Exposure of rod outer segments to serum is not responsible for abnormal disk membrane morphogenesis in a model of retinal detachment.

PURPOSE: The sclerad surface of the retina is exposed to serum proteins in several retinal pathologies that result in degeneration of photoreceptor outer segments. Abnormal disk membrane morphogenesis is observed in rod photoreceptors of detached Xenopus retinas when they are cultured in serum-containing medium. Retinas that remain attached to the pigment epithelium layer produce normal disks. Experiments were conducted to determine whether abnormal disk morphogenesis in detached, cultured retinas is due to the presence of serum in the microenvironment of the rod outer segments. METHODS: Detached retinas and retinas attached to the retinal pigment epithelium in eyecups were cultured in either serum-containing or serum-free medium, and the morphology of the disk lamellae formed in vitro retinas was evaluated. Using protein extraction and immunochemical methods, the presence of albumin in the microenvironment of the outer segments was confirmed for retinas incubated in serum-containing medium. RESULTS: There were no obvious differences in the abnormal disk-like lamellae formed in detached retinas when the retinas were incubated either in serum-containing or in serum-free culture medium. Proteins extracted from detached retinas cultured in serum-containing medium showed a prominent band at 63 kDa that co-localized primarily with outer segment-enriched fractions. Immunolabeling showed that the band was serum albumin. CONCLUSIONS: Rod cells in detached retinas formed abnormal disk-like lamellar membranes in either serum-containing or serum-free medium. This suggests that exposing outer segments to serum albumin or other serum components is not responsible for the abnormal in vitro disk membrane morphogenesis seen in detached retinas.

Disk membrane initiation and insertion are not required for axial disk displacement in Xenopus laevis rod outer segments.

PURPOSE: Mechanisms that maintain the close coupling between the formation of photoreceptor disk membranes and the displacement of disk membranes toward the pigment epithelium are poorly understood. This study was designed to determine whether the axial displacement of disk membranes requires the assembly and insertion of new disk lamellae. METHODS: Retinal detachment and treatment with cytochalasin D were employed to interrupt the normal formation of disk membranes in cultured Xenopus laevis retinas. The effect of disrupting disk initiation and assembly upon disk displacement was documented and quantified. RESULTS: Isolating retinas from the retinal pigment epithelium prevented the normal morphogenesis of disks, but previously formed disks moved distally at a rate that is greater than or equal to the rate in attached retinas or in vivo. Treatment of attached retinas in eyecups with cytochalasin D blocked initiation of new disks and resulted in the formation of ectopic, disk-like membranes, but it did not stop axial displacement of previously formed disks. Rod cells in retinas that were cultured while slightly elevated from the retinal pigment epithelium sometimes formed disks of a smaller diameter than normal, even though the rate of initiation and displacement of disks was the same as in vivo. CONCLUSIONS: Observations on detached retinas and or retinas treated with cytochalasin D suggest that disk displacement does not depend upon normal disk formation and that the motive mechanism does not involve filamentous actin. The formation of small diameter disks in elevated retinas suggests that disk initiation and displacement is independent of the completion of normal diameter disks.

Characterization of a truncated form of arrestin isolated from bovine rod outer segments.

The inactivation of photolyzed rhodopsin requires phosphorylation of the receptor and binding of a 48-kDa regulatory protein, arrestin. By binding to phosphorylated photolyzed rhodopsin, arrestin inhibits G protein (Gt) activation and blocks premature dephosphorylation, thereby preventing the reentry of photolyzed rhodopsin into the phototransduction pathway. In this study, we isolated a 44-kDa form of arrestin, called p44, from fresh bovine rod outer segments and characterized its structure and function. A partial primary structure of p44 was established by a combination of mass spectrometry and automated Edman degradation of proteolytic peptides. The amino acid sequence was found to be identical with arrestin, except that the C-terminal 35 residues (positions 370-404) are replaced by a single alanine. p44 appeared to be generated by alternative mRNA splicing, because intron 15 interrupts within the nucleotide codon for 369Ser in the arrestin gene. Functionally, p44 binds avidly to photolyzed or phosphorylated and photolyzed rhodopsin. As a consequence of its relatively high affinity for bleached rhodopsin, p44 blocks Gt activation. The binding characteristics of p44 set it apart from tryptic forms of arrestin (truncated at the N- and C-termini), which require phosphorylation of rhodopsin for tight binding. We propose that p44 is a novel splice variant of arrestin that could be involved in the regulation of Gt activation.

Mechanism of rhodopsin kinase activation.

The role of the cytoplasmic loops and C-terminal region of bovine rhodopsin (Rho) in binding and activating rhodopsin kinase was investigated. The ability of various enzymatically truncated forms of photolyzed rhodopsin (Rho*) to stimulate rhodopsin kinase activity was quantified. Following endopeptidase Asp-N cleavage of all phosphorylation sites on the C-terminal, the resulting truncated Rho* (329G-Rho*) was not phosphorylated by rhodopsin kinase. This suggests that rhodopsin kinase only phosphorylates C-terminal sites of Rho*. However 329G-Rho* could bind rhodopsin kinase and stimulate phosphorylation of exogenous peptide. Kinase stimulation was investigated for other truncated forms of Rho* in which the C-terminal region was either partially or completely eliminated, and the V-VI loop was either cleaved or left intact (339K-Rho*, 341E239E-Rho*, 329G239E-Rho*, 327P240S-Rho*). Results suggest that the V-VI loop is crucial for kinase binding (similar to the binding of GT). Mastoparan, a model peptide for G-protein-coupled receptors, was found to stimulate rhodopsin kinase in a mechanism similar to that of truncated Rho*. We conclude that rhodopsin kinase binds to the cytoplasmic loops of Rho* to cause a stimulation of its catalytic activity.

Retinal detachment prevents normal assembly of disk membranes in vitro.

The effects of retinal detachment upon disk membrane assembly in rod outer segments were assessed in Xenopus laevis retinas that had been maintained in eyecup cultures for up to 4 days. In these cultures, assembly of disk membranes occurred at a normal rate in regions of the retina that remained attached to the retinal pigment epithelium. In regions of the retina that were detached from the pigment epithelium, the assembly of new disk membranes either was abnormal or was inhibited. This result cannot be attributed to reduced access of cells in the detached retina to oxygen and metabolites. The experiments described here suggest that the apposition of the retina with the pigment epithelium is a necessary condition for normal disk membrane assembly in Xenopus retinas. This effect may be mediated by contact between the rod outer segments and the pigment epithelium, or by trophic factors in the subretinal space.

Temperature-dependent birefringence patterns in Xenopus rod outer segments.

Results reported here show that the birefringence of disk membranes in Xenopus rod photoreceptors depends upon the temperature at which the disks were assembled. Portions of the outer segments produced by the assembly of new disks while frogs are exposed to constant darkness and an ambient temperature of 25 degrees C have a higher birefringence than portions assembled at 18 degrees C. The intensity of birefringence reflects molecular-level structure and macromolecular-level organization of the disk membranes. It has been shown previously that increasing the ambient temperature enhances the rate of assembly of disk membranes in Xenopus rod outer segments. It also has been shown that disk membranes are assembled most frequently in the hours following light onset, and that disks assembled in the light have a lower birefringence than disks assembled in the dark. Therefore, although light and increased temperature both enhance the rate of disk membrane assembly, they have opposite effects upon the birefringence of Xenopus rod outer segments.

Axial diffusion of retinol in isolated frog rod outer segments following substantial bleaches of visual pigment.

Partial bleaches of rhodopsin were made in either the proximal or distal halves of isolated Rana pipiens rod outer segments. The fluorescence of all-trans retinol was recorded 15, 60 and 120 min following 17% and 63% bleaches. Some of the retinol that formed remained immobilized in the bleached halves of the outer segments, while another portion was slowly mobilized and diffused along the cell axis.

Lengths of immunolabeled ciliary microtubules in frog photoreceptor outer segments.

Monoclonal anti-tubulin antibodies were used to label microtubules in the connecting cilia and outer segments of retinal photoreceptors isolated from Rana pipiens. In paraformaldehyde-fixed rods from frogs maintained on diurnal light cycles, the anti-tubulin labeling of ciliary microtubules (mean length = 27 micron) typically extends to slightly over half the length of the outer segments (mean length = 46 micron). Rod outer segments from frogs kept in constant darkness for 3-4 weeks are longer (mean length = 53 micron) than rod outer segments from frogs maintained in cyclic lighting. However, the distribution of fractional lengths of anti-tubulin labeling of ciliary microtubules is the same for both lighting regimens. Incubating retinas in 1.0 mM colchicine prior to outer-segment fixation has no effect on the length of immunolabeling of ciliary microtubules, suggesting that post-mortem elongation artifacts are not significant. Incubating retinas in 10 microM taxol prior to fixation significantly increases the length of stained ciliary microtubules, suggesting that taxol either promotes post-mortem assembly of microtubules, or that taxol reduces post-mortem disassembly. The mean position of the end of anti-tubulin-labeled ciliary microtubules does not correspond to the position of disk shedding for any of the experimental conditions employed.

Distribution and axial diffusion of retinol in bleached rod outer segments of frogs (Rana pipiens).

Isolated retinas and rod outer segments from frogs (Rana pipiens) were exposed to light that produced axially uniform total bleaches of rhodopsin. Using fluorescence video microscopy, it was shown that the formation and equilibrium distribution of all-trans-retinol, the final chromophore product of rhodopsin bleaching is axially uniform. This result shows that the rate and amount of oxidoreductase-mediated reduction of all-trans-retinal to all-trans-retinol is not affected by the relative age of the disk membranes to which the enzymes are bound. Therefore previously reported axial differences in regeneration of rhodopsin and recovery of photocurrent after exposure to bright light probably are not due to axial differences in the formation of rhodopsin photoproducts. In addition, measurements on individual rod cells show that there is no significant redistribution of retinol for up to 2 hr following localized partial bleaches of rhodopsin. This raises the perplexing question of how retinol is shuttled between disk membranes and the pigment epithelium during visual pigment regeneration following substantial bleaches.

Rhodopsin lateral diffusion as a function of rod outer segment disk membrane axial position.

Rhodopsin lateral diffusion was measured at three points along the axis of frog outer segments using the method of absorbance recovery after photobleaching. Mean recovery times were slightly longer in distal disk membranes than in proximal disks. A small reduction of pigment mobility with disk age may reflect subtle changes in membrane composition.

Shedding is correlated with disk membrane axial position rather than disk age in Xenopus laevis rod outer segments.

Disk membrane synthesis and displacement rates in Xenopus laevis retinal rod outer segments, as measured by light-dark dependent birefringence band periods, are proportional to incubation temperature. Outer segment length, however, is approximately the same for frogs raised at different temperatures. Therefore the age of disks shed from outer segment distal tips is a function of temperature. Unless the rate of disk synthesis coincidentally has the same temperature dependence as putative disk membrane aging processes, this implies that position on the outer segment axis rather than disk age is a sufficient condition for disk membrane shedding in Xenopus laevis maintained under diurnal light-dark conditions.

Modeling the rod outer segment birefringence change correlated with metarhodopsin II formation.

A rapid birefringence loss associated with metarhodopsin II formation, delta (delta n) MII, is produced when frog rod outer segments are exposed to a bleaching light flash. To analyze the nature of the underlying structure change, measurements of delta (delta n) MII were made in rod outer segments perfused with glycerol solutions to increase the refractive index of the cytoplasmic and intradisk spaces. Comparisons of experimental results with computed changes in the form birefringence component using two- and three-dielectric outer segment models for several putative structure changes were made. It is concluded that delta (delta n) MII can be due to either a change in the intrinsic birefringence component caused by the reorientation of anisotropic molecules, or to a change in the form birefringence component caused by small changes in the cytoplasmic and/or intradisk volumes.

Concurrent birefringence and forward light-scattering measurements of flash-bleached rod outer segments.

A microretardometer--nephelometer was constructed to measure birefringence and forward-direction light scattering concurrently in single retinal rod outer segments (ROS). The relative contributions of light-induced birefringence and light-scattering changes to observed far-red transmission changes measured normal to the cell axis were evaluated in isolated, whole, flash-bleached ROS. No light-scattering transients were found corresponding to the previously reported rapid birefringence loss associated with the metarhodopsin I lead to metarhodopsin II reaction even under conditions in which such a light-scattering change has been reported for outer-segment fragments.

Light cycle--dependent axial variations in frog rod outer segment structure.

Polarized light microscopy reveals that the structural parameters of Rana pipiens rod outer segments (ROS) are not uniform along the cell axis. In addition to a pronounced birefringence (delta n) gradient found in the basal half of most ROS, periodic delta n bands are seen in approximately 10% of intact ROS isolated by agitating retinas in frog Ringer's solution. These small delta n differences appear as very faint light and dark striations that have a period and width that depends on the duration of light and dark exposure. In ROS from frogs kept on a 14 hr light/10 hr dark cycle at 20 degrees to 22.5 degrees C, the band period for a light-dark band pair is 1.0 to 1.6 micron. Portions of ROS produced during total darkness or constant light are free of distinct periodic bands. Quantitative delta n measurements show that the ROS sections generated in the dark have a relatively higher delta n than those produced in light. Band contrast is irreversibly enhanced when ROS are treated with the calcium ionophore A23187 in the presence of calcium-free saline solution. These results indicate that the synthesis of some calcium-sensitive ROS component is different when the frog is in the dark than when exposed to light.

Structural interpretation of the birefringence gradient in retinal rod outer segments.

The birefringence of frog retinal rod outer segments is analyzed in terms of a three-dielectric layer model. The possibility that the birefringence gradient found in such cells is due to changes in the disk membrane-pair spacing is investigated using previously published glycerol imbibition data (Kaplan et al., 1978. Biophys. J. 23: 59-70). The higher net birefringence of the basal end compared to the midpoint of rod outer segments can be accounted for by a smaller negative form birefringence term due to either a smaller or larger intradiskal space, depending upon the assumed relative solids contents of the intradiskal and cytoplasmic spaces.

Birefringence measurements of structural inhomogeneities in Rana pipiens rod outer segments.

The birefringence (deltan) of Rana pipiens rod outer segments (ROS) reveals microstructure inhomogeneities not seen with other techniques. In the basal 20-30-micron length of the ROS there is a nearly linear axial gradient in deltan of approximately equal to -2 x 10(-5)/micron. No consistent deltan gradients are found in the distal 20-30 micron of the ROS. Using glycerol imbibition to separate the intrinsic and form birefringence components, we found that the basal deltan gradient was principally due to a gradient of the intrinsic birefringence component. The disk membrane volume fraction decreases uniformly from the basal end to the distal end, while the disk membrane refractive index increases. The contributions of these changes to the form birefringence approximately cancel, so that the form component is fairly uniform along the ROS axis. Because its axial distance from the inner segment is a measure of the time since a disk membrane was formed, these gradients may reflect a disk membrane aging process. Occasionally a highly birefringent, 2-micron-wide band is seen at the basal end ot the ROS, possibly where the envelope membrane folds to form new disk membranes.

Slow bleach-induced birefringence changes in rod outer segments.

1. Time resolved birefringence measurements reveal complex structural changes in rod outer segments following a bleaching flash. 2. The detailed character of the observed changes depends upon osmotic integrity of the outer segment envelope membrane. Rod outer segments with intact envelope membranes show a fast initial loss of intrinsic birefringence simultaneous with the formation of metarhodopsin II followed by a slower secondary loss. A subsequent birefringence increase to higher than the dark-adapted level is partially correlated with the formation of retinol. Rod outer segments with ruptured envelope membranes show the initial and secondary losses, but the subsequent recovery only reaches the dark-adapted level. Retinol does not form in such organelles. 3. The slow birefringence changes in intact rod outer segments were qualitatively different when Na+ salts were replaced by equimolar K+ salts in the bathing medium. 4. Glycerol appears to influence both the rate and magnitude of metarhodopsin III formation, and of the spectrally silent secondary birefringence loss.

Modification of optical responses associated with the action potential of lobster giant axons.

The sources of optical retardation changes and light scattering changes occurring during the action potential propagation of lobster giant axons have been investigated. A technique has been developed for resolving the total transmitted-light intensity change into a retardation change component, dI-r, and a forward direction light scattering change, dI-s. Trypsin, pronase, neuraminidase and hyaluronidase all reduce the magnitude of dI-r without diminishing the action potential, probably by cleaving charged saccharides. Dithiothreitol has no effect. This suggests that glycoproteins and hyaluronic acid polymers at the surface of the axon are involved in the optical responses, either by being passively realigned or by contributing to compression and expansion forces as the membrane electric field changes. Large dI-s responses are generated by trypsin and pronase treatment. The modifying effects of these proteases may be due to modification of the membrane or to increases in the refractive index of the medium surrounding the axon, since similar large dI-s, responses are produced by increasing the refractive index with sucrose. Since large reductions in dIr can be produced without concurrent reductions in the action potential, a significant portion of the optical retardation responses cannot be attributable to structural changes that are causally related to membrane ionic permeability changes during the action potential.