Density Of Intramembrane Particles Research Articles

Evaluation of fracture planes and cell morphology in complementary fractures of cultured cells in the frozen-hydrated state by field-emission secondary electron microscopy: feasibility for ion localization and fluorescence imaging studies.

We have employed field-emission secondary electron microscopy (FESEM) for morphological evaluation of freeze-fractured frozen-hydrated renal epithelial LLC-PK1 cells prepared with our simple cryogenic sandwich-fracture method that does not require any high-vacuum freeze-fracture instrumentation (Chandra et al. (1986) J. Microsc. 144. 15-37). The cells fractured on the substrate side of the sandwich were matched one-to-one with their corresponding complementary fractured faces on the other side of the sandwich. The FESEM analysis of the frozen-hydrated cells revealed three types of fracture: (i) apical membrane fracture that produces groups of cells together on the substrate fractured at the ectoplasmic face of the plasma membrane; (ii) basal membrane fracture that produces basal plasma membrane-halves on the substrate; and (iii) cross-fracture that passes randomly through the cells. The ectoplasmic face (E-face) and protoplasmic face (P-face) of the membrane were recognized based on the density of intramembranous particles. Feasibility of fractured cells was shown for intracellular ion localization with ion microscopy, and fluorescence imaging with laser scanning confocal microscopy. Ion microscopy imaging of freeze-dried cells fractured at the apical membrane revealed well-preserved intracellular ionic composition of even the most diffusible ions (total concentrations of K+, Na+ and Ca2+). Structurally damaged cells revealed lower K+ and higher Na+ and Ca2+ contents than in well-preserved cells. Frozen-freeze-dried cells also allowed imaging of fluorescently labelled mitochondria with a laser scanning confocal microscope. Since these cells are prepared without washing away the nutrient medium or using any chemical pretreatment to affect their native chemical and structural makeup, the characterization of fracture faces introduces ideal sample types for chemical and morphological studies with ion and electron microscopes and other techniques such as laser scanning confocal microscopy, atomic force microscopy and near-field scanning optical microscopy.

Distribution of intramembranous particle size in the muscle plasma membrane of the mdx mouse

We investigated whether the reduced intramembranous particles (IMP) in the muscle plasma membrane in mdx mice reflects a preferential depletion of a particular size of the IMP. The experiments were performed using the freeze-fracture method to analyze the frequency distribution of the size of IMP, the density of orthogonal array and caveolae in the extensor digitorum longus (EDL) and soleus (SOL) muscles obtained from mdx and control mice. We detected a reduced density of IMP and orthogonal array, and the increased density of caveolae in EDL muscle but not in SOL in the mdx mouse compared with those of the same muscles in control animals. The reduction of IMP was, however, not limited to any specific size of IMP. Our results suggest that the dystrophin associated glycoprotein present in the membrane does not reflect a specific size of IMP. Therefore, our findings indicate that the mechanism of reduced IMP in dystrophinopathy may be different from that of diminished dystrophin binding glycoprotein associated with dystrophin deficiency in Duchenne muscular dystrophy.

A comparative freeze‐fracture study of plasma membrane of dystrophic skeletal muscles in dy/dy mice with merosin (laminin 2) deficiency and mdx mice with dystrophin deficiency

The intramembranous particle (IMP), orthogonal array (OA) and orthogonal array subunit particle (OASP) densities of skeletal muscle plasma membranes of merosin deficient dy/dy mice and their control mice at 7, 14 and 28 days after birth were analysed by freeze-fracture electron microscopy. Similar studies were performed on dystrophin-deficient mdx mice with mild muscle weakness at 28 days after birth for the comparison with those of dy/dy mice with severe muscle weakness at the same age. In the pre-clinical stage of dy/dy mice at 14 days after birth, the membranes showed a significantly decreased density of OAs (P<0.01 by Wilcoxon rank-sum test) as compared with control mice, while those in the clinical stage of dy/dy mice at 28 days after birth showed normal IMP density but a marked depletion of OA density (P<0.01). Moreover, at 28 days after birth, the reduction of OAs in the plasma membranes of dy/dy mice was more marked than that of mdx mice (P<0.05 by Wilcoxon rank-sum test). These results provided us with the information that the OA density was affected more severely with merosin deficiency than with dystrophin deficiency, and again supported our previously proposed concept that the clinical severity in muscular dystrophies correlated with the OA density.

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.

Analysis of the membrane structures involved in autophagy in yeast by freeze-replica method.

Under starvation conditions, the yeast S. cerevisiae sequesters its own cytoplasmic components by forming autophagosomes with double membrane in the cytoplasm. The autophagosome then fuses with the vacuolar membrane and delivers its own cytoplasmic components into the vacuole in the form of an autophagic body with a single membrane (Baba, M., Takeshige, K., Baba, N., and Ohsumi, Y. 1994. J. Cell Biol., 124: 903-913). We examined membrane structures involved in the autophagy induced by nitrogen-starvation by using freeze-replica method. The most conspicuous characteristic of the autophagic body is that the intramembrane particles were rarely detected on either the protoplasmic or exoplasmic face of its fracture membrane. This morphological feature of the fractured face was clearly different from other intracellular organelles. Next we examined the autophagosomal membrane. The inner membrane of autophagosome was also intramembrane particle-free, and its morphological feature was identical to the membrane of autophagic body. At the fusion site between autophagosome and vacuole we obtained direct evidence that two different membranes, the outer membrane of autophagosome and vacuolar membrane, became continuous by using freeze-etching technique. From these results we concluded that the autophagic body originated from the inner membrane of the autophagosome, and its membrane reflects an intrinsic feature of autophagosomal membrane. The outer membrane of autophagosome had only a few intramembrane particles and may be differentiated from the inner membrane. In cells under nitrogen-starvation condition, the density of intramembrane particles of vacuolar membrane decreased beyond that in control cells.

Freeze–fracture analysis of muscle plasma membrane in Becker's muscular dystrophy

The intramembranous particle (IMP), orthogonal array (OA) and orthogonal array subunit particle (OASP) densities in skeletal muscle plasma membranes from eight patients with Becker's muscular dystrophy (BMD) were analysed by the freeze-fracture technique. The results showed almost normal IMP density with the significant decrease of OA and OASP densities in BMD. The group mean densities +/- SE of IMPs on the protoplasmic faces with and without OASPs, and on extracellular faces/microns 2 were 2137 +/- 207, 1839 +/- 68 and 895 +/- 108, respectively in controls; whereas those of BMD were 1989 +/- 259, 1837 +/- 203 and 900 +/- 239, respectively (P > 0.1 by two-tailed t-test). The group median density of OAs and their pits/microns 2 was 4.89 with mid-ranges (25-75% values of the counts) of 2.66-10.18 in controls; whereas that in BMD was 2.15 with mid-ranges of 1.14-4.31 (P < 0.01 by Wilcoxon rank-sum test). The group mean density +/- SE of OASPs in controls was 15.99 +/- 1.83; whereas that in BMD was 13.47 +/- 1.07 (P < 0.01 by two-tailed t-test). However, the diminution of OA and OASP densities in BMD muscle plasma membranes was not as severe as in Duchenne's muscular dystrophy. There was a relationship between OA density and clinical severity in BMD patients; the decrease of OA density in a severe BMD patient was more marked than that in mildly affected BMD patients. Therefore, it seems that marked depletion of OA density may lead to the severe disability in muscular dystrophies.

Astrocytes alter their polarity in organotypic slice cultures of rat visual cortex.

The ultrastructure of astrocytes in an organotypic slice culture of the rat visual cortex was investigated using ultrathin sections and freeze-fracture replicas. After a culture period of 9-15 days, a glial scaffold formed that separated the bulk of the slice neuropil from the medium and the underlying plasma clot. However, the glial cells and processes did not build a dense barrier but allowed the outgrowth of neurites. A basal lamina covering the medium-oriented surface of the astrocytes was not found. In freeze-fracture replicas, orthogonal arrays of particles (OAP) were characteristic components of astrocytic membranes. The OAP density in membranes bordering the medium was 35 +/- 13 OAP/microns 2, corresponding to 2.5% of this membrane area; the OAP density in membranes within the slice neuropil was 22 +/- 12 OAP/microns 2, corresponding to 1.4% of this membrane area. Although the difference was significant, it was greatly reduced when comparing OAP densities in endfoot and non-endfoot membranes in vivo. Another node of polarity was recognized in astrocytes of the organotypic slice culture. In membranes of astrocytes bordering upon the medium, the density of non-OAP intramembranous particles (IMP) was clearly higher (1130 +/- 136 IMP/microns 2) than in membranes of astrocytes in the center of the slice (700 +/- 172 IMP/microns 2). This pronounced IMP-related polarity was observed neither in vivo nor in cultured astrocytes. The present study suggests, together with data from the literature, that the distribution of astrocytic OAP across the cell surface is influenced by the existence of a basal lamina and neuronal activity, and that astrocytes possess a more remarkable plasticity of membrane structure than previously suspected.

Cytological characterization of isolated sperm cells of perennial ryegrass (Lolium perenne L.)

The cytoplasmic content and the distribution of intramembrane particles (IMPs) of the plasma membrane of isolated sperm cells of perennial ryegrass (Lolium perenne L.) have been characterized using flow cytometry, transmission electron microscopy, confocal scanning laser microscopy and freeze-fracture studies. The isolated haploid sperm cells contain a variety of cell organelles with the exception of microtubules. Proplastids and plastids with starch were observed, although only rarely. Vacuoles containing remnants of organelles and stacked lamellae of endoplasmic reticulum with cytoplasmic inclusions were observed frequently, indicating that autophagy takes place. The number of mitochondria varies from 11 to 26 with an average of 17. Generally, the nucleus has a lobed shape and displays various interphasic stages of chromatin condensation. The analysis of the number of mitochondria and the nuclear state did not show evidence of sperm cell dimorphism. The cytological variability observed, could be explained by differences in developmental stages already present in vivo at the moment of isolation. No correlation between the number of mitochondria and the nuclear cross-sectioned area and/or the condensation state of the chromatin could be found. The density of intramembrane particles of the plasma membrane on the exoplasmic fracture face is more than twice that on the protoplasmic fracture face. That is the opposite of what was found for sporophytic cells of perennial ryegrass. These results are discussed in relation to the potential use of these cells for biotechnology and developmental studies.

A freeze-fracture study of mesophyll cell membranes in the embryonic Phaseolus leaf, in the dry seed and during the initial stages of germination

Using rapid-freezing and freeze-fracturing techniques, we have examined cellular organisation within mesophyll cells in primary leaves of Phaseolus, in the dry seed and over a period of 48 hr from the onset of germination. The rapidly-frozen, unetched image reveals a dynamic cytoplasm with membrane shape and role able to change and fluctuate with the increase in cell metabolism. The ER in this tissue is highly mobile, becoming progressively associated with protein and lipid bodies, then plastids, mitochondria and nuclei as cytoplasmic requirements change. The ER thus provides an efficient transport of substrates to metabolic sites within the cytoplasm. Nuclei and plastics change shape in early germination and the protein body membrane changes in role and character to that of a vacuolar tonoplast. The freeze-fractured image emphasizes the considerable movement that occurs within the cytoplasm during germination, with flexible and mobile membranes of the various organelles able to associate and interact with others. We have also measured the density of intramembrane particles on most of the membranes within the mesophyll parenchyma cell, during this period of early germination. Using our method of rapid-freezing, we find consistently higher IMP density on the protoplasmic face of each membrane. The counts provide a set of comparative values for membranes of a specific tissue, and for a specific cryofixation technique.

Aminoglycoside antibiotics alter the paracellular transport properties of cultured human proximal tubule cells.

Cell cultures retaining properties of the human proximal tubule were utilized to determine whether or not the aminoglycoside antibiotics alter paracellular transport. The transepithelial resistance (RT) of the human proximal tubule (HPT) cell monolayers was determined by Ussing chamber analysis of cells grown on permeable supports. This analysis revealed that RT was reduced as a result of aminoglycoside exposure and that the reduction corresponded to the known clinical nephrotoxicities of the aminoglycosides. Variation in the aminoglycoside concentration necessary to elicit this response was documented using 14 individual cell isolates. Ultrastructural analysis provided evidence indicating that the alterations in RT were not associated with general damage to the HPT cells. An examination of the structure of the tight junctions by freeze-fracture analysis demonstrated only minimal alteration of the sealing strands as a result of aminoglycoside exposure. Consequently, the reductions in RT were not directly associated with discernible tight junction structural alterations. Alteration in the paracellular route of transport, as indicated by altered RT values, was clearly documented as a result of aminoglycoside exposure. In addition, this alteration was accompanied by an increased density of intramembrane particles within the apical cell membrane.

Freeze-fracture and thin section study of the rough ER-Golgi interface in the pancreatic acinar cell. Resemblance between the intramembranal architecture of the outermost Golgi cisterna and the post-rough ER vesicular and tubular elements

Post-ER membranous structures are clearly observed in pancreases fixed with aldehydes and subsequently with reduced osmium. Close to the transitional rough ER, clusters of vesicles of approximately 56 nm diameter are consistently present. In some cells, tortuous tubules appear enmeshed by the approximately 56 nm vesicles and by irregular, vesicular formations. In freeze-fracture replicas, the membranes of the bulges and tubules that protrude from the transitional rough ER differ from those of the donor compartment. These protrusions are herein designated as the budding chamber of the transitional rough ER. Quantitative and qualitative observations performed previously and in the present study show that the P and E freeze-fracture faces of the outermost Golgi cisternal membrane possess patterns of texture that are unique among membranes. The P-face exhibits a very high density of intramembranous particles of dimensions among the smallest yet described; E-faces show rugosities and an unusually high density of intramembranous particles of normal size. The membranes of the budding chamber, the putative transport vesicles of approximately 56 nm diameter, the sinuous tubules and the vesicles of irregular size and shape exhibit P and E fracture faces with textures indistinguishable from those of the corresponding P and E faces of the outermost Golgi cisterna.

Duplication of 10 nucleotides in the erythroid band 3 (AE1) gene in a kindred with hereditary spherocytosis and band 3 protein deficiency (band 3PRAGUE).

We describe a duplication of 10 nucleotides (2,455-2,464) in the band 3 gene in a kindred with autosomal dominant hereditary spherocytosis and a partial deficiency of the band 3 protein that is reflected by decreased rate of transmembrane sulfate flux and decreased density of intramembrane particles. The mutant allele potentially encodes an abnormal band 3 protein with a 3.5-kD COOH-terminal truncation; however, we did not detect the mutant protein in the membrane of mature red blood cells. Since the mRNA levels for the mutant and normal alleles are similar and since the band 3 content is the same in the light and dense red cell fractions, we conclude that the mutant band 3 is either not inserted into the plasma membrane or lost from the membrane prior to the release of red blood cells into circulation. We further show that the decrease in band 3 content principally involves the dimeric laterally and rotationally mobile fraction of the band 3 protein, while the laterally immobile and rotationally restricted band 3 fraction is left essentially intact. We propose that the decreased density of intramembrane particles decreases the stability of the membrane lipid bilayer and causes release of lipid microvesicles that leads to surface area deficiency and spherocytosis.

Tetrameric assembly of CHIP28 water channels in liposomes and cell membranes: a freeze-fracture study.

Channel forming integral protein of 28 kD (CHIP28) functions as a water channel in erythrocytes, kidney proximal tubule and thin descending limb of Henle. CHIP28 morphology was examined by freeze-fracture EM in proteoliposomes reconstituted with purified CHIP28, CHO cells stably transfected with CHIP28k cDNA, and rat kidney tubules. Liposomes reconstituted with HPLC-purified CHIP28 from human erythrocytes had a high osmotic water permeability (Pf0.04 cm/s) that was inhibited by HgCl2. Freeze-fracture replicas showed a fairly uniform set of intramembrane particles (IMPs); no IMPs were observed in liposomes without incorporated protein. By rotary shadowing, the IMPs had a diameter of 8.5 +/- 1.3 nm (mean +/- SD); many IMPs consisted of a distinct arrangement of four smaller subunits surrounding a central depression. IMPs of similar size and appearance were seen on the P-face of plasma membranes from CHIP28k-transfected (but not mock-transfected) CHO cells, rat thin descending limb (TDL) of Henle, and S3 segment of proximal straight tubules. A distinctive network of complementary IMP imprints was observed on the E-face of CHIP28-containing plasma membranes. The densities of IMPs in the size range of CHIP28 IMPs, determined by non-linear regression, were (in IMPs/microns 2): 2,494 in CHO cells, 5,785 in TDL, and 1,928 in proximal straight tubules; predicted Pf, based on the CHIP28 single channel water permeability of 3.6 x 10(-14) cm3/S (10 degrees C), was in good agreement with measured Pf of 0.027 cm/S, 0.075 cm/S, and 0.031 cm/S, respectively, in these cell types. Assuming that each CHIP28 monomer is a right cylindrical pore of length 5 nm and density 1.3 g/cm3, the monomer diameter would be 3.2 nm; a symmetrical arrangement of four cylinders would have a greatest diameter of 7.2 nm, which after correction for the thickness of platinum deposit, is similar to the measured IMP diameter of approximately 8.5 nm. These results provide a morphological signature for CHIP28 water channels and evidence for a tetrameric assembly of CHIP28 monomers in reconstituted proteoliposomes and cell membranes.

Two structural adaptations for regulating transmitter release at lobster neuromuscular synapses

The distal accessory flexor muscle (DAFM) in the lobster (Homarus americanus) walking leg consists of 5 muscle fiber bundles. All five bundles, one proximal, one distal, and 3 medial, are innervated by one excitatory and one inhibitory motor neuron. Both neurons release more transmitter on the distal bundle than on the proximal bundle. The aim of our studies was to investigate the structural basis of this differentiation. Thin sections cut at 50 microns intervals showed a similar number of excitatory synapses on the two bundles. Freeze-fracture views of excitatory synapses showed that synapse size, active zone number per synapse, and intramembrane particle density in the postsynaptic membrane are similar proximally and distally. Active zones at synapses on the distal bundle are larger and contain about 50% more large intramembrane particles, which are thought to include the voltage-gated Ca2+ channels that couple the action potential to transmitter release, than their counterparts on the most proximal bundle. This difference in channel number appears to produce a disproportionate increase in the probability of transmitter release sufficient to account for most of the proximal-distal disparity in the amplitude of the excitatory postsynaptic potential. In contrast, staining the inhibitor for antibodies to the inhibitory neurotransmitter, GABA, showed that it forms more varicosities on the distal bundle than on the proximal bundle. Because most of the synapses are located in the varicosities, differences in synapse number likely regulate the proximal-distal disparity in the amount of inhibitory transmitter released. Therefore, the regional differentiation in the amount of transmitter released in the DAFM appears to be based on two distinct mechanisms. In the inhibitor, transmitter release appears to be regulated differentially by differences in synapse number. In the excitor, transmitter release appears to be regulated differentially from a similar number of synapses by differences in active zone structure.

Freeze fracture analysis of apical membranes in cochlear cultures: differences between basal and apical-coil outer hair cells and effects of neomycin.

Previous studies have shown that exposure of cochlear cultures to the ototoxic aminoglycoside antibiotic neomycin leads to the rapid formation of numerous membrane filled protrusions on the apical surface of the hair cells but not on the surrounding supporting cells, and that hair cells in basal-coil cultures are more sensitive to these effects of neomycin than those in the distal end of apical-coil cultures. Freeze-fracture has been used to examine and compare the apical surfaces of hair cells and supporting cells in basal and apical-coil cultures in order to look for features that may explain the differential sensitivity of the various cell types to neomycin, and to characterize the membrane type that forms in response to neomycin and compare it with the normal apical membrane of the hair cell. The apical surface of the highly responsive basal-coil outer hair cells differs significantly from the apical surfaces of apical-coil outer hair cells and supporting cells in both regions of the cochlea in both surface area and the number and density of endocytotic vesicles associated with this surface. Basal-coil hair cells have an average of 120 +/- 39 vesicles per cell surface and a density of 3.5 +/- 0.89 vesicles per microns 2, whereas apical-coil hair cells have 14.8 +/- 15.8 vesicles/cell surface and density of 0.73 +/- 0.72 vesicles per microns 2. There are no significant differences in intramembrane particle (IMP) density on the apical surfaces of all the cell types examined, and qualitative observations of filipin-treated specimens indicate that cholesterol densities are also similar. The membrane that accumulates in response to neomycin treatment at the apical pole of the hair cell is IMP free, does not respond to filipin, and fractures in a manner that is indicative of a high content of unsaturated phospholipid in a fluid phase, and is therefore different in several respects from the normal apical surface of the hair cell. The results of this study suggest that apical surface associated endocytotic vesicle numbers may determine the differential sensitivity of apical and basal-coil hair cells to neomycin, and that neomycin may interfere with some aspect of phospholipid metabolism or membrane turnover in sensory hair cells.

Freeze-fracture characterization of cell types at the surface of the taste organ of the frog,Rana esculenta

The ultrastructure and distribution of intramembrane particles in the chemoreceptor surface of the frog taste organ have been studied by means of freeze-fracture. Sustentacular, wing, mucous cells and two different types of putative taste cells were found to reach the free surface of this chemoreceptor. Each of these cell types was characterized by a different pattern and density of intramembrane particles in the free surface. Wing cells displayed a relatively low number of large intramembrane particles (11.1 +/- 1.4 nm in diameter). Particles of similar size were also present in a much higher concentration in the membrane of cylinder-ending putative taste cells. In microvilli-ending putative taste cells, mucous cells, and sustentacular cells, small intramembrane particles were observed (6.8 +/- 0.78, 6.9 +/- 1.3, 7.2 +/- 0.7 nm in diameter, respectively). The density of these particles was higher in the sustentacular cells than in the other two cell types. These data provide evidence that there are two morphologically distinct types of putative taste cells in the frog taste organ, demonstrating that they are characterized by different pattern of intramembrane particles in their free surface. Furthermore, the present results support previous findings indicating that wing and sustentacular elements represent two different cell types.

Freeze-fracture planes of methanogen membranes correlate with the content of tetraether lipids.

Methanospirillum hungatei GP1 contained 50% of its ether core lipids (polar lipids less head groups) as tetraether lipids, and its plasma membrane failed to fracture along its hydrophobic domain during freeze-etching. The membrane of Methanosaeta ("Methanothrix") concilii did not contain tetraether lipids and easily fractured to reveal typical intramembranous particles. Methanococcus jannaschii grown at 50 degrees C contained 20% tetraether core lipids, which increased to 45% when cells were grown at 70 degrees C. The frequency of membrane fracture was reduced as the membrane-spanning tetraether lipids approached 45%. As the tetraether lipid content increased, and while fracture was still possible, the particle density in the membrane increased; these added particles could be tetraether lipid complexes torn from the opposing membrane face. The diether membrane (no tetraether lipid) of Methanococcus voltae easily fractured, and the intramembranous particle density was low. Protein-free liposomes containing tetraether core lipids (ca. 45%) also did not fracture, whereas those made up exclusively of diether lipids did split, indicating that tetraether lipids add considerable vertical stability to the membrane. At tetraether lipid concentrations below 45%, liposome bilayers fractured to reveal small intramembranous particles which we interpret to be tetraether lipid complexes.

Evaluation of the Intramembrane Particle Count in Biopsied Myocardium from Patients with Idiopathic Cardiomyopathy.

To clarify the abnormalities of myocardial membrane structure in cardiomyopathy, the density of intramembranous particles (IMP) of mitochondria, sarcoplasmic reticulum and sarcolemma, and the number of caveolar necks was evaluated by freeze-fracture morphology. The subjects consisted of 17 patients with hypertrophic cardiomyopathy (HCM), 6 patients with dilated cardiomyopathy (DCM), and 7 patients as controls. The results showed that the numbers of IMPs on the membrane faces of mitochondria, sarcoplasmic reticulum and sarcolemma were significantly decreased in HCM and DCM, and that they were decreased more markedly in DCM than in HCM. The number of caveolar necks was significantly decreased in DCM. Thus, the decreased numbers of IMPs on myocardial membranes and the decrease in caveolar necks may be important characteristics resulting from abnormal myocardial membrane metabolism in cardiomyopathies, HCM and DCM.

Freeze-fracture study of the site of attachment of Cryptosporidium muris in gastric glands.

The mode and organization of the attachment site of Cryptosporidium muris to gastric glands of stomach were investigated by the freeze-fracture method. Cryptosporidium muris was enveloped by a double membrane, of host plasma membrane origin, which formed the parasitophorous vacuole. The outer membrane of the double membrane was continuous with host plasma membrane, while the inner membrane was connected with the anterior part of the parasite plasma membrane at the annular ring. The density of intramembranous particles (IMP) was severely altered at the above two junctures. The parasitophorous outer membrane showed low IMP-density when compared to the host plasma membrane, although both membranes were continuous at the dense band. The inner membrane had few IMP, whereas the parasite plasma membrane showed numerous IMP, although both membranes were continuous at the annular ring. The size of dense band and annular ring was similar in diameter. The feeder organelle was clearly visible as membrane folds in freeze-fracture and some of them were connected with small vesicles of cytoplasm, indicating that the feeder organelle may play an important role for incorporation of nutrients from the host cell.

Cataractogenesis of diethylmatrate-theated rat lens in vitro

The density of intramembrane particles (IMP) of the arterial smooth muscle cell (SMC) plasmalemma was studied by feeze-fracture methods in rabbits fed an atherogenic diet. After 3 weeks of diet, no change in the IMP E face density was observed but the IMPS of the P face decreased by about 50%. Among the various functions of IMPs, they are also believed to be responsible for mechanical adhesion between adjacent cells and cell-cell interactions. That is why the modification of their frequency observed in this study may be assumed to induce the non-recognition of SMC between each other. Thus, they become free from each other and migrate into the subendothelial space. This is one of the important initial conditions leading to atherosclerosis. Therefore, by this freeze-fracture study, our hypothesis about the involvement of SMC in the earlier stages of atherogenesis is confirmed.