Aggregation Of Intramembranous Particles Research Articles

Thermally induced transformation of mammalian red blood cells during hyperthermia

The structural and transport characteristics of membranes are mainly determined by the state of the cytoskeleton. The characteristic changes in morphology of human (adult donor and cord) and rat Red Blood Cells (RBC) and of their membrane, induced by hyperthermia (46–51 °C) have been analyzed. Two different types of morphological changes have been observed to take place during hyperthermia in all studied RBC groups. We have observed either formation and exfoliation of spiculas from membrane, resulting in the formation of large (4–5 μm) sphere-like cell body and small (0.5–1.5 μm) vesicles or cell fragmentation with formation of large (3–3.5 μm) vesicles. The two distinct phenomena are likely to be determined by the heterogeneity of the RBC population in terms of cell age. There was noted the difference of cord RBC from the donor ones in temperature value of transformation beginning, as well as the character of deformation and vesicle formation, that may testify to their less thermoresistance. The ultrastructure of the membrane, studied with the freeze-fracturing technique, testifies to an irreversible character of membrane changes. The aggregation of intramembrane particles (IMPs) as a continuous network testifies to the strengthening of the interactions between denatured spectrin and bilayer integral components.

The relationship between intramembranous particles and aquaporin molecules in the plasma membranes of normal rat skeletal muscles: a fracture-label study

The plasma membrane of skeletal muscles contains water channels such as aquaporin 4 (AQP4), aquaporin 3 (AQP3) and aquaporin 7 (AQP7). In dehydrated mice, we have recently reported the altered distribution of the aggregations of intramembranous particles (IMPs), such as orthogonal array (a crystal-like structure) and IMP cluster (a rosette-like structure) on the freeze-fractured skeletal muscle plasma membranes. In this fracture-label study, we first tested whether the orthogonal arrays (OAs) were composed of AQP4 in skeletal muscles and further analyzed the relationship between IMPs including IMP clusters and AQP3 molecules. As a result, many of the gold particles indicating AQP4 was associated with OAs (79%) by our fracture-label technique. On the other hand, approximately 50% of gold particles indicating AQP3 were associated with IMP clusters. Thus we confirmed that the OAs are composed of AQP4 in skeletal muscles, and further demonstrated that some of the IMP clusters are composed of AQP3 and may participate in maintaining osmotic homeostasis in skeletal myofibers. The fracture-label method is useful in investigating the molecular identification of membrane proteins such as AQP3 and AQP4.

Alterations of molecular architectures in skeletal muscle plasma membranes of dehydrated and water-loaded mice

It is likely that orthogonal arrays (OAs) and caveolae seen on the replicas of freeze-fractured muscle plasma membranes are involved in maintaining osmotic homeostasis. Therefore, using the freeze-fracture technique, we examined the ultrastructural changes in OAs and caveolae of the skeletal muscle plasma membrane of dehydrated and water-loaded mice. In the muscle plasma membranes of 6 dehydrated and 6 water-loaded mice, caveolar distribution was not altered, and the densities of caveolae and OAs did not show statistically significant differences when compared with those in 6 control mice, although the skeletal muscles of water-loaded mice sometimes had muscle plasma membranes with extremely numerous OAs. In contrast, the muscle plasma membranes of dehydrated mice often revealed changes in the distribution of OAs, which existed in a group at the confined area of the muscle plasma membranes and were frequently accompanied by the aggregations of intramembranous particles (IMPs) around OAs. Thus, on the basis of the present study, we suggest that OAs in skeletal muscles as well as those in brain may play an important role in maintaining osmotic homeostasis of these organs under abnormal water balance.

Are there gap junctions between chief (glomus, type I) cells in the carotid body chemoreceptor? A review.

Since the dye- and electronic couplings between the carotid body chief cells have been demonstrated, the detection and localization of the gap junctions in the carotid body is crucial to understanding the functional mechanism of chemoreception. However, conventional electron microscopy has been unsuccessful in unquestionably detecting ultrastructural features equivalent to the gap junctions, such as close (2 nm in width) membrane appositions in ultrathin sections and aggregations of intramembranous particles in freeze-fracture replicas of the carotid body. We previously reported using a modified electron microscopic study by chemically fixed and subsequent rapid freezing and freeze-substitution method a number of close membrane appositions comparable to the gap junctions. However, we later found that the freeze-substitution also induces numerous close apposition of the membrane in sites where the gap junctions are not known to occur, indicating that the modified electron microscopy by freeze-substitution is not always confirmative in the detection of the gap junction. With regard to the molecular evidence for the gap junction in the carotid body, there have so far been few data on the immunohistochemical demonstration on connexin 32 and 43 in cultured chief cells, but not in the in situ cells.

Microdomain arrangement of the SERCA-type Ca2+ pump (Ca2+-ATPase) in subplasmalemmal calcium stores of paramecium cells.

We localized SERCA pumps to the inner region of alveolar sac membranes, facing the cell interior, by combining ultrastructural and biochemical methods. Immunogold labeling largely predominated in the inner alveolar sac region which displayed aggregates of intramembrane particles (IMPs). On image analysis, these represented oligomeric arrangements of approximately 8-nm large IMP subunits, suggesting formation of SERCA aggregates (as known from sarcoplasmic reticulum). We found not only monomers of typical molecular size ( approximately 106 kD) but also oligomeric forms on Western blots (using anti-SERCA antibodies, also against endogenous SERCA from alveolar sacs) and on electrophoresis gelautoradiographs of 32P-labeled phosphoenzyme intermediates. Selective enrichment of SERCA-pump molecules in the inner alveolar sac membrane region may eliminate Ca2+ after centripetal spread observed during exocytosis activation, while the plasmalemmal Ca2+ pump may maintain or reestablish [Ca2+] in the narrow subplasmalemmal space between the outer alveolar sac membrane region and the cell membrane. We show for the first time the microzonal arrangement of SERCA molecules in a Ca2+ store of a secretory system, an intensely discussed issue in stimulus-secretion coupling research.

Intramembranous structure of synaptic membranes with special reference to spinules in the rat sensorimotor cortex.

The intramembranous structure of the synaptic contact zone at presynaptic and postsynaptic membranes in the rat sensorimotor cortex was examined by means of the freeze-etching technique. In axospinous synapses, the synaptic contact zone is characterized by perforated and nonperforated aggregates of intramembranous particles at the extracellular half or E-face of the postsynaptic membrane. On some perforated synaptic contact zones, both synaptic membranes are marked by so called spinules. These invaginations of the postsynaptic membrane and the parallel presynaptic membrane into the axon terminal are situated at the particle free zones among the postsynaptic E-face intramembranous particle aggregates or in close proximity to it. Intramembranous characteristics of the spinules at both freeze-etched faces of presynaptic and postsynaptic membranes and their density of perforated axospinous synapses were analysed. The results are discussed in terms of plasticity at the synaptic contact zone of the axospinous synapses of the sensorimotor cortex in the rat.

Freeze-fracture analysis of plasma membranes of CHO cells stably expressing aquaporins 1-5.

Several studies suggest that aquaporin water channels can be identified in membranes by freeze-fracture electron microscopy. For this report, Chinese Hamster ovary cells were stably transfected with cDNAs encoding aquaporins 1-5. Measurement of the osmotic water permeability of the cells confirmed that functional protein was expressed and delivered to the plasma membrane. By freeze-fracture electron microscopy, a 20% increase in intramembrane particle (IMP) density was found in plasma membranes of cells expressing AQP2, 3 and 5, and a 100% increase was measured in AQP1-expressing cells, when compared to mock-transfected cells. On membranes of cells expressing AQP4, large aggregates of IMPs were organized into orthogonal arrays, which occupied 10-20% of the membrane surface. IMP aggregates were never seen in AQP2-transfected cells. Hexagonally packed IMP clusters were detected in approximately 5% of the membranes from AQP3-expressing cells. Particle size-distribution analysis of rotary shadowed IMPs showed a significant shift from 13. 5 (control cells) to 8.5 nm or less in AQP-expressing cells; size distribution analysis of unidirectionally shadowed IMPs also showed a significant change when compared to control. Some IMPs in AQP expressing cells had features consistent with the idea that aquaporins are assembled as tetramers. The results demonstrate that in transfected CHO cells, AQP transfection modifies the general appearance and number of IMPs on the plasma membrane, and show that only AQP4 assembles into well-defined IMP arrays.

Prostaglandin-dependent osmotic water permeability of the frog and trout urinary bladder

Washout of autacoids from serosal Ringer solution, using a repeated change of the solution of the frog and trout urinary bladder, was accompanied by a pronounced rise in the osmotic water permeability: the water transport in the frog rose from 0.05±0.02 to 1.21±0.26 μl min −1·cm −2, in the trout, from 0.041±0.011 to 0.26±0.034 μl min −1·cm −2. Such an increase in the osmotic water permeability in the trout and frog urinary bladder occurred in the background of a decrease in the prostaglandin E 2 concentration in the serosal Ringer solution. This permeability increase was accompanied by the formation of aggregates of intramembranous particles in the apical plasma membrane of the trout and frog urinary bladder. A decrease in the osmotic water permeability was achieved by the addition to the serosal Ringer solution of 10 −8 M prostaglandin. Experiments on the frog urinary bladder have shown that prostaglandins E 1, I 2 and F 2 α also decrease the osmotic water permeability. Vasotocin increased the osmotic water permeability in the frog urinary bladder but did not affect the osmotic water permeability of the trout urinary bladder. The data obtained indicates a role of the endogenous prostaglandin production in maintaining the low osmotic water permeability in the frog and trout urinary bladder. A suggestion is made that in the vertebrate evolution, colonisation of the fresh-water was connected with the maintenance of the low osmotic water permeability via participation of prostaglandins, whereas the vasotocin hydroosmotic effect developed in the vertebrate evolution later and provided for the possibility of the water absorption, osmotic homeostasis and animal migration from fresh-water to the land.

Ultrastructural correlates of the antidiuretic hormone-dependent and antidiuretic hormone-independent increase of osmotic water permeability in the frog urinary bladder epithelium.

Electron and confocal microscopy, using immunocytochemical methods, was employed to assess osmotic water permeability of the frog (Rana temporaria) urinary bladder during transcellular water transport, induced by antidiuretic hormone (ADH) or by wash-out of autacoids from serosal, ADH-free Ringer solution. The increase of osmotic water permeability of the urinary bladder was accompanied by relevant ultrastructural changes, the most remarkable being: (1) the appearance of aggregates of intramembranous particles in the apical membrane of granular cells, and the extent of the membrane area covered by the aggregates proportional to that of the water flow; (2) redistribution of actin filaments in the cytoplasm of granular cells; judging from the anti-actin label density, the number of actin filaments in the apical region of cytoplasm was reduced by 2.5-4 times compared with normal; (3) a decrease in the total electron density of the cytoplasm due to the increased water content of granular cells.

Role of prostaglandin E 2 in regulation of low and high water osmotic permeability in frog urinary bladder

The water osmotic permeability of frog urinary bladder was found to be increased from 0.08±0.01 to 1.28±0.20 μl/min cm 2 when serosal bathing medium was changed 4 times for a fresh Ringer solution. High epithelium permeability is accompanied by an increased content of cyclic AMP in the bladder tissue (by 42%, P<0.01), higher activity of both basal and forskolin-stimulated membrane adenylate cyclase (AC) (by 109% and 74%, respectively, P<0.05) and by appearance of aggregates of intramembranous particles in the apical membrane. The water flow was inhibited by 10 −9–10 −5 M prostaglandin E 2 (PGE 2); the inhibitory effect was eliminated in the presence of 10 −4 M N-ethylmaleimide. The increase of water permeability due to changes of the bathing medium was accompanied by a decrease of serosal PGE 2 concentration from 14.8±1.0 in the 1st solution to 0.6±0.1 nM in the 5th. 10 −6 M PGE 2 in vitro inhibited the activity of membrane AC from highly permeable bladders by 33.4% ( P<0.02). Pretreatment of the membranes with 10 μg/ml pertussis toxin (PT) completely reversed this effect (+149%, P<0.01). A significant activation of AC was also observed under 10 −10 M PGE 2 (by 196%). These data demonstrate that the water permeability could be markedly increased independently of ADH, suggesting that the trigger role in activation of water transport is played by a decreased level of PGE 2 which could stimulate AC.

Structural transitions in erythrocyte membranes in hereditary hemochromatosis

Changes in the thermal transition parameters of the backbone proteins of the erythrocyte plasma membrane, particularly of spectrin and membrane domain of band3 protein, in patients with hereditary hemochromatosis are demonstrated by differential scanning microcalorimetry. Electron microscopy (the freeze-fracture technique) reveals aggregation of intramembrane particles and their enlargement in erythrocyte membranes from patients in comparison with membranes from healthy donors. These structural changes in the erythrocyte membranes are assumed to contribute to hemolysis and iron overload of the organism.

Cellulose microfibrils, cell motility, and plasma membrane protein organization change in parallel during culmination in Dictyostelium discoideum.

Prestalk cells of Dictyostelium discoideum contribute cellulose to two distinct structures, the stalk tube and the stalk cell wall, during culmination. This paper demonstrates by freeze fracture electron microscopy that two distinct types of intramembrane particle aggregates, which can be characterized as cellulose microfibril terminal complexes, occur in the plasma membranes of cells synthesizing these different forms of cellulose. The same terminal complexes were observed in situ in developing culminants and in vitro in monolayer cells induced to synthesize the two types of cellulose. We propose that cessation of cell motility is associated with a change in packing and intramembrane mobility of the particle aggregates, which cause a change in the nature of the cellulose synthesized. The terminal complexes are compared to those described in other organisms and related to the previous hypothesis of two modes of cellulose synthesis in Dictyostelium.

Structure of the synaptic junctions in the rat sensorimotor cortex. Freeze-etching study of axodendritic synapses.

The intramembranous structure of axodendritic synapses in the rat sensorimotor cortex was studied by means of freeze-etched replicas and thin sections. In thin sections, symmetric synaptic junctions were located on dendritic shafts. Examination of freeze-etched preparations supported the notion that the postsynaptic dendritic shaft membrane exhibited the same structure as the surrounding non-junctional membrane on both fracture faces. In thin sections, asymmetric perforated and non-perforated synapses were found on dendritic spines and dendritic shafts. In freeze-etched replicas, the postsynaptic membrane of the dendritic spine and some of the dendritic shaft synapses were characterized by perforated and non-perforated aggregates of intramembranous particles at the extracellular half (E-face). These aggregates are accepted as a freeze-etch equivalent of perforated and non-perforated asymmetric synapses seen in thin sections. The intramembranous characteristics of axodendritic synapses in the rat sensorimotor cortex are discussed in terms of synaptic contact zone plasticity.

The Mercurial Insensitive Water Channel (AQP-4) Forms Orthogonal Arrays in Stably Transfected Chinese Hamster Ovary Cells

The mercurial insensitive water channel (MIWC, AQP-4) is a water-selective transporter expressed at the basolateral plasma membrane of principal cells in kidney collecting duct, airway epithelium, and gastric parietal cells, as well as in astrocytes and skeletal muscle plasmalemma. Because these sites correspond to membranes where orthogonal arrays of particles (OAPs) have been observed by freeze-fracture electron microscopy, we tested the hypothesis that MIWC forms OAPs. Chinese hamster ovary cells were stably transfected with the coding sequence of rat MIWC under a cytomegalovirus promoter. Immunostaining of clonal cell populations showed MIWC expression at the plasma membrane. A single band at 31 kDa was detected on immunoblot. Cell fractionation by sucrose gradient centrifugation indicated strong MIWC expression in plasma membrane fractions with lesser expression in Golgi. Functional analysis by stopped-flow light scattering showed high mercurial insensitive water permeability in plasma membrane vesicles. Freeze-fracture electron microscopy showed distinct OAPs on the plasma membrane P-face of MIWC-expressing cells with morphology indistinguishable from that in basolateral membrane of kidney collecting duct; the E-face showed corresponding linear grooves (spacing, approximately 8 nm) in transfected cells and collecting duct. OAPs were not observed in control (empty vector-transfected) cells or CHIP28 (AQP1)-transfected cells in which disorganized intramembrane particle aggregates were found. These results provide direct evidence that a molecular water channel can spontaneously assemble in regular arrays.

Effects of extracellular matrix macromolecules on the differentiation of plasma membrane structure in cultured astrocytes.

Orthogonal aggregates of small intramembrane particles, termed "assemblies," are concentrated especially in the cell membranes of astrocytic processes that form the glia limitans at the outer surface of the brain and the perivascular sheath surrounding the parenchymal blood vessels. As an initial step to clarifying the totally unknown biochemical nature of this intramembrane structure, we have devised a culture system which enhances the differentiation of assemblies in secondary cultures of astrocytes derived from neonatal mouse neopallium. Since assemblies are most concentrated in the plasma membranes attaching to the basement membrane, we expected that extracellular matrix molecules constituting the basement membrane would be suitable candidates for our aim. We report here that a mixture of type IV collagen, laminin, and fibronectin, major components of the extracellular matrix, has the potency to increase assembly density in cultured astrocytes. We also report that, in freeze-fracture electron microscopy of cultured cells, one can satisfactorily preserve membrane structure and reliably obtain large replicas by inoculating cells on aluminium foil and peeling it from the cells in a freeze-fracture apparatus.

Aggregation of myocardial sarcolemmal transmembrane proteins is not hindered by an interaction with the cytoskeleton. Possible implications for ischemia and reperfusion

Heart myocytes subjected to ischemia show a clustering of the sarcolemmal proteins. In the erythrocyte membrane, a system in which intramembranous particle (IMP) aggregation is extensively studied, it is found that an IMP aggregation can in principle only occur upon removal of the membrane skeleton of spectrin and actin by rather drastic experimental conditions. With regard to phospholipid composition and topology the sarcolemma and the erythrocyte membrane show large similarities and therefore it was proposed that a loss of the interaction of the IMPs and the cytoskeleton is also a prerequisite for the sarcolemmal IMP aggregation (Verkleij et al., 1990). Freezing myocardial tissue, both from adult and neonatal rat, from temperatures lower than 37 degrees C resulted in an aggregation of the sarcolemmal IMPs. The aggregation is proportional to the degree of lowering of the temperature at which the tissue is cryofixed. This in contrast to the erythrocyte membrane, where lowering the temperature only induces moderate IMP aggregation. The IMP aggregation in the sarcolemma is reversible upon a subsequent increase in incubation temperature. The results clearly demonstrate that the interaction between the sarcolemmal proteins does not hinder aggregation of the IMPs, as proposed previously, and suggest that loosening of this complex does not have to proceed the aggregation of the sarcolemmal intramembranous particles during ischemia.

Association State of Human Red Blood Cell Band 3 and its Interaction With Ankyrin

We have studied the association state of band 3, the anion channel and predominant transmembrane protein of the human red blood cell, and the anomalous stoichiometry and dynamics of its interaction with ankyrin, which acts as a link to the spectrin of the membrane skeletal network. Band 3 exists in benign nonionic detergent solutions as a dimer. Tetramer is formed irreversibly in the course of manipulations, particularly in ion-exchange chromatography. The dimer in solution binds ankyrin without self-associating. In ankyrin-free inside-out membrane vesicles and when incorporated into phosphatidylcholine liposomes, only some 10% to 15% of band 3 chains bind ankyrin at saturation. Moreover, in liposomes this was independent of protein:lipid ratio between 1:2 and 1:40. The bound fraction of band 3 remains with the detergent-extracted membrane cytoskeleton, but is released if the ankyrin has been cleaved with chymotrypsin before detergent treatment; thus, the attachment to the membrane cytoskeleton is entirely through ankyrin and not through other constituents such as protein 4.1. The ratio of band 3 to ankyrin in this complex implies that it consists of two chains of band 3 and one chain of ankyrin, at least after detergent extraction. The bound and free populations of band 3 exchange freely in the membrane. In the artificial liposome membrane binding of ankyrin to band 3 dimers cause association of the band 3 into higher aggregates, as seen in freeze-fracture electron microscopy. Successive manipulations of the red blood cell membrane, which are involved in the preparation of ghosts, of inside-out vesicles, and of inside-out vesicles stripped of peripheral proteins are accompanied by progressive aggregation of intramembrane particles, as judged by freeze-fracture electron microscopy. Thus the intramembrane particles are evidently stabilized in the intact cell by the peripheral protein network and the cytosolic milieu. Aggregation may be expected to limit the number of functional ankyrin binding sites. However, although extraneous ankyrin binds to the unoccupied binding site on the spectrin tetramers in intact ghost membranes, little or no ankyrin can bind to the unoccupied band 3 dimers in situ, perhaps by reason of occlusion of binding sites by the membrane skeletal network.

Effect of the lipopeptide antibiotic, iturin A, on morphology and membrane ultrastructure of yeast cells

The effects of iturin A, at fungicidal concentrations, on yeast cells were studied by scanning electron microscopy and by transmission electron microscopy. A depression, observed in each iturin A-treated cell, was the consequence of the release of electrolytes and other cytoplasmic components. Iturin A passes through the cell wall and disrupts the plasma membrane with the formation of small vesicles and the aggregation of intramembranous particles. Moreover, iturin A passes through the plasma membrane and interacts with the nuclear membrane and probably with membranes of other cytoplasmic organelles.

Effect of the lipopeptide antibiotic, iturin A, on morphology and membrane ultrastructure of yeast cells

The effects of iturin A, at fungicidal concentrations, on yeast cells were studied by scanning electron microscopy and by transmission electron microscopy. A depression, observed in each iturin A-treated cell, was the consequence of the release of electrolytes and other cytoplasmic components. Iturin A passes through the cell wall and disrupts the plasma membrane with the formation of small vesicles and the aggregation of intramembranous particles. Moreover, iturin A passes through the plasma membrane and interacts with the nuclear membrane and probably with membranes of other cytoplasmic organelles.

Lobster swimmerets: Muscle fiber composition and membrane definition of excitatory synapses in a predominantly fast system

AbstractThe abdomen of the lobster Homarus americanus has four pairs of segmental appendages, the swimmerets, whose rhythmic beating functions in swimming, righting responses, creating water currents, and aerating eggs. There are 24 separate muscles in each swimmeret and 21 of these are fast, based on their relatively short (4 μm) sarcomeres and six thin filaments orbiting a single thick filament. Three of the 24 muscles are slow with relatively long (7–12 μm) sarcomeres and 8–11 thin filaments surrounding a thick filament. Of the eight functional groups amongst which the 24 muscles are distributed, six groups have only fast muscles, one group has a mixture of fast and slow muscles, and one group only slow muscles. Thus the number and distribution of fast muscles in the swimmeret makes it a predominantly fast system. Motor innervation of two fast muscles in the power‐stroke group reveals numerous excitatory nerve terminals (recognized by circular synaptic vesicles) with well‐defined synaptic contacts and presynaptic dense bars or active zones. Synaptic contacts appear in the P‐face of the fractured nerve membrane as circular plateaus with sparsely distributed particles and one or two small aggregations of large intramembranous particles (putative calcium channels) representing active zones. In the complementary E‐face, synapses assume spherical outlines and active zones appear as shallow depressions. Attachment sites of synaptic vesicles appear as large pits in P‐face or protuberances in E‐face leaflets along the edge of the active zones. The surface features of excitatory synapses of fast swimmeret muscles are similar to those on other crustacean fast and slow muscles. The packing density of large intramembranous particles in the active zones also resembles that in other crustacean muscles thereby suggesting an optimal packing density for these ion channels. © 1995 Wiley‐Liss, Inc.