Morphological changes in the zonula adhaerens during embryonic development of chick retinal pigment epithelial cells

Summary Retinal pigment epithelial cells from chicks at various stages of development were examined by transmission electron microscopy to determine how the adult form of the zonula adhaerens, composed of subunits termed zonula adhaerens complexes, is acquired. During early stages of development, between embryonic day 4 and embryonic day 7, the intermembrane discs of zonula adhaerens complexes appear to be formed from material already present between the junctional membranes of the zonulae adhaerentes. In contrast, the cytoplasmic plaque material of the zonulae adhaerentes is difficult to detect before hatching; it is seen as a dense band along the junctional membranes at hatching and as individual subunits in register with the intermembrane discs in adult retinal pigment epithelial cells. After embryonic day 16, when the zonulae adhaerentes increase dramatically in size, single zonula adhaerens complexes are also present basal to the zonulae adhaerentes along the lateral cell membrane. This suggests that, during later stages of development, the junctions grow in size and/or turn over by the addition of pre-assembled zonula adhaerens complexes.Abbreviations CMB Circumferential microfilament bundle - ZA Zonula adhaerens - ZAC Zonula adhaerens complex - RPE Retinal pigment epithelium     

A quantitative study of intramembrane changes during cell junctional breakdown in the dystrophic rat retinal pigment epithelium

Previous electron microscope freeze-fracture and tracer studies have revealed that intercellular junctions in the retinal pigment epithelium (RPE) of Royal College of Surgeons (RCS) rats with inherited retinal dystrophy [5] break down between three and six postnatal weeks [6, 7]. In this study quantitative computer techniques were used to analyze the freeze-fracture changes in the dystrophic RPE. The following parameters were measured: length of tight junctional strands/micron2; number of tight junctional strand anastomoses/micron2; number of gap junctional aggregates/micron2; area of gap junctional aggregates/micron2; and density of background intramembrane particles/micron2. At three postnatal weeks, the dystrophic junctional complex membrane is similar to normal, but at 10 weeks and later there are dramatic decreases in tight junctional strand length/micron2 and number of anastomoses/micron2, as well as in the number/micron2 and area of gap junctions/micron2, while the density of background particles/micron2 is dramatically increased. Correlational analysis revealed that changes in gap and tight junctions were significantly related to each other and to the increase in background particle density. The diameter of background particles within the normal and post-breakdown dystrophic junctions was measured in order to see whether the dispersal of gap and tight junctional particles (8-10 nm) into the surrounding membrane contributes to the increased particle density. These measures showed that background particles in all size ranges were more numerous in the dystrophic RPE, but that the largest increase was in the smallest diameter particles (6-7 nm). Thus, while gap and tight junctional sized particles contribute to the increase, particles from other sources may also be involved. Particle density of apical and basal membranes in the normal and in the 10 week and older dystrophic RPE was analyzed to study the effects of tight junctional breakdown on the distribution of intramembrane particles. These measures showed that particle density was greater basally than apically in the normal RPE and that particle density in both membranes decreased slightly in the dystrophic RPE, but that their ratio remained unchanged. It has been shown previously that even a single intact tight junctional strand is sufficient to maintain differences in particle density between apical and basal surfaces [14, 15] and in the majority of abnormal dystrophic junctional complexes at least one tight junctional strand remains intact.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Modulation of cytoskeletal organization during insect follicle cell morphogenesis

Follicle cell morphogenesis during Rhodnius oogenesis involves extensive changes in lateral follicle cell shape, creating a patent epithelium. Cytoskeletal elements are involved in this cell shape change as assessed by investigating the relative abundance, orientation and dynamics of the follicle cell microtubule and microfilament cytoskeleton. Anti-tubulin immunofluorescence and transmission electron microscopy revealed the cytoskeletal organization from pre-follicular to post-vitellogenic follicle stages. A well-developed cylindrical arrangement of longitudinally orientated microtubules is present beneath the plasmalemma of the non-patent pre-vitellogenic and apical vitellogenic follicle cells. In contrast patent lateral vitellogenic follicle cells contain a dispersed distribution of microtubules in both longitudinal and cross-sectional planes. Prominent microfilament bands are not abundant in the pre-vitellogenic or apical vitellogenic follicle cells. The lateral vitellogenic follicle cells do however contain a prominent band of microfilaments in the subplasmalemmal area and in the projections connecting to adjacent cells and the apical microvilli. The changes in cytoskeletal arrangement in lateral follicle cells during vitellogenesis emphasize a third essential component, in addition to juvenile hormone stimulated [NA(+)K(+)] ATPase cell shrinkage, and cell junctional modulation, for the formation of a patent follicle cell epithelium in Rhodnius.     

Ultrastructural and immunocytochemical analysis of the circumferential microfilament bundle in avian retinal pigmented epithelial cells in vitro

Summary The dedifferentiated phenotype of pigmented epithelial cells in vitro is bipotential and is effected by environmental alterations mediated by the cell surface and associated cytoskeleton. We have begun an investigation into the role that contractile microfilaments play in maintaining cell contact and cell shape in retinal pigmented epithelial cells in vitro. In this paper, we report a structural analysis of the intersection of the circumferential microfilament bundle with the cell membrane of cultured pigmented epithelial cells from chick retina. Techniques of electron microscopy, including freezefracturing and deep-etching, reveal that microfilaments of this bundle associate with a junctional complex in the apical cell compartment and with membrane domains which are not components of the junction. Microfilaments link with the cell membrane either at their termini or along the membrane-apposed surface of the circumferential bundle. Furthermore, we report the immunocytochemical localization of filamin (a high molecular weight actin-binding protein, which forms fiber bundles and sheet-like structures when bound with Factin in solution) in the circumferential/microf相似文献   

Preservation and phallotoxin-staining of the microfilament system in Amoeba proteus

The spatial organization of the microfilament system as the main component of the cytoskeleton in Amoeba proteus was preserved by a glutaraldehyde-lysine-fixation and visualized with fluorescent phallotoxins (NBD- phallacidin , R-phalloidin). Results obtained by means of this method coincide exactly with observations gained from immunocytochemical, ultrastructural and molecular cytochemical studies, i.e., the microfilament system is mainly displayed beneath the cell membrane, at the hyalo - granuloplasmic border and around the cell nucleus. The preparation procedure employed is suitable for the rapid demonstration of cytoplasmic microfilaments in cells difficult to preserve by chemical fixation.     

Development of Sertoli cell junctional specializations and the distribution of the tight-junction-associated protein ZO-1 in the mouse testis

Basally located tight junctions between Sertoli cells in the postpubertal testis are the largest and most complex junctional complexes known. They form at puberty and are thought to be the major structural component of the "blood-testis" barrier. We have now examined the development of these structures in the immature mouse testis in conjunction with immunolocalization of the tight-junction-associated protein ZO-1 (zonula occludens 1). In testes from 5-day-old mice, tight junctional complexes are absent and ZO-1 is distributed generally over the apicolateral, but not basal, Sertoli cell membrane. As cytoskeletal and reticular elements characteristic of the mature junction are recruited to the developing junctions, between 7 and 14 days, ZO-1 becomes progressively restricted to tight junctional regions. Immunogold labeling of ZO-1 on Sertoli cell plasma membrane preparations revealed specific localization to the cytoplasmic surface of tight junctional regions. In the mature animal, ZO-1 is similarly associated with tight junctional complexes in the basal aspects of the epithelium. In addition, it is also localized to Sertoli cell ectoplasmic specializations adjacent to early elongating, but not late, spermatids just prior to sperm release. Although these structures are not tight junctions, they do have a similar cytoskeletal arrangement, suggesting that ZO-1 interacts with the submembrane cytoskeleton. These results show that, in the immature mouse testis, ZO-1 is present on the Sertoli cell plasma membrane in the absence of recognizable tight junctions. In the presence of tight junctions, however, ZO-1 is found only at the sites of junctional specializations associated with tight junctions and with elongating spermatids.     

Retinal epithelial fine structure in the vervet monkey (Cercopithecus aethiops)

The morphology of the retinal pigment epithelium (RPE) and closely associated Bruch's membrane (complexus basalis) and choriocapillaris have been investigated by electron microscopy in the vervet monkey (Cercopithecus aethiops). The RPE is composed of a single layer of cuboidal cells joined laterally by apically-located junctional complexes. Basally (sclerally) these cells display numerous infoldings while apically (vitreally) abundant processes enclose and interdigitate with rod outer segments. Internally the large vesicular nucleus is centrally located and smooth endoplasmic reticulum, mitochondria and lysosome-like bodies, are plentiful. Rough endoplasmic reticulum, polysomes and melanosomes while present are not abundant. Phagosomes of outer segment discs are noted in various stages of uptake and degradation. The choriocapillaris is highly fenestrated over large areas. Bruch's membrane shows the typical pentalaminate structure noted in other mammalian species without a tapetum lucidum.     

Concentric intermediate filament lattice links to specialized Z-band junctional complexes in sonic muscle fibers of the type I male midshipman fish

Type I male midshipman fish produce high-frequency hums for prolonged durations using sonic muscle fibers, each of which contains a hollow tube of radially oriented thin and flat myofibrils that display extraordinarily wide ( approximately 1.2 microm) Z bands. We have revealed an elaborate cytoskeletal network of desmin filaments associated with the contractile cylinder that form interconnected concentric ring structures in the core and periphery at the level of the Z bands. Stretch and release of single fibers revealed reversible length changes in the elastic desmin lattice. This lattice is linked to Z bands via novel intracellular desmosome-like junctional complexes that collectively form a ring, termed the "Z corset," around the periphery and within the core of the cylinder. The junctional complex consists of regularly spaced parallel approximately 900-nm-long cytoskeletal rods, or "Z bars," interconnected with slender (3-4 nm) plectin-positive filaments. Z bars are linked to the Z band by plectin filaments and on the opposite side to a dense mesh of desmin filaments. Adjacent Z bands are linked by slender filaments that appear to suspend sarcotubules. We propose that the highly reinforced elastic desmin cytoskeleton and the unique Z band junctions are structural adaptations that enable the muscles' high-frequency and high-endurance activity.     

Visualization of the hexagonal lattice in the erythrocyte membrane skeleton

The isolated membrane skeleton of human erythrocytes was studied by high resolution negative staining electron microscopy. When the skeletal meshwork is spread onto a thin carbon film, clear images of a primarily hexagonal lattice of junctional F-actin complexes crosslinked by spectrin filaments are obtained. The regularly ordered network extends over the entire membrane skeleton. Some of the junctional complexes are arranged in the form of pentagons and septagons, approximately 3 and 8%, respectively. At least five forms of spectrin crosslinks are detected in the spread skeleton including a single spectrin tetramer linking two junctional complexes, three-armed Y-shaped spectrin molecules linking three junctional complexes, three-armed spectrin molecules connecting two junctional complexes with two arms bound to one complex and the third arm bound to the adjacent complex, double spectrin filaments linking two junctional complexes, and four-armed spectrin molecules linking two junctional complexes. Of these, the crosslinks of single spectrin tetramers and three-armed molecules are the most abundant and represent 84 and 11% of the total crosslinks, respectively. These observations are compatible with the presence of spectrin tetramers and oligomers in the erythrocyte membrane skeleton. Globular structures (9-12 nm in diameter) are attached to the majority of the spectrin tetramers or higher order oligomer-like molecules, approximately 80 nm from the distal ends of the spectrin tetramers. These globular structures are ankyrinor ankyrin/band 3-containing complexes, since they are absent when ankyrin and residual band 3 are extracted from the skeleton under hypertonic conditions.     

Distribution of sterol-specific complexes in a continually shearing region of a plasma membrane and at procaryotic-eucaryotic cell junctions

A narrow zone of plasma membrane between the head and body of a protozoan from termites undergoes continual in-plane shear because the head rotates continuously in the same direction relative to the cell body (Tamm, S.L., and S. Tamm, 1974, Proc. Natl. Acad. Sci. USA 71:4589- 4593). Using filipin and digitonin as cytochemical probes for cholesterol and related 3-beta-hydroxysterols, we found a high level of sterol-specific complexes, visible as membrane lesions in thin sections, in both shearing and nonshearing regions of the membrane, indicating no difference in sterol content. This confirmed previous observations that any region of the fluid membrane can undergo shear, but that this occurs only at certain locations due to cell geometry and proximity to rotating cytoskeletal structures. Filipin and digitonin did not disrupt the plasma membrane at the junctions with ectosymbiotic rod and fusiform bacteria (i.e., membrane pockets and ridges). However, pepsin degradation of dense material coating the junctional membranes resulted in a positive response of these regions to filipin. Fluorescence microscopy revealed a bright halo around each rod bacterium, due to filipin-sterol binding in the sides of the membrane pockets, but no fluorescence at the bottom of the pockets; the same fluorescence pattern was found in pepsin-treated cells despite the presence of sterols throughout the pocket membrane, as shown by electron microscopy. These findings indicate that (a) regional constraints may restrict the ability of filipin to interact with sterols or form visible membrane lesions, and (b) a negative response to filipin, assayed by either electron or fluorescence microscopy, is not sufficient to demonstrate low membrane sterol concentration, particularly in membrane domains characterized by closely associated proteins.     

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium (hfRPE) cultures with morphological, physiological and genetic characteristics of native human RPE. These hfRPE cell cultures exhibit heavy pigmentation, and electron microscopy show extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied mammalian models of native RPE, including human. These results were extended by the development of therapeutic interventions in several animal models of human eye disease. We have focused on strategies for the removal of abnormal fluid accumulation in the retina or subretinal space. The extracellular subretinal space separates the photoreceptor outer segments and the apical membrane of the RPE and is critical for maintenance of retinal attachments and a whole host of RPE/retina interactions.     

Microfilament disruption in a noncycling organized tissue, the corneal endothelium, initiates mitosis

The adult corneal endothelium represents a noncycling cell population that resides as a monolayer on its basement membrane, Descemet's membrane. Evidence is presented for the first time, showing that mitotic regulation in this organized tissue, residing on its natural basement membrane, is coupled to microfilament integrity. When mitotically quiescent rat corneal endothelia are organ cultured in medium containing serum and cytochalasin B, low levels of mitosis are initiated. Supplementing the culture medium with either insulin or IGF-2 augments this response and results in increased cell density within the tissue monolayer. Fluorescence microscopy of actin using TRITC-conjugated phalloidin revealed that cellular circumferential microfilament bundles appear unaffected by cytochalasin B treatment, whereas the cytoplasmic microfilaments appear to be completely disrupted. These results suggest the possibility that the actin cytoskeleton is involved with the regulation of cell growth in the corneal endothelium.     

Fine Structure of the Retinal Pigment Epithelium of the River Lamprey (Lampetra fluviatilis,Cyclostomi)

The retinal pigment epithelium of Lampetra fluviatilis was studied by electron microscopy. The epithelial cells differ in many details from those of gnathostomes. The lateral cell membranes are difficult to distinguish. The smooth endoplasmic reticulum is well developed; in some animals undulated membrane complexes, comprising systems of tightly fused double membrane plates, are related to the endoplasmic reticulum. Myeloid bodies are common and well developed, but pigment granules are comparatively sparse. The intercellular space between pigment epithelium and photoreceptors is rather wide. There are only a few inclusion bodies with membranous contents. The importance of the pigment epithelium in the retinal metabolic exchange is discussed in view of the fine structure of the cells. Compared with that of hagfishes, the lamprey retina is well developed. However, any comparison must be made against the background of a diphyletic development of the two groups.     

Microfilament-associated proteins in tissue culture cells viewed by stereo immunofluorescence microscopy.

Stereo immunofluorescence microscopy avoids the problem of juxtaposition of structures often encountered in normal fluorescence microscopy. The procedure has been used in conjunction with antibodies against microfilament associated proteins to reveal the arrangement of microfilaments in a rat mammary cell line both in the fully spread state and in cells during the process of spreading on the substratum. use of antibodies to myosin, tropomyosin, alpha-actinin and filamin emphasizes that at early times during the spreading process these proteins are abundantly present underneath the upper plasma membrane, suggesting that the cortical layer present underneath this membrane may be contractile. In addition the results emphasize that even in well spread cells microfilament bundles are expressed both above and below the nucleus, in agreement with the assumption that microfilaments may form a supporting layer underneath the plasma membrane.     

Polymerized actin in lymphoid cells: functional interpretation

Nitrobenzoxadiazol (NBD) phallacidin, an active fluorescent derivative of the actin-binding mushroom toxin phallacidin provides a convenient actin-specific fluorescent label for cellular cytoskeleton structures. Topographical fluorescent microscopy images of lymphoid cells obtained with NBD-phallacidin staining reveal that the major feature of the cellular cytoskeleton characterized by actin are mainly associated with cell membrane, a pattern that correlates strikingly with their DNAse 1 inhibition. Such actin pools may thus be involved in a membrane-associated protein network controlling membrane viscoplastic deformation and cell motility.     

Electron microscopic contrast of the cytoskeleton and junctional complexes of intestinal epithelial cells by ethanolic phosphotungstic acid

After glutaraldehyde fixation and treatment with ethanolic phosphotungstic acid (E-PTA) before plastic embedding, sections of rat large intestine showed a characteristic electron contrasting pattern in epithelial cells. The axis of microvilli, terminal web, a thin band below the luminal plasma membrane, centrioles and junctional complexes (tight junctions, adherens junctions, and desmosomes) appeared highly contrasted. In addition to protein components of microfilaments and intermediate filaments, proteins from the junctional complexes could also be implicated in the contrasting reaction with E-PTA. Mitochondrial membranes, chromatin masses, and nucleoli of enterocytes showed considerable electron density, whereas no reaction was found in the glycocalyx and mucin content of goblet cells. The clear visualization of cytoskeleton elements and junctional complexes by E-PTA contrasting represents a simple and valuable method for studies on the normal and pathological organization of these structures in epithelial cells.     

Extracellular matrix fibers containing fibronectin and basement membrane heparan sulfate proteoglycan coalign with focal contacts and microfilament bundles in stationary fibroblasts

Double-label immunofluorescence microscopy and immunoelectron microscopy were performed on stationary cultures of Nil 8 fibroblasts to determine if fibronectin and basement membrane heparan sulfate proteoglycans play coordinated roles in cell-to-substrate adhesion. Relationships between subcellular matrix fibers containing fibronectin plus proteoglycan, and focal contacts associated with microfilament bundles, were studied simultaneously using interference reflection microscopy, differential interference contrast microscopy, and immunofluorescence microscopy. Cells maintained in 0.3% FBS were doubly stained with monospecific anti-fibronectin IgG and antibodies against a basement membrane proteoglycan purified from the EHS (Engelbreth-Holm-Swarm) tumor. Coincident patterns of fibronectin and proteoglycan-containing fibers were found to codistribute with focal contacts and microfilament bundles in both early (6-h) and late (24-h) cultures. The early cells showed doubly-stained fibers colinear with substrate adhesion sites in 43% of the sample, while 100% of the later cells exhibited these coaligned matrix-cytoskeletal attachment complexes. Immunoelectron microscopy showed that both of these antigens were situated in the same type of extracellular matrix fiber that appeared to be loosely associated with the cell surface membrane. We hypothesize that the appearance of proteoglycan in subcellular matrix fibers of these fibroblasts might stabilize fibronectin-containing cell-to-substrate contacts.     

Spectroscopic and molecular characterization of the oligomeric antenna of the diatom Phaeodactylum tricornutum

The photosynthetic antenna system of diatoms contains fucoxanthin chlorophyll a/c binding proteins (FCPs), which are membrane intrinsic proteins showing high homology to the light harvesting complexes (LHC) of higher plants. In the present study, we used a mild solubilization of P. tricornutum thylakoid membranes in combination with sucrose density gradient centrifugation or gelfiltration and obtained an oligomeric FCP complex (FCPo). The spectroscopic characteristics and pigment stoichiometries of the FCPo complex were comparable to FCP complexes that were isolated after solubilization with higher detergent per chlorophyll ratios. The excitation energy transfer between the FCP-bound pigments was more efficient in the oligomeric FCPo complexes, indicating that these complexes may represent the native form of the diatom antenna system in the thylakoid membrane. Determination of the molecular masses of the two different FCP fractions by gelfiltration revealed that the FCP complexes consisted of trimers, whereas the FCPo complexes were either composed of six monomers or two tightly associated trimers. In contrast to vascular plants, stable functional monomers could not be isolated in P. tricornutum. Both types of FCP complexes showed two protein bands in SDS-gels with apparent molecular masses of 18 and 19 kDa, respectively. Sequence analysis by MS/MS revealed that the 19 kDa protein corresponded to the fcpC and fcpD genes, whereas the 18 kDa band contained the protein of the fcpE gene. The presence of an oligomeric antenna in diatoms is in line with the oligomeric organization of antenna complexes in different photoautotrophic groups.     

Nuclear surface complex as observed with the high resolution scanning electron microscope. Visualization of the membrane surfaces of the neclear envelope and the nuclear cortex from Xenopus laevis oocytes

The nuclear envelope and associated structures from Xenopus laevis oocytes (stage VI) have been examined with the high resolution scanning electron microscope (SEM). The features of the inner and outer surfaces of the nuclear surface complex were revealed by manual isolation , whereas the membranes facing the perinuclear space (the space between the inner and outer nuclear membranes) were observed by fracturing the nuclear envelope in this plane and splaying the corresponding regions apart. Pore complexes were observed on all four membrane surfaces of this double-membraned structure. The densely packed pore complexes (55/micron2) are often clustered into triplets with shared walls (outer diameter = 90 nm; inner diameter = 25 nm; wall thickness = aproximately 30 nm), and project aproximately 20 nm above each membrane except where they are flush with the innermost surface. The pore complex appears to be an aggregate of four 30-nm subunits. The nuclear cortex, a fibrous layer (300 nm thickness) associated with the inner surface of the nuclear envelope, has been revealed by rapid fixation. This cortical layer is interrupted by funnel-shaped intranuclear channels (120-640 nm diam) which narrow towards the pore complexes. Chains of particles, arranged in spirals, are inserted into these intranuclear channels. The fibers associated with the innermost face of the nuclear envelope can be extraced with 0.6 MKI to reveal the pore complexes. A model of the nuclear surface complex, compiled from the visualization of all the membrane faces and the nuclear cortex, demonstrates relations between the intranuclear channels (3.2/micron2) and the numerous pore complexes, and the possibility of their role in nucleocytoplasmic interactions.