The cytoskeleton of chick retinal pigment epithelial cells in situ

Summary Gelatin-coated slides were used to obtain en face preparations of retinal pigment epithelium (RPE) from 6-to 21-day-old chick embryos in order to study the distribution of F-actin in microfilaments (MF) and the MF-associated proteins, myosin, tropomyosin,-actinin and vinculin in situ at different stages of development by fluorescence microscopy. The epithelial sheets were fixed in formaldehyde and then extracted in a solution containing 0.1% Triton X-100. NBD-Phallacidin was used to visualize the F-actin in MF, and antisera against myosin, tropomyosin,-actinin and vinculin were used to determine the distribution of these four MF-associated proteins. F-actin, myosin, tropomyosin,-actinin and vinculin were present in cortical rings around the apical ends of the RPE cells throughout this period of development. Of these proteins, only F-actin was identified in the apical processes of RPE cells. The increase in the amount of F-actin could be followed as the length and the number of apical processes increased with age and maturation of RPE cells. F-actin was first detected in numerous short apical processes on the surface of each RPE cell on day 12. From day 12 to day 17, they were at an intermediate stage of elongation and from day 17 onward all of the RPE cells had long F-actin-containing apical processes. These results indicate that the F-actin-containing MF assemble much later in the apical processes than in the cortical rings. Also the cortical rings and apical processes of RPE cells resemble those in absorptive intestinal cells in that the cortical rings in both cell types contain MF associated with myosin, tropomyosin,-actinin and vinculin while the MF in the apical processes and microvilli lack these MF associated proteins, and both of these structures lack talin. In addition to apical processes and cortical rings, stained fibers were also observed at a level below the cortical rings. The simple and highly reproducible en face method described is useful for determining changes in the organization of cytoskeletal components and other macromolecules in RPE cells and other epithelial cells in situ.     

Apical polarity of Na,K-ATPase in retinal pigment epithelium is linked to a reversal of the ankyrin-fodrin submembrane cytoskeleton

In striking contrast to most other transporting epithelia (e.g., urinary or digestive systems), where Na,K-ATPase is expressed basolaterally, the retinal pigment epithelium (RPE) cells display Na,K-ATPase pumps on the apical membrane. We report here studies aimed to identify the mechanisms underlying this polarity "reversal" of the RPE Na,K-ATPase. By immunofluorescence on thin frozen sections, both alpha and beta subunits were localized on the apical surface of both freshly isolated rat RPE monolayers and RPE monolayers grown in culture. The polarity of the RPE cell is not completely reversed, however, since aminopeptidase, an apically located protein in kidney epithelia, was also found on the apical surface of RPE cells. We used subunit- and isoform-specific cDNA probes to determine that RPE Na,K-ATPase has the same isoform (alpha 1) as the one found in kidney. Ankyrin and fodrin, proteins of the basolateral membrane cytoskeleton of kidney epithelial cells known to be associated with the Na,K-ATPase (Nelson, W. J., and R. W. Hammerton. 1989. J. Cell Biol. 110:349-357) also displayed a reversed apical localization in RPE and were intimately associated to Na,K-ATPase, as revealed by cross-linking experiments. These results indicate that an entire membrane-cytoskeleton complex is assembled with opposite polarity in RPE cells. We discuss our observations in the context of current knowledge on protein sorting mechanisms in epithelial cells.     

Bipolar assembly of caveolae in retinal pigment epithelium

Caveolae and their associated structural proteins, the caveolins, are specialized plasmalemmal microdomains involved in endocytosis and compartmentalization of cell signaling. We examined the expression and distribution of caveolae and caveolins in retinal pigment epithelium (RPE), which plays key roles in retinal support, visual cycle, and acts as the main barrier between blood and retina. Electron microscopic observation of rat RPE, in situ primary cultures of rat and human RPE and a rat RPE cell line (RPE-J) demonstrated in all cases the presence of caveolae in both apical and basolateral domains of the plasma membrane. Caveolae were rare in RPE in situ but were frequent in primary RPE cultures and in RPE-J cells, which correlated with increased levels in the expression of caveolin-1 and -2. The bipolar distribution of caveolae in RPE is striking, as all other epithelial cells examined to date (liver, kidney, thyroid, and intestinal) assemble caveolae only at the basolateral side. This might be related to the nonpolar distribution of both caveolin-1 and 2 in RPE because caveolin-2 is basolateral and caveolin-1 nonpolar in other epithelial cells. The bipolar localization of plasmalemmal caveolae in RPE cells may reflect specialized roles in signaling and trafficking important for visual function. caveolin; raft microdomains; membrane traffic; normal rat kidney     

Comparative study of a new ultrastructural specialization of the mammalian retinal pigment epithelium: basal intracytoplasmic tubules

We recently described basal intracytoplasmic tubules that arise from the basal plasma membrane of rat retinal pigment epithelial cells (RPE). They are a previously undescribed ultrastructural specialization of the basal plasma membrane in addition to the well-known basal folds. This report describes similar tubules in the RPE cells of the rabbit, hamster, and kitten. As in the rat, the tubules in the hamster and kitten ramify singly through the basal cytoplasm and bear no special relationship to any other organelle. In the rabbit, however, stacks of closely apposed tubules frequently abut the large lipid droplets in their RPE cells. Although the function of the tubules is unknown, their occurrence in several different mammals suggests they are involved in the function of RPE cells in general.     

Association of stomatin with lipid-protein complexes in the plasma membrane and the endocytic compartment

Membrane protein - microvilli - lipid raft - GPI-anchored protein - epithelial cell The 31 kDa integral membrane protein stomatin (protein 7.2b) has a monotopic structure and a cytofacial orientation. We have shown previously that stomatin is located in plasma membrane protruding structures and forms high-order homo-oligomers in the human epithelial cell line UAC, suggesting that this protein has a structural function in the cortical morphogenesis of the cells. It is also present in a pool of juxtanuclear vesicles. In this study, we show that stomatin colocalizes with the GPI-anchored proteins placental alkaline phosphatase (PLAP) and membrane folate receptor alpha (MFRalpha) endogenously expressed in UAC cells. This observation enabled us to demonstrate two different aspects of stomatin. First, using anti-PLAP antibody internalization, we show that the peri-centrosomal vesicles containing stomatin correspond to a subset of endosomes, which can also be labeled with the late endosomal/lysosomal marker LAMP-2. Secondly, we found that stomatin is partially present in detergent-insoluble membrane domains and co-patches with PLAP on the plasma membrane, after cross-linking of PLAP by antibodies. These data indicate that stomatin and GPI-anchored proteins are linked through lipid rafts and undergo the same sorting events. We propose that stomatin, through its affinity for lipid rafts, functions in concentrating GPI-anchored proteins in membrane microvillar structures. Consistent with this hypothesis, we found that stomatin is expressed exclusively in microvilli of the apical membrane in polarized Madin-Darby canine kidney (MDCK) cells.     

In vivo gene transfer as a means to study the physiology and morphogenesis of the retinal pigment epithelium in the rat

Our understanding of the morphogenesis of epithelial phenotypes has been greatly advanced by the use of in vitro cell culture systems. However, cell cultures often do not faithfully reconstitute many of the differentiated properties of the cell from which they are derived and cannot be used to examine complex physiologic interactions between adjacent tissues. This is particularly true of the retinal pigment epithelium (RPE). Many plasma membrane proteins, in vivo, exhibit a reversed polarity with respect to other epithelia, and RPE-derived cell lines seldom exhibit these same polarity properties. Furthermore, the interaction between the RPE cell and the neuorsensory retina, or the underlying blood supply, the choroid, is absent in cell culture. Most epithelia are difficult to isolate and study in vivo. The RPE is an exception to this. We have explored several aspects of RPE protein transport properties, vision-related physiology, and disease-related pathophysiology in the eye using in vivo gene transfer and electrophysiologic techniques. By injecting replication-defective adenoviruses into the subretinal space of rat eyes, we have been able to easily direct the expression of a test protein and follow its sorting and physiologic effects on RPE cells and adjacent tissues. Due to binding and internalization of adenoviral vectors to integrins found on the RPE apical plasma membrane, expression in a healthy eye is essentially confined to the RPE cell, even under control of a cytomegalovirus promotor. The use of varying amounts of adenoviral vector allows for determination of dose-responsive effects and the comparison of multiple mutants of a protein. In addition, there are substantial savings with respect to time and money in comparison to standard transgenic approaches.     

Interaction of biological membranes with the cytoskeletal framework of living cells

The review is focused on the molecular structure and function of the proteins composing the actin-based cytokeletal cortex, located at the cytoplasmic face of plasma membranes of eucaryotic cells, which stabilizes integral membrane proteins in separate domains of cell membranes. It includes a survey of the molecular properties of teh proteins of the erythrocyte membrane skeleton such as spectrin, ankyrin, protein 4.1, and adducin. The properties of the immunological counterparts of erythroid cortical proteins found in nonerythroid tissues and cells are compared. The structural organization and function of the newly discovered class of calcium-binding proteins, nonerythroid peripheral membrane proteins, calpactins, are also described. Finally, the discussion of some experimental models illustrates that the membrane skeleton of living cells is actively involved in a wide variety of essential biological functions ranging from differentiation, to maintenance of cell polarity and cell shape, and regulation of exocytotic processes.     

Apical-basal polarity in Drosophila neuroblasts is independent of vesicular trafficking

The possession of apical-basal polarity is a common feature of epithelia and neural stem cells, so-called neuroblasts (NBs). In Drosophila, an evolutionarily conserved protein complex consisting of atypical protein kinase C and the scaffolding proteins Bazooka/PAR-3 and PAR-6 controls the polarity of both cell types. The components of this complex localize to the apical junctional region of epithelial cells and form an apical crescent in NBs. In epithelia, the PAR proteins interact with the cellular machinery for polarized exocytosis and endocytosis, both of which are essential for the establishment of plasma membrane polarity. In NBs, many cortical proteins show a strongly polarized subcellular localization, but there is little evidence for the existence of distinct apical and basolateral plasma membrane domains, raising the question of whether vesicular trafficking is required for polarization of NBs. We analyzed the polarity of NBs mutant for essential regulators of the main exocytic and endocytic pathways. Surprisingly, we found that none of these mutations affected NB polarity, demonstrating that NB cortical polarity is independent of plasma membrane polarity and that the PAR proteins function in a cell type-specific manner.     

Plasticity in epithelial cell phenotype: modulation by expression of different cadherin cell adhesion molecules

A primary function of cadherins is to regulate cell adhesion. Here, we demonstrate a broader function of cadherins in the differentiation of specialized epithelial cell phenotypes. In situ, the rat retinal pigment epithelium (RPE) forms cell-cell contacts within its monolayer, and at the apical membrane with the neural retina; Na+, K(+)-ATPase and the membrane cytoskeleton are restricted to the apical membrane. In vitro, RPE cells (RPE-J cell line) express an endogenous cadherin, form adherens junctions and a tight monolayer, but Na+,K(+)-ATPase is localized to both apical and basal-lateral membranes. Expression of E- cadherin in RPE-J cells results in restriction and accumulation of both Na+,K(+)-ATPase and the membrane cytoskeleton at the lateral membrane; these changes correlate with the synthesis of a different ankyrin isoform. In contrast to both RPE in situ and RPE-J cells that do not form desmosomes, E-cadherin expression in RPE-J cells induces accumulation of desmoglein mRNA, and assembly of desmosome-keratin complexes at cell-cell contacts. These results demonstrate that cadherins directly affect epithelial cell phenotype by remodeling the distributions of constitutively expressed proteins and by induced accumulation of specific proteins, which together lead to the generation of structurally and functionally distinct epithelial cell types.     

Sorting nexin 27 regulates basal and stimulated brush border trafficking of NHE3

Sorting nexin 27 (SNX27) contains a PDZ domain that is phylogenetically related to the PDZ domains of the NHERF proteins. Studies on nonepithelial cells have shown that this protein is located in endosomes, where it regulates trafficking of cargo proteins in a PDZ domain–dependent manner. However, the role of SNX27 in trafficking of cargo proteins in epithelial cells has not been adequately explored. Here we show that SNX27 directly interacts with NHE3 (C-terminus) primarily through the SNX27 PDZ domain. A combination of knockdown and reconstitution experiments with wild type and a PDZ domain mutant (GYGF → GAGA) of SNX27 demonstrate that the PDZ domain of SNX27 is required to maintain basal NHE3 activity and surface expression of NHE3 in polarized epithelial cells. Biotinylation-based recycling and degradation studies in intestinal epithelial cells show that SNX27 is required for the exocytosis (not endocytosis) of NHE3 from early endosome to plasma membrane. SNX27 is also required to regulate the retention of NHE3 on the plasma membrane. The findings of the present study extend our understanding of PDZ-mediated recycling of cargo proteins from endosome to plasma membrane in epithelial cells.     

Subunits in zonulae adhaerentes and striations in the associated circumferential microfilament bundles in chicken retinal pigment epithelial cells in situ

This study shows that the zonula adhaerens in chicken retinal pigment epithelial (RPE) cells in situ consists of independent subunits which are composed of extracellular intermembrane discs sandwiched between cytoplasmic plaques. These zonula adhaerens complexes (ZACs) are hexagonally arranged within the junction. Previous immunocytochemical studies suggest that the zonula adhaerens region, composed of ZACs, contains the actin associated proteins vinculin and alpha-actinin. The intermembrane discs of ZACs likely mediate cell-to-cell adhesion whereas the cytoplasmic plaques are probably involved in binding the microfilaments of the relatively large circumferential microfilament bundles (CMBs), associated with the zonula adhaerens, to the cell membrane. The CMBs of chicken RPE cells in situ show striations similar to those found in stress fibers of other cell types and in CMBs of cultured epithelial cells. The observation that in the striated regions of CMBs the adjacent junctional membranes tend to follow an undulating path suggests that the CMBs are attached intermittently to the cell membrane and are contractile. The structural similarities between CMBs and stress fibers and the fact that they share similar actin associated proteins support the view that CMBs and stress fibers are related structures.     

The human AC133 hematopoietic stem cell antigen is also expressed in epithelial cells and targeted to plasma membrane protrusions

The human AC133 antigen and mouse prominin are structurally related plasma membrane proteins. However, their tissue distribution is distinct, with the AC133 antigen being found on hematopoietic stem and progenitor cells and prominin on various epithelial cells. To determine whether the human AC133 antigen and mouse prominin are orthologues or distinct members of a protein family, we examined the human epithelial cell line Caco-2 for the possible expression of the AC133 antigen. By both immunofluorescence and immunoprecipitation, the AC133 antigen was found to be expressed on the surface of Caco-2 cells. Interestingly, immunoreactivity for the AC133 antigen, but not its mRNA level, was down-regulated upon differentiation of Caco-2 cells. The AC133 antigen was specifically located at the apical rather than basolateral plasma membrane. An apical localization of the AC133 antigen was also observed in various human embryonic epithelia including the neural tube, gut, and kidney. Electron microscopy revealed that, within the apical plasma membrane of Caco-2 cells, the AC133 antigen was confined to microvilli and absent from the planar, intermicrovillar regions. This specific subcellular localization did not depend on an epithelial phenotype, because the AC133 antigen on hematopoietic stem cells, as well as that ectopically expressed in fibroblasts, was selectively found in plasma membrane protrusions. Hence, the human AC133 antigen shows the features characteristic of mouse prominin in epithelial and transfected non-epithelial cells, i.e. a selective association with apical microvilli and plasma membrane protrusions, respectively. Conversely, flow cytometry of murine CD34(+) bone marrow progenitors revealed the cell surface expression of prominin. Taken together, the data strongly suggest that the AC133 antigen is the human orthologue of prominin.     

Remodelling of the retinal pigment epithelium in response to intraepithelial capillaries: evidence that capillaries influence the polarity of epithelium

Summary Light- and urethane-induced retinopathies in rats are characterized by loss of photoreceptors. Retinal capillaries subsequently become incorporated into the normally avascular retinal pigment epithelium. These models provided an opportunity to study the response of epithelial cells to closely apposed capillaries, in order to determine if capillaries contribute to the polar organization of epithelial cells. Pigment epithelial cells reorganized their lateral plasma membrane where the latter faced intraepithelial capillaries. This normally flat, undifferentiated membrane developed attachment sites, folds and intracytoplasmic tubules, and exhibited endocytosis and putative basal lamina secretion. These structural and functional specializations are normally restricted to the basal plasma membrane — the normal vascular front of the cell facing the dense meshwork of capillaries constituting the choriocapillaris. We conclude that RPE cells, and perhaps epithelia in general, polarize in response to an adjacent capillary bed.     

Adhesiveness and distribution of vinculin and spectrin in retinal pigmented epithelial cells during growth and differentiation in vitro

Colonies of chick retinal pigmented epithelial (RPE) cells offer an excellent model system for studying the organization of cytoskeleton in sheets of differentiating epithelial cells. The cells occupying the center of the colony resemble RPE cells in vivo and are cuboidal, pigmented, and relatively nonadherent while those toward the periphery gradually become flatter, nonpigmented, motile, and strongly adherent to the substratum. Immunofluorescence microscopy with antiserum against chicken erythrocyte alpha-spectrin reveals that this protein is present in the cortex of RPE cells in all parts of the colony. It is neither concentrated in, nor excluded from the regions occupied by the major microfilament bundles, and its distribution is not related to the adhesion patterns visualized by surface reflection interference microscopy. In contrast, the distribution of vinculin is closely correlated with the adhesiveness of RPE cells in different parts of the colony. Immunofluorescence microscopy reveals that in the RPE cells vinculin may be diffusely distributed in the cytoplasm; present in a cortical band outlining the cell borders; and present in focal contacts and adhesions. The distribution of vinculin is affected by the length of time the colonies grow in culture, by the degree of cell packing and by the adhesiveness of cells to the substratum. In RPE cells grown in vitro for short periods (less than or equal to 3 days) vinculin is found in focal contacts and adhesions in both the undifferentiated, well spread peripheral cells as well as in the differentiated, polygonally packed central cells of the colony. In RPE cells cultured for longer periods (greater than or equal to 14 days) vinculin is present in focal contacts and adhesions only in strongly adherent, undifferentiated cells at the edge of the colony. In packed central cells of both short- and long-term cultures vinculin is found in the cortical band which circumscribes the apical ends of cells at the level of the adherens type intercellular junctions. Its appearance in the cortical bands does not depend on the length of time the colonies are grown in vitro but on the presence of cell-cell contacts resulting from an increased degree of cell packing within the central part of the colony. These results are discussed in relation to the development and the role of extracellular matrix in determining the adhesiveness of RPE cells in vitro.     

Suction pressure can induce uncoupling of the plasma membrane from cortical actin

We tested the hypothesis that a pressure difference can cause blebbing associated with uncoupling of the plasma membrane from the cortical actin, a phenomenon found earlier in locomoting blebbing Walker carcinosarcoma cells. Untreated, initially spherical Walker carcinosarcoma cells were exposed to suction pressure by partial aspiration into micropipettes. The suction pressure required to induce blebbing was in the range of 0.9-3 cm H2O, i.e., somewhat lower than the increase in intracellular pressure measured before formation of protrusions in Amoeba proteus (Yanai et al., Cell Motil. Cytoskeleton 33, 22-29, 1996). The response was temperature-dependent, blebbing occurring more frequently at 37 degrees C than at room temperature. Blebbing was associated with formation of cytoplasmic actin layers, restriction rings and/or of gaps in the plasma membrane-associated cortical actin. The results support the view that blebbing associated with uncoupling of cortical actin and plasma membrane as observed in locomoting cells can be caused by a pressure gradient.     

The transmembrane domain of the respiratory syncytial virus F protein is an orientation-independent apical plasma membrane sorting sequence

The processes that facilitate transport of integral membrane proteins though the secretory pathway and subsequently target them to particular cellular membranes are relevant to almost every field of biology. These transport processes involve integration of proteins into the membrane of the endoplasmic reticulum (ER), passage from the ER to the Golgi, and post-Golgi trafficking. The respiratory syncytial virus (RSV) fusion (F) protein is a type I integral membrane protein that is uniformly distributed on the surface of infected nonpolarized cells and localizes to the apical plasma membrane of polarized epithelial cells. We expressed wild-type or altered RSV F proteins to gain a better understanding of secretory transport and plasma membrane targeting of type I membrane proteins in polarized and nonpolarized epithelial cells. Our findings reveal a novel, orientation-independent apical plasma membrane targeting function for the transmembrane domain of the RSV F protein in polarized epithelial cells. This work provides a basis for a more complete understanding of the role of the transmembrane domain and cytoplasmic tail of viral type I integral membrane proteins in secretory transport and plasma membrane targeting in polarized and nonpolarized cells.     

Retinal pigment epithelial fine structure in the bobtail goanna (Tiliqua rugosa)

The retinal pigment epithelium (RPE), the choriocapillaris and Bruch's membrane (complexus basalis) have been studied by light and electron microscopy in the bobtail goanna (Tiliqua rugosa) an Australian diurnal lizard. The RPE consists of a single layer of cuboidal cells which display very deep and tortuous basal (choroidal) infoldings as well as numerous apical (vitreal) processes which interdigitate with the photoreceptor cells. The lateral cell borders are relatively smooth and joined by basally located tight junctions. Internally smooth endoplasmic reticulum is abundant while rough endoplasmic reticulum is not. The RPE cell nucleus is large and vesicular and basally located in the light-adapted state. Polysomes, mitochondria and myeloid bodies are present and widely distributed. Melanosomes are plentiful in the apical region of the epithelial cells in light-adaptation. Bruch's membrane is pentalaminate with the basal lamina of the choriocapillaris being exceptionally thick. The choriocapillaris is a single layer of large-caliber capillaries with thin but only moderately fenestrated endothelium. Numerous dense granules are always present within these endothelial cells.     

Apical Polarity of N-CAM and EMMPRIN in Retinal Pigment Epithelium Resulting from Suppression of Basolateral Signal Recognition

Retinal pigment epithelial (RPE) cells apically polarize proteins that are basolateral in other epithelia. This reversal may be generated by the association of RPE with photoreceptors and the interphotoreceptor matrix, postnatal expansion of the RPE apical surface, and/or changes in RPE sorting machinery. We compared two proteins exhibiting reversed, apical polarities in RPE cells, neural cell adhesion molecule (N-CAM; 140-kD isoform) and extracellular matrix metalloproteinase inducer (EMMPRIN), with the cognate apical marker, p75-neurotrophin receptor (p75-NTR). N-CAM and p75-NTR were apically localized from birth to adulthood, contrasting with a basolateral to apical switch of EMMPRIN in developing postnatal rat RPE. Morphometric analysis demonstrated that this switch cannot be attributed to expansion of the apical surface of maturing RPE because the basolateral membrane expanded proportionally, maintaining a 3:1 apical/basolateral ratio. Kinetic analysis of polarized surface delivery in MDCK and RPE-J cells showed that EMMPRIN has a basolateral signal in its cytoplasmic tail recognized by both cell lines. In contrast, the basolateral signal of N-CAM is recognized by MDCK cells but not RPE-J cells. Deletion of N-CAM''s basolateral signal did not prevent its apical localization in vivo. The data demonstrate that the apical polarity of EMMPRIN and N-CAM in mature RPE results from suppressed decoding of specific basolateral signals resulting in randomized delivery to the cell surface.     

Effects of a tumour promoter, 12-0-tetradecanoyl-phorbol-13-acetate (TPA), on expression of differentiated phenotype in the chick retinal pigmented epithelial cells and on their interactions with the native basement membrane and with artificial substrata

Chick retinal pigmented epithelial (RPE) cells grown in vitro on basement membrane matrices from the Engelbreth-Holm-Swarm tumour (BM-matrigel) do not spread, and they maintain their differentiated phenotype, most notably the heavy pigmentation. Maintenance of the differentiated phenotype by RPE cells on BM-matrigel is promoted not only by the biochemical composition of the gel but also by its mechanical properties, i.e., its low rigidity prevents cell spreading. In this report, RPE cells on BM-matrigel were treated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) to promote the transformed phenotype and diminish cell traction. In contrast to most cell types TPA treatment induced RPE cells to increase their spread area. TPA promoted RPE cell spreading on BM-matrigel and changed the spatial organization of actin and actin-associated proteins in the cytoskeleton-ECM linkage complexes, uncoupling actin from its extracellular counterpart. TPA did not affect other components of the cytoskeleton in RPE cells. TPA also affected labile adhesions i.e., focal contacts and adherens junctions in statu nascendi, but preformed, stable adherens junctions were resistant to TPA. TPA enhanced proliferation, blocked melanogenesis and thus inhibited differentiation of RPE cells grown on either artificial substrata or their natural basement membrane.     

Changes in retinal pigment epithelial cell autofluorescence and protein expression associated with phagocytosis of rod outer segments in vitro.

The accumulation of autofluorescent lipofuscin was quantified in cultured human retinal pigment epithelial (RPE) cells phagocytosing bovine rod outer segments (BROS) and the expression of proteins in these cells was investigated. Results showed a steady increase in autofluorescence of RPE cells over a 4-week period as measured by fluorophotometric flow cytometry. A significantly greater increase in autofluorescence was found in the cultured RPE cells from a 7-year-old donor compared with those from a 47-year-old donor. Within both groups the BROS-challenged cells had significantly higher fluorescence readings than the control cells which were not challenged. Autoradiography of 35S-labelled proteins separated by polyacrylamide gel electrophoresis (PAGE) revealed a small distinct band at 102 kDa in BROS-challenged RPE cells of both bovine and human origin that did not appear in control or microsphere-phagocytosing RPE cells. The intensity of the signal was unrelated to the duration of the challenge period.