Vertebrate Rod Outer Segments Research Articles

High-resolution atomic force microscopy imaging of rhodopsin in rod outer segment disk membranes.

Atomic force microscopy (AFM) is a powerful imaging technique that allows recording topographical information of membrane proteins under near-physiological conditions. Remarkable results have been obtained on membrane proteins that were reconstituted into lipid bilayers. High-resolution AFM imaging of native disk membranes from vertebrate rod outer segments has unveiled the higher-order oligomeric state of the G protein-coupled receptor rhodopsin, which is highly expressed in disk membranes. Based on AFM imaging, it has been demonstrated that rhodopsin assembles in rows of dimers and paracrystals and that the rhodopsin dimer is the fundamental building block of higher-order structures.

Will the Rod Bend or Break? Analyzing the Structural Resilience of Cellular Organelles

Will the Rod Bend or Break? Analyzing the Structural Resilience of Cellular Organelles

Regulation of Phosphatidic Acid Metabolism by Sphingolipids in the Central Nervous System

This paper explores the way ceramide, sphingosine, ceramide 1-phosphate, and sphingosine 1-phosphate modulate the generation of second lipid messengers from phosphatidic acid in two experimental models of the central nervous system: in vertebrate rod outer segments prepared from dark-adapted retinas as well as in rod outer segments prepared from light-adapted retinas and in rat cerebral cortex synaptosomes under physiological aging conditions. Particular attention is paid to lipid phosphate phosphatase, diacylglycerol lipase, and monoacylglycerol lipase. Based on the findings reported in this paper, it can be concluded that proteins related to phototransduction phenomena are involved in the effects derived from sphingosine 1-phosphate/sphingosine or ceramide 1-phosphate/ceramide and that age-related changes occur in the metabolism of phosphatidic acid from cerebral cortex synaptosomes in the presence of either sphingosine 1-phosphate/sphingosine or ceramide 1-phosphate/ceramide. The present paper demonstrates, in two different models of central nervous system, how sphingolipids influence phosphatidic acid metabolism under different physiological conditions such as light and aging.

Lipid second messengers and related enzymes in vertebrate rod outer segments

Rod outer segments (ROSs) are specialized light-sensitive organelles in vertebrate photoreceptor cells. Lipids in ROS are of considerable importance, not only in providing an adequate environment for efficient phototransduction, but also in originating the second messengers involved in signal transduction. ROSs have the ability to adapt the sensitivity and speed of their responses to ever-changing conditions of ambient illumination. A major contributor to this adaptation is the light-driven translocation of key signaling proteins into and out of ROS. The present review shows how generation of the second lipid messengers from phosphatidylcholine, phosphatidic acid, and diacylglycerol is modulated by the different illumination states in the vertebrate retina. Findings suggest that the light-induced translocation of phototransduction proteins influences the enzymatic activities of phospholipase D, lipid phosphate phosphatase, diacylglyceride lipase, and diacylglyceride kinase, all of which are responsible for the generation of the second messenger molecules.

Phosphoinositide 3-kinase signaling in the vertebrate retina

The phosphoinositide (PI) cycle, discovered over 50 years ago by Mabel and Lowell Hokin, describes a series of biochemical reactions that occur on the inner leaflet of the plasma membrane of cells in response to receptor activation by extracellular stimuli. Studies from our laboratory have shown that the retina and rod outer segments (ROSs) have active PI metabolism. Biochemical studies revealed that the ROSs contain the enzymes necessary for phosphorylation of phosphoinositides. We showed that light stimulates various components of the PI cycle in the vertebrate ROS, including diacylglycerol kinase, PI synthetase, phosphatidylinositol phosphate kinase, phospholipase C, and phosphoinositide 3-kinase (PI3K). This article describes recent studies on the PI3K-generated PI lipid second messengers in the control and regulation of PI-binding proteins in the vertebrate retina.

Calcium-Sensitive Downregulation of the Transduction Chain in Rod Photoreceptors of the Rat Retina

In vertebrate rod outer segments phototransduction is suggested to be modulated by intracellular Ca. We aimed at verifying this hypothesis by recording saturated photosignals in the rat retina after single and double flashes of light and determining the time t c to the beginning of the signal recovery. The time course of Ca i after a flash was calculated from a change of the spatial Ca 2+ concentration profile recorded in the space between the rods. After single flashes t c increased linearly with the logarithm of flash intensity, confirming the assumption that t c is determined by deactivation of a single species X* in the phototransduction cascade. The photoresponse was shortened up to 45% if the test flash was preceded by a conditioning preflash. The shortening depended on the reduction of Ca i induced by the preflash. The data suggest that the phototransduction gain determining the amount of activated X* is regulated by a Ca i-dependent mechanism in a short time period (<800 ms) after the test flash. Lowering of Ca i by a preflash reduced the gain up to 20% compared to its value in a dark-adapted rod. The relation between phototransduction gain and Ca i revealed a K 1/2 value close to the dark level of Ca i.

32] Identification of phosphorylation sites within vertebrate and invertebrate rhodopsin

Publisher Summary Studies of vertebrate and invertebrate rhodopsin have been applicable to our understanding of amplification and desensitization processes of Gprotein-coupled receptors in general. In vertebrate rod outer segments, photoisomerization of rhodopsin (Rho*) initiates activation of hundreds of G-protein (G t ) molecules, which in turn stimulate cGMP phosphodiesterase, resulting in closure of cGMP gated channels in response to the decrease in cytosolic cGMP concentrations. To study the enzymatic and functional roles of Rho phosphorylation, it was necessary to develop protocols for the identification of phosphorylation sites within vertebrate and invertebrate Rho. This chapter describes a method that employs mass spectrometry in conjunction with specific proteolysis, and should be applicable to the study of phosphorylation in other systems. Rho phosphorylation is carried out on rod outer segment (ROS) membranes isolated from fresh bovine retinas by discontinuous sucrose density gradient centrifugation in 10 mM Bis-Tris propane (BTP) buffer, pH 7.5, containing 5 mM MgCl2 and 1 mM ATP at 30° for 0–45 minutes under illumination from a 150-W lamp at a distance of 10 cm. Phosphorylated forms of the peptide could, in principle, be analyzed by tandem mass spectrometry (MS/MS) to identify the specific residues phosphorylated.

Intracellular spreading of second messengers

Intracellular spreading of second messengers

X-ray crystal structure of arrestin from bovine rod outer segments.

Retinal arrestin is the essential protein for the termination of the light response in vertebrate rod outer segments. It plays an important role in quenching the light-induced enzyme cascade by its ability to bind to phosphorylated light-activated rhodopsin (P-Rh*). Arrestins are found in various G-protein-coupled amplification cascades. Here we report on the three-dimensional structure of bovine arrestin (relative molecular mass, 45,300) at 3.3 A resolution. The crystal structure comprises two domains of antiparallel beta-sheets connected through a hinge region and one short alpha-helix on the back of the amino-terminal fold. The binding region for phosphorylated light-activated rhodopsin is located at the N-terminal domain, as indicated by the docking of the photoreceptor to the three-dimensional structure of arrestin. This agrees with the interpretation of binding studies on partially digested and mutated arrestin.

Crystallization and preliminary X-ray analysis of arrestin from bovine rod outer segment

We present the first X-ray study of a member of the arrestin family, the bovine retinal arrestin. Arrestin is essential for the fine regulation and termination of the light-induced enzyme cascade in vertebrate rod outer segments. It plays an important role in quenching phototransduction by its ability to preferentially bind to phosphorylated light-activated rhodopsin. The crystals diffract between 3 Å and 3.5 Å (space group P2 12 12, cell dimensions a=169.17 Å, b=185.53 Å, c=90.93 Å, T=100 K). The asymmetric unit contains four molecules with a solvent content of 68.5% by volume.

Cytochemical localization of guanylate cyclase in photoreceptor cells of the mouse.

Light-stimulated excitation causes a decrease of the cGMP concentration in vertebrate photoreceptor cells. The cGMP content is restored by the catalytic action of a guanylate cyclase (EC 4.6.1.2). The spatial distribution of guanylate cyclase was determined cytochemically in rod visual cells of the mouse. In retinal tissue of the mouse guanylate cyclase was found throughout the photoreceptor cells, in the outer and the inner segments, and was especially prominent in the cilia and in elongations of cilia extending into the outer segments. A reaction product of adenylate cyclase (EC 4.6.1.1) could not be demonstrated in vertebrate rod outer segments. The relatively high amount of guanylate cyclase in the inner segments and the cilia may contribute-at least in part-to the actual concentration and the time course of concentration changes of the cGMP concentration in rod outer segments.

Modulation of guanylate cyclase and phosphodiesterase by monovalent cations and nucleoside triphosphates in light-sensitive excised patches of rod outer segments

Excised inside-out patches of vertebrate rod outer segment can support phototransduction. I have examined how ionic and metabolic conditions influence the functional properties of light-sensitive patches from Gekko gekko. I find that such patches retain a variable level of basal phosphodiesterase activity, which lowers the cyclic guanosine monophosphate (cGMP) concentration reaching the channels and reduces the dark current. The dose/response relationship for channel opening by cGMP varies among patches and this variability is only reduced by working in darkness with the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX), suggesting that it is only partially due to phosphodiesterase activity. MgATP or MgGTP, but not Mg or ATP separately, increase this activity but a kinase does not appear to be involved. Intracellular monovalent cations also influence dark current intensity and light response kinetics. With 5 mM MgGTP, 1 mM IBMX, and 144 mM Li+, Na+, K+, or Rb+, dark current intensity and recovery time follow the respective sequences K+ > Rb+ > Na+ > Li+ and K+ < Rb+ < Li+ < Na+. Without IBMX, a dark current develops with K+ but not with Na+. These effects are not due to altered channel permeability (P) [PLi+:Na+:K+:Rb+:guanidinium)/PNa+ = 0.84:1.00:1.01:1.09:0.42], or differential Mg2+ block, but to modulation of guanylate cyclase, which overcomes phosphodiesterase when the major cation is K+ but not when it is Na+.

Distinctive subtypes of bovine phospholipase C that have preferential expression in the retina and high homology to the norpA gene product of Drosophila.

The Drosophila norpA gene encodes a phospholipase C involved in phototransduction. However, phospholipase C apparently is not directly involved in phototransduction in vertebrate photoreceptors, although light-activated phospholipase C activity has been reported in vertebrate rod outer segments. Conserved regions of norpA cDNA were used to isolate bovine cDNAs that would encode four alternative forms of phospholipase C of the beta class that are highly homologous to the norpA protein and expressed preferentially in the retina. Two of the variants are highly unusual in that they lack much of the N-terminal region present in all other known phospholipases C. The sequence conservation between these proteins and the norpA protein is higher than that between any other known phospholipases C. GTPase sequence motifs found in proteins of the GTPase superfamily are found conserved in all four variants of the bovine retinal protein as well as the norpA protein but not in other phospholipases C. Results suggest that these proteins together with the norpA protein constitute a distinctive subfamily of phospholipases C that are closely related in structure, function, and tissue distribution. Mutations in the norpA gene, in addition to blocking phototransduction, cause light-dependent degeneration of photoreceptors. In view of the strong similarity in structure and tissue distribution, a defect in these proteins may have similar consequences in the mammalian retina.

The γ-subunit of the principal G-protein from squid ( Loligo forbesi) photoreceptors contains a novel N-terminal sequence

The squid ( Loligo forbesi) visual system presents as accessible a system for study of G-protein mediated signal transduction as the vertebrate rod outer segment with the added advantage that the major G-protein is a member of the Gq-class. Here the cDNA clone encoding the γ-subunit of this G-protein is reported, thereby completing the molecular cloning of the heterotrimeric G-protein. The deduced protein structure of G-γ has relatively little sequence identity with known mammalian counterparts particularly in comparison with the relatively high degree found for both the α- and β-subunits of this protein. In particular, the N-terminus of the squid visual G-γ contains a repetitive, highly charged region, rich in lysine and glutamate, that has no parallel in other G-proteins. The amino acid sequence of a number of peptides derived by chemical cleavage of G-γ accounted for much of the protein sequence predicted from the cDNA, including the unusual N-terminal region.

Enrichment of polyunsaturated fatty acids from rat retinal pigment epithelium to rod outer segments.

Polyunsaturated fatty acids (PUFA), especially docosahexaenoic acid (DHA, 22:6n-3), are enriched in phospholipids of vertebrate rod outer segments (ROS). Retinal ROS can incorporate 22 carbon (C-22) PUFA from the plasma pool where C-20 PUFA are predominant. In this study, we analyzed the fatty acid composition of retinal pigment epithelium (RPE) and ROS from rats fed different fatty acid supplements to determine whether this enrichment is at the photoreceptor-RPE boundary or the RPE-choriocapillaris boundary. Long Evans rats were raised from birth for 13-14 weeks on a diet supplemented with 10% (wt/wt) hydrogenated coconut oil (COC; 0.2% 18:2n-6, no 18:3n-3), safflower oil (SAF; 73.8% 18:2n-6, 0.1% 18:3n-3), or linseed oil (LIN; 16.4% 18:2n-6, 52.2% 18:3n-3). These diets were chosen because they increased plasma levels of 20:3n-9, 20:4n-6, and 20:5n-3, respectively. These three fatty acids served as metabolic markers. Plasma levels of 22:6n-3 were reduced by the COC and SAF diets. The RPE incorporated 20:3n-9, 20:4n-6, and 20:5n-3 from the plasma. However, the levels of 20:3n-9 and 20:5n-3 were very low in ROS and 20:4n-6 was not significantly elevated in the ROS of the SAF diet group. The relative amount of total C-20 PUFA in phospholipids in RPE was similar to that found in plasma and was about 4-16 times (depending on different lipid classes) that in the ROS. In contrast, C-22 PUFA (22:6n-3 and 22:5n-6) showed a step-wise, average 3-5 fold increase in concentration from the plasma to the RPE to the ROS.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphoinositide metabolism in frog rod outer segments

Previous studies have shown that vertebrate rod outer segments (ROS) have a light activated phospholipase C which hydrolyzes phosphatidylinositol-4,5-bisphosphate (PIP 2). Three different experimental approaches have been used to test the hypothesis that the phosphatidylinositol (PI) biosynthetic cycle is present in ROS and that PIP 2 can be regenerated from DG independent of rod inner segments. In the first study, enzyme activities of the PI cycle were assayed simultaneously in the presence of CTP, myo-inositol and [γ- 32P]ATP using endogenous lipids as substrates. Under these conditions, broken (leaky) ROS prepared by continuous sucrose gradient centrifugation showed PI, PIP and DG kinase activities similar to those found in intact ROS and non-ROS membranes, whereas PI synthetase activity was much lower in the leaky ROS than in the other two fractions. The relative distribution of PI synthetase specific activity in the three membrane preparations was similar to that of the microsomal enzyme marker cytochrome c reductase. ROS prepared by discontinuous sucrose gradient centrifugation showed only 2–3% of whole homogenate PI synthetase or phosphatidyl: cytidyl transferase activities, and the distribution of activities was the same as for microsomal and mitochondrial marker enzymes. In the second study, whole retinas were incubated with myo-[2- 3H]inositol or [2- 3H]glycerol in vitro, and the time course of incorporation of radioactivity into PI and other phospholipids was determined for ROS and three other retinal fractions. Over a 10-hr period, the rate of incorporation of myo-[2- 3H]inositol or [2- 3H]glycerol into PI in ROS was lowest among the various retinal fractions. In the third study, chemical analysis of the molecular species composition of PI, DG and phosphatidic acid (PA) from ROS shows that PA is substantially different from PI and DG, the latter two being quite similar. These results are consistent with a precursor-product relationship between PI and DG, but not with the conversion of DG to PA or of PA to PI. Taken together, these three studies indicate that ROS do not have PI synthetase or phosphatidyl: cytidyl transferase activities, but do have DG, PI and PIP kinase activities. Thus, the PI in ROS lost through rapid turnover must be replaced with molecules derived from de novo synthesis in the inner segment of the photoreceptor cell.

Excised patches of plasma membrane from vertebrate rod outer segments retain a functional phototransduction enzymatic cascade.

Ion channels in excised patches of plasma membrane are generally considered to be isolated from any intracellular regulation mechanisms. For example, in excised patches of vertebrate rod outer segment plasma membrane, the cGMP-activated cation channels have traditionally been studied in room light because the enzyme cascade linking photon absorption to channel closure was assumed to be inoperative. To investigate the possibility that, in fact, such excised patches retain a functional phototransduction enzymatic cascade, this same preparation was studied in darkness. Patches excised in the dark were found to retain the light sensitivity of their cGMP-induced conductance and the ability to synthesize cGMP. In the presence of guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]), a nonhydrolyzable GTP analog, light suppresses the cGMP-induced conductance irreversibly. Furthermore, inhibitors of phosphodiesterase activity reduce light sensitivity, whereas activated phosphodiesterase or activated transducin does not directly affect the channels. These results (i) establish that excised patches from rod outer segment retain functional phototransduction enzymes, (ii) support the classical view that channel opening is modulated by phosphodiesterase-mediated cGMP hydrolysis, and, most surprisingly, (iii) demonstrate that diffusion in excised patches is so restricted that local enzymes can induce variations in the concentration of small molecules. The indication that excised patches are not as simple as usually surmised opens the possibility of using them to study other intracellular transduction mechanisms.

Effects of pro and anti-oxidizing conditions on phospholipid metabolism within isolated vertebrate rod outer segments

Effects of pro and anti-oxidizing conditions on phospholipid metabolism within isolated vertebrate rod outer segments

βγ-Subunit of Bovine Transducin Composed of Two Components with Distinctive γ-Subunits

Abstract During the process of transduction of a photon signal in vertebrate rod outer segments, transducin, a guanine nucleotide binding protein, mediates between a photobleaching intermediate of rhodopsin and a cGMP-phosphodiesterase. We report here that the beta gamma-subunit of bovine transducin (T beta gamma) characterized so far consists of two components (T beta gamma-1 and T beta gamma-2), which can be separated by anion exchange chromatography under nondenaturing conditions. Both components consisted of two polypeptides of Mr 36,000 (T beta) and about 8,000 (T gamma) in sodium dodecyl sulfate polyacrylamide (13%) gel electrophoresis. On a further analysis by 8 M urea/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, T gamma subunits of T beta gamma-1 and T beta gamma-2 showed Mr values of 8,000 (T gamma-1) and 6,000 (T gamma-2), respectively. Amino acid compositions of both T gamma-1 and T gamma-2 roughly corresponded with that of T gamma previously reported and were quite different from that of gamma-subunit of cGMP-phosphodiesterase. Western blot analysis of freshly isolated rod outer segments by an antiserum raised against a mixture of T beta gamma-1 and T beta gamma-2 revealed the presence of both components in the membranes of a starting material. This observation excludes the possibility that one of the components might be produced artificially in the course of the purification. In the presence of a photobleaching intermediate of either unphosphorylated or phosphorylated rhodopsin, the binding of guanosine 5'-(beta, gamma-imido)triphosphate (GppNHp) to the alpha-subunit of transducin (T alpha) was remarkably enhanced with increasing concentrations of purified T beta gamma-2. On the contrary, T beta gamma-1 retained little ability, if any, to enhance the GppNHp binding to T alpha; the ability of T beta gamma-1 was at least 30 times lower than that of T beta gamma-2. Such a low activity of T beta gamma-1 was attributed to inability for coupling of T alpha with a photobleaching intermediate of rhodopsin. These results indicate that T gamma-2 is essential for the GTP binding of transducin. The role of T gamma-1 in vertebrate photoreceptor cells was discussed.

The activation of the cyclic-GMP phosphodiesterase via metarhodopsin I: a new model for vertebrate transduction.

Light-scattering spectrophotometry has been used to study rapid photo-induced molecular-cellular changes in vertebrate rod outer segments. Here, we discuss the temporal profiles of the nucleotide-independent P signal as a function of photobleaching, pH dependence in membrane-permeable and -impermeable buffers, angular and wavelength dependence, and cyclic-GMP phosphodiesterase inhibitors. On the basis of these observations, we suggest that (i) the P signal is coupled with the metarhodopsin I photo-intermediate and (ii) processes involved in the P signal invoke activation of cyclic-GMP phosphodiesterase. Furthermore, temperature-dependence studies indicate that the G protein does not participate in the scheme until the metarhodopsin II stage has been reached. This latter finding suggests that GTP-dependent processes are involved principally in the recovery of the system following light absorption. Our results point to a new model for phototransduction in vertebrate vision.