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.     

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     

Gap junctions between sertoli and germ cells of rat seminiferous tubules

Ultrastructural observations of rat seminiferous tubules show clearly the presence of plasma membrane junctions between Sertoli and germ cells in the basal and adluminal compartments. Results obtained from the freeze fracture and thin section techniques were correlated in order to elucidate the nature of these intercellular junctions. We suggest that these intercellular membrane specializations are gap junctions which occur within regions of plasma membrane that also exhibit adherens-like modifications.     

Comparison of actin-filament bundles in myoid cells and Sertoli cells of the rat,golden hamster and mouse

Testes of adult rats, golden hamsters and mice were fixed with paraformaldehyde. Seminiferous tubules were then isolated by collagenase dissociation, stained with fluorescent phallotoxin, and viewed in a confocal laser microscope to observe actin filaments. Bundles of actin filaments in the myoid cells, especially in the rat, were arranged at right angles to each other in relation to the longitudinal axis of the tubule. In the hamster, circumferentially directed bundles were more frequent than longitudinally directed bundles. The actin bundles in the mouse were thinner than those in the rat and hamster, and their lattice network was less prominent. Nuclei of the myoid cells were elliptical and their short diameters were parallel to the long axis of the seminiferous tubules in the animals examined. Areas of myoid cells and of basal junctional portions of Sertoli cells were measured and compared in all animals studied. There were significant differences in the areas among the three species. The golden hamster showed the largest value for myoid-cell area, and the mean value for Sertoli-cell area was highest in the mouse.     

Endocytosis in the rat retinal pigment epithelium

Endocytosis in the retinal pigment epithelium (RPE) of rats was studied using horseradish peroxidase, microperoxidase and ferritin tracers. Tracer uptake was mediated by coated pits and coated vesicles. Coated pits formed at two discrete regions at the RPE plasma membrane: that portion of basal membrane directly opposing Bruch's membrane, and at the bases of the apical lamellae and villi. Two populations of coated vesicles were identified and distinguished by size, location and function. Large coated vesicles (91.8 +/- 14.7 nm in diameter) were located near the cell surface and incorporated tracer. Small coated vesicles (64.5 +/- 15.7 nm diameter) located more deeply within the cell were not tracer-labeled, and were often fused with the endoplasmic reticulum or the Golgi apparatus. Observations of the endocytic pathway in rat RPE cells are presented. Tracer was also found in organelles of the lysosomal system, e.g. the multivesicular body, but was not identified in the smooth endoplasmic reticulum or Golgi apparatus.     

Ezrin, a membrane-organizing protein, as a polarization marker of the retinal pigment epithelium in vertebrates

Immunoreactivity for ezrin, a membrane-organizing phosphoprotein that tethers actin microfilaments to cell membrane proteins, was evaluated as a polarization marker in the intraocular neuroepithelial cells of vertebrates, especially in the retinal pigment epithelium (RPE). Six fetal human eyes representing the 14th-28th gestational weeks, 9 normal adult eyes, 12 eyes with intraocular tumors, and 26 eyes from 15 other vertebrate species were analyzed by immunohistochemistry using the avidin-biotinylated peroxidase complex (ABC) method and monoclonal antibody (mAb) 3C12 to ezrin. The apical cytoplasm and microvilli of the human RPE always reacted with mAb 3C12, but the basal cytoplasm was labeled in reactive RPE only. In autopsy eyes and if fixation was delayed, ezrin immunoreactivity in RPE was more diffuse. Developing RPE became gradually immunoreactive from the 14th week of gestation onward. The microvilli of the baboon, pig, raccoon dog, cow, and rat RPE cells were likewise labeled, and their basal cytoplasm was variably immunoreactive as well, but the microvilli of the avian RPE did not react with the antibody used. In all six mammals mentioned, both layers of the ciliary epithelium and the anterior iris epithelium reacted for ezrin, and the posterior epithelium was weakly labeled in pig, cow, and rat eyes. Normal peripheral and reactive human retina, and normal baboon, pig, raccoon dog, cow, rat, black grouse, and jay eyes, showed immunoreaction for ezrin in Müller cells, usually in their microvilli. Ezrin is widely found in RPE and anterior segment neuroepithelia of the mammalian eye, in which it may segregate membrane proteins to specific membrane surfaces, especially to the apical microvilli of the RPE, which intimately interact with outer segments of photoreceptor cells. The ezrin gene on human chromosome 6q25-26 is consequently a candidate gene for causing retinal degenerations.     

Distribution of glucose transporters and insulin receptors in the plasma membrane and transverse tubules of skeletal muscle

The distribution of glucose transporters and of insulin receptors on the surface membranes of skeletal muscle was studied, using isolated plasma membranes and transverse tubule preparations. (i) Plasma membranes from rabbit skeletal muscle were prepared according to Seiler and Fleischer (1982, J. Biol. Chem. 257, 13862-13871), and transverse tubules from rabbit skeletal muscle were prepared according to Rosemblatt et al. (1981, J. Biol. Chem. 256, 8140-8148) as modified by Hidalgo et al. (1983, J. Biol. Chem. 258, 13937-13945). The membranes were identified by the abundance of nitrendipine receptors in the transverse tubules, and their relative absence from the plasma membranes. (ii) Plasma membranes and transverse tubules were also isolated from rat skeletal muscle, according to a novel procedure that isolates both fractions from the same common homogenate. (iii) Glucose transporters were detected by D-glucose protectable binding of the specific inhibitor [3H]cytochalasin B, and insulin receptors were detected by saturable binding of 125I-insulin. The concentration of glucose transporters was about threefold (rabbit) or fivefold (rat) higher in the transverse tubule membrane compared to the plasma membrane, whereas the insulin receptor concentration was about the same in both membranes. These results indicate that the glucose transporters on the surface of the muscle are preferentially segregated to the transverse tubules, and this poses interesting consequences on the functional response of glucose transport to insulin in skeletal muscle.     

Ezrin promotes morphogenesis of apical microvilli and basal infoldings in retinal pigment epithelium

Ezrin, a member of the ezrin/radixin/moesin (ERM) family, localizes to microvilli of epithelia in vivo, where it bridges actin filaments and plasma membrane proteins. Here, we demonstrate two specific morphogenetic roles of ezrin in the retinal pigment epithelium (RPE), i.e., the formation of very long apical microvilli and of elaborate basal infoldings typical of these cells, and characterize the role of ezrin in these processes using antisense and transfection approaches. In the adult rat RPE, only ezrin (no moesin or radixin) was detected at high levels by immunofluorescence and immunoelectron microscopy at microvilli and basal infoldings. At the time when these morphological differentiations develop, in the first two weeks after birth, ezrin levels increased fourfold to adult levels. Addition of ezrin antisense oligonucleotides to primary cultures of rat RPE drastically decreased both apical microvilli and basal infoldings. Transfection of ezrin cDNA into the RPE-J cell line, which has only trace amounts of ezrin and moesin, sparse and stubby apical microvilli, and no basal infoldings, induced maturation of microvilli and the formation of basal infoldings without changing moesin expression levels. Taken together, the results indicate that ezrin is a major determinant in the maturation of surface differentiations of RPE independently of other ERM family members.     

Intramembrane particle structures in epithelial cells of the toad urinary bladder: a quantitative freeze-fracture study

Freeze-fracture electron microscopy reveals intramembrane particle arrays in basal membranes of granular epithelial cells as well as both upper and lower plasma membranes of the underlying basal cells in the toad urinary bladder. These particle arrays are morphologically indistinguishable from the luminal membrane aggregates which are known to be associated with antidiuretic hormone (ADH)-stimulated water transport. In both granular and basal cells particle arrays are frequently located in and/or around the openings of vesicular and/or tubular structures fused to the plasma membranes, suggesting that they may be transferred from the cytoplasm by membrane fusion. Quantification of cytoplasmic aggrephores in control granular cells shows that they can be numerous and as close to the basolateral membrane as they are with the luminal membrane, to which they are known to fuse and deliver aggregates upon ADH stimulation. Aggrephore-like tubules were also found in the basal cells. Particle array densities were quantified for 6 pairs of control and ADH-stimulated hemibladders. At least 1440 microns 2 area of plasma membrane for each membrane domain was examined. Results indicate that the presence of these particle arrays in granular and basal cell membranes is highly variable and that exposure to ADH does not cause a statistically significant increase in their frequency.     

Immunocytochemical localization of Na+,K(+)-ATPase in rat retinal pigment epithelial cells

We investigated quantitatively the ultrastructural localization of the alpha-subunit of Na+,K(+)-ATPase in rat retinal pigment epithelial cells by the protein A-gold technique, using an affinity-purified antibody against the alpha-subunit of rat kidney Na+,K(+)-ATPase. Immunoblot analysis showed that the antibody bound specifically to the alpha- and alpha(+)-subunits of Na+,K(+)-ATPase in the whole retina [the sensory retina plus retinal pigment epithelium (RPE)]. Rat eyes were fixed by perfusion with 4% paraformaldehyde containing 1% glutaraldehyde and embedded in Lowicryl K4M. Ultra-thin sections were incubated with affinity-purified antibody against the alpha-subunit of rat kidney Na+,K(+)-ATPase and subsequently with protein A-gold complex. Light microscopy with a silver enhancement procedure revealed Na+,K(+)-ATPase localized to both the apical and the basal plasma membrane domains of the RPE. Quantitative immunocytochemical analysis by electron microscopy showed a higher density of gold particles on the apical surface than on the basolateral one. Microvilli are so well developed on the apical surface of the RPE that the apical surface profile is much longer than the basolateral one. This means that Na+,K(+)-ATPase is mainly located on the apical surface of the RPE cells.     

Biogenesis of transverse tubules in skeletal muscle in vitro

The transverse (T) tubules of skeletal muscle are membrane tubules that are continuous with the plasma membrane and penetrate the mature muscle fiber radially to carry surface membrane depolarization to the sites of excitation-contraction coupling. We have studied the development of the T-tubule system in cultured amphibian and mammalian muscle cells using a fluorescent lipid probe and antibodies against T-tubules and plasma membranes. Both the lipid probe and the T-tubule antibody recognized an extensive tubular membrane system which subsequently differentiated into the T-system. At all developmental stages, the molecular composition of the T-system was distinct from that of the plasma membrane, suggesting that during myogenesis T-tubules and the plasma membrane form independently from each other and that exchange of membrane proteins between the two continuous compartments is restricted. In rat muscle cultures, T-tubule-specific antigens were first expressed in terminally differentiated myoblasts. Prior to myoblast fusion the antigens appeared as punctate label throughout the cytoplasm. Shortly after fusion the T-tubule-specific antibody labeled a tubular membrane system that extended from the perinuclear region and penetrated most parts of the cells. In contrast, the lipid probe, which labels the T-tubules by virtue of their direct continuity with the plasma membrane, only labeled short tubules extending from the plasma membrane into the periphery of the myotubes at the early stage in development. Thus, the assembly of the T-tubules appears to begin before their connections with the plasma membrane are established.     

The ultrastructure of Malpighian tubules and hindgut of Frankliniella occidentalis (Pergande) (Thysanoptera : Thripidae)

The ultrastructure of the western flower thrips, Frankliniella occidentalis (Pergande) (Order : Thysanoptera), has 4 Malpighian tubules that are free of the intestine as they leave their junction at the pyloric region. The tubules consist of an epithelium with a single type of microvillated cells; proximally, the cells are lined by a thin cuticle. Numerous mitochondria, basal infoldings of the plasma membrane and vesicles with varying densities suggest active transit of fluid in the cell for osmoregulation. Two of the Malpighian tubules are bent posteriorly and closely adhere to the hindgut in the region of the rectal pads where the 2 epithelia are separated only by a basal lamina. The ultrastructure of this region suggests possible fluid reabsorption from the gut lumen.     

Acetylcholinesterase and butyrylcholinesterase in the gut mucosal cells of various mammal species: distribution along the intestine and molecular forms

1. A cholinesterase activity was shown to be present in the homogenates of the gut mucosal cells from seven mammal species examined. 2. The distribution of the cholinesterase activity in the mucosal cells along the intestine differs from one species to another. This distribution is not correlated with that of the aminopeptidase which is a specific marker of the enterocyte plasma membranes. 3. Except rabbit, all the other species contain a (G4) globular tetrameric form and either a (G1) monomeric form (pig, ox) or a (G2) dimeric form (mouse, rat, sheep). Both (G1) and (G2) forms are found with the (G4) form in the mucosal cells of kitten and cat. The solubility characteristics of these various forms were studied by sucrose gradient centrifugations in the presence and the absence of 1% Triton X-100. 4. The mucosal cells from the studied species essentially possess either acetylcholinesterase (rabbit, kitten, cat) or butyrylcholinesterase (ox, pig, sheep, rat, mouse). These findings indicate that both enzymes probably present identical physiological functions in this cell type.     

II. Ultrastructure of duct cell granules in mammalian submaxillary glands

Summary Discrete, PAS-positive granules of relatively uniform electron-density and size characterise the intercalated duct cells of mammalian submaxillary glands. Smaller, electron-dense organelles are seen in the cells at the junction of the intercalary-striated duct region in the guinea-pig. The large granules of variable electron-density which are observed in the proximal, modified intercalary cells in the rabbit closely resemble the granules in the acinar cells of the guinea-pig. Several populations of granules differing in size are found in the striated granular tubules of the rat and hamster; the organelles in the rat show two grades of electron-density whereas those in the hamster are uniformly dense. Numerous small granules with compactly arranged intragranular material occupy the apical part of the striated ducts of the cat, dog and rabbit. The chemical composition of each population of duct cell granules is unknown. The question whether granules containing kallikrein, trypsin-like enzymes and amylase are stored in the duct cells is discussed.We wish to thank The Wellcome Trust for a travel grant to attend the International Symposium on Vasopeptides (Florence, July 1971) where this work was briefly reported.     

Identification and partial characterization of five major membrane glycoproteins of BHK fibroblasts

Summary Five major membrane glycoproteins of the BHK-B4 hamster fibroblast plasma membrane have been identified by binding specific rabbit antibodies to the cell surface and by recovering the detergent solubilized immunocomplexes with Protein A-Sepharose immunoadsorption. These glycoproteins, designated as gp45, gp65, gp95, gp130 and gp140, are exposed at the cell surface since: (i) they were accessible to antibodies in intact viable cells; (ii) they were radioiodinated by the lactoperoxidase-glucose oxidase procedure; and (iii) they were cleaved by proteolytic enzymes in conditions affecting only the cell surface. Among these glycoproteins the gp130 is the predominant component and its exposed portion is characterized by lack of sensitivity to trypsin cleavage. Glycoproteins of different molecular weight, but immunologically related to the major hamster membrane glycoproteins, have been detected at the surface of both rat and mouse fibroblasts.     

Retinal epithelial fine structure in the southern fiddler ray (Trygonorhina fasciata).

The morphology of the retinal epithelium (RPE), choriocapillaris and Bruch's membrane (complexus basalis) has been investigated by light and electron microscopy in an elasmobranch, the southern fiddler ray or guitarfish (Trygonorhina fasciata). The RPE consists of a single layer of cuboidal cells which display basal (scleral) infoldings as well as numerous apical (vitreal) finger-like processes which interdigitate with the photoreceptor outer segments. The lateral cell borders are relatively smooth and are joined in the mid-region by a series of tight junctions. Internally the RPE nucleus is large, vesicular and centrally located. Smooth endoplasmic reticulum (SER) is abundant while rough endoplasmic reticulum (RER) is scarce. Polysomes are however widespread and mitochondria are plentiful. Two unusual organelles are also noted. One consists of a membrane bound array of tubules while the other is a membrane bound structure consisting of a granular matrix with again an internal tubular array. This species possesses a choroidally located tapetum lucidum in the superior fundus and over this tapetal area, melanosomes are absent from the RPE cells. In non-tapetal locations a few melanosomes are present that do not appear to undergo photomechanical movements. Bruch's membrane is a pentalaminate structure with an almost continuous central elastic layer (lamina densa). The choriocapillaris forms a single layer of capillaries with a thin but only minimally fenestrated endothelium facing Bruch's membrane.     

Immunocytochemical localization of a urate oxidase immunoreactive protein in the plasma membranes and membranes of the secretory/endocytic compartments of digestive gland cells of the mussel Mytilus galloprovincialis

The subcellular compartmentalization of urate oxidase (UOX) in the digestive glands of mussels, Mytilus galloprovincialis Lmk, was studied by means of immunoblotting and immunocytochemistry, using an antibody raised in rabbit against rat liver UOX. Western blot analysis of subcellular fractions revealed an immunoreactive polypeptide with a molecular weight similar to the corresponding mammalian hepatic protein. This crossreactive polypeptide of 32 kDa was particle-bound yet not peroxisome-associated. In paraffin sections the antiserum specifically labeled the plasma membrane of the digestive gland epithelial cells and discrete regions within the perinuclear and apical portions of the digestive tubules and duct cells. By electron microscopy gold particles representing antigenic sites were found on the microvilli and the lateral plasma membrane as well as the membranes of the secretory/ endocytic compartments, that is, the Golgi complex, secretory and some endocytic vesicle membranes. Since the peroxisomal UOX-antibody exhibits a comparable immunoreactivity towards a urate-transporter channel protein in rat kidney proximal tubules and has been used for its molecular cloning (Leal-Pinto et al., 1997, J. Biol. Chem. 272, 617-625), we suggest that the membrane protein identified in mussel digestive glands could represent a homologous urate-transporter protein.     

The renal cortical lymphatic system in the rat, hamster, and rabbit

Rat, hamster, and rabbit renal cortical lymphatics were examined by light and electron microscopy. Rat and hamster kidneys possessed both intra- and interlobular lymphatics that were structurally similar at the light microscopic level. Ultrastructural examination of the hamster lymphatic endothelium, however, revealed an unusual arrangement of cytoplasmic extensions not seen in the other two species. The intralobular lymphatics were related primarily to tubules, afferent arterioles, and renal corpuscles and were consistent with lymph formation from both plasma filtrate and tubular reabsorbate. Interlobular lymphatics were seen in connective tissue associated with the interlobular blood vessels. Rabbit cortex contained only interlobular lymphatics. Cross-sectional area, maximum diameter, volume density, and profile density were determined by stereological measurements using a computer-based image analyzer. The morphological data from the rat were used, in combination with published values for lymph flow, to calculate the rate of lymph formation per unit area of endothelium in lymphatics of the renal cortex. Among kidneys fixed by retrograde perfusion, the cortical lymphatic system was most extensive in maximum diameter, volume density, and profile density. It was smallest in the rabbit and intermediate in the rat. Lower volume and profile density were found for rat kidneys fixed by the dripping technique. It was concluded that: tubular reabsorbate probably contributes to renal lymph in the rat and hamster, but not in the rabbit; significant differences exist in the extent of the renal lymphatic systems among the three species, with the hamster kidney having the richest network and the rabbit the poorest; the method of fixation influences the measured size and density of renal cortical lymphatics; and the estimated rate of lymph formation in the kidney of the rat is roughly comparable to that in the dog.     

Species-specific difference in distribution of voltage-gated L-type Ca(2+) channels of cardiac myocytes

Ca²⁺influx via sarcolemmal voltage-dependent Ca²⁺ channels(L-type Ca²⁺ channels) is the fundamental step inexcitation-contraction (E-C) coupling in cardiac myocytes.Physiological and pharmacological studies reveal species-specificdifferences in E-C coupling resulting from a difference in thecontribution of Ca²⁺ influx and intracellularCa²⁺ release to activation of contraction. We investigatedthe distribution of L-type Ca²⁺ channels in isolatedcardiac myocytes from rabbit and rat ventricle by correlativeimmunoconfocal and immunogold electron microscopy. Immunofluorescence labeling revealed discrete spots in the surface plasma membrane and transverse (T) tubules in rabbit myocytes. In ratmyocytes, labeling appeared more intense in T tubules than in thesurface sarcolemma. Immunogold electron microscopy extended thesefindings, showing that the number of gold particles in the surfaceplasma membrane was significantly higher in rabbit than rat myocytes.In rabbit myocyte plasma membrane, the gold particles were distributedas clusters in both regions that were associated with junctionalsarcoplasmic reticulum and those that were not. The findings areconsistent with the idea that influx of Ca²⁺ via surfacesarcolemmal Ca²⁺ channels contributes to intracellularCa²⁺ to a greater degree in rabbit than in rat myocytes.