Photoreceptor Cell-specific Protein Research Articles

Expression Patterns of Germ Cell-specific Phosducin-like 2 during Testicular and Ovarian Development in Chickens

Phosducin (PDC) is a photoreceptor cell-specific protein that is phosphorylated by cyclic nucleotide-dependent protein kinase. PDC and PDC-like proteins (PDCL, PDCL2, and PDCL3) are members of a conserved family of small thioredoxin-like proteins that modulate the β- and γ-subunits of G-proteins. In mammals, Pdc, Pdcl, and Pdcl3 genes show ubiquitous expression; however, Pdcl2 gene expression is limited to the testis and ovary. The aim of the present study was to examine the expression patterns of chicken Pdcl2 (cPdcl2) during testicular and ovarian development. Protein sequence comparisons performed using the CLUSTAL X program revealed that the amino acid sequences and potential phosphorylation sites of cPDCL2 and mammalian PDCL2 proteins were highly conserved. Quantitative real-time PCR analysis revealed that cPdcl2 was differentially expressed in the testis and ovary. Specifically, cPdcl2 expression was detected at low levels in the ovary at all time points. In the testis, cPdcl2 expression was detected at low levels until 5 weeks of age. At 8 weeks of age, however, cPdcl2 showed increased expression levels in the testis. Using in situ hybridization, we detected high levels of cPdcl2 expression in the testis, particularly in the spermatocytes and round spermatids. In summary, our data describe expression patterns of germ cell-specific Pdcl2 during testicular and ovarian development in chickens.

Neurocalcin-immunoreactive cells in the rat hippocampus are GABAergic interneurons.

Neurocalcin (NC) is a recently described calcium-binding protein isolated and characterized from bovine brain. NC belongs to the neural calcium-sensor proteins defined by the photoreceptor cell-specific protein recoverin that have been proposed to be involved in the regulation of calcium-dependent phosphorylation in signal transduction pathways. We analyzed the distribution and morphology of the NC-immunoreactive (IR) neurons in the rat dorsal hippocampus and the coexistence of NC with GABA and different neurochemical markers which label perisomatic inhibitory cells [parvalbumin (PV) and cholecystokinin (CCK)], mid-proximal dendritic inhibitory cells [calbindin D28k (CB)], distal dendritic inhibitory cells [somatostatin (SOM) and neuropeptide Y (NPY)], and interneurons specialized to innervate other interneurons [calretinin (CR) and vasoactive intestinal polypeptide (VIP)]. NC-IR cells were present in all layers of the dentate gyrus and hippocampal fields. In the dentate gyrus, NC-IR cells were concentrated in the granule cell layer, especially in the hilar border, whereas in the CA fields they were most frequently found in the stratum radiatum. NC-IR cells were morphologically heterogeneous and exhibited distinctive features of non-principal cells. In the dentate gyrus, pyramidal-like, multipolar and fusiform (horizontal and vertical) cells were found. In the CA3 region most NC-IR cells were multipolar, but vertical and horizontal fusiform cells also appeared. In the CA1 region, where NC-IR cells showed most frequently vertically arranged dendrites, multipolar, bitufted and fusiform (vertical and horizontal) cells could be distinguished. All the NC-IR cells were found to be GABA-IR in all hippocampal layers and regions, and they represented about 19% of the GABA-positive cells. NC/CB, NC/CR and NC/VIP double-labeled cells were found in all hippocampal regions, and represented 29%, 24% and 18% of the NC-IR cells, respectively. NC and CCK did not coexist in the dentate gyrus; however, 9% of the NC-IR cells in the CA fields also contained CCK. No coexistence of NC with PV, SOM or NPY was found in any hippocampal region. We conclude that NC is exclusively expressed by interneurons in the rat hippocampus. NC-IR cells are a morphologically and neurochemically heterogeneous subset of GABAergic non-principal cells, which, on the basis of the known termination pattern of the colocalizing markers, are also functionally heterogeneous and are mainly involved in feed-forward dendritic inhibition in the commissural-associational and Schaffer collateral termination zones (CB containing cells), in innervation of other interneurons (CR- and VIP-containing cells), and in perisomatic inhibition (CCK-containing cells). NC is never present in perisomatic inhibitory PV-containing cells, or in feed-back distal dendritic inhibitory SOM/NPY-containing cells.

Crx, a Novel Otx-like Paired-Homeodomain Protein, Binds to and Transactivates Photoreceptor Cell-Specific Genes

The otd/ Otx gene family encodes paired-like homeodomain proteins that are involved in the regulation of anterior head structure and sensory organ development. Using the yeast one-hybrid screen with a bait containing the Ret 4 site from the bovine rhodopsin promoter, we have cloned a new member of the family, Crx (Cone rod homeobox). Crx encodes a 299 amino acid residue protein with a paired-like homeodomain near its N terminus. In the adult, it is expressed predominantly in photoreceptors and pinealocytes. In the developing mouse retina, it is expressed by embryonic day 12.5 (E12.5). Recombinant Crx binds in vitro not only to the Ret 4 site but also to the Ret 1 and BAT-1 sites. In transient transfection studies, Crx transactivates rhodopsin promoter-reporter constructs. Its activity is synergistic with that of Nrl. Crx also binds to and transactivates the genes for several other photoreceptor cell-specific proteins (interphotoreceptor retinoid-binding protein, β-phosphodiesterase, and arrestin). Human Crx maps to chromosome 19q13.3, the site of a cone rod dystrophy (CORDII). These studies implicate Crx as a potentially important regulator of photoreceptor cell development and gene expression and also identify it as a candidate gene for CORDII and other retinal diseases.

Crystallization and preliminary x‐ray crystallographic studies of recombinant bovine neurocalcin δ

Neurocalcins are novel brain-specific proteins that belong to a new subclass of the EF-hand super-family of calcium binding proteins, defined by the photoreceptor cell-specific protein recoverin (Terasawa et al., J. Biol. Chem. 267:19596-19599, 1992). Here we report the purification and crystallization of unmyristoylated recombinant bovine neurocalcin delta from Escherichia coli. Crystals of a bovine neurocalcin delta have been grown by macro-seeding at room temperature through vapor phase equilibration using the hanging drop technique with ammonium sulfate as the precipitating agent. The crystals diffract to at least 2.5 A resolution and belong to monoclinic space group P21 with unit cell dimensions a = 42.734 A, b = 94.343 A, c = 50.696 A, and beta = 98.37 degrees. The asymmetric unit contains two molecules, with corresponding crystal volume per protein mass (Vm) of 2.29 A3/Da and solvent fraction of 45% by volume, exhibiting an approximate 222 point symmetry.

Calcium and Membrane Binding Properties of Bovine Neurocalcin δ Expressed in Escherichia coli

Calcium and Membrane Binding Properties of Bovine Neurocalcin δ Expressed in Escherichia coli

Mutations in calphotin, the gene encoding a Drosophila photoreceptor cell-specific calcium-binding protein, reveal roles in cellular morphogenesis and survival.

Calphotin is a Drosophila photoreceptor cell-specific protein expressed very early in eye development, at the time when cell-type decisions are being made. Calphotin is a very hydrophobic and proline-rich protein which lacks obvious transmembrane domains. The cDNA encoding Calphotin was mapped to a region removed by a set of existing chromosomal deletions. Mutations that alter photoreceptor cell structure and development were isolated that fail to complement these deletions. These mutations fall into two classes. Class I mutations alter the structure of the rhabdomere, a photoreceptor cell organelle specialized for phototransduction. Class II mutations have rough eyes, due to misorientation of the rhabdomeres and photoreceptor cell death. Transformation rescue of these phenotypes in transgenic flies bearing calphotin genomic DNA indicates that both classes of mutations are in the calphotin gene. Analysis of these mutations suggest that Calphotin plays important roles in both rhabdomere development and in photoreceptor cell survival.

A Drosophila photoreceptor cell-specific protein, calphotin, binds calcium and contains a leucine zipper.

The calphotin protein, encoded by the calphotin (cap) gene, is expressed in the soma and axons of all Drosophila photoreceptor cells. It is expressed early in photo-receptor cell development, at the time when cell-type decisions are being made. Expression of calphotin is not altered by the glass mutation, which blocks photoreceptor cell development. The calphotin protein binds calcium and contains a long C-terminal leucine zipper. Potential implications of these properties are discussed.

Chromosomal assignment of the recoverin gene and cancer-associated retinopathy

The deduced amino acid sequence of the recently cloned mouse 23kD photoreceptor cell-specific protein showed it to be identical to the recoverin protein and the CAR (cancer-associated retinopathy) protein. DNA sequence variants were found in the mouse recoverin gene (Rcvrn), and segregation analysis of restriction fragment length variants in recombinant inbred strains of mice assigned Rcvrn to mouse Chromosome (Chr) 11, between Sparc (3.7 map units) and Zfp-3 (2.3 map units). These results demonstrate a close linkage of recoverin to the tumor suppressor gene, Trp53. On the basis of these data, knowledge of the function of recoverin, and the characteristics of CAR, an experimentally testable model is presented to explain the molecular basis for CAR.

Cloning and sequencing of the 23 kDa mouse photoreceptor cell-specific protein

The 23 kDa protein was localized by immunocytochemistry to photoreceptor cells of the mouse retina, and bovine and mouse cDNA clones were isolated and sequenced. The deduced amino acid sequences showed that the mouse 23 kDa protein is 91% identical to the bovine protein, and is the same as S-modulin, the CAR (cancer-associated retinopathy) protein and recoverin, the Ca 2+-dependent activator of photoreceptor guanylate cyclase. The amino acid sequence reveals two Ca 2+ binding sites, no internal repeats, 59% homology to the chicken visinin protein and 40% homology to calmodulin while Northern analysis demonstrated a single 1.0 kb mRNA species in bovine and mouse retina.

Photoreceptor cell specific proteins of snake pineal

The pineal body of lower vertebrates is saccular and directly photoreceptive. The pineal gland of mammals is parenchymal and not directly photoreceptive. The parenchymal morphology of snake pineal raises questions of direct photoreceptivity of snake pineal and of correspondence of molecular homology with morphological homology. S-antigen and rhodopsin are highly conserved photoreceptor cell specific proteins. We used site-specific monoclonal antibodies (MAb) to study S-antigen and rhodopsin of snake pineal. Immunohistochemical reactivity of snake retina and pineal was compared to that of trout, guinea pig, and rat. MAb's to S-antigen reacted with each pineal and retina tested, but reactivity of individual MAb's with snake tissue was more similar to that with trout than with rat or guinea pig tissue. MAb's to rhodopsin did not react with snake pineal, although they did react with the photoreceptive trout pineal body. MAb's to rhodopsin reacted with retina of each species. These results suggest that although snake pineal is morphologically similar to mammalian pineal, and like mammalian pineal is probably not directly photoreceptive, it does have S-antigen homology with lower vertebrates such as trout.

Decrease of Opsin Content in the Developing Rat Photoreceptor Cells by Systemic Administration of L-Glutamate.

L-Glutamate, a putative photoreceptor cell neurotransmitter, causes thinning of the inner layers of the retina and has been used for preparing biologically fractionated photoreceptor cells. However, it is possible that absence of the inner retinal layers may affect the remaining retina, and/or glutamate may directly affect photoreceptor cells. We evaluated quantitatively the effects of L-glutamate on the developing photoreceptor cells by measuring the rod photoreceptor cell-specific protein, opsin. We purified rat rhodopsin and used it as the standard for measuring opsin content of rat retinas with competitive enzyme-linked immunosorbent assay. Various concentrations of glutamate were injected into 7-day-old rats, and the effects of the amino acid concentration on opsin expression were determined on postnatal day 14. Inner layers of the retina degenerated when 10 microliters or 15 microliters of 2.4 M glutamate/gram body weight was administered subcutaneously. Opsin content of these glutamate-treated retinas decreased significantly compared with control retinas. We administered glutamate to rats at various stages of development and determined the effects by light microscopy on postnatal day 14. The administration of glutamate resulted in no degeneration of the inner retina if injected on postnatal day 1 or 2, degeneration of the inner retina between day 3 to 7, and again, no degeneration after postnatal day 13. Opsin content decreased significantly when glutamate was administered between postnatal day 1 to 7, but not after day 13, the day the blood-retinal barrier seems to reach maturity. Our findings indicate that systemic administration of L-glutamate affects the expression of opsin in the developing rod photoreceptor cells.

EM immunocytochemical localization of rhodopsin and IRBP during retinal development

Two rod photoreceptor cell-specific proteins, rhodopsin and interphotoreceptor retinoid binding protein (IRBP), were localized during fetal development of the bovine retina using immuncicrytcichemistry. Rhodopsin is the light sensitive protein that, when activated, begins the process of transducing light energy to an electrical response. IRBP is a carrier protein that shuttles light-isomerized vitamin A (retinol) between the rod outer segment and the overlying retinal pigment epithelium where it is recycled to its light-sensitive form. Rhodopsin has been previously imimmocytochemically localized to rod (but not cone) photoreceptor outer segment membranes. IRBP has been localized to the subretinal space using immunocytochemistry.Retinas were obtained from fetuses at approximately 4, 5, 6, 7, and 8 months of gestation, fixed in 4% paraformaldehyde and 0.5% glutaraldehyde, and embedded at 40°C in epoxy resin. Thin sections were mounted on Ni grids and incubated with antibodies raised against the purified antigens: a mouse monoclonal anti-rhodopsin or a rabbit polyclonal anti-IRBP. A second antibody (Goat-anti-mouse or Goat-anti-rabbit) was conjugated with 15 nm Au particles and reacted with the sections. Control incubations were made using pre-immune rabbit serum and mouse monoclonals made against other tissue or bacterial sources.

The Drosophila ninaC locus encodes two photoreceptor cell specific proteins with domains homologous to protein kinases and the myosin heavy chain head

The fruit fly Drosophila melanogaster has been extensively used to identify genes required for photoreceptor cell function. We show that the ninaC gene, originating isolated as a Drosophila visual mutation with an electrophysiological phenotype, encodes two novel cytoskeletal proteins. We identified the DNA sequences encoding the ninaC genes by rescuing the electrophysiological phenotype using P-element-mediated germ line transformation. The ninaC locus is expressed as two extensively overlapping mRNAs encoding proteins of 1135 and 1501 amino acids. Both proteins contain a putative protein kinase domain joined to a domain homologous to the head region of the myosin heavy chain and are spatially restricted to photoreceptor cells.

Immunoelectron microscopy of retinoblastoma

The histogenesis of retinoblastoma is uncertain. The derivation of this tumor has been attributed to neuronal and/or glial cells, by routine transmission electron microscopy and light microscopic immunocytochemical staining. Immunoelectron microscopy can provide more precise localization of retinal proteins. We investigated the immunoelectron microscopic localization of photoreceptor cell specific proteins to determine their distribution in eyes enucleated for retinoblastoma.Surgically excised tissues were fixed in 1% glutaraldehyde 2%- paraformaldehyde in 0.1M phosphate buffer for 2 h at 4°C, dehydrated in 50%, 75% and 90% dimethyl-formamide and embedded in Lowicryl. Polymerization of Lowicryl was performed by exposure to UV light for 24 h at 4°, followed by polymerization at room temperature for 48-72 hr. Thin sections were incubated for 1 h at room temperature with primary antibodies against interphotoreceptor retinoid-binding protein (IRBP) (1:200 dilution), S-antigen (1:500 dilution) and opsin (1:500 dilution). Appropriate secondary antibodies either to mouse or rabbit IgG (1:20 dilution) were linked to colloidal gold (10-20 nm) and incubated for 30 min.