patologia , Feminino , Técnicas de Inativação de Genes , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Retina/diagnóstico por imagem , Retina/patologia , Canais de Potássio Shab/genética , Transmissão Sináptica , Visão Ocular/fisiologiaRESUMO
Vertebrate vision is accomplished through light-sensitive photopigments consisting of an opsin protein bound to a chromophore. In dim light, vertebrates generally rely on a single rod opsin [rhodopsin 1 (RH1)] for obtaining visual information. By inspecting 101 fish genomes, we found that three deep-sea teleost lineages have independently expanded their RH1 gene repertoires. Among these, the silver spinyfin (Diretmus argenteus) stands out as having the highest number of visual opsins in vertebrates (two cone opsins and 38 rod opsins). Spinyfins express up to 14 RH1s (including the most blueshifted rod photopigments known), which cover the range of the residual daylight as well as the bioluminescence spectrum present in the deep sea. Our findings present molecular and functional evidence for the recurrent evolution of multiple rod opsin-based vision in vertebrates.
Assuntos
Evolução Molecular , Proteínas de Peixes/fisiologia , Peixes/fisiologia , Opsinas de Bastonetes/fisiologia , Visão Ocular/fisiologia , Animais , Escuridão , Proteínas de Peixes/classificação , Proteínas de Peixes/genética , Peixes/genética , Variação Genética , Genoma , Filogenia , Opsinas de Bastonetes/classificação , Opsinas de Bastonetes/genética , Visão Ocular/genéticaRESUMO
Most vertebrates have a duplex retina comprising two photoreceptor types, rods for dim-light (scotopic) vision and cones for bright-light (photopic) and color vision. However, deep-sea fishes are only active in dim-light conditions; hence, most species have lost their cones in favor of a simplex retina composed exclusively of rods. Although the pearlsides, Maurolicus spp., have such a pure rod retina, their behavior is at odds with this simplex visual system. Contrary to other deep-sea fishes, pearlsides are mostly active during dusk and dawn close to the surface, where light levels are intermediate (twilight or mesopic) and require the use of both rod and cone photoreceptors. This study elucidates this paradox by demonstrating that the pearlside retina does not have rod photoreceptors only; instead, it is composed almost exclusively of transmuted cone photoreceptors. These transmuted cells combine the morphological characteristics of a rod photoreceptor with a cone opsin and a cone phototransduction cascade to form a unique photoreceptor type, a rod-like cone, specifically tuned to the light conditions of the pearlsides' habitat (blue-shifted light at mesopic intensities). Combining properties of both rods and cones into a single cell type, instead of using two photoreceptor types that do not function at their full potential under mesopic conditions, is likely to be the most efficient and economical solution to optimize visual performance. These results challenge the standing paradigm of the function and evolution of the vertebrate duplex retina and emphasize the need for a more comprehensive evaluation of visual systems in general.
Assuntos
Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/química , Animais , Arrestina/classificação , Arrestina/genética , Evolução Biológica , Proteínas de Peixes/classificação , Proteínas de Peixes/genética , Peixes , Opsinas/classificação , Opsinas/genética , Filogenia , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/química , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Transcriptoma , Transducina/classificação , Transducina/genéticaRESUMO
PI3KC2α is a broadly expressed lipid kinase with critical functions during embryonic development but poorly defined roles in adult physiology. Here we utilize multiple mouse genetic models to uncover a role for PI3KC2α in regulating the internal membrane reserve structure of megakaryocytes (demarcation membrane system) and platelets (open canalicular system) that results in dysregulated platelet adhesion under haemodynamic shear stress. Structural alterations in the platelet internal membrane lead to enhanced membrane tether formation that is associated with accelerated, yet highly unstable, thrombus formation in vitro and in vivo. Notably, agonist-induced 3-phosphorylated phosphoinositide production and cellular activation are normal in PI3KC2α-deficient platelets. These findings demonstrate an important role for PI3KC2α in regulating shear-dependent platelet adhesion via regulation of membrane structure, rather than acute signalling. These studies provide a link between the open canalicular system and platelet adhesive function that has relevance to the primary haemostatic and prothrombotic function of platelets.