Effects of light and cGMP on membrane fluidity of frog rod outer segments.

Abstract—We have used a spin-labeled fatty acid to detect changes induced by light and by cGMP in isolated rod outer segment membranes. We chose a spin probe (5-doxylstearic acid) which has the nitroxide group placed on the hydrocarbon chain, so the probe should reside somewhat inside the hydrophobic region of the membrane. We found that light exposures which bleached the rhodopsin also produced a small change in the EPR spectra. The spectral changes are consistent with a small increase in membrane fluidity. Light exposures which bleach rhodopsin are known to activate a phosphodiesterase that markedly decreases the cGMP level in rod outer segment. Therefore, we attempted to vary cGMP levels directly by adding Bu₂-cGMP, or indirectly, by adding IBMX, CDTA or ATP to try to inhibit the phosphodiesterase. In each case where the cGMP level is expected to increase, we observed spectral changes in the dark which suggested a small decrease in membrane fluidity. Thus, all of our results with this probe are consistent with the idea that changing the level of cGMP produces changes in membrane fluidity. The light-induced spectral changes we observed required the presence of ATP, and were inhibited by 2mM Ca²⁺, or by the chelator of divalent cations, CDTA.     

Distribution and translocation of photoreceptor Gbetagamma-phosducin system in medaka retina

Unlike other vertebrates, teleosts have rod- and cone-specific phosducins (PD-R and PD-C) in the retina. To evaluate the teleost Gbetagamma-PD systems, we isolated cDNAs encoding medaka Gbeta1 and GbetaC, which selectively expressed rods and cones. Immunohistochemical studies showed that the strong reactivity of GbetaC but not PD-C was detected in cone outer segments. In rod outer segments (ROS), PD-R reactivity was stronger in light-adapted retina than in dark-adapted retina. Western blot analyses of fractions torn from the cryosections showed that the PD-R concentration was low in dark-adapted ROS. It is suggested that PD-R is translocated to ROS and effectively downregulates the phototransduction cascade in light-adapted rods.     

All‐Trans Retinal Mediates Light‐Induced Oxidation in Single Living Rod Photoreceptors†

All‐trans retinal is a potent photosensitizer that is released in photoreceptor outer segments by the photoactivated visual pigment following the detection of light. Photoreceptor outer segments also contain high concentrations of polyunsaturated fatty acids, and are thus particularly susceptible to oxidative damage such as that initiated by light via a photosensitizer. Upon its release, all‐trans retinal is reduced within the outer segment to all‐trans retinol, through a reaction requiring metabolic input in the form of NADPH. The phototoxic potential of physiologically generated all‐trans retinal was examined in single living rod photoreceptors obtained from frog (Rana pipiens) retinas. Light‐induced oxidation was measured with fluorescence imaging using an oxidation‐sensitive indicator dye from the shift in fluorescence between the intact and oxidized forms. Light‐induced oxidation was highest in metabolically compromised rod outer segments following photoactivation of the visual pigment rhodopsin, and after a time interval, sufficiently long to ensure the release of all‐trans retinal. Furthermore, light‐induced oxidation increased with the concentration of exogenously added all‐trans retinal. The results show that the all‐trans retinal generated during the detection of light can mediate light‐induced oxidation. Its removal through reduction to all‐trans retinol protects photoreceptor outer segments against light‐induced oxidative damage.     

Salamander blue-sensitive cones lost during metamorphosis

The tiger salamander lives in shallow water with bright light in the aquatic phase, and in dim tunnels or caves in the terrestrial phase. In the aquatic phase, there are five types of photoreceptors--two types of rods and three types of cones. Our previous studies showed that the green rods and blue-sensitive cones contain the same visual pigment and have the same absorbance spectra; however, the green rods have a larger photon-catch area and thus have higher light sensitivity than the blue-sensitive cones. Here we show that after metamorphosis, the terrestrial salamander looses the blue-sensitive cones, while the density of the green rods increases. Moreover, the size of the green rod outer segments is increased in the terrestrial phase, compared to that in the aquatic phase. This switch from the blue-sensitive cones to the green rods may represent an adaptation to the dim light environment of the terrestrial phase.     

Catalytic nanomotors: autonomous movement of striped nanorods

Rod-shaped particles, 370 nm in diameter and consisting of 1 microm long Pt and Au segments, move autonomously in aqueous hydrogen peroxide solutions by catalyzing the formation of oxygen at the Pt end. In 2-3% hydrogen peroxide solution, these rods move predominantly along their axis in the direction of the Pt end at speeds of up to 10 body lengths per second. The dimensions of the rods and their speeds are similar to those of multiflagellar bacteria. The force along the rod axis, which is on the order of 10(-14) N, is generated by the oxygen concentration gradient, which in turn produces an interfacial tension force that balances the drag force at steady state. By solving the convection-diffusion equation in the frame of the moving rod, it was found that the interfacial tension force scales approximately as SR(2)gamma/muDL, where S is the area-normalized oxygen evolution rate, gamma is the liquid-vapor interfacial tension, R is the rod radius, mu is the viscosity, D is the diffusion coefficient of oxygen, and L is the length of the rod. Experiments in ethanol-water solutions confirmed that the velocity depends linearly with the product Sgamma, and scaling experiments showed a strong dependence of the velocity on R and L. The direction of motion implies that the gold surface is hydrophobic under the conditions of the experiment. Tapping-mode AFM images of rods in air-saturated water show soft features that are not apparent in images acquired in air. These features are postulated to be nanobubbles, which if present in hydrogen peroxide solutions, would account for the observed direction of motion.     

Rhodopsin phosphorylation suggests biochemical heterogeneities of retinal rod disks.

Frogs (Rana pipiens) were injected subcutaneously with (3H)-leucine and allowed to incorporate the radioactive amino acid into newly assembled disks in the retinal rod outer segment. The labeled disks served as a temporal marker for following the turnover of rod outer segments. Animals were killed at different times after injection and outer segments were isolated and phosphorylated with ATP in the light. The visual pigment (as isorhodopsin) was regenerated with 9-cis retinal, extracted, and chromatographed on epichlorohydrin triethanolamine cellulose so that phosphorylated pigment could be separated from unphosphorylated pigment. The ratio of (3H)-radioactivity of phosphorylated pigment to that of unphosphorylated pigment was then plotted against the time after injection. The ratio was high when (3H)-labeled disks were largely associated with the basal region of the rod and decreased as the labeled disks moved toward the rod apical region. The results were interpreted as suggesting that newer disks are phosphorylated preferentially to older disks. Papain digestion of (3H)-labeled disks indicated that rhodopsin in newer disks is more susceptible to proteolysis than that in older disks.     

Biochemical steps in visual transduction: roles for nucleotides and calcium ions

Abstract— Recent studies from this laboratory permit the suggestion of a scheme for describing molecular mechanisms which may regulate excitation and adaptation in amphibian rod photoreceptor cells. The experiments have studied several chemical changes which occur upon illumination of isolated rod outer segments: (1) activation of cyclic GMP phosphodiesterase which is sensitive to calcium concentration, (2) a resulting rapid drop in cyclic GMP levels which has stoichiometry and time course appropriate for the internal transmitter presumed to mediate between photon absorption in the disc membrane system and the permeability decrease in the plasma membrane, (3) a dephosphorylation of two small proteins whose phosphorylation is controlled by cyclic GMP levels, and (4) a slower hydrolysis of GTP which may drive efflux of calcium from the outer segment. It is suggested that the rapid decrease in sodium conductance which follows illumination is caused by the dephosphorylation of the two small proteins, with their dephosphorylation being controlled by the cyclic GMP decrease. In a slower reaction light activates a GTP-dependent extrusion of calcium from the cytoplasmic space. This lowering of internal calcium causes desensitization of the light-sensitive phosphodiesterase enzyme responsible for the cyclic GMP decrease, so that its intensity-response function now resembles that of the light-adapted rod photoreceptor. Thus, changes in plasma membrane conductance are regulated by cyclic GMP, and the sensitivity of the system is controlled by slower calcium movements which set the light-sensitivity of the phosphodiesterase enzyme. Finally, the light-initiated phosphorylation of rhodopsin also appears to play a role, with phosphorylated rhodopsin causing desensitization of the phosphodiesterase enzyme.     

Monte Carlo study of defect fluctuations and reptation in polymer melts

Defect fluctuations in highly distorted polymer chains were simulated by Monte Carlo calculation. The NMR autocorrelation function was derived and described by the superposition of three exponential functions with time constants spread over two orders of magnitude. As a consequence of defect diffusion, longitudinal chain diffusion (reptation) can be expected in polymer melts. By simulating the mean-square displacement of a segment, it was found that after sufficiently long times, compared with defect density correlation times, a linear relationship holds fairly well. As a rule of thumb, it can be stated that the linear Einstein equation is valid for times much greater than 10³ mean step times in practical cases (chain length: several thousand segments, defect concentration: 10–20%), or, in other words, for mean-square displacements greater than a few diffusion step lengths. A long-time chain-diffusion coefficient depending on the molecular weight and on the defect concentration could be derived. Effects on the low-field NMR relaxation behavior are derived and discussed.     

Detection of molecular processes in the intact retina by ATR-FTIR spectromicroscopy

We used Fourier transform infrared spectromicroscopy in the attenuated total reflection configuration to study biochemical events associated with the response to light of an intact retina. We show that the technique is suitable for the detection in real time of molecular processes occurring in rod outer segments induced by light absorption. Two-dimensional correlation analysis was applied to the identification and interpretation of specific spectral changes associated to the evolution of the system. The technique allows us to observe an extensive protein translocation, which we interpret as arising from the release of transducin from the disk membrane and its redistribution from the outer segment towards the inner segment of rod cells. These results are in full agreement with our current understanding of retinal physiology and validate the technique as a useful tool for the study of complex molecular processes in intact tissue. Figure Spectral changes in the mid infrared region following exposure of an intact retina to light     

Tokay gecko photoreceptors achieve rod-like physiology with cone-like proteins

The retinal photoreceptors of the nocturnal Tokay gecko (Gekko gekko) consist exclusively of rods by the criteria of morphology and key features of their light responses. Unlike cones, they display robust photoresponses and have relatively slow recovery times. Nonetheless, the major and minor visual pigments identified in gecko rods are of the cone type by sequence and spectroscopic behavior. In the ongoing search for the molecular bases for the physiological differences between cones and rods, we have characterized the molecular biology and biochemistry of the gecko rod phototransduction cascade. We have cloned cDNAs encoding all or part of major protein components of the phototransduction cascade by RT-PCR with degenerate oligonucleotides designed to amplify cone- or rod-like sequences. For all proteins examined we obtained only cone-like and never rod-like sequences. The proteins identified include transducin alpha (Galphat), phosphodiesterase (PDE6) catalytic and inhibitory subunits, cyclic nucleotide-gated channel (CNGalpha) and arrestin. We also cloned cDNA encoding gecko RGS9-1 (Regulator of G Protein Signaling 9, splice variant 1), which is expressed in both rods and cones of all species studied but is typically found at 10-fold higher concentrations in cones, and found that gecko rods contain slightly lower RGS9-1 levels than mammalian rods. Furthermore, we found that the levels of GTPase accelerating protein (GAP) activity and cyclic GMP (cGMP) phosphodiesterase activity were similar in gecko and mammalian rods. These results place substantial constraints on the critical changes needed to convert a cone into a rod in the course of evolution: The many features of phototransduction molecules conserved between those expressed in gecko rods and those expressed in cones cannot explain the physiological differences, whereas the higher levels of RGS9-1 and GAP activity in cones are likely among the essential requirements for the rapid photoresponses of cones.     

LIGHT-SENSITIVITY MODULATING PROTEIN IN FROG RODS

Cyclic GMP is the second messenger in the phototransduction mechanism in rod photoreceptors. Light-induced activation of cGMP phosphodiesterase (PDE), the hydrolyzing enzyme of cGMP, reduces cytoplasmic cGMP concentration to close the cGMP-activated channel and thereby causes a hyperpolarizing light response. Ca2+ concentration decreases during light-adaptation and this decrease is thought to be at least one of the underlying mechanisms of light-adaptation. Our previous electrophysiological work suggested that PDE in frog rod photoreceptors is regulated by this Ca2+ concentration decrease. In the present work, we isolated a protein that binds to disk membranes at high Ca2+ concentrations. In the presence of this protein (a 26 kDa protein), PDE light sensitivity becomes high at high Ca2+ concentrations. The effect was observed at physiological ranges of Ca2+ concentrations. Thus we could explain high light-sensitivity of photoreceptors under the dark-adapted condition. According to its function, we termed the 26 kDa protein 'sensitivity-modulating protein' or 'S-modulin'. During the purification we noticed that there are additional mechanisms present that may contribute to light-adaptation in frog rod photoreceptors.     

Effects of Nematic Ordering on the Relaxation Spectrum of a Polymer Network with Included rod‐like Particles: Mean‐Field Approximation

Summary: Equilibrium and local dynamic properties of ordered polymer networks with included rod‐like particles are considered using a simplified network model. Lagrange multipliers in the equations of motion of rigid rods are replaced by their averaged values. This approximation corresponds to modelling rod‐like particles by elastic Gaussian springs with mean‐square lengths independent of the orientational order. Nematic‐like interactions between network segments and rods are taken into account in terms of the Maier‐Saupe mean‐field approximation. Nematic ordering of rods induces network segments ordering and changes the relaxation spectrum of the network. The relaxation spectrum of the ordered network splits into two main branches for parallel and perpendicular components of chain segments with respect to the director. Relaxation times of a polymer network are calculated as functions of the wave number for the corresponding normal mode and of the order parameter taking into account both the dynamic factor (determined by friction effects) and the statistical factor (related to mean‐square fluctuations of segment projections). We compare the relaxation spectra of ordered unstretched polymer networks with fixed boundaries with those for polymer networks at free boundaries. A polymer network with free boundaries is stretched along the director. This produces additional fine structure of the two main branches in the relaxation spectrum.

Cell of a three‐chain network model with included rods.     

PHOTORECEPTOR CELL TYPES IN THE RETINA OF VARIOUS VERTEBRATE SPECIES: IMMUNOCYTOCHEMISTRY WITH ANTIBODIES AGAINST RHODOPSIN AND IODOPSIN

Types of photoreceptor cells in the retinas of 36 species of vertebrates (5 classes, 14 orders) were investigated immunocytochemically with monoclonal antibodies against chicken iodopsin (Io-mAb) and antiserum against bovine rhodopsin (Rh-As). In mammals, Rh-As labeled the outer segments of some photoreceptor cells in striped squirrels (a diurnal mammal) and those of most photoreceptor cells in mice (a nocturnal mammal), while Io-mAb labeled any photoreceptor cells in either of them. In all species of birds studied, Io-mAb labeled the principal and accessory members of double cones and single cones with a red oil droplet. Rh-As labeled single cones with a yellow or clear oil droplet in addition to rods. In turtles, both Rh-As and Io-mAb labeled single cones with a red or clear oil droplet and the principal (with a yellow oil droplet) and accessory members of double cones. This suggests that the visual pigments in these cones of turtles have common epitopes with bovine rhodopsin and chicken iodopsin. In Japanese grass lizards, single cones with a yellow oil droplet and double cones were immunoreactive to both Rh-As and Io-mAb. In snakes, rods and cones could not be distinguished but both positively and negatively stained cells were observed by the use of each antibody. In geckos, however, all photoreceptor cells were immunonegative to Io-mAb. In all species studied in amphibians, Rh-As labeled rods but not cones. Neither rods nor cones reacted with Io-mAb. In fishes, almost all species studied had well developed cones, and some of these cones were labeled by Rh-As. However, Io-mAb labeled the outer segments of some cones only in loaches. Rh-As labeled photoreceptor cells in all species of fishes studied. Thus, Rh-As recognized the outer segments of rods in all species studied from fishes to mammals, whereas the epitope recognized by Io-mAb is conserved in some species of fishes, most species of reptiles and all species of birds studied.     

ORIENTATION OF RETINYLIDENE CHROMOPHORE OF HYPSORHODOPSIN IN FROG RETINA

Abstract— Hypsorhodopsin and bathorhodopsin were formed in the frog retina by irradiating rhodopsin at liquid He temperature (9 K) with orange light (> 520 nm) and blue light (437 nm), respectively. Hypsorhodopsin was converted to bathorhodopsin in the retina by warming above 32 K in the dark. Similar phenomena were observed in the rod outer segment suspension. A difference spectrum between hypsorhodopsin and bathorhodopsin in the retina produced by warming was almost identical with that in the rod outer segment suspension. This suggests that the transition dipole moment of hypsorhodopsin is parallel to the disk membrane plane which is also parallel to that of bathorhodopsin.     

Diffusion of spheres in crowded suspensions of rods

Translational tracer diffusion of spherical macromolecules in crowded suspensions of rodlike colloids is investigated. Experiments are done using several kinds of spherical tracers in fd-virus suspensions. A wide range of size ratios L/2a of the length L of the rods and the diameter 2a of the tracer sphere is covered by combining several experimental methods: fluorescence correlation spectroscopy for small tracer spheres, dynamic light scattering for intermediate sized spheres, and video microscopy for large spheres. Fluorescence correlation spectroscopy is shown to measure long-time diffusion only for relatively small tracer spheres. Scaling of diffusion coefficients with a/xi, predicted for static networks, is not found for our dynamical network of rods (with xi the mesh size of the network). Self-diffusion of tracer spheres in the dynamical network of freely suspended rods is thus fundamentally different as compared to cross-linked networks. A theory is developed for the rod-concentration dependence of the translational diffusion coefficient at low rod concentrations for freely suspended rods. The proposed theory is based on a variational solution of the appropriate Smoluchowski equation without hydrodynamic interactions. The theory can, in principle, be further developed to describe diffusion through dynamical networks at higher rod concentrations with the inclusion of hydrodynamic interactions. Quantitative agreement with the experiments is found for large tracer spheres, and qualitative agreement for smaller spheres. This is probably due to the increasing importance of hydrodynamic interactions as compared to direct interactions as the size of the tracer sphere decreases.     

Stepped strips from self-organization of oligo(p-phenylene) rods with lateral dendritic chains

We have demonstrated that the rod segments with lateral dendritic chains self-assemble into unique stepped strips in which the rods are aligned parallel to the strip long axis. This unique organization of the rod segments arises from a balance between the energetic gain of a parallel arrangement of the rods and the resulting entropic penalty associated with stretching of the lateral flexible chains.     

LIGHT INDUCED INTERACTION BETWEEN RHODOPSIN AND GTP DEPENDENT PROCESSES IN ROD OUTER SEGMENTS—I. KINETIC ANALYSES OF LIGHT SCATTERING TRANSIENTS

Abstract— A fast light-induced light scattering transient in the presence of Mg²⁺ and GTP is observed from bovine rod outer segment suspension. This transient is studied at various temperatures, degrees of photo-bleaching and GTP concentration. The kinetic analysis of this signal at low photo-bleaching (∼ 5%) reveals two different processes which are consecutive rather than simultaneous. The rate of both processes depends upon the extent of photo-bleaching and indicates that each process requires a photolyzed rhodopsin intermediate. At low temperature, the rate constant of both the processes increases with the extent of photo-bleaching while at higher temperatures it decreases. The calculated activation energy of both processes decreases with increasing photo-bleaching. It has also been observed that with decreasing GTP concentration, the rates of both processes decrease indicating that the two processes though first order are most likely bimolecular, first order in both GTP and the photolytic rhodopsin intermediate (metarhodopsin II)     

Synthesis and self-organization of rod–dendron and dendron–rod–dendron molecules

We synthesized molecules containing one or two dendritic segments and a rigid-rod-like segment with their structures in the solid state. The molecules with rod–dendron or dendron–rod–dendron architecture had biphenyl ester rigid segments and 3,4,5 tris(n-dodecyloxy)benzoate of first or second generation as their dendritic segments. The variables investigated included the rod segment length as well as dendron generation, and all materials obtained were characterized by optical microscopy, differential scanning calorimetry, and X-ray scattering. Depending on the size of the rod segment and generation number of the dendritic segment, molecules organized into smectic, columnar, or cubic phases, and the symmetries observed were dominated by the anisotropic rod–rod interactions. © 2003 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 41: 3501–3518, 2003     

Magnetic manipulation and optical imaging of an active plasmonic single-particle Fe-Au nanorod

Superparamagnetic microbeads play an important role in a number of scientific and biotechnology applications including single-molecule force measurements, affinity separation, and in vivo and in vitro diagnostics. Magneto-optically active nanorods composed of single-crystalline Au and polycrystalline Fe segments were synthesized with diameters of 60 or 295 nm using templated electrodeposition. The Fe section was magnetically soft and had a saturation magnetization of approximately 200 emu/g, resulting in a 10-fold increase in magnetization relative to that iron oxide nanoparticles. The strong plasmonic response of the Au segment of the rod in both the longitudinal and transverse directions made it possible to detect the orientation of a single rod in a polarized light microscope with nanometer resolution. These nanorods provide significantly improved physical properties over iron oxide superparamagnetic beads, making it possible to simultaneously manipulate and monitor the orientation of biomolecules with well-defined forces at the nanometer scale.     

Flow properties of freshly prepared ettringite suspensions in water at 25 degrees C

The rheology of a complex, heterogeneous mineral colloid was rationalised using models devised for model rod systems. Mixing a calcium hydroxide slurry with an aluminium sulphate solution produces a suspension of rod-shaped ettringite particles. Ettringite rod suspensions exhibit non-Newtonian flow behaviour, which depends on the shape of the particles, their size distribution, concentration and surface properties as well as the suspension medium characteristics. We have measured the shear viscosity of suspensions of ettringite rods with a median aspect ratio, r(i) approximately 8, at 25 degrees C as a function of particle volume fraction, phi, in the range 0.0001-0.08. It was found that the viscosity of the suspensions increased with phi, and showed a marked change of slope at phi approximately 0.01, which we identified as the minimum overlap concentration phi(*). Above phi(*), the system is in the semi-dilute regime. At phi>phi(*), when Pe(rot)>1, hydrodynamic interactions between rods become increasingly significant, and we observe shear-thinning behaviour. The high effective hydrodynamic volume of rotating rods, resulting in much lower values of the maximum packing fraction, phi(c), than for spheres, dominates the rheological behaviour of ettringite suspensions.