Similarity of junctions between plasma membranes and endoplasmic reticulum in muscle and neurons

The structure of membranes at junctions between the plasma membrane and underlying cisterns of endoplasmic reticulum in amphioxus muscle and mouse cerebellar neurons was studied using the freeze-fracture technique. In amphioxus muscle, subsurface cisterns of sarcoplasmic reticulum form junctions with the surface membrane at the level of the sarcomere I bands. On the protoplasmic leaflet of the sarcolemma overlying these junctions were aggregates of large particles. On the protoplasmic leaflet of the membranes of cerebellar basket, stellate and Purkinie cells there were similar aggregates of large particles. In both tissues, the corresponding external membrane halves had arrays of pits apparently complementary to the aggregates of large particles. Cross fractures through junctions showed that the particle aggregates in neuronal and muscle membranes were consistently located over intracellular cisterns closely applied to the plasma membrane. Thus, a similar plasma membrane specialization is found at subsurface cisterns in mammalian neurons and amphioxus muscle. This similarity supports the hypothesis that subsurface cisterns in neurons, like those in muscle, couple some intracellular activity to the electrical activity of the plasma membrane.     

Fine structural analysis of membrane changes during reaggregation culture of chick optic tectum

Thin section and freeze-fracture electron microscopy have been used to characterize the changes in membrane morphology of reaggregating cultures of chick optic tectum. The cells are rounded and freely dispersed at 0 hr after dissociation. Between 2 and 6 hr the cells become closely apposed on all sides by other cells and form small aggregates. At this time punta adhaerentia junctions and focal densities are seen along the membranes of neighboring cells. Between 1 and 5 days in vitro (DIV) neurites containing growth cone regions are present. At 5 DIV the first synaptic contacts are observed. Between 7 and 14 DIV, the number of synaptic contacts increase and fewer growth cone regions are observed. As early as 7 DIV profiles are observed which strongly resemble both astrocytic and oligodendroglial cell somata and processes. Freeze-fracture analysis of aggregates at 0–4 hr reveals a sparse particle distribution on the P and E faces of apposed cells. By 1 DIV small clusters of loosely packed, large sized particles are seen on the P face of apposed cell membranes which may represent junctional contacts. Apparent coated vesicle fusion sites are common on the P face at 1–2 DIV. By 7 DIV, E face particle arrays are seen on cell bodies and neurites which correspond to specializations characteristic of excitatory synaptic junctions. By 8–10 DIV particle arrays are seen on the P face of post-synaptic membrane which may represent inhibitory synaptic contacts. Other types of particle specializations seen in freeze-fracture replicas include: specializations characteristic of gap junctions between cells and orthogonal assemblies of particles thought to be characteristic of astrocytes.     

Quick-freeze,deep-etch visualization of the ‘cytoskeletal spring’ of cochlear outer hair cells

Summary The lateral membrane system of the cochlear outer hair cell, consisting of the lateral plasma membrane, pillars, filamentous lattice and subsurface cisternae, is considered to be involved in the contractile movement of the isolated cochlear outer hair cell. The filamentous lattice, called the cytoskeletal spring, has been identified in the demembranated cochlear outer hair cell treated with the detergent Triton X-100. In this study, the quick-freeze, deep-etch method was applied to demonstrate the three-dimensional organization of both the filamentous and membranous structures of the lateral membrane system of cochlear outer hair cells. Treatment with saponin revealed that the inner leaflet of the lateral plasma membrane of the cochlear outer hair cell possesses more membrane particles than the outer leaflets, and that the pillars are closely associated with membrane particles in the inner leaflet of the lateral membrane. The presence of filamentous bridges between the filamentous lattice and the subsurface cisternae was also detected. We propose that the lateral membrane system in the cochlear outer hair cell may play an important role in the tuning mechanisms within the cochlea in normal hearing.     

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)     

Distribution and mobility of lectin receptors on synaptic membranes of identified neurons in the central nervous system

The distribution and mobility of concanavalin A (Con A) and Ricinus communis agglutinin (RCA) receptors (binding sites) on the external surfaces of Purkinje, hippocampal pyramidal, and granule cells and their attached boutons were studied using ferritin-lectin conjugates. Dendritic fields of these cells were isolated by microdissection and gently homogenized. Cell fragments and pre- and postsynaptic membranes were labeled with the ferritin-lectin conjugates at a variety of temperatures, and the distribution of lectin receptors was determined by electron microscopy. Both classes of these lectin receptors were concentrated at nearly all open and partially open postsynaptic junctional membranes of asymmetric-type synapses on all three neuron types. Con A receptors were most concentrated at the junctional membrane region, indicating that the mature neuron has a specialized nonrandom organization of carbohydrates on its outer surface. Lectin receptors located on postsynaptic junctional membranes appeared to be restricted in their mobility compared to similar classes of receptors on extrajunctional membrane regions. Labeling with ferritin-RCA and - Con A at 37 degrees C produced clustering of lectin receptors on nonjunctional surfaces; however, Con A and RCA receptors retained their nonrandom topographic distribution on the postsynaptic junctional surface. The restricted mobility of lectin receptors was an inherent property of the postsynaptic membrane since the presynaptic membrane was absent. It is proposed that structures in the postsynaptic density may be transmembrane-linked to postsynaptic receptors and thereby determine topographic distribution and limit diffusion of specialized synaptic molecules. Speicalized receptor displays may play an important role in the formation and maintenance of specific synaptic contacts.     

Chloroplast membrane structure. Intramembranous particles of different sizes make contact in stacked membrane regions.

The supramolecular architecture of stacked thylakoid membrane regions of class II spinach chloroplasts has been investigated by means of freeze-fracture electron microscopy. Such membranes contain two basic types of intramembranous particles: laarge particles, which are found on the fracture face of the lumenal membrane leaflet (Bs face), and smaller ones which are found on the fracture face of the external leaflet (Cs face). By analyzing thylakoid membranes containing geometrical arrangements of intramembranous particles it is shown (a) that within the plane of each membrane approximately two small particles are associated with each large particle, and (b) that normal thylakoid stacking involves the connection of large particles of one membrane to small particles of the other and vice versa. If the two types of particles are related to Photosystems I and II, as suggested by circumstantial evidence, then our observations provide support for the idea that maximum Photosystem I-photosystem II interaction is obtained by intermembrane subunit interaction in grana stacks. To this end, our results suggest that stacking should enhance the quantum yield at very low light intensities.     

Hormonal modulation of ovarian interstitial cells with particular reference to gap junctions

Thin-section and freeze-fracture studies on the rat ovarian interstitial cells revealed reductions in the size and the number of gap junctions after pituitary ablation. Small gap junctions, however, persist as long as 90 days after hypophysectomy even though regressive cytoplasmic changes are completed 75 d earlier. Administration of exogenous human chorionic gonadotrophin (HCG) results in the restoration of the normal interstitial cell morphology which is accompanied by amplification of junctional membrane. Within 24 h of hormone application, gap junction growth is characterized by the appearance of formation plaques. These studies suggest that the effect of hormone on interstitial cell gap junctions is to modulate the junctional surface area.     

Freeze-fracture studies of frog atrial fibres.

The freeze-fracturing technique was used to characterize the junctional devices involved in the electrical coupling of frog atrial fibres. These fibres are connected by a type of junction which can be interpreted as a morphological variant of the "gap junction" or "nexus". The most characteristic features are rows of 9-nm junctional particles forming single or anastomosed circular profiles on the inner membrane face, and corresponding pits on the outer membrane face. Very seldom aggregates consisting of few geometrically disposed 9-nm particles are found. The significance of the junctional structures in the atrial fibres is discussed, with respect to present knowledge about junctional features of gap junctions in various tissues, including embryonic ones.     

Fusion images and intramembrane particle aggregates during the action of antidiuretic hormone

Summary Antidiuretic hormone (ADH) causes the appearance of water-conducting particle aggregates in the luminal membrane of receptor cells in amphibian bladder and skin, and in the mammalian collecting duct. The aggregates originate from cytoplasmic tubules that fuse with the luminal membrane during ADH stimulation. We have studied the process of fusion and the structure of the particle aggregates by a rapid-freeze technique that renders chemical fixation and glycerol protection unnecessary. Our findings differ in some important respects from previously published work. Aggregate particles, in our study, partition equally between the external (EF) and protoplasmic (PF) membrane leaflets, rather than remaining in the protoplasmic leaflet exlcusively. By including the entire population of fusion images in our survey, we have found that aggregate delivery in ADH-treated cells proceeds preferentially from small fusion images whose diameter is significantly less than the 0.12 m characteristic of the carrier tubules themselves. We have also found that, even in unstimulated preparations, fusion images are numerous, being mostly of small diameter. ADH stimulation produces a moderate increase in the number of fusion images and a significant increase in fusion-image diameter. These findings indicate that the individual particles are mobile within the membrane, lacking interparticle linkage. In addition, contact of cytoplasmic tubules with the luminal membrane may take place even in the absence of ADH, producing small fusion images which are not associated with aggregate delivery to the luminal membrane.Faculty Scholar, Josiah Macey Jr. Foundation     

Loss and reappearance of gap junctions in regenerating liver

Changes in intercellular junctional morphology associated with rat liver regeneration were examined in a freeze-fracture study. After a two-thirds partial hepatectomy, both gap junctions and zonulae occludentes were drastically altered. Between 0 and 20 h after partial hepatectomy, the junctions appeared virtually unchanged. 28 h after partial hepatectomy, however, the large gap junctions usually located close to the bile canaliculi and the small gap junctions enmeshed within the strands of the zonulae occudentes completely disappeared. Although the zonulae occludentes bordering the bile canaliculi apparently remained intact, numerous strands could now be found oriented perpendicular to the canaliculi. In some instances, the membrane outside the canaliculi was extensively filled with isolated junctional strands, often forming very complex configurations. About 40 h after partial hepatectomy, very many small gap junctions reappeared in close association with the zonulae occludentes. Subsequently, gap junctions increased in size and decreased in number until about 48 h after partial hepatectomy when gap junctions were indistinguishable in size and number from those of control animals. The zonulae occludentes were again predominantly located around the canalicular margins. These studies provide further evidence for the growth of gap junctions by the accretion of particles and of small gap junctions to form large maculae.     

Ultrastructural and chemical analysis of the outer membrane leaflet of the human red blood cell

Structural and biochemical analysis of the outer membrane leaflet of human erythrocytes freeze-fractured on positively charged supports showed that glycophorin A is its major constituent. Two classes of intramembrane particles can be discriminated on the external fracture face: those which are high but small in diameter and those which are low and large or elongated. The presence of small amount of band 3 protein in the outer membrane leaflet cannot be ruled out; it could be contained in the class of 'high' intramembrane particles on the external fracture face.     

Glial cells maintain synaptic structure and function and promote development of the neuromuscular junction in vivo

To investigate the in vivo role of glial cells in synaptic function, maintenance, and development, we have developed an approach to selectively ablate perisynaptic Schwann cells (PSCs), the glial cells at the neuromuscular junction (NMJ), en masse from live frog muscles. In adults, following acute PSC ablation, synaptic structure and function were not altered. However, 1 week after PSC ablation, presynaptic function decreased by approximately half, while postsynaptic function was unchanged. Retraction of nerve terminals increased over 10-fold at PSC-ablated NMJs. Furthermore, nerve-evoked muscle twitch tension was reduced. In tadpoles, repeated in vivo observations revealed that PSC processes lead nerve terminal growth. In the absence of PSCs, growth and addition of synapses was dramatically reduced, and existing synapses underwent widespread retraction. Our findings provide in vivo evidence that glial cells maintain presynaptic structure and function at adult synapses and are vital for the growth and stability of developing synapses.     

Acetylcholine receptor aggregation parallels the deposition of a basal lamina proteoglycan during development of the neuromuscular junction

To determine the time course of synaptic differentiation, we made successive observations on identified, nerve-contacted muscle cells developing in culture. The cultures had either been stained with fluorescent alpha-bungarotoxin, or were maintained in the presence of a fluorescent monoclonal antibody. These probes are directed at acetylcholine receptors (AChR) and a basal lamina proteoglycan, substances that show nearly congruent surface organizations at the adult neuromuscular junction. In other experiments individual muscle cells developing in culture were selected at different stages of AChR accumulation and examined in the electron microscope after serial sectioning along the entire path of nerve-muscle contact. The results indicate that the nerve-induced formation of AChR aggregates and adjacent plaques of proteoglycan is closely coupled throughout early stages of synapse formation. Developing junctional accumulations of AChR and proteoglycan appeared and grew progressively, throughout a perineural zone that extended along the muscle surface for several micrometers on either side of the nerve process. Unlike junctional AChR accumulations, which disappeared within a day of denervation, both junctional and extrajunctional proteoglycan deposits were stable in size and morphology. Junctional proteoglycan deposits appeared to correspond to discrete ultrastructural plaques of basal lamina, which were initially separated by broad expanses of lamina-free muscle surface. The extent of this basal lamina, and a corresponding thickening of the postsynaptic membrane, also increased during the accumulation of AChR and proteoglycan along the path of nerve contact. Presynaptic differentiation of synaptic vesicle clusters became detectable at the developing neuromuscular junction only after the formation of postsynaptic plaques containing both AChR and proteoglycan. It is concluded that motor nerves induce a gradual formation and growth of AChR aggregates and stable basal lamina proteoglycan deposits on the muscle surface during development of the neuromuscular junction.     

A freeze-fracture study of membrane events during neurohypophysial secretion

Freeze-fracture was used to study the membrane events taking place during neurosecretory granule discharge (exocytosis) and subsequent membrane internalization (endocytosis) in axons of neurohypophyses from control and water-deprived rats. En face views of the cytoplasmic leaflet (P face) of the split axolemma reveal circular depressions that represent the secretory granule membranes fused with the plasma membrane during exocytosis. These depressions often contain granule core material in the process of extrusion into the extracellular space. The membrane surrounding some of the exocytotic openings shows a decreased number of intramembrane particles (mean diameter, 8 nm) which are elsewhere more numerous and evenly distrubuted on the fracture face. Endocytotic sites appear as smaller plasma membrane invaginations, with associated intramembrane particles. Moreover, such invaginations often contain large particles (mean diameter, 12 nm) that appear as clusters on en face views of the membrane leaflet. Quantitative analysis indicates that the number of exocytotic images increases significantly in glands from water-deprived rats. Concomitantly, the number of endocytotic figures per unit area of membrane is raised as is the number of clusters of large particles. The observations demonstrate that, in the neurohypophysis, it is possible to distinguish exocytosis morphologically from endocytosis and that the two events can be assessed quantitatively.     

Changes occurring in plasma membranes and intercellular junctions during the process of carcinogenesis and in squamous cell carcinoma

A 0.5% mineral-oil solution of 9.10-dimethyl-1.2-benzanthracene (DMBA) was applied to artificial cecal pouches in the lower lips of rats. Ultrastructural studies were made of plasma membranes and intercellular junctions during the process of malignant transformation in the oral mucosal epithelium and after squamous cell carcinoma had been induced by the carcinogen. After the administration of DMBA, the inner leaflet of the membranes where the microfilaments are attached showed high electron density and intramembranous particles on the P-face of basal cells decreased to about half that of controls. However, on the E-face the number of intramembranous particles increased by approximately 10% compared with controls. Though the normal size range for intramembranous particles was 9-12 nm, the administration of DMBA caused aggregations of from three to six particles on the P-face. In squamous cell carcinomas, only the outer leaflet of the membranes showed high electron density; the number of intramembranous particles was 30% higher on the P-face and approximately three times higher on the E-face compared with controls and the morphology of the intramembranous particles, which formed irregular aggregates of from five to 20 particles, was specific. In animals treated with DMBA, the number of gap junctions decreased by between 50% and 70%, although no structural changes occurred. In squamous cell carcinomas, the area of gap junctions was about 50% lower and the number of gap junctions about 40% lower than in controls. Changes in the number and area of desmosomes were similar to those of gap junctions both in the DMBA-treated animals and in squamous cell carcinomas.     

LUMENAL PLASMA MEMBRANE OF THE URINARY BLADDER : I. Three-Dimensional Reconstruction from Freeze-Etch Images

To determine the three-dimensional structure of the lumenal membrane of transitional epithelium, a study was made of sectioned, negatively stained, and freeze-etched specimens from intact epithelium and membrane fractions from rabbit urinary bladder. Particulate membrane components are confined to plaque regions within which the unit membrane is asymmetric, having a thicker outer leaflet. Transversely fractured freeze-etched plaques display a thick (~80 A), particulate lumenal leaflet and a thin (~40 A) cytoplasmic one. Four different faces of the two leaflets can be distinguished: two complementary, split, inner membrane faces exposed by freeze-cleaving the bilayer and two external (lumenal and cytoplasmic) membrane surfaces revealed by deep-etching. On the split, inner face of the lumenal leaflet appear polygonal plaques of hexagonally arranged particles. These fit into holes observed on the complementary, split, innerface of the cytoplasmic leaflet. The particles, which have a center-to-center spacing of ~160 A, also seem to protrude from the external surface of the lumenal leaflet, where their subunits (~50 A in diameter) are revealed by freeze-etching and negative staining. The plaques are separated from each other by smooth-surfaced regions, which cleave like simple lipid bilayers. Since the array of plaque particles covers only ~73% of the membrane surface area, whereas 27% is taken up by particle-free interplaque regions, the presence of particles cannot in itself entirely account for the permeability barrier of the lumenal membrane. Although no particles are observed protruding from the cytoplasmic surface of the membrane, cytoplasmic filaments are attached to it by short, cross-bridge-like filaments that seem to contact the particles within the membrane. These long cytoplasmic filaments cross-link adjacent plaques. Therefore, we suggest that at least one function of the particles is to serve as anchoring sites for cytoplasmic filaments, which limit the expansion of the lumenal membrane during distention of the bladder, thereby preventing it from rupturing. The particle-free interplaque regions probably function as hinge areas between the stiff plaques, allowing the membrane to fold up when the bladder is contracted.     

Cytoskeletal organization at the postsynaptic complex

Postsynaptic densities and the adjacent cytoskeleton were examined in deep-etched, unfixed slices of guinea pig anteroventral cochlear nucleus. The postsynaptic density seen in conventional thin sections corresponds to a meshwork of 4-nm filaments associated with intramembrane particles at the postsynaptic active zone of inhibitory as well as excitatory synapses. These filaments intermesh with a lattice of 8- to 9-nm microfilaments, tentatively identified as F- actin, that is concentrated under the postsynaptic density. We postulate that the meshwork of 4-nm filaments anchors receptors to the adjacent microfilament lattice; this extended postsynaptic complex may limit the mobility of receptors and help maintain the curvature of the postsynaptic membrane.     

Effect of castration and testosterone administration on the neuromuscular junction in the levator ani muscle of the rat

Summary The ultrastructure of the neuromuscular junction (n.m.j.) of the androgen-sensitive levator ani muscle was studied in normal adult male rats, in 8-month-old rats castrated at the age of one month and in castrated rats treated with testosterone propionate (TP). Castration does not result in significant changes of the n.m.j. The density of synaptic vesicles and the postsynaptic junctional folds remain practically normal in spite of marked atrophy of the muscle. TP administration for 7 days results in marked changes in preand postsynaptic structures. There is slow progressive depletion of synaptic vesicles, appearance of cisternae and coated vesicles in axon terminals, and coalescence of coated vesicles with the plasma membrane. Coated vesicles are also found inside Schwann cells and among junctional folds. Dense core vesicles appear both in the axon terminals and in the postsynaptic area. Collateral sprouting of terminal axons with the formation of new immature junctions is observed. After 35 days of TP administration depletion of synaptic vesicles continues. Glycogen -particles, mostly freely dispersed, occasionally seen in axon terminals 7 days after TP administration, subsequently increase in number. In the endplate zone of the muscle fibre increased protein synthesis is indicated by a rapid increase in ribosomes and irregularly located myofilaments and myofibrils. The appearance of n.m.j. after testosterone administration resembles that described after nerve stimulation; the degree of change is however less pronounced.The authors wish to acknowledge the skillful technical assistance of Mrs. L. Vedralová     

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.     

Postsynaptic membranes in the electric tissue of Narcine: II. A freeze-fracture study of nicotinic receptor molecules

The ventral, postsynaptic membranes of the electroplaques of Narcine were found to containe intramembranous particles similar in location, packing density (about 5700/micron 2), transmembrane position and end appearance to nicotinic acetylcholine receptor-channel molecules. In fixed tissue the particles were limited to the cytoplasmic lamina, while in unfixed tissue an equivalent number were found symmetrically in both laminae. Four populations of particle diameters were observed in each unfixed lamina, even though other morphological evidence indicates the presence of large number of molecules of uniform structure, and biochemical studies of isolated postsynaptic membranes indicate that at least 70% of the membrane protein is receptor-channel protein. Intramembranous particles in dorsal, non-innervated electroplaque membranes, presumably representing Na+, K+-associated ATPase and other channel proteins, were found to have similar characteristics to particles in ventral membranes. Receptor-channel molecules cannot, therefore, be distinguished from other intrinsic membrane proteins by freeze-replication alone.