Retinal cone cells exhibit distinctive photoresponse with a more restrained sensitivity to light and a more rapid shutoff kinetics than those of rods. To understand the molecular basis for these characteristics of cone responses, we focused on the opsin deactivation process initiated by G protein-coupled receptor kinase (GRK) 1 and GRK7 in the zebrafish, an animal model suitable for studies on retinal physiology and biochemistry. Screening of the ocular cDNAs identified two homologs for each of GRK1 (1A and 1B) and GRK7 (7-1 and 7-2), and they were classified into three GRK subfamilies, 1 A, 1B and 7 by phylogenetic analysis. In situ hybridization and immunohistochemical studies localized both GRK1B and GRK7-1 in the cone outer segments and GRK1A in the rod outer segments. The opsin/GRKs molar ratio was estimated to be 569 in the rod and 153 in the cone. The recombinant GRKs phosphorylated light-activated rhodopsin, and the V max value of the major cone subtype, GRK7-1, was 32-fold higher than that of the rod kinase, GRK1A. The reinforced activity of the cone kinase should provide a strengthened shutoff mechanism of the light-signaling in the cone and contribute to the characteristics of the cone responses by reducing signal amplification efficiency.
Phosphorylation of tau, a heat-stable neuron-specific microtubule-associated protein, by cdk5 was stimulated in the presence of microtubules (MTs). This stimulation was due to an increased phosphorylation rate and there was no increase in total amount of phosphorylation. Two-dimensional phosphopeptide map analysis showed that MTs stimulated phosphorylation of a specific peptide. Using Western blotting with antibodies that the recognized phosphorylation-dependent epitopes within tau, the phosphorylation sites stimulated by the presence of MTs were found to be Ser202 and Thr205 (numbered according to the human tau isoform containing 441 residues). MT-dependent phosphorylation at Thr205 was observed in situ in rat cerebrum primary cultured neurons. Stimulated phosphorylation at Ser202 and Thr205 decreased the MT-nucleation activity of tau, which is in contrast to MT-independent phosphorylation at Ser235 and Ser404.
We detected rhodopsin gene expression in the pigeon lateral septum, a photosensitive deep brain region that is responsible for the photoperiodic gonadal response. The nucleotide sequence of the deep brain rhodopsin cDNA clone exactly matched that of the retinal one, indicating that a single rhodopsin gene is transcribed in the two tissues. Immunohistochemical analysis localized rhodopsin in the cerebrospinal fluid-contacting neurons, which have been assumed to be photoreceptive cells in the deep brain. Pigeon rhodopsin seems to play dual important roles in the visual and non-visual systems, the latter of which contributes to the photoperiodic response.
Total mass of mitochondria increases during cell proliferation and differentiation through mitochondrial biogenesis, which includes mitochondrial proliferation and growth. During the mitochondrial growth, individual mitochondria have been considered to be enlarged independently of mitochondrial fusion. However, molecular basis for this enlarging process has been poorly understood. Cone photoreceptor cells in the retina possess large mitochondria, so-called mega-mitochondria that have been considered to arise via the enlarging process. Here we show that ES1 is a novel mitochondria-enlarging factor contributing to form mega-mitochondria in cones. ES1 is specifically expressed in cones and localized to mitochondria including mega-mitochondria. Knockdown of ES1 markedly reduced the mitochondrial size in cones. In contrast, ectopic expression of ES1 in rods significantly increased both the size of individual mitochondria and the total mass of the mitochondrial cluster without changing the number of them. RNA-seq analysis showed that ERRα and its downstream mitochondrial genes were significantly up-regulated in the ES1-expressing rods, suggesting facilitation of mitochondrial enlargement via ERRα-dependent processes. Furthermore, higher energy state was detected in the ES1-expressing rods, indicating that the enlarged mitochondria by ES1 are capable of producing high energy. ES1 is the mitochondrial protein that is first found to promote enlargement of individual mitochondria.
Non-technical summary When rod and cone photoreceptors in the eye respond to light, they need to recover, and the first step in recovery involves a protein called G-protein receptor kinase (GRK). Rods, which underlie night vision, employ a variant called GRK1, whereas cones, which mediate day vision, typically employ a variant called GRK7. We have engineered rod cells in the zebrafish retina that additionally express the cone variant, GRK7. By recording electrically from these modified rods, we have found that they are less sensitive to light than normal rods, in that regard mimicking cones. We have also found evidence to suggest that the size of the cell's response to a single photon (the smallest particle of light) is normal when recovery is mediated by GRK1, but is small (and hence somewhat cone-like) when mediated by GRK7. These results help us understand the differences between rod and cone photoreceptors.Abstract To investigate the roles of G-protein receptor kinases (GRKs) in the light responses of vertebrate photoreceptors, we generated transgenic zebrafish lines, the rods of which express either cone GRK (GRK7) or rod GRK (GRK1) in addition to the endogenous GRK1, and we then measured the electrophysiological characteristics of single-cell responses and the behavioural responses of intact animals. Our study establishes the zebrafish expression system as a convenient platform for the investigation of specific components of the phototransduction cascade. The addition of GRK1 led to minor changes in rod responses. However, exogenous GRK7 in GRK7-tg animals led to lowered rod sensitivity, as occurs in cones, but surprisingly to slower response kinetics. Examination of responses to long series of very dim flashes suggested the possibility that the GRK7-tg rods generated two classes of single-photon response, perhaps corresponding to the interaction of activated rhodopsin with GRK1 (giving a standard response) or with GRK7 (giving a very small response). Behavioural measurement of optokinetic responses (OKR) in intact GRK7-tg zebrafish larvae showed that the overall rod visual pathway was less sensitive, in accord with the lowered sensitivity of the rods. These results help provide an understanding for the molecular basis of the electrophysiological differences between cones and rods.F. Vogalis, T. Shiraki and D. Kojima contributed equally to this work.
Photoreceptive areas responsible for the regulation of photoperiodicity have been localized in the brain of some avian species. We found that immunoreactivities to rhodopsin and the alpha-subunit of rod-type transducin (Gt(1)alpha) were colocalized in cerebrospinal fluid (CSF) contacting neurons in the pigeon lateral septum, a possible site for photoreception. Furthermore, RT-PCR analyses showed specific gene expression of both cGMP-phosphodiesterase beta-subunit and cone-type cGMP-gated cation channel alpha-subunit in this region. These results suggest that several components in rod/cone photoreceptors compositely contribute to the deep encephalic phototransduction cascade.
Rods and cones are functionally and morphologically distinct. We previously identified N-myc downstream-regulated gene 1b (ndrg1b) in carp as a cone-specific gene. Here, we show that NDRG1b and its paralog, NDRG1a-1, contribute to photoreceptor outer segment (OS) formation in zebrafish. In adult zebrafish photoreceptors, NDRG1a-1 was localized in the entire cone plasma membranes, and also in rod plasma membranes except its OS. NDRG1b was expressed specifically in cones in the entire plasma membranes. In a developing retina, NDRG1a-1 was expressed in the photoreceptor layer, and NDRG1b in the photoreceptor layer plus inner nuclear layer. Based on our primary knockdown study suggesting that both proteins are involved in normal rod and cone OS development, NDRG1a-1 was overexpressed or NDRG1b was ectopically expressed in rods. These forced-expression studies in the transgenic fish confirmed the effect of these proteins on the OS morphology: rod OS morphology changed from cylindrical to tapered shape. These taper-shaped rod OSs were not stained with N,N’-didansyl cystine that effectively labels infolded membrane structure of cone OS. The result shows that rod OS membrane structure is preserved in these taper-shaped OSs and therefore, suggests that tapered OS morphology is not related to the infolded membrane structure in cone OS.
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