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.     

Gap junction dynamics: reversible effects of hydrogen ions

Reversible crystallization of intramembrane particle packings is induced in gap junctions isolated from calf lens fibers by exposure to 3 x 10(-7) M or higher [H+] (pH 6.5 or lower). The changes from disordered to crystalline particle packings induced by low pH are similar to those produced in junctions of intact cells by uncoupling treatments, indicating that H+, like divalent cations, could be an uncoupling agent. The freeze-fracture appearance of both control and low pH-treated gap junctions is not altered by glutaraldehyde fixation and cryoprotective treatment, as suggested by experiments in which gap junctions of both intact cells and isolated fractions are freeze- fractured after rapid freezing to liquid N2 temperature according to Heuser et al. (13). In junctions exposed to low pH, the particles most often form orthogonal and rhombic arrays, frequently fused with each other. A number of structural characteristics of these arrays suggest that the particles of lens fiber gap junctions may be shaped as tetrameres.     

A freeze-fracture study of the digestive tract of the parasitic nematode Trichinella

Freeze-fracture preparations of the esophagus and intestine of larvae and adults of the nematode Trichinella spiralis illustrate the distribution of intramembranous particles in membranes of a number of cell types, and several specializations were found. Esophageal glands are prominently linked by gap junctions, but gap junctions were not found between intestinal cells. Muscle cells of the esophagus have rectilinear arrays of particles, thought to be points of adherence of the muscles to the esophageal epithelium. Clusters of particles are associated with these arrays and particle-free areas (probably Z bodies) also occur. Intestinal cells have small particles in their microvilli, large particles in the cells' apical membranes, and intermediate size particles, similar to membranes of other cells, in the lateral and basal membranes. Apical smooth septate junctions and tricellular junctions occur between intestinal cells.     

Intercellular junctions and rhombic particle arrays in the developing and adult dorsal ocelli of the honeybee

Intercellular junctions and particle arrays in the developing and mature dorsal ocelli of the honeybee Apis mellifera have been studied with conventional and freeze-fracture electron microscopy. Four types of junctions are found in the lentigenic and retinogenic part during development. These are desmosomes, septate junctions, tight junctions, and gap junctions. Gap junctions and septate junctions are found between differentiating photoreceptor cells only as long as the rhabdoms are beginning to form. Their disappearance after differentiation indicates that they could play a part in cell determination. Desmosomes connect photoreceptor cells into the early imaginai stage and then disappear. Other junctions, once they have formed, remain for the life of the animal, but can change considerably in structure, distribution and frequency. The cells of the perineurium surrounding the ocellus are connected by septate and gap junctions, which may be the basis of the blood-eye barrier. Rhombic particle arrays on the E-face of the glial membrane attached to the photoreceptor cell membrane first appear in small groups one day before emergence. In the further course of life these arrays become more extensive and apparent. Their significance may be to play some role in receptor function.     

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.     

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.     

Gap junctions and rhombic particle arrays in the freeze-fractured mouse oocyte-follicle cell complex

Intact follicles as well as defolliculated oocytes of the mouse were studied by freeze-fracture electron microscopy. In intact follicles the oocyte plasma membrane shows two prominent types of intra-membrane particle array:gap junctions and yet undescribed rhombic particle arrays. The gap junctions vary in size (from 5 to 500 IMPs) and shape. Occasionally they are organized in so-called formation plaques. The rhombic particle arrays consist of 25 IMPs on an average, the IMP diameter is 10.5 nm, the mean IMP distance is 19.8 nm and the acute angle in the array is 81.3 degrees. After defolliculation the gap junctions disassemble and change transiently into linear IMP arrays. The rhombic particle arrays persist indicating that they are of a non-junctional nature. The possible function of the rhombic particle arrays is discussed in relation to similar membrane specializations in excitable cells.     

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.     

Gap junctions between pancreatic B-cells are modulated by cyclic AMP

Gap junction morphology was studied on freeze fracture replicas of pancreatic islet tissue, using morphometric techniques. In rat islets in situ, 60 percent of the connexions were polygonally packed in gap junctions, whereas the remaining part occurred in linear strands. After collagenase isolation, the islets presented similar numbers of gap junctions but contained virtually no linear strands. The distribution of connexions over polygonal or linear arrays also varied with the culture conditions: at 11.2 mM glucose, a higher percentage of particles occurred in gap junctions than at 5.6 mM glucose; this was also the case in other conditions with elevated cellular cyclic AMP levels. The total number of connexions increased when islets were cultured with dibutyryl cyclic AMP or with a phosphodiesterase inhibitor; conditions with an augmented number of gap junctions also displayed an elevated islet cyclic AMP content. A similar association was noted in newly formed aggregates of pancreatic B-cells purified by autofluorescence-activated. cell sorting. These results indicate that the number of classically defined gap junctions is not only dependent on the total number of connexions but also on their organization within the membrane. It is suggested that the distribution of connexions over polygonal and linear arrays follows a dynamic equilibrium varying with the extracellular conditions. Cyclic AMP appears to modulate the number of gap junctions between pancreatic B-cells both through an induction of new connexions and through an assembly of linearly organized particles into polygonal arrays.     

Morphometric analysis of gap junctions in rat myocardium after hyperkalemia

The effect of membrane depolarization was investigated on gap junctions from isolated rat hearts perfused with a modified Krebs-Henseleit solution containing 16 mM K+. After freeze-fracturing, the configuration of the junctional particles in the ventricular myocardium was analysed by measurements of connexon densities and centre-to-centre distances between neighbouring particles. Both in control and hyperkalemic tissue, the gap junctions occur on the intercalated discs as round or oval aggregates of connexons which are closely and regularly packed in small, criss-cross-oriented arrays separated by particle-free aisles. Within the arrays, the mean (+/- SD) centre-to-centre distances between particles from control and hyperkalemic tissue, i.e. 9.17 +/- 1.52 and 9.15 +/- 1.51 mm, respectively, are not significantly different. Similarly, comparison of particle densities after control and high-K+ perfusion, i.e. 8,490 +/- 600 and 8,420 +/- 620 particles/microns 2, respectively, reveals no difference in the proportion of the particle arrays to the empty aisles. The apparently unaltered gap junctional morphology after depolarization of the sarcolemma by high-K+ perfusion provides support for the electrophysiological finding that the conductance of cardiac gap junctions is insensitive to membrane potential.     

Role of gap junctions in fluid secretion of lacrimal glands

In glands such as the liver and pancreas, gap junctions containing connexin 26 and 32 (Cx26 and Cx32, respectively) couple the secretory cells. Uncoupling these junctions compromises the secretory function of these glands. Lacrimal glands also contain extensive arrays of gap junctions consisting of Cx26 and Cx32. We wanted to determine the role of these junctions in fluid secretion. In Cx32-deficient mice, immunocytochemistry showed that, in the male lacrimal gland, the remaining Cx26 was found evenly distributed in the membrane whereas there was little in the membranes of female glands. Western blot analysis of Cx26 showed that female Cx32-deficient mice expressed Cx26. Patch-clamp analyses of acinar cell coupling showed that the cell pairs from male glands were coupled whereas those from female glands were not. Stimulated fluid production by the glands from Cx32-deficient mice was abnormally low in female glands compared with controls at low topical doses of carbachol. The protein secretory response to different doses of carbachol was the same in all animals. These data suggest that gap junctions are essential for optimal fluid secretion in lacrimal glands.     

ASSEMBLY OF GAP JUNCTIONS DURING AMPHIBIAN NEURULATION

Sequential thin-section, tracer (K-pyroantimonate, lanthanum, ruthenium red, and horseradish peroxidase), and freeze-fracture studies were conducted on embryos and larvae of Rana pipiens to determine the steps involved in gap junction assembly during neurulation. The zonulae occludentes, which join contiguous neuroepithelial cells, fragment into solitary domains as the neural groove deepens. These plaque-like contacts also become permeable to a variety of tracers at this juncture. Where the ridges of these domains intersect, numerous 85-Å participles apparently pile up against tight junctional remnants, creating arrays recognizable as gap junctions. With neural fold closure, the remaining tight junctional elements disappear and are replaced by macular gap junctions. Well below the junctional complex, gap junctions form independent of any visible, preexisting structure. Small, variegated clusters, containing 4–30 particles located in flat, particle-free regions, characterize this area. The number of particles within these arrays increases and they subsequently blend together into a polygonally packed aggregate resembling a gap junction. The assembly process in both apical and basal regions conforms with the concept of translational movement of particles within a fluid plasma membrane.     

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.     

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.     

Molecular portrait of lens gap junction protein MP70

A 70-kDa membrane protein (MP70) is a component of the lens fiber gap junctions. Its membrane topology and its N-terminal sequence are similar to those of the connexin family of proteins. Some features of MP70 containing fiber gap junctions are, however, distinct from gap junctions in other mammalian tissues: (i) Lens connexons form crystalline arrays only after cleavage of junctional proteins in vitro. These hexagonal arrays have a periodicity of 13.6 nm which is significantly larger than the 8- 9-nm spacing of liver and heart gap junctions. (ii) Lens fiber gap junctions dissociate in low concentrations of nonionic detergent and this provides an avenue to purify MP70 directly from a membrane mixture. Isolated MP70 in the form of 17 S structures has an appearance consistent with connexon pairs. (iii) The C-terminal half of MP70 is cleaved in situ by a lens endogenous calcium-dependent protease. The processed from MP38 remains in the membrane and is abundant in the central region of the lens. A testable hypothesis for MP70 function is presented.     

Molecular organization of gap junction membrane channels

Gap junctions regulate a variety of cell functions by creating a conduit between two apposing tissue cells. Gap junctions are unique among membrane channels. Not only do the constituent membrane channels span two cell membranes, but the intercellular channels pack into discrete cell-cell contact areas formingin vivo closely packed arrays. Gap junction membrane channels can be isolated either as two-dimensional crystals, individual intercellular channels, or individual hemichannels. The family of gap junction proteins, the connexins, create a family of gap junctions channels and structures. Each channel has distinct physiological properties but a similar overall structure. This review focuses on three aspects of gap junction structure: (1) the molecular structure of the gap junction membrane channel and hemichannel, (2) the packing of the intercellular channels into arrays, and (3) the ways that different connexins can combine into gap junction channel structures with distinct physiological properties. The physiological implications of the different structural forms are discussed.     

Short and reversible uncoupling evokes little change in the gap junctions of pancreatic acinar cells

Three different preparations of mouse pancreatic fragments where all the cells tested electrophysiologically showed (a) complete electrical coupling (control), (b) complete uncoupling (after 1-to 2-min exposure to 100% CO2), or (c) complete recoupling (1-2 min after removal of 100% CO2) were fixed, with the electrodes in situ, with 0.2% glutaraldehyde and freeze-fractured for quantitative analysis of acinar cell gap junctions. No obvious difference was observed between gap junctions of coupled and uncoupled acinar cells. However, quantitation revealed a small (2.3-5.6%) increase in particle diameter and spacing within junctions of uncoupled cells. Such increase was rapidly reversed upon cell recoupling. In all preparations, most of the gap junctions were made up of disordered arrays of particles but a few of them showed a more tight packing of their particles of which most had lost the usual globular appearance. These "amorphous" gap junctions had larger particle diameter but smaller particle spacing than the other gap junctions and these parameters were not modified during cell uncoupling. However, "amorphous" gap junctions were more frequent in the latter condition.     

Gap junction connexon configuration in rapidly frozen myocardium and isolated intercalated disks

By using two ultrarapid freezing techniques, we have captured the structure of rat and rabbit cardiac gap junctions in a condition closer to that existing in vivo than to that previously achieved. Our results, which include those from fully functional hearts frozen in situ in the living animal, show that the junctions characteristically consist of multiple small hexagonal arrays of connexons. In tissue frozen 10 min after animal death, however, unordered arrays are common. Examination of junction structure at intervals up to 40 min after death reveals a variety of configurations including dispersed and close-packed unordered arrays, and hexagonal arrays. By use of an isolated intercalated disk preparation, we show that the configuration of cardiac gap junctions in vitro cannot be altered by factors normally considered to induce functional uncoupling. These experiments demonstrate that, contrary to the conclusions of some earlier studies (Baldwin, K. M., 1979, J. Cell Biol., 82:66-75; Peracchia, C., and L. L. Peracchia, 1980, J. Cell Biol., 87:708-718), the arrangement of gap junction connexons, in cardiac tissue at least, cannot be used as a reliable guide to the functional state of the junctions.     

The role of gap junctions in trophoblastic cell fusion in the guinea-pig placenta

Summary Fusion of cytotrophoblast cells in the guinea-pig placenta occurs at regions of plasma membrane interdigitation where the cells are attached to one another by complex arrays of gap junctions and desmosomes. Fusion begins at the gap junctions, which are lost in this process. The desmosomes play no obvious part in the fusion mechanism and remain after fusion as sites of attachment of syncytiotrophoblast membrane to itself. It is proposed that a major role of gap junctions in placental development is to bring trophoblast plasma membranes into a close relationship which may act as a starting point for cell fusion.     

Morphological variations in gap junctions of ovarian granulosa cells.

Granulosa cells in ovarian follicles of rat, mouse, rabbit and hamster were studied by lanthanum tracer and freeze-fracture techniques. Abundant gap junctions exhibited striking intraspecific variation in size and pattern of particle aggregation. The smaller gap junctions showed close packing of the intramembranous A face particles. In large gap junctions, ranging up to 6 mu in diameter, particles were packed in rectilinear arrays separated by a labyrinthine network of particle-free 'aisles'. Small clusters of particles in a particle-poor circumferential zone suggested enlargement of junctions by peripheral accretion. Linear intramembranous structures, resembling those of occluding junctions, occasionally bounded large gap junctions. Spherical intracytoplasmic structures limited by gap junctional membranes were shown by tracer studies to arise by invagination of the cell surface. These were intrepreted as a means of disposal of junctions by interiorization.