Hepatocyte growth factor/scatter factor stimulates Ca2+-activated membrane K+ current and migration of MDCK II cells.

Hepatocyte growth factor/scatter factor (HGF/SF) stimulates migration of various cells and has been linked via Met tyrosine kinase-signaling to transformation and the metastatic phenotype. Migration of transformed MDCK-F cells depends on activation of a charybdotoxin-sensitive, volume-activated membrane K+ current. Thus, we used patch-clamp electrophysiology and transwell migration assays to determine whether HGF/SF stimulation of MDCK II cell migration depends on the activation of membrane K+ currents. HGF/SF activated a membrane K+ current that increased over 24 hr, and which could be modulated by increasing intracellular calcium concentration, [Ca2+]i. Charybdotoxin (ChTX, 50 nM), iberiotoxin (IbTX, 100 nM), stichodactyla toxin (Stk, 100 nM) and clotrimazole (CLT, 1 mM) all inhibited this current. HGF/SF (100 scatter units/ml) significantly increased MDCK II cell migration over 8 hr compared to control cells. Addition of ChTX (50 nM), IbTX (100 nM), Stk (100 nM) or CLT (1 microM) inhibited the HGF/SF-stimulated MDCK II cell migration. We conclude that the activation of membrane Ca2+-activated K+current is necessary for HGF/SF stimulation of MDCK II cell.

Long hours and little sleep: work schedules of residents in obstetrics and gynecology.

OBJECTIVE: To investigate residents' work schedules and their attitudes toward limiting their hours. METHODS: An anonymous survey regarding resident work hours and call schedules was administered to the 4674 obstetric-gynecologic residents who took the year 2000 Council on Resident Education in Obstetrics and Gynecology in-training examination. RESULTS: A total of 4510 surveys were analyzed (96.5%). Three of four (75.5%) respondents reported working between 61 and 100 hours each week. Most (71.3%) reported sleeping less than 3 hours while on night call. Eight of ten reported having postcall clinical responsibilities. The reported number of hours on call declined and the reported number of hours of sleep increased with year of residency. Three of four residents wanted limits on their work hours. Residents who reported longer on-call hours or less sleep during night shift were significantly more likely to want a restriction on work hours. Fatigue was the most commonly selected reason (77.6%) followed by "need more personal time" (76.3%), and "fear of compromising quality of care" (59.8%). Women were more concerned about fatigue than were men. Among residents who did not want work hour restrictions, "additional surgical experience" was the most commonly selected reason (69.0%). CONCLUSION: Residents in obstetrics and gynecology report working long hours, and experiencing periods of little sleep. Most want their work hours to be limited. Fatigue is a major concern among residents that want their hours limited. A sizable minority worries that such limits might also limit their experience.

Survival of the retinal pigment epithelium in vitro: comparison of freshly isolated and subcultured cells.

Cells of the retinal pigment epithelium (RPE) are generated prenatally and generally survive the lifetime of the individual without undergoing proliferation or replacement. Therefore, the mechanisms promoting individual RPE cell survival and longevity in vivo may be distinct from, or a limited subset of, the mechanisms known to promote survival in proliferative cells in culture. To identify specific factors that sustain cell viability independent of effects on cell division, we studied RPE cells in low-density suspension culture, in which cell proliferation is inhibited. Single cells from Xenopus laevis eyes were plated onto a non-adhesive surface in protein-free medium, then assayed for survival using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cell viability in these cultures was essentially undiminished over the initial 2 days. However, by approximately 1 week in culture, only an average of 53% of the cells remained alive. Plating cells on a fibronectin-coated substratum significantly enhanced survival, such that the number of cells alive at 1 week was 80-90% of the initial level. Essentially identical results were obtained with laminin- or collagen IV-coated substrata, or with insulin (5 microg ml(-1)) in the medium. The absence of cell division in these cultures was confirmed by cell counting and BrdU incorporation experiments. Interestingly, in suspension cultures derived from monolayers previously established on microporous membrane filters, cells lost viability much faster (average of 80% dead at 3 days), and showed a relatively greater response to extracellular matrix proteins (five-fold increase in cell survival at 3 days). Enhanced RPE survival in response to fibronectin required spreading of the cell on a substratum, rather than mere adherence, as there was a high correlation between the percentage of spread cells and the percentage that were MTT-positive (r=0.940). Cell spreading apparently enhanced survival by preventing the initiation of programmed cell death: unattached non-viable cells in culture exhibited morphological features expected of apoptosis, as well as positive staining by the TUNEL reaction. These studies demonstrate that, of several factors shown to maintain or increase cell number in proliferating cultures, some have their effect, at least in part, by promoting the survival of individual cells. The increased susceptibility of subcultured RPE to cell death has implications for clinical transplantation applications that may require manipulation of RPE in vitro.

Melatonin receptor RNA expression in Xenopus retina.

Melatonin is an indolamine hormone presumably synthesized by retinal photoreceptors, and may act as a paracrine signal of darkness within the retina. Previous studies have suggested that melatonin, acting through specific receptors, may be involved in cyclic retinal functions such as photoreceptor outer segment disc shedding and phagocytosis, and modulation of neurotransmitter release in the inner retina. The goal of this study was to determine if melatonin receptor mRNA is expressed in the neural retina and retinal pigment epithelium (RPE) of Xenopus laevis. Sheets of RPE, devoid of contaminating cells, were obtained from Xenopus eyes, and epithelial cultures were subsequently established on microporous membrane filters in a defined medium. Total RNA was isolated from whole brain, neural retina, fresh RPE sheets, and cultured RPE cells. RNA expression of the three known Xenopus melatonin receptor subtypes (MEL1A, 1B, and 1C) was determined by reverse-transcription/polymerase chain reaction (RT/PCR) amplification, followed by Southern hybridization with RNA probes. PCR-amplified cDNA encoding melatonin receptor subtypes 1B and 1C, but not 1A, were detected in reverse-transcribed RNA obtained from brain, neural retina and RPE. RPE cells grown in culture for two weeks also demonstrated 1B and 1C receptor RNA expression. This study suggests that RNA encoding the 1B and 1C melatonin receptor subtypes is expressed in the neural retina and RPE of Xenopus retina, and the expression persists in RPE cells when grown in culture. The expression of melatonin receptor RNA in the RPE may reflect a regulatory role for melatonin in some diurnal events that occur in this tissue, such as phagocytosis of photoreceptor outer segment membranes, and intracellular migration of pigment granules.

Serum inhibits tight junction formation in cultured pigment epithelial cells.

PURPOSE: These experiments were designed to characterize tight junction formation by retinal pigment epithelial (RPE) cells in vitro and to compare the effects on this process of hormonally defined medium (HDM) and serum-containing medium. METHODS: Formation of RPE tight junctions was analyzed in freshly isolated rat RPE cells maintained either in HDM or serum-containing medium. Junctions were evaluated functionally by measuring transepithelial electrical resistance (TER) and permeability and structurally by immunolocalization of the junction-associated actin microfilaments. Calcium dependency of the junctions was determined by reducing media calcium concentration. RESULTS: RPE cells cultured in serum-free HDM developed calcium-dependent tight junctions, which exhibited TER levels > 150 omega cm2 and low paracellular permeability. Serum-containing media inhibited tight junction formation as indicated by significant reductions in TER and increases in permeability. Junction-associated actin microfilaments and cell density were unchanged. CONCLUSIONS: Tight junction formation by RPE cells is inhibited by serum. This activity may play an important role in responses of the RPE layer to injury, contributing to the pathologic progression of blood-retinal barrier dysfunction.

Evidence for beta 1-integrins on both apical and basal surfaces of Xenopus retinal pigment epithelium.

The retinal pigment epithelium (RPE) is a transporting epithelium with polarized membrane domains. A unique characteristic of these cells is that their apical surface does not face a lumenal space, but is directly apposed to a layer of neurons (photoreceptors) and their associated extracellular matrix. Because the interaction occurring at this site is important for retinal attachment and particle phagocytosis, an attempt was made to identify epithelial molecules which potentially could mediate cell-cell or cell-matrix adhesion. In the present report, the subcellular localization of beta 1-integrins, the main receptors for extracellular matrix ligands, has been examined within Xenopus RPE. Several previously characterized antibodies were used in this analysis including: two rabbit polyclonal antibodies directed against purified chick muscle fibronectin receptor (pAbs No. 3818 and No. 2999), and a monoclonal antibody specific for Xenopus beta 1-integrin subunit (mAb 8C8). In Western blots of whole epithelial cell extracts, each of the antibodies intensely labeled a 115 kDa band, consistent with beta 1-integrin reactivity. One of the reagents (pAb No. 3818) also weakly stained unidentified bands of 50 and 100 kDa. Pre-clearing experiments demonstrated that pAb No. 3818 and mAb 8C8 both recognize the same detergent-soluble integrin: when cell extracts were depleted of beta 1-integrin by immunoprecipitation with mAb 8C8, the 115 kDa antigen recognized by pAb No. 3818 was not observed. Consistent with their similar immunochemical reactivities, each of the antibodies produced equivalent immunocytochemical staining of many eyecup tissues, including extraocular skeletal muscle cells, scleral and choroidal fibroblasts and vascular endothelium of the choroid and neural retina. In the native RPE, and isolated sheets of epithelium, however, qualitative differences in labeling between these antibodies were evident. Analysis by confocal microscopy showed that, while all three antibodies stained the basal surface of the epithelium, pAb No. 3818 also strongly labeled the apical microvillar surface. As the adjacent photoreceptors did not cross-react with this antibody in control experiments, the apical RPE staining could not be accounted for as contamination with retinal tissues during isolation. Furthermore, when the apical cell surface was selectively biotinylated in situ, and biotinylated proteins precipitated by streptavidin-agarose, beta 1-integrin was detected by immunoblotting with both mAb 8C8 and pAb No. 3818. This domain-specific material, however, represented only a fraction of the whole cell surface integrin: substantially greater amounts of tagged molecules could be detected when isolated epithelial sheets were biotinylated, most likely representing the basal protein. Based on these results, it can be concluded that beta 1-integrin is present in both basal and apical RPE plasma membranes. Molecules present in the apical membrane may represent components of adhesion receptors responsible for retina-epithelium interactions.

Retinal pigment epithelial cells from dystrophic rats form normal tight junctions in vitro.

PURPOSE: In the genetically defective Royal College of Surgeons (RCS) rat model for retinal degeneration, a breakdown occurs in the retinal pigment epithelial (RPE) cell tight junctions just as the photoreceptors begin to degenerate. These experiments sought to determine the impact of the RPE genetic defect on this alteration in the RPE cell tight junctions. METHODS: Retinal pigment epithelial cell cultures prepared from RCS and control rats were treated with hormonally defined medium (HDM), base medium conditioned by RCS or control retinas, or unconditioned base medium. The tight junctions formed by these cultures were assayed functionally by measuring transepithelial electrical resistance and permeability. Junction structure was evaluated by immunolocalization of the tight junction protein zonula occludens 1 and of the junction-associated actin microfilaments. RESULTS: Retinal pigment epithelial cultures from dystrophic rats formed structurally and functionally normal tight junctions when maintained in hormonally defined medium. The junctions remained stable when the medium bathing the apical surface was switched to base medium preconditioned by normal retinas. In contrast, cultures treated with medium preconditioned by degenerating dystrophic retinas or with unconditioned medium exhibited a breakdown in their tight junctions. CONCLUSIONS: Retinal pigment epithelial cells isolated from dystrophic RCS rats can form tight junctions normally in vitro. Normal, but not dystrophic, retinas release factors that support RPE tight junctions. Therefore, the junctional abnormality seen in dystrophic rat RPE cells in vivo is probably caused by the loss of trophic factors normally provided by the healthy neural retina rather than by a direct effect of the genetic defect on the tight junctions.

Autoradiographic and biochemical assessment of rod outer segment renewal in the vitiligo (C57BL/6-mivit/mivit) mouse model of retinal degeneration.

Rod outer segment renewal was assessed in vitiligo (C57BL/6-mivit/mivit) mice using autoradiographic and biochemical methods. This process was examined because the number of phagosomes is reduced in this mouse. Rod outer segment renewal was detectable in the mivit/mivit retina. Within 24 hr of intraperitoneal injection of 3H leucine, there was a distinct band of radioactivity present at the junction of the ROS and RIS in mutant mice that was similar to controls. The displacement of the radioactive band progressed normally in the peripheral regions of the vitiligo retina, but did not in the posterior retina. Morphometric analysis of the posterior region of mutant retinas, indicated that the band of radioactivity became less distinct between 1 and 3 days post-injection. In vitiligo retinas it remained at 2.23 microns, whereas in controls, the band migrated 6.16 microns from the ROS base. When posterior regions of retinas were evaluated 8 days post-injection, there was no band discernible in the vitiligo retinas, but a very dense band at the ROS apex in controls. Assessment of incorporation of radioactivity into rhodopsin using SDS-PAGE indicated a progressive displacement of radio-labeled rhodopsin through the RER, but not as complete a progression through the outer segments. The elongation of the outer segments in the posterior regions of the mutant retina suggests impaired shedding. This, plus the lack of attachment in the posterior retina of photoreceptor cells to RPE in this mouse, seem to be likely causes for the decreased number of phagosomes.

Membrane polarity of the Na(+)-K+ pump in primary cultures of Xenopus retinal pigment epithelium.

The retinal pigment epithelium is a transporting epithelium that helps regulate the volume and composition of the subretinal space surrounding photoreceptor outer segments. The capacity of the RPE to actively transport Na+ and K+ between the retina and the blood supply depends on the localization of the Na+, K(+)-ATPase to the apical membrane, but in culture this polar distribution can be lost. Using primary cultures of Xenopus RPE, we examined the anatomical and functional polarity of this electrogenic pump. Confluent monolayers were established on Matrigel-coated microporous filters and cultured for 2-4 weeks in serum-free defined medium. Electrogenic pump activity at the apical and basolateral membranes was assayed by mounting the monolayer and filter in an Ussing chamber and exposing one or the other surface to ouabain while recording the apical (Vap) and basolateral (Vba) membrane potentials with an intracellular microelectrode. The addition of 0.2 mM ouabain to the apical bath caused Vap to rapidly depolarize by about 4 mV, consistent with the inhibition of a hyperpolarizing pump current at that membrane. When ouabain was added to the basal bath, however, it had no effect on Vba, suggesting the absence of a functional Na(+)-K+ pump on the basolateral membrane. To confirm these electrophysiological results, we examined the distribution of the Na+, K(+)-ATPase catalytic component using an antiserum specific for the bovine kidney alpha subunit. Antibody labeling of cultures was highly polarized, with strong reaction present on the apical microvilli, but not the basolateral cell surfaces. The findings of this study indicate that the Na(+)-K+ pump in monolayers of Xenopus RPE, as in native RPE, is located mainly in the apical membrane, providing evidence of a functionally intact transport pathway in these primary cultures.

Reduction of phagosomes in the vitiligo (C57BL/6-mivit/mivit) mouse model of retinal degeneration.

PURPOSE: The vitiligo (C57BL/6-mivit/mivit) mouse has a slowly progressing retinal degeneration, in which photoreceptor cell nuclei are gradually lost and the retinal pigment epithelium (RPE) is unevenly pigmented. The purpose of the present study was to assess the phagocytic ability of the RPE in the vitiligo mouse by determining whether and when a phagocytic burst occurs in affected mice and whether the number of phagosomes varies between control and affected animals. METHODS: Eyes of control and vitiligo mice 4 to 20 weeks of age were embedded in Spurr. Thin sections were cut and examined by electron microscopy to confirm the presence of phagosomes, particularly in the affected animals. Thick (1 micron) sections were cut, and quantitative morphometry was performed at the light microscope level. The length of RPE was determined, and phagosomes were counted in RPE cytoplasmic and microvillous areas. Data were expressed as phagosomes per 1000 microns. RESULTS: The vitiligo mouse has a peak phagocytic episode approximately 2 hours after light onset. The number of phagosomes in 4-week-old affected mice was significantly less than that in controls (13 phagosomes per 1000 microns compared to 30 phagosomes per 1000 microns). By week 8, the number was reduced to approximately 5 per 1000 microns. Phagosome number was not reduced further between weeks 8 and 20 in the affected animal. Macrophage-like cells containing pigment granules and phagosomes were observed in the subretinal space in areas where the rod outer segments had been separated from the RPE. CONCLUSIONS: The vitiligo mouse RPE contains phagosomes, but there are significantly fewer than in controls. It is not known whether a defect in RPE phagocytosis is the direct cause of the retinal defect in this model.

Reattachment of retinas to cultured pigment epithelial monolayers from Xenopus laevis.

PURPOSE: The authors aimed to establish an in vitro model of the retinal pigment epithelium (RPE) suitable for studies of neural retina-epithelium interactions. METHODS: Sheets of RPE were obtained from Xenopus laevis eyes by Dispase treatment of intact globes. After dispersal in trypsin-EDTA solution, cells were plated onto Matrigel-coated microporous membrane filters and grown in a serum-free defined medium. RESULTS: Confluent cell monolayers obtained after 7 to 10 days of culture appeared by electron microscopy to be morphologically polarized, established junctional complexes, and exhibited transepithelial resistances ranging from 300 to 500 omega.cm2. As did the native epithelium, these cells formed cytoskeletons consisting of circumferential bands of actin myofilaments at their periphery and whorls of cytokeratin intermediate filaments throughout the cytoplasm. Co-culture of freshly isolated neural retinas with monolayers resulted in the apparent reattachment of photoreceptors to the RPE within 3 hours. CONCLUSIONS: Primary cultures of amphibian RPE that express phenotypic characteristics of the epithelium in situ are also capable of establishing adhesive interactions with isolated neural retinas.

Reconstitution of the photoreceptor-pigment epithelium interface: L-glutamate stimulation of adhesive interactions and rod disc shedding after recombination of dissociated Xenopus laevis eyecups.

In order to investigate adhesive interactions between photoreceptor and pigment epithelial cells, we have mechanically separated neural retinas from Xenopus laevis eyecups and then recombined the tissues in vitro. When tissue pairs are incubated in a defined medium, cell-cell contact is achieved within 3 hr. However, the average proportion of reassembled eyecups in which photoreceptor outer segments interdigitate with epithelial microvilli is limited. Furthermore, rod disc shedding does not take place in these cultures, even following a dark to light transition. When recombined tissues are placed in medium supplemented with 12 mM L-glutamate, retinal reattachment is enhanced and there is a four-fold increase in epithelial phagosome content. The positive effect of excitatory amino acid exposure on shedding, however, is restricted to regions where visual and epithelial cells interdigitate. These results indicate that re-establishment of cell contact may be necessary for shedding of apical disc membranes prior to their engulfment by the epithelium. While reattachment is not affected by pre-incubation of separated tissues in normal medium, rod photoreceptors fail to undergo membrane turnover in response to L-glutamate if a delay of 1 hr or more is interposed between isolation of the retina and its recombination with the pigment epithelium. This is probably due to a decline in retinal function in culture, since a similar preincubation of the pigment epithelium prior to reassembly with a freshly isolated retina does not inhibit the shedding response.(ABSTRACT TRUNCATED AT 250 WORDS)

An improved method for isolation and culture of pigment epithelial cells from rat retina.

An improved method for isolation and culture of pigment epithelial cells from normal rat retinas is described. Following a brief incubation in the neutral protease Dispase, large epithelial sheets can be harvested rapidly. The separation of the choroid from the pigment epithelium prior to retinal detachment greatly reduces the risk of contamination of the cultures with choroidal cells. Growth of pigment epithelial cells on Matrigel-coated microporous filters in hormonally-defined medium allows for development of high levels of transepithelial electrical resistance as well as for preservation of the differentiated pigment epithelial cell phenotype. This method should be useful for studies of pigment epithelial cell permeability and structural differentiation in vitro.

Cytochalasin D inhibits L-glutamate-induced disc shedding without altering L-glutamate-induced increase in adhesiveness.

Excitatory amino acid-stimulated disc shedding is correlated with the appearance of microfilament-rich ensheathing processes of the retinal pigment epithelium (RPE) and increased apparent adhesiveness between photoreceptors and RPE in explanted eyecups of Xenopus laevis. We have compared the time course of disc shedding and increased retinal adherence during L-glutamate treatment. Increased adherence was measured on the basis of the tendency of the apical RPE domains to partition with isolated neural retinas. In medium supplemented with L-glutamate (12 mM) or kainate (100 microM), a glutamate analog, the time course of increased partitioning of melanin pigment-rich cell fragments which contain ensheathing processes differs, even though the kinetics of induced disc shedding is the same in either case. Co-treatment with cytochalasin D (5 microM) completely blocks L-glutamate-induced disc shedding, as well as formation of microfilament-rich ensheathing processes, even though it has little effect upon apparent adhesiveness. The virtually complete dissociation of the effects of L-glutamate on disc shedding from that on increased adhesiveness of photoreceptors to RPE suggests that increased retinal adherence and pseudopod formation may be unrelated causally.

Membrane turnover in rod photoreceptors: ensheathment and phagocytosis of outer segment distal tips by pseudopodia of the retinal pigment epithelium.

We have documented the ultrastructural changes that occur within the photoreceptor outer segment and the retinal pigment epithelium (RPE) during photosensitive membrane turnover. We employed an in vitro eyecup preparation from Xenopus laevis in which a large shedding event was induced by adding the excitatory amino acid L-aspartate (Greenberger & Besharse 1985; J. comp. Neurol. 239, 361-372). We found that during L-aspartate-induced shedding the RPE cells formed, on their apical domains, previously undescribed processes that were directly involved in disc phagocytosis. These processes are structurally similar to processes formed by macrophages during phagocytosis and are accordingly referred to as pseudopodia. Pseudopodia were distinguishable from the apical villous process normally extended from the RPE in that they were closely applied to the surface of the outer segment, had a cytoplasmic matrix of low electron density that was devoid of most cellular organelles and were enriched in thin (7 nm diameter) filaments. Filament size, specific pseudopodial staining with the actin-specific probe rhodamine phalloidin and inhibition of pseudopod formation by cytochalasin D suggested that the thin filaments were composed of actin. Pseudopodial formation also occurs during a normal light-initiated shedding event. However, the low frequency of shedding, the asynchrony of the individual shedding events and the transient appearance of the pseudopodia prevented a full appreciation of their role during normal disc shedding. Associated with massive shedding and pseudopodial formation, there was an increased adherence between retina and RPE. During L-aspartate treatment, the apical portions of the RPE cells partitioned with the distal outer segment during retinal isolation. This effect was directly related to the development of pseudopodia and may reflect alteration of surface features of the rod outer segment (ROS)-RPE interface related to phagocytosis. Our observations show that transiently forming pseudopodia are the organelles of phagocytosis and that they may play a role in disc detachment as well.

Tunicamycin-induced dysgenesis of retinal rod outer segment membranes. II. Quantitative freeze-fracture analysis.

Tunicamycin (TM), a selective inhibitor of dolichylphosphate-dependent oligosaccharide biosynthesis, effectively blocks glycosylation, but not synthesis, of opsin, the rod visual pigment apoglycoprotein. In parallel with this inhibition, vesicular membrane material accumulates in the compartment between rod inner and outer segments (the intersegmental space) in TM-treated retinas (Fliesler et al, J Cell Biol 100:574-587, 1985). Freeze-fracture analysis was applied to isolated Xenopus laevis retinas which were incubated in the presence or absence of TM in order to clarify the relationship of those intersegmental membranous structures to others in the rod outer segment (ROS) assembly pathway. The membranes in the intersegmental space display characteristics similar to those of the ROS, but distinct from those of the inner segment. They exhibit densely particulate convex protoplasmic face (PF)-leaflets and relatively particle-free concave exoplasmic face (EF)-leaflets, similar to comparable leaflets of the ROS plasmalemma and nascent (immature) disc membranes. Quantitative analysis further demonstrates that the size distribution and densities of intramembrane particles (IMPs) in PF-leaflets of the intersegmental membranes are indistinguishable from those of ROS membranes, suggesting that both membrane systems contain similar integral proteins (i.e., opsin). Finally, the topology of the intersegmental membranes is most closely related to that of the ROS plasmalemma or nascent disc membranes, suggesting that they arise as a result of aberrant disc morphogenesis, rather than by breakdown of mature discs. Overall, the data support the conclusion that the tubulo-vesicular membranes in the intersegmental space represent newly assembled, opsin-containing material which has been efficiently compartmentalized in preparation for disc morphogenesis, but is incapable of forming normal, topologically closed discs.

Membrane assembly in retinal photoreceptors. II. Immunocytochemical analysis of freeze-fractured rod photoreceptor membranes using anti-opsin antibodies.

We have used a cytochemical technique for labeling freeze-fractured tissues (Pinto da Silva, P., C. Parkison, and N. Dwyer (1981) Proc. Natl. Acad. Sci. U.S.A. 78: 343-347) to examine the distribution of immunoreactive opsin in rod photoreceptor membranes. Aldehyde-fixed retinas of African clawed frogs (Xenopus laevis) embedded in a cross-linked protein matrix were frozen and fractured at -196 degrees C, then thawed and labeled with biotinylated sheep anti-cow opsin IgG followed by avidin-ferritin. In thin sections of plastic-embedded retinas, rod outer segment (ROS) disc membranes exposed by fracturing bound specific antibody intensely and relatively uniformly. However, they differed from membranes of the inner segment as well as those of erythrocytes in that protoplasmic face leaflets did not assume an interrupted bilayer appearance and disc exoplasmic face leaflets were apparently lost during thawing. The disposition of opsin immunoreactivity in the cell membrane was highly asymmetric. Although ROS plasma membranes from which discs are elaborated labeled heavily with anti-opsin after cleavage, fractures passing along inner segment plasma membranes bound very little antibody. In cross-fractures exposing inner segment cytoplasm, we found specific labeling of Golgi complex elements, as well as both perimitochondrial and periciliary vesicles. The latter are presumed to be the vehicle shuttling newly synthesized membrane to the ROS for disc assembly. These results suggest that opsin-containing membrane is sorted out within the cell, being transported from synthetic sites to the immediate periciliary zone where localized insertion into the cell membrane takes place. Furthermore, the close correspondence of the present immunocytochemical analysis with the distribution of opsin deduced from prior quantitative freeze-fracture analysis (Besharse, J. C., and K. H. Pfenninger (1980) J. Cell Biol. 87: 451-463) offers the possibility that fracture-label may be generally useful for study of patterned membrane topography in neuronal cells.

Membrane assembly in retinal photoreceptors. III. Distinct membrane domains of the connecting cilium of developing rods.

To investigate the putative role of the photoreceptor connecting cilium in the delivery of opsin to forming discs and in the maintenance of membrane domains (Besharse, J. C., and K. H. Pfenninger (1980) J. Cell Biol. 87: 451-463), we have studied developing photoreceptors of neonatal rats during the period of initial disc formation using conventional freeze-fracture, immunocytochemistry, and lectin cytochemistry. Specific anti-opsin-binding sites were localized in the distal cilium, the developing outer segment plasma membrane, and at focal sites on the inner segment plasma membrane at all developmental stages examined, including the period prior to the onset of disc morphogenesis. The proximal ciliary shaft generally lacked anti-opsin-binding sites or exhibited them in extremely low density. The distribution of anti-opsin-binding sites corresponded in a general way to the distribution of large intramembranous particles (IMPs) in freeze-fracture replicas like those seen in the rod outer segment (ROS). The proximal zone corresponded in freeze-fracture images to a zone of consecutive horizontal rows of intramembrane particles (ciliary necklaces) and axoneme-membrane cross-linkers. Although protoplasmic face leaflet IMPs similar to those of the distal cilium and outer segment were less abundant in the inner segment and proximal cilium than in the distal cilium and ROS, they were detected in these zones at low frequency. Cytochemistry with concanavalin A and wheatgerm agglutinin revealed the presence of a well developed glycocalyx in the proximal zone. Although opsin binds both lectins, the results suggest heterogeneity among the glycoconjugates of the three membrane domains. Our data define distinct membrane domains of the developing photoreceptor cilium that have important implications for the mechanisms for delivering and sequestering opsin in the outer segment. They also establish that the mechanism of opsin delivery to the distal zone occurs well in advance of the period of disc morphogenesis.

Rhodopsin chromophore exchanges among opsin molecules in the dark.

Turnover of rhodopsin chromophore in vertebrate visual cells has been explored by light microscope autoradiography (LMARG) and radiobiochemical techniques. Retinol-binding protein (RBP) was isolated from human serum, its native ligand removed and replaced with [3H]-retinol. After reconstitution, [3H]-retinol-RBP was reassociated with prealbumin (PA), and the protein complex injected intravenously into dark-adapted animals. After selected intervals in the dark, animals were killed, and ocular tissues dissected under infrared illumination. Eyecups from frogs and mice were fixed (4 C) and after in situ reduction of the chromophore-protein linkage of rhodopsin with borane dimethyl amine (BDMA), processed histologically to retain lipids, or alternatively to extract them with chloroform-methanol (C-M), and LMARG performed. Rhodopsin was purified from detergent-solubilized mouse retinas by Concanavalin A (Con A) affinity chromatography and analyzed for radioactivity. Autoradiographic labeling of frog rod outer segments (ROS) was first detectable at 1 day postinjection, increasing over the duration of the experiment. At all times, label was distributed throughout the organelle in a diffuse pattern, although in certain cases a band of silver grains was also evident at the proximal end of the ROS, the site of new membrane assembly. Similar autoradiographic patterns were noted in mouse rods, although the kinetics of labeling differed in certain respects. In biochemical experiments, incorporation of [3H]-retinol into mouse rhodopsin was seen to occur very rapidly (less than 30 min), without an appreciable lag period. We interpret the diffuse labeling of ROS to result from an exchange in the dark of [3H]-vitamin A aldehyde for unlabeled opsin-bound chromophore, whereas the formation of a reaction band no doubt reflects the continual renewal of ROS membrane occurring in the dark. With respect to the former, the turnover of chromophore qualitatively resembles that found for membrane fatty acids.