Disc shedding in rodlike and conelike photoreceptors of tree squirrels.

Electron microscopic observations suggest that the rodlike and conelike photoreceptors of diurnal tree squirrels shed outer segment discs. Twenty-four hours after injection of triated L-leucine, the rodlike photoreceptors show a band of radioactivity at the base of the outer segment. The conelike photoreceptor outer segments show only a pattern of diffuse labeling. These results strongly suggest that disc shedding can occur in photoreceptor outer segments in which proteins are diffusely renewed.

Crystal structure of alpha 1: implications for protein design.

X-ray diffraction shows the structure of a synthetic protein model, formed from noncovalent self-association of a 12-residue peptide and of sulfate ions at low pH. This peptide is a fragment of a 16-residue polypeptide that was designed to form an amphiphilic alpha helix with a ridge of Leu residues along one helical face. By interdigitation of the leucines of four such helices, the design called for self-association into a four-alpha-helical bundle. The crystal structure (2.7 angstrom resolution; R factor = 0.215) reveals a structure more complex than the design, with both a tetramer and a hexamer. The alpha-helical tetramer with leucine interior has more oblique crossing angles than most four-alpha-helical bundles; the hexamer has a globular hydrophobic core of 12 leucine residues and three associated sulfate ions. Computational analysis suggests that the hexameric association is tighter than the tetrameric one. The consistency of the structure with the design is discussed, as well as the divergence.

v-fps causes transformation by inducing tyrosine phosphorylation and activation of the PDGFbeta receptor.

The v-fps oncogene encodes an activated tyrosine kinase which is capable of transforming fibroblasts. In this report, we provide evidence that within a few minutes of activation of the tyrosine kinase activity of v-Fps, tyrosine phosphorylation of the platelet derived growth factor (PDGF) beta receptor is observed. Further, sustained expression of activated v-Fps results in a down-regulation of the PDGF receptor both at the level of the mRNA (approximately 4-8-fold), but even more markedly at the level of the receptor protein (> 100-fold). The kinase activity of the v-Fps oncoprotein was found to be required for both the induction of PDGF receptor tyrosine phosphorylation and ultimately the reduced receptor protein levels. Tyrosine phosphorylation of a kinase inactive PDGF receptor was also demonstrated in cells which also express v-fps, but this was not sufficient to induce transformation. Only cells expressing both v-fps and a wild type PDGF receptor were able to form colonies in soft agar. These findings suggest that wild type v-fps may use tyrosine phosphorylation of the PDGFbeta receptor to constitutively activate the kinase activity of the receptor, resulting in a sustained proliferative signal and fibroblast transformation.

Charges, hydrogen bonds, and correlated motions in the 1 A resolution refined structure of the mating pheromone Er-1 from Euplotes raikovi.

A detailed description is given of the structure of the small protein mating pheromone Er-1 at atomic resolution. Emphasis is placed on the locations of charges and hydrogen bonds. The model includes all the protein atoms, anisotropic displacement parameters, four disordered side chains, 22 water molecules, a disordered ethanol, and "riding" hydrogen atoms. Analysis of the model revealed that this dense crystal is perfused by hydrogen-bonding networks of solvent and protein atoms. The termini of helices are capped by hydrogen bonding to solvent and protein atoms, and to symmetry-related molecules. An examination of the valencies and charges of the hydrogen-bonding groups suggests that three of the "water" molecules capping the C termini of two helices, and one other, may instead be NH4 ions. Water molecules mediate all but one of the interhelical hydrogen bonds, and many of the lattice interactions. Regions of the molecule where the atomic vibrations deviate from isotropy are identified. There is almost no overall libration of the molecule allowed by the packing, but the side-chains vibrate relative to the backbone. Four side-chains display alternate conformations. Indirect evidence is presented that the switches between their conformations are correlated and driven by protonation of acidic side-chains. These structural features are discussed in the context of function and stability. Equipped with the analysis of the model, we review the course and results of the refinement of the model against 1 A X-ray diffraction data to a crystallographic R-factor of 12.92%.

An interfacial mechanism and a class of inhibitors inferred from two crystal structures of the Mycobacterium tuberculosis 30 kDa major secretory protein (Antigen 85B), a mycolyl transferase.

The Mycobacterium tuberculosis 30 kDa major secretory protein (antigen 85B) is the most abundant protein exported by M. tuberculosis, as well as a potent immunoprotective antigen and a leading drug target. A mycolyl transferase of 285 residues, it is closely related to two other mycolyl transferases, each of molecular mass 32 kDa: antigen 85A and antigen 85C. All three catalyze transfer of the fatty acid mycolate from one trehalose monomycolate to another, resulting in trehalose dimycolate and free trehalose, thus helping to build the bacterial cell wall. We have determined two crystal structures of M. tuberculosis antigen 85B (ag85B), initially by molecular replacement using antigen 85C as a probe. The apo ag85B model is refined against 1.8 A data, to an R-factor of 0.196 (R(free) is 0.276), and includes all residues except the N-terminal Phe. The active site immobilizes a molecule of the cryoprotectant 2-methyl-2,4-pentanediol. Crystal growth with addition of trehalose resulted in a second ag85B crystal structure (1.9 A resolution; R-factor is 0.195; R(free) is 0.285). Trehalose binds in two sites at opposite ends of the active-site cleft. In our proposed mechanism model, the trehalose at the active site Ser126 represents the trehalose liberated by temporary esterification of Ser126, while the other trehalose represents the incoming trehalose monomycolate just prior to swinging over to the first trehalose site to displace the mycolate from its serine ester. Our proposed interfacial mechanism minimizes aqueous exposure of the apolar mycolates. Based on the trehalose-bound structure, we suggest a new class of antituberculous drugs, made by connecting two trehalose molecules by an amphipathic linker.

An integrated hypothesis that considers drusen as biomarkers of immune-mediated processes at the RPE-Bruch's membrane interface in aging and age-related macular degeneration.

Age-related macular degeneration (AMD) is a blinding disease that afflicts millions of adults in the Western world. Although it has been proposed that a threshold event occurs during normal aging which leads to AMD, the sequelae of biochemical, cellular, and/or molecular events leading to the development of AMD are poorly understood. Although available data provide strong evidence that a significant proportion of AMD has a genetic basis, no gene(s) has yet been identified that causes a significant proportion of AMD. Moreover, no major molecular pathways involved in the etiology of this disease have been elucidated.Drusen, pathological deposits that form between the retinal pigmented epithelium (RPE) and Bruch's membrane, are significant risk factors for the development of AMD. In our view, the development of testable new hypotheses of drusen origins has been hindered significantly by the absence of a comprehensive profile of their molecular composition. In this review, we describe an integrated ultrastructural, histochemical, molecular biological, and biochemical approach to identify specific molecular pathways associated with drusen biogenesis. The implicit assumption underlying these recent investigations has been that a thorough understanding of the composition of drusen and source(s) of drusen-associated material is likely to provide fresh insight into the pathobiology underlying AMD. Significantly, these studies have revealed that proteins associated with inflammation and immune-mediated processes are prevalent among drusen-associated constituents. Transcripts that encode a number of these molecules have been detected in retinal, RPE, and choroidal cells. These data have also lead to the observations that dendritic cells, potent antigen-presenting cells, are intimately associated with drusen development and that complement activation is a key pathway that is active both within drusen and along the RPE-choroid interface. We propose herein a unifying hypothesis of drusen biogenesis that attempts to incorporate a large body of new and previously published structural, histochemical, and molecular data pertaining to drusen composition and development. This theory is put forth with the acknowledgment that numerous AMD genotypes may exist. Thus, only some aspects of the proposed hypothesis may be involved in any given AMD genotype. Importantly, this hypothesis invokes, for the first time, the potential for a direct role of cell- and immune-mediated processes in drusen biogenesis. We acknowledge that the proposed hypothesis clearly represents a paradigm shift in our conceptualization pertaining to pathways that participate in the development of drusen and age-related macular degeneration. It is our hope that other investigators will test, validate and/or refute various aspects of this hypothesis, and in so doing, increase our overall understanding of the biological pathways associated with early AMD.

Packed protein bilayers in the 0.90 A resolution structure of a designed alpha helical bundle.

A 12-residue peptide designed to form an alpha-helix and self-associate into an antiparallel 4-alpha-helical bundle yields a 0.9 A crystal structure revealing unanticipated features. The structure was determined by direct phasing with the "Shake-and-Bake" program, and contains four crystallographically distinct 12-mer peptide molecules plus solvent for a total of 479 atoms. The crystal is formed from nearly ideal alpha-helices hydrogen bonded head-to-tail into columns, which in turn pack side-by-side into sheets spanning the width of the crystal. Within each sheet, the alpha-helices run antiparallel and are closely spaced (9-10 A center-to-center). The sheets are more loosely packed against each other (13-14 A between helix centers). Each sheet is amphiphilic: apolar leucine side chains project from one face, charged lysine and glutamate side chains from the other face. The sheets are stacked with two polar faces opposing and two apolar faces opposing. The result is a periodic biomaterial composed of packed protein bilayers, with alternating polar and apolar interfaces. All of the 30 water molecules in the unit cell lie in the polar interface or between the stacked termini of helices. A section through the sheet reveals that the helices packed at the apolar interface resemble the four-alpha-helical bundle of the design, but the helices overhang parts of the adjacent bundles, and the helix crossing angles are less steep than intended (7-11 degrees rather than 18 degrees).

Centrosymmetric bilayers in the 0.75 A resolution structure of a designed alpha-helical peptide, D,L-Alpha-1.

We report the 0.75 A crystal structure of a racemic mixture of the 12-residue designed peptide "Alpha-1" (Acetyl-ELLKKLLEELKG), the L-enantiomer of which is described in the accompanying paper. Equivalent solutions of the centrosymmetric bilayers were determined by two direct phasing programs in space groups P1 and P1bar. The unit cell contains two L-alpha-helices and two D-alpha-helices. The columnar-sheet bilayer motif seen in L-Alpha-1 is maintained in the D,L-Alpha-1 structure except that each sheet of head-to-tail helices is composed of one enantiomer and is related to its neighboring sheets by inversion symmetry. Comparison to the L-Alpha-1 structure provides further insight into peptide design. The high resolution and small asymmetric unit allowed building an intricate model (R = 13.1%, Rfree = 14.5%) that incorporates much of the discrete disorder of peptide and solvent. Ethanolamine and 2-methyl-2,4-pentanediol (MPD) molecules bind near helix termini. Rigid body analysis identifies sites of restricted displacements and torsions. Side-chain discrete disorder propagates into the backbone of one helix but not the other. Although no side chain in Alpha-1 is rigid, the environments in the crystal restrict some of them to no or only one active torsion.

Disc morphogenesis in vertebrate photoreceptors.

Electron microscopic examination of the bases of adult rod and cone outer segments (rhesus monkey, ground squirrel, and grey squirrel) has led to a new model of disc morphogenesis. In this model the disc surfaces and disc rims develop by separate mechanisms and from separate regions of the membrane of the inner face of the cilium. This membrane is alternately specified into regions that will form either the disc surfaces or the disc rims. The disc surfaces develop by an evagination or outpouching of the ciliary membrane. The two surfaces of an evagination, scleral and vitreal, each form one of the surfaces of adjacent discs. The disc rim is initially specified as a region of ciliary membrane between adjacent disc-surface evaginations. This region grows bilaterally around the circumferences of adjacent discs, zippering together the apposed surfaces to form the rim and completed disc. At the same time it seals the plasma-membrane edges of the evaginations, which have become detached from the surfaces. Incisures form in rod discs by infolding of the rim and surfaces together, and they begin to form before the rim is completed around the disc perimeter. When a number of new discs are developing simultaneously the ciliary membrane at the base of an outer segment consists of a stack of rim forming and surface forming growth points. This model provides, in addition, for the continuous renewal of outer-segment plasma membrane. It also establishes a developmental basis for the structural uniqueness of the disc rim. Finally, it indicates an evolutionary relationship between the discs of vertebrate visual cells and the membrane specializations of invertebrate visual cells.

Vitronectin receptor expression and distribution at the photoreceptor-retinal pigment epithelial interface.

Laser scanning confocal microscopy was employed to map the distribution of integrin immunoreactivity at the photoreceptor-retinal pigment epithelial (RPE) interface of the primate retina, and to determine its relationship to the actin cytoskeleton. Immunolabeling using a polyclonal antibody to the human vitronectin receptor (VnR), a heterodimer containing the alpha v subunit in combination with either the beta 3 or beta 5 subunits, is detected primarily on the apical surface of the retinal pigment epithelium (RPE) in vivo and in vitro. It is also associated with the photoreceptor inner and outer segment cell surfaces. In contrast, immunolabeling using a polyclonal antibody to the human fibronectin receptor (FnR), a heterodimer containing the alpha 5 and beta 1 subunits, is detected principally on the basolateral surface of the RPE and is virtually absent in photoreceptors. A partial three-dimensional reconstruction of the anti-VnR labeling pattern in cone photoreceptors reveals cell surface labeling that originates at the level of the myoid just distal to the outer limiting membrane. It extends distally toward the ellipsoid and terminates at the level of the cone outer segment. Approximately 20-22 immunoreactive foci are distributed evenly around the perimeter of the cone ellipsoid. These foci correspond in number and location to the calycal processes that protrude from the distal portion of the ellipsoid. A double-labeling procedure, employing VnR antibody and a fluorescently labeled phallotoxin (phalloidin), was used to identify regions of VnR co-distribution with filamentous actin (F-actin). One such region includes the VnR-immunoreactive foci at the margins of the cone inner segments and the actin cables that course through the photoreceptor ellipsoid and terminate within the calycal processes. A second zone of co-distribution coincides with the actin-containing, circumferential bundle at the lateral borders of the RPE cells, and a third zone is associated with the apical microvilli of the RPE that ensheath cone outer segments. In order to help identify the specific subunits underlying VnR (alpha v beta 3/5) immunoreactivity, Northern blots of retinal-RPE RNA were probed with alpha 32P-cDNAs to the human alpha v, beta 3, and beta 5 subunits and additional immunolocalization studies were performed using integrin human alpha or beta subunit-specific antisera. The results from these studies strongly suggest that one or more integrins, containing the alpha v and/or beta 5 subunits, are expressed by the photoreceptors and RPE.(ABSTRACT TRUNCATED AT 400 WORDS)

Rod photoreceptors and scotopic vision in ground aquirrels.

Ground squirrel retinas contain a relatively small complement of rods (5--10% of all photoreceptors) which are thought to provide the basis for a weak scotopic visual capacity. In a previous investigation of the California ground squirrel (Spermophilus beecheyi) involving the recording of a retinal gross potential, the electroretinogram (ERG), electrophysiological evidence for a viable scotopic signal could be obtained from some, but not all of the ground squirrels examined. To further pursue the possibility that there is a structural/functional discrepancy in the relationship between rod photoreceptors and scotopic vision in the ground squirrel, several experiments involving electrophysiological, behavioral, and anatomical observations have been conducted. We found that although about one-third of the ERGs recorded from a large sample of California ground squirrels lack those characteristics which would indicate the presence of a viable scotopic signal, the retinas of all the squirrels appear to contain the same small population of rod photoreceptors. Additional experiments on the golden-mantled ground squirrel (Spermophilus lateralis), including behavioral as well as ERG measurements and anatomical observations, lead to this same conclusion.

Scotopic and photopic vision in the California ground squirrel: physiological and anatomical evidence.

The California ground squirrel is a highly diurnal species previously thought to have an all-cone retina. This issue was re-examined in physiological and anatomical experiments. The electroretinogram (ERG) was used to measure the spectral sensitivity of the eye under different conditions of adaptation. The occurrence of a Purkinje shift could be demonstrated, although there was some indication that not all members of this species show such a shift. Spectral sensitivity of the dark-adapted eye of this squirrel is close to that predicted by a typical mammalian rhodopsin. Light adaptation produces a shift in spectral sensitivity to a peak location of about 525 nm. It was shown that two mechanisms having different spectral sensitivities contribute to the photopically recorded ERG. The degree to which these two mechanisms contribute to the ERG was found to be strikingly different from the degree to which the two contribute to visual behavior. Our anatomical results indicate that the retina of the California ground squirrel has two structurally distinct photoreceptors which, on the basis of various criteria, can be classified as cone and rod-like. The rod-like receptors comprise about 6-7% of the total. The two photoreceptor types differ in placement of their inner segments, size of their outer segments, outer segment ultrastructure, and terminal structure and organization.

Structural basis for regulation in gram-negative bacterial citrate synthases.

The citrate synthases of Gram-negative bacteria, unlike those of eukaryotes, are inhibited allosterically by NADH, but the two kinds of citrate synthase are about 30% homologous in amino acid sequence--the two Gram-negative citrate synthase sequences so far determined, from Escherichia coli and Acinetobacter anitratum, are about 70% identical. A model for the NADH-sensitive E. coli citrate synthase has been constructed using sequence homology and the known structure of the pig heart enzyme. The most reactive cysteine in the E. coli enzyme, which probably marks the NADH binding site, has now been identified as Cys-206. The model places this residue far from the active site. An E. coli citrate synthase mutant, from which a stretch of 24 amino acids has been deleted near the active site, still binds NADH normally. Two active site missense mutants of this enzyme, generated by oligonucleotide-directed mutagenesis, have lower affinities for one substrate, oxaloacetate, but also are much less sensitive to 2-oxoglutarate, an oxaloacetate analogue hitherto believed to be an allosteric inhibitor. These results confirm that NADH binds to a truly allosteric site in E. coli citrate synthase, the features of which are still to be defined; while 2-oxoglutarate is really an active-site directed inhibitor, although it may still play a regulatory role in vivo.

The TB structural genomics consortium: a resource for Mycobacterium tuberculosis biology.

The TB Structural Genomics Consortium is an organization devoted to encouraging, coordinating, and facilitating the determination and analysis of structures of proteins from Mycobacterium tuberculosis. The Consortium members hope to work together with other M. tuberculosis researchers to identify M. tuberculosis proteins for which structural information could provide important biological information, to analyze and interpret structures of M. tuberculosis proteins, and to work collaboratively to test ideas about M. tuberculosis protein function that are suggested by structure or related to structural information. This review describes the TB Structural Genomics Consortium and some of the proteins for which the Consortium is in the progress of determining three-dimensional structures.

Rapid upregulation of fibroblast growth factor receptor 1 (flg) by rat photoreceptor cells after injury.

PURPOSE: To determine the mechanism by which basic fibroblast growth factor (bFGF) exerts its neuroprotective effects on degenerating or injured photoreceptors. METHODS: Confocal immunofluorescence microscopy was used to identify sites of bFGF and FGF receptor 1 (FGFR1) expression after focal injury or experimental retinal detachment in adult rats. FGFR1 expression was analyzed immunohistochemically and at the transcription level in single photoreceptor cells, after reverse transcription (RT), using the polymerase chain reaction (PCR). Real time quantitative RT-PCR was used to measure changes in FGFR1 mRNA levels in the retina in response to injury or detachment. RESULTS: Confocal immunofluorescence observations showed that FGFR1 immunoreactivity in the rat retina is concentrated primarily in the perinuclear cytoplasm of photoreceptor cell bodies. Reverse transcription of total RNA derived from dissociated rat photoreceptor cells, followed by amplification of FGFR1 cDNA using the PCR, verified the presence of FGFR1 transcripts in normal rat photoreceptor cells; in contrast, no evidence of bFGF transcription was detected. Collectively, these results provide compelling evidence for FGFR1 gene expression by rat photoreceptors in situ. Within hours after experimental retinal detachment or focal injury, there is a twofold increase in FGFR1 immunoreactivity in the outer nuclear layer that persists for at least 7 days; a similar increase in bFGF immunoreactivity in the interphotoreceptor matrix is also apparent. This increase in FGFR1 protein levels after detachment and injury also was confirmed by western blot analysis. Real time quantitative RT-PCR analyses revealed that a rapid upregulation of FGFR1 mRNA occurred within 12 hours after retinal injury/detachment, but then declined to near baseline levels by 24 hours. CONCLUSIONS: This body of evidence strongly suggests that the photoreceptor rescue effect elicited by retinal injury as well as by injection of exogenous bFGF is mediated, at least in part, by upregulation of the FGFR1 by the photoreceptor cells.

Cytoskeletal redifferentiation of feline, monkey, and human RPE cells in culture.

Retinal pigment epithelial (RPE) cells in vivo have a polarized structure with specialized apical and basal faces. Isolated RPE cells lose but eventually regain their epithelial morphology under appropriate culture conditions. We evaluated the ability of isolated feline, primate, and human RPE cells to regain this morphology in culture with scanning electron, transmission electron, phase contrast, and immunofluorescence microscopy. In culture, isolated RPE cells lose their cuboidal shape, their apical microvilli, and their in vivo cytoskeletal organization. Stress fibers from in these cells; microtubules radiate from the cells' center to their periphery; and vimentin filaments radiate from the cells' nucleus to their periphery. As cultures become confluent, RPE cells aggregate into small groups, gradually regaining a cuboidal shape and acquiring microvilli on their apical surface. Filamentous actin redistributes to the apical face where it presumably forms the cytoskeletal core normally present in RPE microvilli. Stress fibers disappear and are replaced by a circumferential microfilament bundle (CMB). Confluent cells surrounding the colonies of differentiated RPE attain a cuboidal shape but do not show complete cytoskeletal redifferentiation. Such cells, while appearing to be differentiated by phase contrast microscopy, fail to develop a compacted CMB. In these cells, f-actin is organized as a loose peripheral band within the cell cytoplasm. Our observations indicate that confluency cannot be equated with the end stage of morphologic differentiation, and that cytoskeletal organization provides a more accurate gauge of RPE maturation in culture.

Changes in intermediate filament immunolabeling occur in response to retinal detachment and reattachment in primates.

The immunolabeling patterns for vimentin and glial fibrillary acidic protein (GFAP) were studied in five rhesus monkeys that had undergone retinal detachment or detachment and reattachment. Anti-vimentin and anti-GFAP labeling intensity increased in Müller cells after 2 days of detachment. Weak anti-vimentin labeling of the basal RPE cytoplasm, which was absent in control tissue, was detected 2 days after detachment. After detachment for 7 days and reattachment for 7 or 14 days, the pattern and extent of intermediate filament (IF) labeling changed. In Müller cells, the labeling, which in controls was restricted to processes near the vitreal border of the retina, was present in Müller processes spanning the entire retina. In retinal pigment epithelium (RPE) cells, prominent anti-vimentin labeling was identified in the basal and basolateral cytoplasm. The extent of RPE and Müller cell IF labeling in two animals whose retinas had been detached and then reattached for 150 days was different from that found at either the 7- or 14-day reattachment time points. This suggests that the abnormal IF distribution triggered by detachment may be attenuated after a lengthy period of reattachment.

Both rod and cone disc shedding are related to light onset in the cat.

Nineteen domestic cats were entrained to a 12-hr light/12-hr dark lighting cycle and killed at different times of the cycle. The numbers of rod and cone phagosomes in the retinal pigment epithelium (RPE) were quantified by light microscopy. The number of both rod and cone phagosomes shows a large increase within 2 hrs after light onset. Thereafter, the phagosome count remains low until the latter part of the dark period when the number of rod and cone phagosomes increases slightly. In a few cases, substantial variability in phagosome numbers was found between animals killed at the same times in the lighting cycle. Small (0.4-0.8 microns), dense, membrane-bound granules, that have the ultrastructural features of lysosomes, occur in large numbers in the tapetal RPE cells. The granules may contain only a homogeneous matrix or matrix plus membrane fragments or melanin. Variations in the number of such granules in different animals shows no clear diurnal pattern. Phagosome number, size, and length measurements of outer segments are used to provide estimates of cone outer segment turnover time in the cat.

Mammalian cones: disc shedding, phagocytosis, and renewal.

During the past several years we have examined a variety of different mammalian retinas for ultrastructural evidence of cone disc shedding and RPE phagocytosis. In this paper we review our previously published evidence from squirrel and human retinas as well as present new evidence of cone disc shedding in rhesus monkey and cat. All these species show definitive evidence for the shedding of discs from cone outer segments and the phagocytosis of shed discs by apical processes of the RPE; both of these events closely resemble those described for mammalian rods. The occurrence of cone disc shedding leads directly to the conclusion that new membrane must be added to the cone outer segment in order to maintain its length. The successive evaginations, which are observed at the bases of cone outer segments, we consider to be indirect evidence for the addition of new discs. Finally, we propose a model for the structural organization of mammalian cone outer segments.

Identification and localization of a beta-1 receptor from the integrin family in mammalian retinal pigment epithelial cells.

The distribution of an adhesion receptor from the integrin family was mapped in cultured mammalian retinal pigment epithelial cells (RPE), and in primate RPE cells in vivo, using antibodies to the human fibronectin receptor (FnR) in conjunction with indirect immunofluorescence and immunoelectron microscopy. Protein homogenates from human RPE or MG-63 cells were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions. On Western blots of proteins from both cell types, anti-FnR and a monoclonal antibody to the beta 1 subunit of human FnR each recognized a single band with a molecular mass of approximately 115 kDa. After 1-6 weeks in culture human, monkey and feline RPE cells gradually acquired a morphology and cytoskeletal arrangement typical of a mature cuboidal epithelium. The distribution of anti-FnR labeling changed dramatically in accordance with the cells' phenotype. In preconfluent cells, labeling consisted of streaks and flecks of fluorescence at the termini of stress fibers and at putative sites of cell substratum attachment. As the cells became confluent and acquired an epithelioid morphology, the bulk of anti-FnR labeling shifted to the peripheral cytoplasm and appeared as a cross-hatched meshwork. In fully differentiated RPE cells anti-FnR labeling consisted of a dense punctate pattern on or close to the apical cell surface that coincided with the distribution of f-actin as shown by phalloidin staining, and to the distribution of apical microvilli as identified by scanning electron microscopy. In addition, a compacted rim of fluorescence appeared at the cells' lateral margins that was also virtually identical to the phalloidin staining pattern. No basal surface labeling was apparent at this stage. At the ultrastructural level, FnR was localized to the apical surface of nonpermeabilized RPE cells and, in particular, to the plasma membrane of apical microvilli in vitro and in vivo using an indirect, pre-embedding method. The results strongly suggest that: (1) a membrane receptor/s containing the integrin beta 1 subunit is normally present on the plasma membrane of apical microvilli and on the lateral cell surfaces of cultured mammalian RPE cells; and (2) this receptor also is present on the plasmalemma of RPE apical microvilli in vivo.