Synaptic ribbons in the pineal organ of the goldfish: Circadian rhythmicity and the effects of constant light and constant darkness

Summary Synaptic ribbons in photoreceptor cells of the goldfish pineal organ undergo significant daily changes in their length, distance from the plasma membrane, and number per unit area of pineal end-vesicle. The rhythms persist in fish exposed to constant darkness. Constant light abolishes the rhythms in length and distance of synaptic ribbons from the plasmalemma, but has little effect on numerical changes over a 24-h cycle. These findings suggest that synaptic ribbons in the pineal organ of lower vertebrates might be useful as indicators of metabolic activity.     

An ethanolic phosphotungstic acid (EPTA) analysis of photoreceptor and synaptic ultrastructure in the guinea pig retina

Ethanolic phosphotungstic acid (EPTA) has been used to elucidate the structure of certain organelles contained within retinal cells not clearly discernible using conventional preparations. Both synaptic and nonsynaptic components of the guinea pig neural retina have been analyzed. Within the photoreceptor (PR) cell EPTA-stained components include the connecting cilia, their basal bodies, and the root filament system. Cross-striated fibrillar organelles, similar in appearance to the root filaments, are also observed in the nuclear region, the synaptic terminal and other parts of the PR cell. The possible structural continuity and significance of these structures are discussed. Within retinal synapses of both the inner and outer plexiform layers, ribbons and associated paramembranous specializations are stained. The photoreceptor ribbons have a trialaminar structure with filamentous, tufted borders. Synaptic cleft material and postsynaptic densities are also stained. Bipolar cell synapses in the inner plexiform layer contain stained short ribbons as well as closely associated peg-like densities extending towards the presynaptic membrane.     

Prenatal development of "synaptic" ribbons in the guinea pig pineal gland

Pineal "synaptic" ribbons are a heterogeneous population of organelles. "Synaptic" ribbons (SR) sensu stricto, "synaptic" spherules (SS), and intermediate forms (IMF) are present. Their function and origin are unknown, and a knowledge of their prenatal development is lacking. Thus the pineal glands of prenatal, neonatal, and adult guinea pigs were prepared for electron microscopy. "Synaptic" ribbons were studied morphologically and quantitatively. The three categories of "synaptic" ribbons reported in adult pineal glands were also present in prenatal pineal glands. Their structural features, distribution, grouping, and composition patterns are similar to those in adults. "Synaptic" ribbons were first detected in pinealocytes of the distal region of a 42-day postcoitus (PC) pineal gland and were comparable with those in adults. They increased in number with age and reached a peak at 63 days PC, followed by a steep decline at 66 and 67 days PC. By day 69 PC, the numbers increased again and showed a dramatic increase after birth. Several true ribbon synapses were seen at day 63 PC between pinealocyte cell processes or between pinealocyte cell process and pinealocyte cell body. Since true ribbon synapses have not been found in adult guinea pig pinealocytes, their synaptic nature could have been lost during development. No precursors for the "synaptic" ribbons were found. The endoplasmic reticulum cisternae may be the origin for the ribbon vesicles because of their close association with the "synaptic" ribbons.     

Histotypic organization of the rat retina in vitro

Summary Retinae from two- and three-day-old rats were explanted in plasma clots and grown in vitro with the flying coverslip method. After seven to seventeen days in culture, the retinal tissue was fixed with aldehydes and osmium tetroxide and embedded for examination with the electron microscope. Study of cross sections (perpendicular to the coverslip) revealed a histotypic pattern of organization, especially in the thicker regions of the explants. Layering of cells quite similar to that in the intact retina was seen to develop from the relatively primitive, explanted retinal epithelium. However, each layer contained fewer cells than its counterpart in vivo. All major neuronal cell types were distinguished by their location and cytological characteristics. Development of the saccules of sensory cell outer segments was observed to occur in vitro by an infolding of the plasma membrane. Synaptic ribbon complexes developed to the mature form in the outer plexiform layers, while conventional synapses were numerous in the inner plexiform layers. Synaptic ribbons were also seen in bipolar cell axons in the inner plexiform layers. Amacrine and ganglion cells in these regions were relatively sparse. A survey of posterior regions of noncultured three-day-old rat retinae showed no synapses of any sort; therefore the synapses in the cultures formed in vitro. The retina is recommended for studies of synaptogenesis in tissue culture, for it offers several advantages over expiants from other areas of the neuraxis, including a clear layering pattern, many identifiable cell processes with characteristic synaptic relationships between them, and a well-defined sequence of developmental events.Dedicated to Professor Wolfgang Bargmann on the occasion of his 65th birthday.     

Cholesterol depletion induces dynamic confinement of the G-protein coupled serotonin1A receptor in the plasma membrane of living cells

Cholesterol is an essential constituent of eukaryotic membranes and plays a crucial role in membrane organization, dynamics, function, and sorting. It is often found distributed non-randomly in domains or pools in biological and model membranes and is thought to contribute to a segregated distribution of membrane constituents. Signal transduction events mediated by seven transmembrane domain G-protein coupled receptors (GPCRs) are the primary means by which cells communicate with and respond to their external environment. We analyzed the role of cholesterol in the plasma membrane organization of the G-protein coupled serotonin_1A receptor by fluorescence recovery after photobleaching (FRAP) measurements with varying bleach spot sizes. Our results show that lateral diffusion parameters of serotonin_1A receptors in normal cells are consistent with models describing diffusion of molecules in a homogenous membrane. Interestingly, these characteristics are altered in cholesterol-depleted cells in a manner that is consistent with dynamic confinement of serotonin_1A receptors in the plasma membrane. Importantly, analysis of ligand binding and downstream signaling of the serotonin_1A receptor suggests that receptor function is affected in a significantly different manner when intact cells or isolated membranes are depleted of cholesterol. These results assume significance in the context of interpreting effects of cholesterol depletion on diffusion characteristics of membrane proteins in particular, and cholesterol-dependent cellular processes in general.     

Membrane Dynamics of the Contractile Vacuole Complex of Paramecium1

ABSTRACT. Membrane dynamics of the contractile vacuole complex of Paramecium were investigated using conventional electron microscopy of cells so that the vacuoles were serial-sectioned longitudinally and transversely. During systole, vacuolar membrane collapses first into flattened cisternae which undergo further modification into a mass of interconnected small membrane tubules. These tubules retain their connections with the radiating microtubular ribbons; consequently they are found only in the poleward hemisphere. Permanent connections between ampullae and the collapsed vacuole membrane could not be verified nor was a sphincter-like mechanism for closing such a junction observed. Membranes of the ampullae and the collecting canals also collapse to varying extents into arrays of tubules that remain bound to microtubular ribbons during diastole. Thus vacuole, ampullae, and collecting canal membranes all assume tubular forms when internal volume is at a minimum. Having failed to observe a microfilamentous encasement of the vacuole, we suggest that an alternative mechanism for the “contractile” function should be sought. One such is based on fluid volume increase and fluid flow within transiently interconnected tubular membrane systems that cycle between a tubular and a planar membrane form as internal volume is periodically increased and reduced. The driving force for this mechanism might best be sought in the molecular structure of the membranes of the contractile vacuole complex.     

Seasonal ultrastructural alterations in the plasma membrane produced by slow freezing in cortical tissues of mulberry (Morus bombyciz Koidz. cv. Goroji)

Seasonal alterations in the ultrastructure of the plasma membrane produced by slow freezing were examined in cortical parenchyma cells of mulberry twigs (Morus bombyciz Koidz. cv. Goroji) grown in northern Japan. In freezing-sensitive summer, freezing produced distinct aparticulate domains with accompanying inverted hexagonal_II (H_II) phase transitions in the plasma membrane. In autumn and spring, during cold acclimation and deacclimation, freezing produced aparticulate domains in the plasma membrane without accompanying H_ii phase transitions. In winter, when the twigs were freezing-tolerant, freezing did not produce ultrastructural alterations in the plasma membrane. A significant relationship was recognized between the percentages of cells with aparticulate domains in the plasma membrane, regardless of the presence or absence of H_II phase transitions, and the occurrence of freezing injury throughout all seasons and at all freezing temperatures tested in each season. The aparticulate domains in the plasma membranes were shown to be produced by the close apposition of membranes due to freezing-induced dehydration and deformation of cells. Although the precise mechanisms that cause injury as a result of the formation of aparticulate domains in the plasma membrane remain unclear, our results indicate that the development of cold acclimation paralleled the process whereby cells developed the ability to reduce and finally to prevent the formation of aparticulate domains in the plasma membrane that would otherwise result from freezing-induced cellular dehydration and deformation that brings membranes into close proximity with one another.     

Boron deficiency-induced impairments of cellular functions in plants

The essentiality of B for growth and development of plants is well-known, but the primary functions of B still remain unknown. Evidence in the literature supports the idea that the major functions of B in growth and development of plants are based on its ability to form complexes with the compounds having cis-diol configurations. In this regard, the formation of B complexes with the constituents of cell walls and plasma membranes as well as with the phenolic compounds seems to be a decisive step affecting the physiological functions of B. Boron seems to be of crucial importance for the maintenance of structural integrity of plasma membranes. This function of B is mainly related to stabilisation of cell membranes by B association with membrane constituents. Possibly, B may also protect plasma membranes against peroxidative damage by toxic O₂ species. In B-deficient plants, plasma membranes are highly leaky and lose their functional integrity. Under B-deficient conditions, substantial changes in ion fluxes and proton pumping activity of the plasma membranes were noted. Impairments in phenol metabolism and increases in levels of phenolics and polyphenoloxidase activity are typical indications of B deficiency, particularly in B deficiency-sensitive plant species, such as Helianthus annuus (sunflower). Enhanced oxidation of phenols is responsible for generation of reactive quinones which subsequently produce extremely toxic O₂ species, thus resulting in the increased risk of a peroxidative damage to vital cell components such as membrane lipids and proteins. In B-deficient tissues, enhancement in levels of toxic O₂ species may also occur as a result of impairments in photosynthesis and antioxidative defence systems. Recent evidence shows that the levels of ascorbic acid, non-protein SH-compounds (mainly glutathione) and glutathione reductase, the major defence systems of cells against toxic O₂ species, are reduced in response to B deficiency. There is also increasing evidence that, in the heterocyst cells of cyanobacteria, B is involved in protection of nitrogenase activity against O₂ damage.     

Isolation and partial characterization of rat brain synaptic plasma membranes

Abstract— Synaptic plasma membranes from the cortices of adult rat brain were isolated from synaptosomes prepared by flotation of a washed mitochondrial pellet (P2) in a discontinuous Ficoll-sucrose gradient. Contamination of the synaptosome fraction by microsomes was estimated by enzymic and chemical analysis to be less than 15 per cent. (2) The purified synaptosome fraction was subjected to osmotic shock, subfractionated on a discontinuous sucrose gradient and the distribution of enzymic and chemical markers for synaptic plasma membranes, microsomal membranes and mitochondria was determined. (3) Comparison of synaptosome subfractions prepared in the presence and absence of 1 mM NaH₂ PO₄/0.1 mM EDTA buffer pH 7.5, indicated that the ionic composition of the isolation medium markedly affected the distribution and enzymic composition of the subfractions. (4) Synaptic plasma membranes prepared in the presence of PO₄/EDTA exhibited a 10-fold enrichment in [Na⁺+ K⁺] ATPase and were characterized by less than 15 and 10 per cent contamination by microsomes and mitochondria respectively. (5) The polypeptide composition of the purified synaptic plasma membranes was compared with the microsomes and mitochondria by polyacrylamide gel electrophoresis in sodium dodecyl sulphate. No differences between the protein and glycoprotein composition of the synaptic plasma membranes and microsomes were detected. The mitochondria, in contrast, possessed a unique protein composition.     

Composition lipidique membranaire durant la préparation de spermatozoïdes à la fécondation

The final modifications that the spermatozoa undergo correspond with the destabilization of their plasma membrane. This indispensable step facilitates the fusion of membranes and primes the signal transduction during fertilization. This destabilization is composed of a series of changes and modulation of the lipids in membranes such as cholestérol, phospholipids and glycolipids. Several differences exist in the lipid composition of the plasma, acrosome, nuclear and mitochondrial membranes of spermatozoa. The principal membrane phospholipids are phosphatidyl choline, phosphatidyl ethanolamine and sphingomyelin. Plasma membrane of sperm is also rich in polyunsaturated fatty acids (PUFA) linked to phospholipids. Such as C₁₈∶2n?6, C₂₀∶4n?6 and large amounts of docosahexaenoic acid (C₂₂∶6n?6). The amount of membrane lipids in human sperm varies considerably between patients. This variation, could influence certain functional properties of the sperm cells such as their ability to undergo capacitation, the acrosome reaction and the fusion between sperm and oocyte membranes. The lipid composition of the human sperm cell can be altered during the process of freezing-thawing. A significant decrease in phospholipids (phosphatidyl choline, phosphatidyl ethanolamine), and PUFA in particular docosahexaenoic acid and arachidonic acid was observed. Human spermatozoa have a molar cholestérol/phopholipid ratio ≤1.0, and reduces during capacitation due to loss of cholestérol. In addition, the decrease in the levels of cholestérol and the methylation of phospholipids is involved in the modification of membrane fluidity and in the maturation of the sperm plasma membrane receptors. Therefore it seems that the methylation is important for the fusion between sperm and oocyte membranes. Intrinsic sperm phospholipase A2 also plays a role in the destabilization of the plasma membrane by producing of lysophospholipid. Therefore this enzyme and free fatty acids are believed to play a role in the acrosome reaction, an indispensable event facilitating the fusion between sperm and oocyte membranes.     

Synaptic ribbons during postnatal development of the pineal gland in the golden hamster (Mesocricetus auratus)

Summary Synaptic ribbons, functionally enigmatic structures of mammalian pinealocytes, were studied during the postnatal development of the pineal gland in the golden hamster (Mesocricetus auratus). On day 4 post partum, synaptic ribbons appear in cells that have already started to differentiate into pinealocytes. Between days 4 and 9, an increase in the number of synaptic ribbons occurs, concomitant with the continuing differentiation of the pineal tissue. Between days 9 and 16, when differentiation of this tissue is almost completed, the number of synaptic ribbons decreases and approaches that characteristic of the adult pineal gland. During development, the synaptic ribbons increase in length, and dense core vesicles are frequently found in the vicinity of these structures. It is assumed that a functional relationship exists between dense core vesicles and the synaptic ribbons, which are considered to be engaged in a certain form of secretory activity of the mammalian pineal gland.Supported by the Deutsche Forschungsgemeinschaft     

Antioxidant Ascorbate Is Stabilized by NADH-Coenzyme Q10 Reductase in the Plasma Membrane

Plasma membranes isolated from K562 cells contain an NADH-ascorbate free radical reductase activity and intact cells show the capacity to reduce the rate of chemical oxidation of ascorbate leading to its stabilization at the extracellular space. Both activities are stimulated by CoQ₁₀ and inhibited by capsaicin and dicumarol. A 34-kDa protein (p34) isolated from pig liver plasma membrane, displaying NADH-CoQ₁₀ reductase activity and its internal sequence being identical to cytochrome b ₅ reductase, increases the NADH-ascorbate free radical reductase activity of K562 cells plasma membranes. Also, the incorporation of this protein into K562 cells by p34-reconstituted liposomes also increased the stabilization of ascorbate by these cells. TPA-induced differentiation of K562 cells increases ascorbate stabilization by whole cells and both NADH-ascorbate free radical reductase and CoQ₁₀ content in isolated plasma membranes. We show here the role of CoQ₁₀ and its NADH-dependent reductase in both plasma membrane NADH-ascorbate free radical reductase and ascorbate stabilization by K562 cells. These data support the idea that besides intracellular cytochrome b ₅-dependent ascorbate regeneration, the extracellular stabilization of ascorbate is mediated by CoQ₁₀ and its NADH-dependent reductase.     

Intermediate stages in the disassembly of synaptic ribbons in cone photoreceptors of the crucian carp,Carassius carassius

Synaptic ribbons are trilaminated plate-shaped presynaptic densities of certain types of receptor cells and neurons. In cone photoreceptors, these structures dissassemble and reassemble in response to light and to a variety of other stimuli. We used the lithium-ionenhanced disassembly and reassembly of synaptic ribbons to characterize structural intermediates in these cyclic changes. A few minutes after exposure of isolated retinas from the crucian carp (Carassius carassius) to lithium, ribbons fragmented into 50-nm-sized dense globular structures. These small spheres were concentrically surrounded by synaptic vesicles attached to them by stalk-like fine bridging filaments. Disassembly always started at the free cytoplasmic edges of the ribbons and proceeded toward the membrane-associated edges. As the disassembly process never started at the membraneanchored site, synaptic ribbons appeared to be polarized structures with functionally different ends. Spheres were subjected to further depolymerization. They disintegrated into clusters of small granular material and disappeared after ca. 45 min of lithium treatment. Spheres were not observed during the reassembly of synaptic ribbons, indicating that the assembly of synaptic ribbons proceeds via smaller subunits.     

The development of the plasma membrane reticular system in the fat body of an insect

Several insect tissues have plasma membranes that are folded inwards to make a subsurface reticulum on faces that are exposed to hemolymph. The infolds have been called plasma membrane reticular systems (RSs) to distinguish them from the somewhat similar structures found in transporting epithelia. They are characterized by having negative charges on the plasma membranes of the entranceways and by the concentration of some hemolymph proteins in their lymph spaces. Their formation and loss in the fat body has been studied by scanning electron microscopy during the fifth stadium of Calpodes ethlius (Lepidoptera, Hesperiidae). Fat body cells begin the fifth stadium arranged in ribbons with the cells linked together by a fringe of processes. In the first stage many more processes form. These partially fuse together in the second stage, leaving a subsurface reticulum connected by narrow entrances to the lateral cell faces and the face below the basal lamina. Both the cell processes and the reticular systems that they enclose are usually axially orientated. The completed RS persists for the second half of the intermoult devoted to larval syntheses when the concentration of hemolymph proteins rises. After protein sequestration prior to pupation the RS is lost and the fat body returns to being a tissue of rounded cells linked by a few enmeshed processes.     

ATP-dependent fusion of synaptic vesicles and synaptosomal plasma membrane in a cell-free system

The final step in exocytosis is the fusion of synaptic vesicle membrane with the synaptosomal plasma membrane, leading to the release of the neurotransmitters. We have reconstituted this fusion event in vitro, using isolated synaptic vesicles and synaptosomal plasma membranes from the bovine brain. The membranes of synaptic vesicles were loaded with the lipid--soluble fluorescent probe octadecylrhodamine B at the concentration that resulted in self-quenching of its fluorescence. The vesicles were then incubated with synaptosomal plasma membranes at 37 degrees C and fusion was measured through the dilution-dependent de-quenching of the fluorescence of the probe. Synaptic vesicles by themselves did not fused with plasma membrane, only addition of ATP induced the fusion. W-7 and trifluoroperasine, the drugs reported to inhibit calmodulin-dependent events, were effective inhibitors of the ATP-induced fusion synaptic vesicles and synaptosomal plasma membranes. Our results indicate that the membrane fusion in the nerve terminals during exocytosis may be under direct control of calmodulin-dependent protein phosphorylation.     

Coenzyme Q distribution in HL-60 human cells depends on the endomembrane system

Coenzyme Q (Q) is an essential factor in the mitochondrial electron chain but also exerts important antioxidant functions in the rest of cell membranes of aerobic organisms. However, the mechanisms of distribution of Q among cell membranes are largely unclear. The aim of the present work is to study the mechanisms of distribution of endogenous Q₁₀ and exogenous Q₉ among cell membranes in human HL-60 cells. Endogenous Q₁₀ synthesized using the radiolabelled precursor [¹⁴C]-pHB was first detected in mitochondria, and it was later incorporated into mitochondria-associated membranes and endoplasmic reticulum (ER). Plasma membrane was the last location to incorporate [¹⁴C]-Q₁₀. Brefeldin A prevented Q₁₀ incorporation in plasma membrane. Exogenous Q₉ was preferably accumulated into the endo-lysosomal fraction but a significant amount was distributed among other cell membranes also depending on the brefeldin-A-sensitive endomembrane system. Our results indicate that mitochondria are the first location for new synthesized Q. Exogenous Q is mainly incorporated into an endo-lysosomal fraction, which is then rapidly incorporated to cell membranes mainly to MAM and mitochondria. We also demonstrate that both endogenous and dietary Q is distributed among endomembranes and plasma membrane by the brefeldin A-sensitive endo-exocytic pathway.     

The role of the plasma membrane in the development of Dictyostelium discoideum. II. Developmental and topographic analysis of polypeptide and glycoprotein composition

Previous workers have shown in a variety of ways that cell contact is required for the differentiation of Dictyostelium discoideum. Because interactions between cells are probably mediated by molecules on their plasma membranes, we have characterized the polypeptide composition of the membrane of cells at different stages of development. At least 55 polypeptides are found in the plasma membrane of vegetative cells. The polypeptide composition of the plasma membranes changes considerably during development. Treatment of intact cells with pronase indicated that many of the altered components appear to be located on the external surface of the plasma membrane where they could participate in interactions between cells. Similar digestion of the isolated membranes destroys most of their polypeptides, indicating that the bulk of the proteins of the plasma membrane are not completely embedded in the membrane. Several polypeptides appear to change in sensitivity to pronase during development. There are several changes in glycoprotein composition which occur between log phase and aggregation phase. An almost complete change in glycoprotein species occurs between aggregation and pre-culmination. Unlike the polypeptides, the glycoproteins are very resistant to pronase treatment in intact cells. However, some are pronase sensitive in isolated membranes.     

Intramembrane particle structures in epithelial cells of the toad urinary bladder: a quantitative freeze-fracture study

Freeze-fracture electron microscopy reveals intramembrane particle arrays in basal membranes of granular epithelial cells as well as both upper and lower plasma membranes of the underlying basal cells in the toad urinary bladder. These particle arrays are morphologically indistinguishable from the luminal membrane aggregates which are known to be associated with antidiuretic hormone (ADH)-stimulated water transport. In both granular and basal cells particle arrays are frequently located in and/or around the openings of vesicular and/or tubular structures fused to the plasma membranes, suggesting that they may be transferred from the cytoplasm by membrane fusion. Quantification of cytoplasmic aggrephores in control granular cells shows that they can be numerous and as close to the basolateral membrane as they are with the luminal membrane, to which they are known to fuse and deliver aggregates upon ADH stimulation. Aggrephore-like tubules were also found in the basal cells. Particle array densities were quantified for 6 pairs of control and ADH-stimulated hemibladders. At least 1440 microns 2 area of plasma membrane for each membrane domain was examined. Results indicate that the presence of these particle arrays in granular and basal cell membranes is highly variable and that exposure to ADH does not cause a statistically significant increase in their frequency.     

Plasma membrane dehydrogenases in rat brain synaptic membranes. Multiplicity and subunit composition

Plasma membrane redox enzymes have been investigated in synaptic membranes from rat brain nerve terminals. UV-Vis spectra of intact synaptic plasma membranes are presented and the presence of ab-type cytochrome, detectable at 77°K and sensitive to NADH or NADPH, is shown. The molecular characterization of rat synaptic NADH-dehydrogenases was further performed on solubilized enzymes using a recently developed nondissociating polyacrylamide gel electrophoresis technique. Synaptic plasma membranes were solubilized with 1% sodium cholate or Triton X-114 and centrifuged. The supernatant retained over 60% of the NADH-dehydrogenase activity, tested with either DCIP or ferricyanide as substrates, together with NADH. Both enzyme activities were insensitive toward rotenone. This extraction procedure also solubilized about 50% of the proteins. When submitted to polyacrylamide gel electrophoresis under nondenaturing conditions and stained for NADH-dehydrogenase activity, five bands of different mobilities were detected. The multiple NADH-dehydrogenases of synaptic plasma membranes were investigated by means of band excision and the five excised bands each submitted to amino acid analysis and to 2-D electrophoresis. The subunit composition of each band was then deduced, together with the molecular weight and pI of each respective subunit. NADH-dehydrogenases have also been purified by means of FPLC on Mono-P (chromatofocusing) followed by gel filtration on Superose 12. NADH-Dehydrogenase IV and V could be purified in their active forms by this approach.Abbreviations DCIP dichlorophenol-indophenol - FPLC fast protein liquid chromatography.