Freeze-fracture studies of gap junctions in the developing and adult amphibian cardiac muscle.

The freeze-fracture technique has been used to characterize the junctional devices involved in the electrical coupling of Ambystoma cardiac tissue. These cells are connected by junctions formed by either linear or circular arrays of particles. Such structures can be interpreted as a special type of gap junction. Gap junctions have also been investigated during the growth and differentiation of two amphibians, Rana and Xenopus. In both genera the earliest stage of junctional assembly is characterized by linear rows of particles. Later, a gradual transformation of these linear rows into circles was found. Finally, in the fully formed gap junctions, these circles appeared to join together into clusters. In summary, in the adult amphibian myocardial cells, three different types of gap junctions can be described. The first type, which has been observed in all embryonic stages and in adults in all three genera, consists of linear or circular arrays of particles: this is the only type of gap junction seen at any age in Xenopus. The second type, consisting of a variable number of anastomosing circles forming regular networks, is never observed in embryonic cells. It is typical of the adult frog heart and may also be seen in Ambystoma. The third type is characteristic only of adult Ambystoma heart and consists of geometrically packed particles identifiable with classic communicating macula. The fact that only the first class of structure is observed in Xenopus heart strongly supports the conclusion that such linear arrays of intramembranous particles really represent true functional electrical junctions.     

Formation of tight and gap junctions in the inner enamel epithelium and preameloblasts in human fetal tooth germs

Human fetal primary tooth germs in the cap stage were fixed with a glutaraldehyde-formaldehyde mixture, and formative processes of tight and gap junctions of the inner enamel epithelium and preameloblasts were examined by means of freeze-fracture replication. Chains of small clusters of particles on the plasma membrane P-face of the inner enamel epithelium and preameloblasts were the initial sign of tight junction formation. After arranging themselves in discontinuous, linear arrays in association with preexisting or forming gap junctions, these particles later began revealing smooth, continuous tight junctional strands on the plasma membrane P-face and corresponding shallow grooves of a similar pattern on the E-face. Although they exhibited evident meshwork structures of various extents at both the proximal and distal ends of cell bodies, they formed no zonulae occludentes. Small assemblies of particles resembling gap junctions were noted at points of cross linkage of tight junctional strands; but large, mature gap junctions no longer continued into the tight junction meshwork structure. Gap junctions first appeared as very small particle clusters on the plasma membrane P-face of the inner enamel epithelium. Later two types of gap junctions were recognized: one consisted of quite densely aggregated particles with occasional particle-free areas, and the other consisted of relatively loosely aggregated particles with particle-free areas and aisles. Gap junction maturation seemed to consist in an increase of particle numbers. Fusion of gap junctions in the forming stage too was recognized. The results of this investigation suggest that, from an early stage in their development, human fetal ameloblasts possess highly differentiated cell-to-cell interrelations.     

A model for de novo synthesis and assembly of tight intercellular junctions. Ultrastructural correlates and experimental verification of the model revealed by freeze-fracture

The structure and function of intercellular tight (occluding) junctions, which constitute the anatomical basis for highly regulated interfaces between tissue compartments such as the blood-testis and blood-brain barriers, are well known. Details of the synthesis and assembly of tight junctions, however, have been difficult to determine primarily because no model for study of these processes has been recognized. Primary cultures of brain capillary endothelial cells are proposed as a model in which events of the synthesis and assembly of tight junctions can be examined by monitoring morphological features of each step in freeze-fracture replicas of the endothelial cell plasma membrane. Examination of replicas of non-confluent monolayers of endothelial cells reveals the following intramembrane structures proposed as 'markers' for the sequential events of synthesis and assembly of zonulae occludentes: development of surface contours consisting of elongate terraces and furrows (valleys) orientated parallel to the axis of cytoplasmic extensions of spreading endothelial cells, appearance of small circular PF face depressions (or volcano-like protrusions on the EF face) that represent cytoplasmic vesicle-plasma membrane fusion sites, which are positioned in linear arrays along the contour furrows, appearance of 13-15 nm intramembrane particles at the perimeter of the vesicle fusion sites, and alignment of these intramembrane particles into the long, parallel, anastomosed strands characteristic of mature tight junctions. These structural features of brain endothelial cells in monolayer culture constitute the morphological expression of: reshaping the cell surface to align future junction-containing regions with those of adjacent cells, delivery and insertion of newly synthesized junctional intramembrane particles into regions of the plasma membrane where tight junctions will form, and aggregation and alignment of tight junction intramembrane particles into the complex interconnected strands of mature zonulae occludentes. The distribution of filipin-sterol complex-free regions on the PF intramembrane fracture face of junction-forming endothelial plasmalemmae corresponds precisely to the furrows, aligned vesicle fusion sites and anastomosed strands of tight junctional elements.(ABSTRACT TRUNCATED AT 400 WORDS)     

Changes in the blood-brain barrier of the central nervous system in the blowfly during development, with special reference to the formation and disaggregation of gap and tight junctions

In the central nervous system (CNS) of pupal Calliphora, dramatic alterations occur in the perineurial and glial gap junctions. Having formed macular plaques by late larval stages, in early pupae cell migration causes the EF intramembranous junctional particles to disaggregate and move apart into linear and then disorganised arrays as shown by freeze-fracture. After nerve and glial cell reorganisation into the adult pattern, the gap junctions begin to reform in the late pupae, again seemingly by particle migration into linear arrays and clusters. Ultimately the particles form numerous macular plaques between both perineurial and glial cells. Statistical analyses support the contention that these are performed EF particles which undergo translateral movement from macular larval junctions into the disaggregated particles of early pupae and that the same particles appear to undergo realignment and reclustering in late pupae to form the mature gap junctions of adults. This is the first report to indicate breakdown and reformation of gap junctions in vivo involving reutilisation of the same intramembranous particles. Perineurial “tight” junctions are not to be found in early pupal stages and their absence can be correlated with the free entry of ionic lanthanum into the CNS observed during that period. In late pupae, when the tight junctional moniliform ridges have apparently reformed, the entry of the tracer lanthanum becomes restricted to the level of the perineurium, penetrating no deeper. This is also the case in the adult, where the blood-brain barrier is maintained. PF particles in the form of short linear ridges and clustered particle arrays in nerve cell membranes are present throughout pupal and adult stages; their continued presence throughout the whole of development suggests some role in neuronal function, as yet unclear.     

In vitro assembly of gap junctions

Gap junction structures were assembled in vitro from octyl-β- -glucopyranoside-solubilized components of lens fiber cell membranes. Individual pore structures (connexons), short double-membrane structures, and other amorphous material were evident in the solubilized mixture. Following the removal of the detergent by dialysis, these connexons associated to form single- and double-layered, two-dimensional hexagonal arrays (unit cell size a = B = 8.5 nm). The formation of larger arrays was dependent on the lipid-to-protein ratio and the presence of Mg²⁺ ions. Crystallographic analysis of electron micrographs revealed that lens junctional connexons consisted of six subunits surrounding a stain-filled channel. Upon further detergent treatment, in vitro assembled gap junctions were insoluble and formed three-dimensional stacks while other components were solubilized. SDS-PAGE and mass data from scanning transmission electron microscopy strongly suggest that a 38-kDa polypeptide, which is a processed form of the lens specific gap junction protein MP70, is a major component of the arrays. The in vitro assembly of gap junctions opens new avenues for the structural analysis of gap junctions and for the study of the intermolecular interactions of connexons during junctional assembly.     

Changes in the blood-brain barrier of the central nervous system in the blowfly during development, with special reference to the formation and disaggregation of gap and tight junctions. I. Larval development

In the central nervous system (CNS) of full-grown larvae of the blowfly Calliphora erythrocephala, the glial-ensheathed nerve cells are completely surrounded by a layer of perineurial cells which form a “blood-brain barrier” between the circulating haemolymph and the CNS. A variety of intercellular junctions, including gap and tight junctions, are found between adjacent perineurial cells and some also between apposing glial cells; these have been characterized by freeze-fracturing as well as by tracer studies and analysis of thin sections. They are found not to be present between such cells in the undifferentiated CNS in the newly hatched larvae, nor are the nerve cells encompassed by glial cells; ionic lanthanum can penetrate to the axonal surfaces at this stage. However, over the 5 days of larval growth and development the glial cells produce attentuated cytoplasmic processes that ensheath the nerve cells, and the perineurium is formed; junctional complexes are assembled and a larval blood-brain barrier is produced which excludes tracers. Freeze-fracture preparations suggest that the inverted gap junctions which develop have done so by migration of individual intramembranous EF particles to form, at first, linear arrays and small clusters and, ultimately, macular aggregations in the perineurium; these lie between the undulating rows of PF particles forming the septate junctions. These septate junctions are formed by the organization of arrays of PF particles into multiple rows. Extensive PF particles fusing into ridges with EF grooves to form perineurial “tight” junctions are also observed, seemingly in the process of development; entry of exogenous lanthanum followed by its exclusion parallels the completion of ridge formation. These ridges are simple linear arrays of particles which may be discontinuous, lying in parallel with one another and the surface. Clustered particle arrays as well as scattered short ridges on the axonal PF, however, appear to be present unchanged throughout larval life; their role may therefore be associated with neural membrane function although there are suggestions that some may form axo-glial junctions. This is the first report on the lateral migration of intramembranous particles as the mode of formation of gap junctions in the nervous system of an invertebrate.     

Junctional structures in digestive epithelia of a cephalopod

Intercellular junctions have been studied in the epithelia of digestive organs of Sepia officinalis (digestive gland, digestive duct appendages and caecum) by conventional staining, lanthanum tracer and freeze-fracturing techniques. In the three organs studied the same junctional complex occurs, consisting of a belt desmosome, a septate junction and gap junctions. The septate junction is of pleated-sheet type and the gap junction has its particles on the P face of the fracture. Circular structures have been found in the digestive gland septate junctions. Neither continuous nor tight junctions have been found. These results show that Cephalopods have junctional structures very close to those of other Molluscs and of Annelids. Some small differences between the septate junctions of the three organs could be related to their different physiology.     

The distribution of orthogonal arrays and their relationship to intercellular junctions in neuroglia of the freeze-fractured hypothalamo-neurohypophysial system

Summary Using freeze-fracture techniques, we have investigated membrane specializations of the glia associated with the hypothalamo-neurohypophysial system of the rat. In the paraventricular (PVN) and supraoptic (SON) nuclei, astrocytes in areas of high neuronal density (i.e., magnocellular regions) display orthogonal arrays of 6–7 nm particles soley near gap junctions, while astrocytes in areas of lower neuronal density (i.e., parvocellular regions) contain additional arrays in membranes not displaying gap junctions. Arrays are especially numerous on astrocytic perivascular end-feet in both nuclei and in the laminations of the pial-glial limitans ventral to the SON. Ependymal cells near the PVN show arrays both on their lateral surfaces (displaying gap junctions) and on their apical surfaces (facing the CSF). Tight junctions are not noted on astrocytes or ependymal cells, but are noted on both the somas and myelin lamellae of oligodendroglia. Both of these latter membranes occasionally contain gap junctions as well; however, orthogonal arrays are never noted on oligodendroglia.The plasma membranes of pituicytes in the neurohypophysis display gap junctions, complex junctions, and tight junctions. Orthogonal arrays are noted near the first two of these, but not near the last. Arrays in the neural lobe appear most dense on membranes adjacent to subpial or perivascular spaces. Pituicyte membranes containing orthogonal arrays appear infrequently near the neural stalk, increasing towards the distal end of the neural lobe. The distribution of orthogonal arrays in this system, as well as in other systems in which they have been noted, suggests a polarization of membrane activity.     

COOH Terminus of Occludin Is Required for Tight Junction Barrier Function in Early Xenopus Embryos

Occludin is the only known integral membrane protein localized at the points of membrane– membrane interaction of the tight junction. We have used the Xenopus embryo as an assay system to examine: (a) whether the expression of mutant occludin in embryos will disrupt the barrier function of tight junctions, and (b) whether there are signals within the occludin structure that are required for targeting to the sites of junctional interaction. mRNAs transcribed from a series of COOH-terminally truncated occludin mutants were microinjected into the antero–dorsal blastomere of eight-cell embryos. 8 h after injection, the full-length and the five COOH-terminally truncated proteins were all detected at tight junctions as defined by colocalization with both endogenous occludin and zonula occludens-1 demonstrating that exogenous occludin correctly targeted to the tight junction. Importantly, our data show that tight junctions containing four of the COOH-terminally truncated occludin proteins were leaky; the intercellular spaces between the apical cells were penetrated by sulfosuccinimidyl-6-(biotinamido) Hexanoate (NHS-LC-biotin). In contrast, embryos injected with mRNAs coding for the full-length, the least truncated, or the soluble COOH terminus remained impermeable to the NHS-LC-biotin tracer. The leakage induced by the mutant occludins could be rescued by coinjection with full-length occludin mRNA. Immunoprecipitation analysis of detergent-solubilized embryo membranes revealed that the exogenous occludin was bound to endogenous Xenopus occludin in vivo, indicating that occludin oligomerized during tight junction assembly. Our data demonstrate that the COOH terminus of occludin is required for the correct assembly of tight junction barrier function. We also provide evidence for the first time that occludin forms oligomers during the normal process of tight junction assembly. Our data suggest that mutant occludins target to the tight junction by virtue of their ability to oligomerize with full-length endogenous molecules.     

Carbon tetrachloride induced proliferation of tight junctions in the rat liver as revealed by freeze-fracturing

Application of carbon tetrachloride produced a progressive proliferation of tight junctions in the rat liver. This system proved to be rapid and highly reproducable and affords the opportunity for tracing the fate of tight junctions in freeze-fracture replicas, facilitating investigations on their formation and function. Beginning on day one carbon tetrachloride treatments resulted in the progressive loosening and fragmentation of the junctional meshwork. After three to four days the membrane outside the zonulae occludentes was extensively filled with proliferated discrete junctional elements often forming complex configurations. From the fifth day on the zonulae occludentes were restricted again predominantly around the bile canaliculus margins. But the junctional meshwork of the zonulae occludentes remained loosened in comparison to those in the control rats. It could be shown that tight junction proliferation on the lateral surface of the plasmalemma occurred both through de novo formation from discrete centers of growth by addition of intramembranous particles and through reorganization of preexistent junctional strands of the fragmented zonulae occludentes bodies. Whereas the large gap junctions close associated with the zonulae occludentes remained more or less unaffected during the experiments, small gap junctions increased in number after five days and were located at the margin or in the tight junction domain. It is assumed that the degeneration of the tight junctions served as a pool for intramembranous particles which form the gap junctions. The results of these observations are discussed in relation to those obtained in other systems.     

Cyclic modulation of Sertoli cell junctional complexes in a seasonal breeder: the mink (Mustela vison)

The development and modulation of Sertoli cell junctions was studied in newborn and adult mink during the active and inactive spermatogenic phases. The techniques used were electron microscopy of freeze-fractured replicas and thin sections of tissues infused with horseradish peroxidase as a junction permeability tracer. In the newborn, freeze-fractured developing junctions had either spherical or fibrillar particles. In addition, junctional domains where particles were associated preferentially with the E-face, and others where particles were associated preferentially with the P-face, were found developing either singly or conjointly within a given membrane segment, thus yielding a heterogeneous junctional segment. Coincidently with the development of a tubular lumen and the establishment of a competent blood-testis barrier, junctional strands were composed primarily of particulate elements associated preferentially with the E-face. In adult mink during active spermatogenesis, cell junctions were found on the entire lateral Sertoli cell plasma membrane from the basal to the luminal pole of the cell. In the basal third of the Sertoli cell, membranous segments that faced a spermatogonium or a migrating spermatocyte displayed forming tight, gap, and adherens junctions. In the middle third, abutting membrane segments localized above germ cells were involved in continuous zonules and in adherens junctions. In the apical or luminal third, the zonules were discontinuous, and the association of junctional particles with the E-face furrow was lost. Gap junctions increased in both size and numbers. Junctional vesicles that appeared as annular gap and tight-junction profiles in thin sections or as hemispheres in freeze-fracture replicas were present. Reflexive tight and gap junctions were formed through the interaction of plasma membrane segments of the same Sertoli cell. Internalized junctional vesicles were also present in mature spermatids. During the inactive spermatogenic phase, cell junctions were localized principally in the basal third of the Sertoli cell; junctional strands resembled those of the newborn mink. During the active spermatogenic phase, continuous zonules were competent in blocking passage of the protein tracer. During the inactive phase the blood-testis barrier was incompetent in blocking entry of the tracer into the seminiferous epithelium. It is proposed that modulation of the Sertoli cell zonules being formed at the base and dismantled at the apex of the seminiferous epithelium follows the direction of germ cell migration and opposes the apicobasal direction of junction formation reported for most epithelia.(ABSTRACT TRUNCATED AT 400 WORDS)     

Vitamin-A-induced mucous metaplasia. An in vitro system for modulating tight and gap junction differentiation

Stratified squamous epithelia from 14-day chick embryo shank skin contain rare tight-junctional strands and only small gap junctions. Exposure of this tissue to retinoic acid (vitamin-A) (20 U/ml) in organ culture, however, induces mucous metaplasia, accompanied by tight-junction formation and gap-junction growth; untreated specimens continue to keratinize. To investigate sequential stages of junctional assembly and growth, we examined thin sections and freeze-fracture replicas at daily intervals for 3 days. During the metaplastic process, tight junctions assemble in midepidermal and upper regions, beginning on day 1 and becoming maximal on day 3. Two tight-junctional patterns could be tentatively identified as contributing to the emergence of fully formed zonulae occludentes: (a) the formation of individual ridges along the margins of gap junctions; (b) de novo generation of continuous ramifying strands by fusion of short strand segments and linear particulate aggregates near cellular apices. Gap junction enlargement, already maximal at day 1, occurs primarily three to four cell layers deep. Growth appears to occur by annexation of islands of 20-40 8.5-nm particles into larger lattices of islands separated by particle-free aisles. Eventually, a single gap junction may occupy much of the exposed membrane face in freeze-fractured tissue, but during apical migration of the cells such junctions disappear. The vitamin- A chick-skin system is presented as a responsive model for the controlled study of junction assembly.     

VARIATIONS IN TIGHT AND GAP JUNCTIONS IN MAMMALIAN TISSUES

The fine structure and distribution of tight (zonula occludens) and gap junctions in epithelia of the rat pancreas, liver, adrenal cortex, epididymis, and duodenum, and in smooth muscle were examined in paraformaldehyde-glutaraldehyde-fixed, tracer-permeated (K-pyroantimonate and lanthanum), and freeze-fractured tissue preparations. While many pentalaminar and septilaminar foci seen in thin-section and tracer preparations can be recognized as corresponding to well-characterized freeze-fracture images of tight and gap junction membrane modifications, many others cannot be unequivocally categorized—nor can all freeze-etched aggregates of membrane particles. Generally, epithelia of exocrine glands (pancreas and liver) have moderate-sized tight junctions and large gap junctions, with many of their gap junctions basal to the junctional complex. In contrast, the adrenal cortex, a ductless gland, may not have a tight junction but does possess large gap junctions. Mucosal epithelia (epididymis and intestine) have extensive tight junctions, but their gap junctions are not as well developed as those of glandular tissue. Smooth muscle contains numerous small gap junctions The incidence, size, and configuration of the junctions we observed correlate well with the known functions of the junctions and of the tissues where they are found.     

In vitro assembly of gap junctions.

Gap junction structures were assembled in vitro from octyl-beta-D-glucopyranoside-solubilized components of lens fiber cell membranes. Individual pore structures (connexons), short double-membrane structures, and other amorphous material were evident in the solubilized mixture. Following the removal of the detergent by dialysis, these connexons associated to form single- and double-layered, two-dimensional hexagonal arrays (unit cell size a = b = 8.5 nm). The formation of larger arrays was dependent on the lipid-to-protein ratio and the presence of Mg2+ ions. Crystallographic analysis of electron micrographs revealed that lens junctional connexons consisted of six subunits surrounding a stain-filled channel. Upon further detergent treatment, in vitro assembled gap junctions were insoluble and formed three-dimensional stacks while other components were solubilized. SDS-PAGE and mass data from scanning transmission electron microscopy strongly suggest that a 38-kDa polypeptide, which is a processed form of the lens specific gap junction protein MP70, is a major component of the arrays. The in vitro assembly of gap junctions opens new avenues for the structural analysis of gap junctions and for the study of the intermolecular interactions of connexons during junctional assembly.     

Gap junctions in the differentiated neural retinae of newly hatched chickens.

Gap junctions in the neural retinae of newly hatched chickens were examined in thin section and by freeze cleaving. Unusual gap junctions containing linear arrays of intramembrane particles are found between principal and accessory cones which form a double cone at the region of the outer limiting membrane. These unusual gap junctions are often continuous with macular aggregates of hexagonally packed intramembrane particles which are characteristic of a typical gap junction. Typical gap junctions are also found in both the outer and the inner plexiform layers and in the outer nuclear layer, but are not so abundant as in the outer limiting membrane region. The sizes of intramembrane particles and their centre-to-centre spacing within the macular aggregate of a gap junction in differentiated neural retinae are slightly larger than those in undifferentiated neural retinae. Tight junctions are not found in differentiated neural retinae.     

Distinct domains of paracingulin are involved in its targeting to the actin cytoskeleton and regulation of apical junction assembly

Paracingulin is an M(r) 150-160 kDa cytoplasmic protein of vertebrate epithelial tight and adherens junctions and comprises globular head, coiled-coil rod, and globular tail domains. Unlike its homologous tight junction protein cingulin, paracingulin has been implicated in the control of junction assembly and has been localized at extrajunctional sites in association with actin filaments. Here we analyze the role of paracingulin domains, and specific regions within the head and rod domains, in the function and localization of paracingulin by inducible overexpression of exogenous proteins in epithelial Madin Darby canine kidney (MDCK) cells and by expression of mutated and chimeric constructs in Rat1 fibroblasts and MDCK cells. The overexpression of the rod + tail domains of paracingulin perturbs the development of the tight junction barrier and Rac1 activation during junction assembly by the calcium switch, indicating that regulation of junction assembly by paracingulin is mediated by these domains. Conversely, only constructs containing the head domain target to junctions in MDCK cells and Rat1 fibroblasts. Furthermore, expression of chimeric cingulin and paracingulin constructs in Rat1 fibroblasts and MDCK cells identifies specific sequences within the head and rod domains of paracingulin as critical for targeting to actin filaments and regulation of junction assembly, respectively. In summary, we characterize the functionally important domains of paracingulin that distinguish it from cingulin.     

Studies on perineural junctional complexes and the sites of uptake of microperoxidase and lanthanum in the cockroach central nervous system

The perineurial junctional complexes in the nerve cord of Periplaneta americana have been shown to consist of septate desmosomes, extensive gap junctions and relatively limited regions of tight junctions. Microperoxidase (M.W. 1,900) undergoes limited intercellular penetration into the septate desmosomes. Lanthanum penetrates both the septate desmosomes and gap junctions. It is concluded that the restricted access of these substances to the underlying extracellular spaces results from the presence of the perineurial tight junctions. These results contrast with those for small peripheral nerves, which lack equivalent junctional complexes, and in which the extracellular spaces are found to be accessible to externally applied lanthanum. The results are discussed in relation to current concepts of the insect blood-brain barrier.     

Development of an apical plasma membrane domain and tight junctions during histogenesis of the mammalian pancreas

The role of tight junctions (zonula occludens) in the formation of apical plasma membrane (PM) domains was investigated in the embryonic rat pancreas. In the present study, lectin-rhodamine (WGA-TRITC and RCAII-TRITC) and lectin-gold (WGA-Au and RCAII-Au) conjugates were used to monitor apical PM domain formation and freeze-fracture analysis was used to monitor tight junction formation in the pancreatic epithelium of embryonic, neonatal, and adult rats. Fluorescent and TEM analysis of WGA and RCAII binding indicated that an apical PM domain is formed as early as Day 13 of gestation in the pancreatic epithelium. While apical WGA binding remained into adult life, RCAII binding was lost by 1 day after birth. In contrast, tight junctions were not observed until Day 14 of gestation. At this time, tight junctions were found to be incomplete in formation and typically consisted of linear arrays of IMPs or discontinuous arrays of sealing strands (focal adherens). Continuous tight junctions were not completely formed until Day 15 of gestation. Continued development of tight junctions during gestation was characterized by (1) an increase in the number of sealing strands and (2) a more parallel arrangement of sealing strands within each junctional complex. By 8 weeks after birth, tight junctions were more loosely organized and contained fewer sealing strands as compared to that observed in the fetus. These results suggest that lateral diffusion of apical PM glycoconjugates may be restricted even in the absence of complete tight junctional complexes during development of the rat pancreas.     

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)     

The supramolecular architecture of junctional microdomains in native lens membranes

Gap junctions formed by connexons and thin junctions formed by lens-specific aquaporin 0 (AQP0) mediate the tight packing of fibre cells necessary for lens transparency. Gap junctions conduct water, ions and metabolites between cells, whereas junctional AQP0 seems to be involved in cell adhesion. High-resolution atomic force microscopy (AFM) showed the supramolecular organization of these proteins in native lens core membranes, in which AQP0 forms two-dimensional arrays that are surrounded by densely packed gap junction channels. These junctional microdomains simultaneously provide adhesion and communication between fibre cells. The AFM topographs also showed that the extracellular loops of AQP0 in junctional microdomains adopt a conformation that closely resembles the structure of junctional AQP0, in which the water pore is thought to be closed. Finally, time-lapse AFM imaging provided insights into AQP0 array formation. This first high-resolution view of a multicomponent eukaryotic membrane shows how membrane proteins self-assemble into functional microdomains.