Actin filament localization in developing and pathologic human corneas

Actin is associated with motility, cell morphology, and cell-substrate adhesion. The molecular probe NBD phallacidin, which reacts with filamentous actin, was used to study the distribution of actin filaments in the corneal and conjunctival epithelium, stroma, and endothelium. Frozen sections of human fetal eyes from 8 weeks to 40 weeks of gestation were reacted with NBD phallacidin. Pathologic tissues included keratoplasty specimens from patients with hereditary posterior polymorphous corneal dystrophy (PPMD) and surgically excised tissues removed for treatment of epithelial down-growth. Normal human cornea was used as a control. Immunofluorescent staining disclosed actin filament distribution in corneal epithelium as early as 9-10 weeks of gestation. Staining increased with maturation until term. Adult human corneal epithelium showed more pronounced staining of the surface layers. Stromal staining was more extensive in earlier stages of gestation and decreased in later stages of gestation, after 20-21 weeks. In pathologic corneas with posterior polymorphous dystrophy, there was localization of actin, as well as keratin, in the abnormal epithelial-like layers lining the posterior cornea. In epithelial downgrowth, actin and keratin were demonstrated in multilayered squamous epithelium on the anterior iris surface. Actin appears to be involved in migration of corneal epithelial and endothelial cells.     

Actin filaments in normal and migrating corneal epithelial cells.

The distribution of actin filaments in the cytoplasm of normal and migrating rat corneal epithelium was studied by in situ labeling with myosin subfragment-one. In normal epithelium, actin filaments are present as an apical network under microplicae of superficial cell layers. This apical actin network is possibly a cytoskeleton responsible for maintenance of the regular array of microplicae. A dramatic difference in distribution of actin filaments was noted between the normal cells and cells migrating to cover an abrasion. In migrating epithelial cells, actin filaments are concentrated is basal regions of the cells. They are present as bundles of parallel filaments near the basal plasmalemma and in networks of filaments in leading edges and cell processes. These basally located actin filaments may play a role in migration of the cells during wound healing.     

Actin filament localization in normal and migrating rabbit corneal epithelium

The molecular probe NBD phallacidin (7-nitro-benz-2-oxa-1, 3-diazolylphallacidin), which reacts specifically with filamentous actin (f-actin), was used to study the distribution of polymerized actin oligmers in normal and migrating rabbit corneal epithelial cells. In the normal cornea, the majority of the NBD phallacidin fluorescence was localized to the cortical or sub-plasma membrane area of the superficial and wing epithelial cells. Following full thickness corneal trephination injury, migrating corneal epithelial cell exhibited a marked increase in the cortical NBD phallacidin fluorescence. Transmission electron microscopy, using fixation techniques which revealed bundles of fine filaments (6nm underlying the plasma membrane of migrating epithelial cells, thus supporting the fluorescent results. These findings suggest that corneal re-epithelialization is characterized by a marked increase in the amount of filamentous actin within the migrating epithelial cells. We conclude that NBD phallacidin may be of value in analyzing changes in actin polymerization during wound healing.     

Lens actin: purification and localization

Actin was purified from the chick lens using DEAE-52 column chromatography followed by hydroxylapatite chromatography. The antibody produced against the purified actin cross-reacted specifically with lens actin from other species in addition to smooth and skeletal muscle actin and labelled the stress bundles of cultured fibroblasts. Actin was localized, using immunological methods, primarily to the plasma membrane of the epithelial and fiber cells of the chick and human lens. Actin filaments were also identified by HMM S-1 labeling in bovine cortical fiber cells. Using this procedure, the actin filaments were found throughout the fiber cell but were mainly concentrated near the plasma membrane and in cell processes. They formed a population distinct from the beaded filaments. The initial DEAE-52 column chromatography was also useful in the initial purification of lens fiber cell intermediate filament protein and two species of beta-crystallins.     

Actin in wound-healing of rabbit corneal endothelium. II. Study by nitrobenzoxadiazole-phallacidin method

The distribution of F-actin in the rabbit corneal endothelial cells was studied in vivo and in culture using nitrobenzoxadiazole-conjugated phallacidin. In the normal cornea, the fluorescence showing the presence of F-actin was observed along the membrane of the endothelial cells, but little fluorescence was seen in the cytoplasm. During wound-healing processes after transcorneal freezing, the endothelial cells migrating to the wound area showed abundant fiber-like fluorescence in the cytoplasm. In about 28 days after the injury, the endothelial cells recovered normal shape and the pattern of actin localization became normal, ie, fiber-like fluorescence localization along the cell membrane. The endothelial cells were cultured for about one week and the explants were removed. After further culture for about two weeks the cultured cells became confluent forming a monolayer. At the center of this monolayer, a small wound was made, and changes in the cell shape and actin distribution were studied. The actin distribution in the undisturbed monolayer cells was similar to that seen in vivo, ie, fiber-like fluorescence along the cell membrane. After the wound production, many cells were seen to migrate toward the wound center, and abundant fluorescent fiber-like structures were observed throughout the cytoplasm. Addition of cytochalasin B to the culture medium suppressed cell migration in a dose-dependent manner. At a high cytochalasin B concentration the fiber-like fluorescence was not formed and scattered fluorescent speckles were observed. Further culture in cytochalasin B-free medium after exposure to this agent permitted a recovery of cell migration and formation of the fiber-like actin fluorescence. It was suggested that polymerization of actin filaments is activated in the migrating cells during wound-healing, and that cytochalasin B reversibly blocks this polymerization, thereby suppressing cell migration. Actin filament polymerization would constitute a significant part of the mechanism underlying cell migration and wound-healing.     

Keratin filaments of corneal epithelial cells

Intermediate filaments with a diameter of approximately 8–10 nm are present in great abundance in the cytoplasm of corneal epithelial cells. In order to study the immunological relationship between these filaments and the tonofilaments of epidermal cells, we prepared an antiserum against keratin proteins isolated from the stratum corneum of human epidermis. Immunofluorescent staining of frozen sections of rabbit and human corneas with this anti-keratin antiserum resulted in the intense staining of the entire corneal epithelium, but no staining of the endothelium or any of the stromal components. Similar staining of cultured rabbit and human corneal epithelial cells revealed a cytoplasmic network of wavy fibers distinct from microfilaments and microtubles. Electron microscopic examination of cultured rabbit corneal epithelial cells showed that they formed a stratified squamous structure with prominent desmosomal cell-cell junctions and wavy bundles of cytoplasmic 8–10 nm filaments. These results suggest that the 8–10 nm filaments of corneal epithelium are immunologically related to tonofilaments of the epidermis and are composed of keratin proteins. Since corneal epithelium is the only keratin-containing cell type in normal cornea, anti-keratin staining provides a simple, specific and sensitive method for identifying corneal epithelial cells. Further characterization of the keratins synthesized by corneal epithelial cells under various in vivo and in vitro conditions may provide useful information concerning the mechanisms of corneal epithelial differentiation.     

Actin filaments in cells of human trabecular meshwork and Schlemm's canal

With subfragment-one of myosin used as the histochemical marker, actin filament distribution was mapped in cells of the aqueous outflow pathway. In uveal, corneal-scleral and juxtacanalicular meshwork cells, bundles of actin filaments were present along the basal cytoplasm. Some of these bundles terminated at adhesion plaques. Juxtacanalicular meshwork cells contained, in addition to bundles, randomly oriented actin filaments at the end of cell extensions. Giant vacuoles in the inner wall of Schlemm's canal did not have actin filaments associated with their membranes; there were, however, intermediate filaments present along the membrane. Bundles of actin filaments were also found within the cells of both inner and external walls of Schlemm's canal. Cells of both the meshwork and inner wall of Schlemm's canal had actin filaments extending out into the cytoplasm from cell junctions. In all areas of the meshwork and especially in the area external to Schlemm's canal, cells were observed which had a cytoplasm, very rich in actin filaments. These cells had characteristics common to smooth muscle cells. The actin filaments of cells of the aqueous outflow pathway could have any or all of several functions. Bundles of filaments in the meshwork could contract to lift and separate trabecular sheets and/or, through their association with adhesion plaques, they could stabilize cells on their collagen substrate, thus acting as cytoskeletal struts. Randomly oriented actin filaments, noted particularly in cells of the juxtacanalicular meshwork, may play a role in phagocytosis. Actin filaments found in association with junctions may be important structures for maintaining cell-to-cell contacts. Lack of actin filaments around giant vacuoles in Schlemm's canal indicates that they do not play a role in shuttling aqueous across the endothelium of the canal. The significance of "actin-rich" cells in the meshwork and those external to Schlemm's canal is unknown.     

Cytochemical localization of actin filaments in endothelial cells of rabbit trabecular meshwork

Localization of actin filaments in the endothelial cells of rabbit trabecular meshwork was studied by the nitrobenzoxadiazole-phallacidin (NBD-ph) staining method for fluorescence microscopy and modified heavy meromyosin (HMM) decoration method for electron microscopy. Endothelial cells stained with NBD-ph exhibited intense fluorescence which was apparently associated with the basal plasma membrane area. By the modified HMM decoration method, labeled actin filaments were readily detected in the prefixed endothelial cells, because of the distinctive arrowhead-like appearance, observed beneath the basal plasma membrane facing the trabecular collagen sheet. The actin filaments were arranged with dual directionality within the bundle. In contrast, intermediate (10nm) filaments in the deeper region of endothelial cells were always unlabeled with HMM. The function of the actin filament bundles in endothelial cells may be to maintain the cell shape and provide contractility of the trabecular meshwork resulting in an alteration of the outflow resistance of aqueous humor drainage.     

Cellular and cytoskeletal dynamics within organ cultures of porcine neuroretina

The aim of this study was to establish a retinal organ culture and to follow the cellular and cytoskeletal changes. For this purpose the authors detached porcine neuroretinas from the underlying pigment epithelium, and incubated them for 2 weeks under standardized conditions. After 3, 6, 10, and 13 days in culture the retinal tissues were fixed, embedded in LR-White resin or paraffin wax and processed for electron-, light-, immunofluorescence- and confocal laser scanning microscopy. Antibodies directed against alpha-tubulin, actin, glial fibrillary acidic protein (GFAP), vimentin, neurofilament(200) and beta-catenin were used to investigate the cytoskeletal changes over a certain period of time. After experimental detachment Müller cells quickly started to hypertrophy showing increased levels of intermediate filaments (i.e. vimentin and GFAP). The actin labelling of photoreceptor cells decreased concomitantly with a rapid degeneration of the outer segments. After 1 week of detachment the Müller cell cytoplasm revealed increasing amounts of actin and tubulin staining. Actin filaments appeared frequently organized in thick bundles across the full width of the retina, whereas increasing levels of tubulin shifted into the outer nuclear region especially concentrated near the outer limiting membrane. A prolonged time of explant culturing resulted in a discontinuous staining of beta-catenin along the adherent junctions of the outer limiting membrane, followed by an outgrowth of Müller cell extensions into the subretinal space. Double staining of tubulin and cones showed that this outgrowth predominantly occurred between cone inner segments. The outer limiting membrane was penetrated by stalk-like structures, highly enriched with tubulin and associated with swollen tips, reaching into the subretina. Electron microscopy demonstrated in detail the focal disruption of the outer limiting membrane by Müller cell extensions and subsequent subretinal gliosis. The cytoskeletal reactions described here were compared with degenerative changes observed after induced retinal detachments.     

Epitope variability of Bcl-2 immunolocalization in the human corneal epithelium.

PURPOSE: To further study the immunological localization of Bcl-2 protein in human corneal epithelium. METHODS: Three anti-human Bcl-2 antibodies, generated against amino acid residues (aa) 4-21 (polyclonal), 1-205 (monoclonal), and 41-54 (monoclonal), were used to localize Bcl-2 protein immunocytochemically in fresh eye bank donor human corneas. RESULTS: In the central corneal epithelium, two anti-Bcl-2 antibodies (aa 4-21 and aa 1-205) showed intense cytoplasmic staining of basal epithelial cells. These antibodies produced similar staining in the limbal epithelium, with gradually less intense staining of wing and superficial cells. By contrast, as previously reported, a monoclonal antibody to aa 41-54 stained nuclei of all epithelial cell layers with the exception of some surface corneal epithelial cells; this antibody also demonstrated very bright anti-Bcl-2 staining of Langerhans cells localized in the peripheral corneal epithelium. CONCLUSION: In our previous study, Bcl-2 protein was immunocytochemically localized to the nuclear compartment of all corneal epithelial cell layers with the use of antibodies specific for the regulatory flexible loop domain of Bcl-2. However, Bcl-2 can also be uniquely localized to the cytoplasm of the corneal epithelium with the use of antibodies generated against aa 4-21 and aa 1-205. Taken together, these results using epitope specific antibodies indicate that different epitopes on the Bcl-2 protein are available for antibody binding within different cells and cellular compartments, suggesting that proliferation and differentiation may lead to changes in the Bcl-2 structure and conformation within different compartments of the epithelial cells themselves.     

Change with age in murine corneal epithelial actin and myosin: immunofluorescent and ELISA analyses

The purpose of this study was to use immunofluorescence and ELISA immunoassay to determine whether the cellular distribution and concentration of corneal epithelial actin and myosin change with chronologic age. Diffuse anti-actin and anti-myosin indirect immunofluorescence was observed within the cytoplasm of the corneal epithelium from mice aged postnatal day (PND) 1-18 months. Additionally, highly fluorescent punctate foci were first observed in cortical cytoplasm consistently for both anti-actin and anti-myosin at PND 14. This fluorescent pattern remained relatively unchanged for the remaining ages examined. An enzyme-linked immunosorbent assay (ELISA) method was used to quantitate the amount of actin and myosin in corneal epithelium from mice aged PND 1 to 24 months. Corneal epithelial sheets were removed from whole eyes and processed for ELISA assay. Actin cellular concentration increased from PND 1-7 and decreased from PND 7-16. These results were statistically significant (p less than .005). No statistically significant difference in actin concentration was found for any of the remaining ages examined (PND 16-24 months). Myosin concentration increased from PND 1-7 and decreased until PND 14. These results also were statistically significant (p less than .005 and p less than .005, respectively). There was no significant change in myosin concentration for any of the remaining ages examined (PND 16-24 months).     

Migration mechanisms: corneal epithelial tissue and dissociated cells

The migratory mechanism of intact bovine corneal epithelial tissue and individual corneal epithelial cells over synthetic surfaces in vitro were compared. In migrating tissue, adhesion between component cells was demonstrated by immunostaining for desmoplakin and identification of desmosomes by electron microscopy. The apparent intermeshing of microtubules within the tissue and interdigitation of cytoplasmic membranes showed the close association of cells. Portions of the advancing edge of the tissue contained actin filaments that were orientated parallel to the leading tissue front. These filaments appeared to span adjacent cells suggesting that migration partially involved the contraction of the actin cable, similar to the 'purse-string' mechanism originally identified in the closure of fetal skin wounds. Intact actin filaments and microtubules were necessary to maintain optimum migration rates for tissue and cells. However, tissue morphology was not dependent on microtubule integrity. During the migration of individual epithelial cells, no staining for desmoplakin was observed and there were clear divisions between the microtubules of adjacent cells. Actin filaments tended to be arranged parallel to the direction of cell movement.Therefore, migration of epithelial tissue sheets over synthetic surfaces occurs by mechanisms that differ from the migration of individual epithelial cells. Model systems based on the migration of intact tissue would give a more realistic assessment of the suitability of a material for biomaterial applications than the use of separate epithelial cells.     

The suprabasal layer of corneal epithelial cells represents the major barrier site to the passive movement of small molecules and trafficking leukocytes

AIMS: To investigate the site of barrier function to the passive diffusion of a small molecule (phalloidin) in the corneal epithelium in the mouse. METHODS: Penetration of phalloidin (molecular weight 1115 daltons) into the cornea was evaluated by studying fluorescent binding of phalloidin to actin in tissue sections, in whole mount preparations, and in the fixed intact globe by confocal microscopy. In addition, the location of tight junction proteins in the individual layers of the corneal epithelium was determined by immunohistochemistry. RESULTS: Phalloidin staining of corneal sections was positive in all corneal layers in tissue sections and in all layers of the corneal epithelium except the suprabasal layer in excised fixed whole mounts of the cornea. However, when phalloidin staining was attempted in intact fixed globes, before excision of the cornea for whole mount preparation, only the most superficial layer of cells was stained indicating that phalloidin could not penetrate the tissue beyond the suprabasal epithelial layer. Detergent (Triton X-100) treatment of the excised cornea and the intact fixed globe, allowed penetration of phalloidin into the suprabasal epithelial layer. Tight junction proteins occludin, ZO-1 and claudin were present in most layers of the cornea but while ZO-1 and occludin were distributed in a typical pericellular pattern, claudin seemed to be particularly prominent in the suprabasal layer and appeared only as a discontinuous punctate pericellular pattern in the superficial layer. Intraepithelial leukocytes were detected in the superficial epithelium and the basal epithelium but not in the suprabasal epithelium. CONCLUSION: The suprabasal epithelium cell layer appears to represent the main barrier site to the passage of small molecules and cells in the mouse cornea and this property may be attributable to prominent claudin expression in this layer.     

连接黏附分子-1和咬合蛋白在人角膜上皮中的表达

目的检测咬合蛋白（0ccludin）和连接黏附分子-l（junction adhesion mo1ecule-l，JAM-1）在正常人角膜上皮各层中的表达。方法培养人角膜上皮细胞提取细胞总RNA，以逆转录后获得的cDNA为模板，PCR扩增目的基因JAM-1及0ccludin。流式细胞仪检测JAM-l蛋白表达。双重免疫荧光观察JAM-l与0ccludin在正常人角膜上皮组织的原位表达。结果RT-PCR在培养人角膜上皮细胞中检测到JAM-l与0ccludin扩增片段；流式细胞仪检测到JAM-l蛋白表达；双重免疫荧光结果显示0ccludin染色主要位于表层上皮层细胞之间；而JAM-l荧光染色不仅见于上皮表层，在整个上皮层细胞之间均可见其荧光反应。结论 0ccludin主要位于正常人角膜上皮表层细胞之间，JAM-1在正常人角膜上皮的全层中均有表达。     

Actin in polygonal arrays of microfilaments and sequestered actin bundles (SABs) in lens epithelial cells of rabbits and mice

The pattern of localization of actin filaments was compared in whole mounts of lens epithelium of rabbit and mouse using the fluorescently-labeled actin-specific probe, rhodamine-phalloidin. In the adult rabbit lens, fluorescent polygonal arrays consisting of central vertices and interconnecting filaments were present in the apical end of each epithelial cell. Electron microscopy confirmed that these arrays lined the cytoplasmic side of the apical membrane. In the mature adult mouse, polygonal arrays were not seen either with fluorescence or electron microscopy. Instead, the actin was packaged in a single, elongated, often curved bundle near the epithelial cell apex, referred to as a "sequestered actin bundle" or SAB. The SAB often appeared attached to the plasma membrane and to approach the perinuclear basket of microfilaments. The significance of the differences in these two patterns of actin is discussed in terms of differences in the accommodative ability and static lens shape in these two animals.     

In vivo and in vitro demonstration of epithelial cell-induced myofibroblast differentiation of keratocytes and an inhibitory effect by amniotic membrane

PURPOSE: To examine the role of epithelial cells in inducing the differentiation of keratocytes into myofibroblasts and to determine whether this effect may be inhibited by amniotic membrane matrix. METHODS: In vivo, a 9-mm diameter, partial-thickness corneal flap was created in 12 rabbit eyes (6 rabbits), which were equally subdivided into three groups. The first group was implanted with one layer of a 6-mm diameter human amniotic membrane, from which the epithelium had been removed by dispase. The second group received an implantation of dispase-treated amniotic membrane with cultured rabbit corneal epithelial cells. The third group received the same implantation as the second group except that the cultured corneal epithelial cells were sandwiched between two layers of membrane. All corneas were removed 2 weeks later and were subjected to Masson trichrome staining and immunofluorescence staining with monoclonal antibodies to alpha-smooth muscle (alpha-SM) actin for myofibroblasts and cytokeratins for epithelial cells. In vitro collagen gels impregnated with different types of human ocular surface fibroblasts were seeded with or without rabbit corneal epithelial cells before testing for gel contraction. RESULTS: Positive staining of alpha-SM actin was noted only in keratocytes adjacent to corneal epithelial cells at the incision site and those grown on the basement membrane side of the amniotic membrane. Negative staining was noted when epithelial cells were removed by dispase or when cultured corneal epithelial cells were sandwiched between two layers of membrane. Gel contraction by fibroblasts was significantly promoted when epithelial cells were seeded on the gel. In the latter situation, positive staining of alpha-SM actin was noted in fibroblasts subjacent to epithelial cells but not in those impregnated in the gel. CONCLUSION: Epithelial cells are capable of inducing the differentiation of adjacent fibroblasts into myofibroblasts; such an induction requires a close epithelial-mesenchymal contact. Amniotic membrane alone does not induce this effect and can help block such induction by epithelial cells.     

连接粘附分子-1在人角膜上皮中的表达

目的：检测连接粘附分子-1（junction adhesion molecule,JAM-1）在正常人角膜上皮各层中的表达及分布特点并与咬合蛋白（occludin）进行比较。 方法：培养人角膜上皮细胞提取细胞总RNA。以逆转录后获得的cDNA为模板PCR扩增目的基因JAM-1及occludin。流式细胞仪检测JAM-1蛋白表达。双重免疫荧光观察JAM-1与occludin在正常人角膜上皮组织的原位表达。 结果：通过RT—PCR在培养人角膜上皮细胞中检测到JAM-1与occludin扩增片段;流式细胞仪检测到JAM-1蛋白表达;双重免疫荧光结果显示occludin染色主要位于表层上皮层细胞之间;而JAM-1荧光染色不仅见于上皮表层,在整个上皮层细胞之间均可见其荧光反应。 结论：Occludin主要位于正常人角膜上皮表层细胞之间,JAM-1在正常人角膜上皮的全层中均有表达。     

Expression of junctional adhesion molecule -1 in human corneal epithelium

AIM: To investigate the expression and distribution of junction adhesion molecule-1(JAM-1) in human corneal epithelium and compare with those of occludin. METHODS: The expression in RNAs of JAM-1 and occludin was revealed by RT-PCR and the presence of protein was analyzed by the FACS method. Double immunofluorescent staining was used to determine the tissue distribution of JAM-1 and occludin in human corneal epithelium. RESULTS: The expression of JAM-1 and occludin was found in cultured human corneal epithelial cells. The double immunofluorescent study showed positive staining for JAM-1 at cell borders in the entire epithelial layer, while relatively extensive staining was seen in the superficial layer, where it coexisted with the expression of occludin. CONCLUSION: JAM-1 is expressed in entire layer of human corneal epithelium encircling the cells.