Electron Microscopy Of Freeze-fracture Replicas Research Articles

Tight-junction strand networks and tightness of the epithelial barrier.

Tight junctions (TJs) are cell-cell junction structures critical for controlling paracellular permeability. On freeze-fracture replica electron microscopy, they appear as a continuous network of fibrils (TJ strands). TJ strands function as zippers that create a physical barrier against paracellular diffusion of molecules. The morphology of the TJ strand network varies greatly between tissues, and in recent years, studies have highlighted the mechanisms regulating the morphology of TJ strand networks and on their relevance to barrier function. In this review, we discuss evidence regarding the components of the TJ strand and the mechanisms for creating the TJ strand network. Furthermore, we discuss and hypothesize how its morphology contributes to the establishment of the epithelial barrier.

Electron Microscopic Detection of Single Membrane Proteins by a Specific Chemical Labeling.

SummaryElectron microscopy (EM) is a technology that enables visualization of single proteins at a nanometer resolution. However, current protein analysis by EM mainly relies on immunolabeling with gold-particle-conjugated antibodies, which is compromised by large size of antibody, precluding precise detection of protein location in biological samples. Here, we develop a specific chemical labeling method for EM detection of proteins at single-molecular level. Rational design of α-helical peptide tag and probe structure provided a complementary reaction pair that enabled specific cysteine conjugation of the tag. The developed chemical labeling with gold-nanoparticle-conjugated probe showed significantly higher labeling efficiency and detectability of high-density clusters of tag-fused G protein-coupled receptors in freeze-fracture replicas compared with immunogold labeling. Furthermore, in ultrathin sections, the spatial resolution of the chemical labeling was significantly higher than that of antibody-mediated labeling. These results demonstrate substantial advantages of the chemical labeling approach for single protein visualization by EM.

Two classes of gap junction channels mediate soma-germline interactions essential for germline proliferation and gametogenesis in Caenorhabditis elegans.

In all animals examined, somatic cells of the gonad control multiple biological processes essential for germline development. Gap junction channels, composed of connexins in vertebrates and innexins in invertebrates, permit direct intercellular communication between cells and frequently form between somatic gonadal cells and germ cells. Gap junctions comprise hexameric hemichannels in apposing cells that dock to form channels for the exchange of small molecules. Here we report essential roles for two classes of gap junction channels, composed of five innexin proteins, in supporting the proliferation of germline stem cells and gametogenesis in the nematode Caenorhabditis elegans. Transmission electron microscopy of freeze-fracture replicas and fluorescence microscopy show that gap junctions between somatic cells and germ cells are more extensive than previously appreciated and are found throughout the gonad. One class of gap junctions, composed of INX-8 and INX-9 in the soma and INX-14 and INX-21 in the germ line, is required for the proliferation and differentiation of germline stem cells. Genetic epistasis experiments establish a role for these gap junction channels in germline proliferation independent of the glp-1/Notch pathway. A second class of gap junctions, composed of somatic INX-8 and INX-9 and germline INX-14 and INX-22, is required for the negative regulation of oocyte meiotic maturation. Rescue of gap junction channel formation in the stem cell niche rescues germline proliferation and uncovers a later channel requirement for embryonic viability. This analysis reveals gap junctions as a central organizing feature of many soma–germline interactions in C. elegans.

Consideration of the reasons why dormant buds of trees have evolved extraorgan freezing as an adaptation for winter survival

Dormant buds of larch (Larix kaempferi) exhibit extraorgan freezing, in which extracellular ice crystals are formed in localized areas far from the primordia, as an adaptive response to freezing temperatures. In order to better understand why dormant buds have evolved such a complicated mechanism, the freezing behavior of isolated primordia from dormant buds of larch were examined using cryo-scanning electron microscopy (cryo-SEM) and by freeze-fracture replica electron microscopy (FR-EM). When isolated primordia were slowly frozen (5°C/day), extracellular ice crystals developed within primordia. Thus, isolated primordia serves as a useful model to examine the freezing response of primordia in which extracellular freezing occurs. Slow freezing (5°C/day) of isolated primordia resulted in a much lower survival rate (25% at −30°C) than in intact buds (100% at −30°C). In whole buds, exhibiting extraorgan freezing, cryo-SEM observations indicated that slow freezing to −30°C resulted in deep supercooling with a slight shrinkage of all primordial cells, whereas in isolated primordia, slow freezing to −30°C resulted in intracellular freezing of about half of the cells with other cells exhibiting extracellular freezing with significant shrinkage and deformation. FR-EM of whole buds subjected to slow freezing revealed the formation of intramembrane particle (IMP)-free areas with long rectangular shapes in the plasma membrane of many primordial cells at temperatures lower than −15°C and in a small number of cells in isolated primordia. In isolated primordia, the number of cells exhibiting intracellular freezing, which in most cases did not reveal membrane fracture faces, corresponded to the numbers of cells exhibiting IMP-free areas in whole buds. We speculate that the formation of IMP-free areas in plasma membranes may induce intracellular freezing when extracellular ice crystals have developed within primordial tissues. FR-EM also revealed the formation of fracture-jump lesions only in the plasma membranes of primordial cells of slowly-frozen, isolated primordia. We propose that such fracture-jump lesions are produced not only by osmotic dehydration but also by the deformation of cells due to the presence of extracellular ice crystals within primordial tissues in isolated primordia. The results indicate that the plasma membrane of primordial cells in ecodormant buds of larch are subject to a phase transition that may give rise to intracellular freezing, as well as increased sensitivity to dehydration, when extracellular ice crystals form within primordial tissues. We propose that extraorgan freezing represents a mechanism that prevents the formation of extracellular ice crystals within primordial tissues, thus lowering the risk of intracellular freezing and dehydrative stress.

Claudin-2–deficient mice are defective in the leaky and cation-selective paracellular permeability properties of renal proximal tubules

Claudin-2 is highly expressed in tight junctions of mouse renal proximal tubules, which possess a leaky epithelium whose unique permeability properties underlie their high rate of NaCl reabsorption. To investigate the role of claudin-2 in paracellular NaCl transport in this nephron segment, we generated knockout mice lacking claudin-2 (Cldn2(-/-)). The Cldn2(-/-) mice displayed normal appearance, activity, growth, and behavior. Light microscopy revealed no gross histological abnormalities in the Cldn2(-/-) kidney. Ultrathin section and freeze-fracture replica electron microscopy revealed that, similar to those of wild types, the proximal tubules of Cldn2(-/-) mice were characterized by poorly developed tight junctions with one or two continuous tight junction strands. In contrast, studies in isolated, perfused S2 segments of proximal tubules showed that net transepithelial reabsorption of Na(+), Cl(-), and water was significantly decreased in Cldn2(-/-) mice and that there was an increase in paracellular shunt resistance without affecting the apical or basolateral membrane resistances. Moreover, deletion of claudin-2 caused a loss of cation (Na(+)) selectivity and therefore relative anion (Cl(-)) selectivity in the proximal tubule paracellular pathway. With free access to water and food, fractional Na(+) and Cl(-) excretions in Cldn2(-/-) mice were similar to those in wild types, but both were greater in Cldn2(-/-) mice after i.v. administration of 2% NaCl. We conclude that claudin-2 constitutes leaky and cation (Na(+))-selective paracellular channels within tight junctions of mouse proximal tubules.

ZO-1 and ZO-2 Independently Determine Where Claudins Are Polymerized in Tight-Junction Strand Formation

A fundamental question in cell and developmental biology is how epithelial cells construct the diffusion barrier allowing them to separate different body compartments. Formation of tight junction (TJ) strands, which are crucial for this barrier, involves the polymerization of claudins, TJ adhesion molecules, in temporal and spatial manners. ZO-1 and ZO-2 are major PDZ-domain-containing TJ proteins and bind directly to claudins, yet their functional roles are poorly understood. We established cultured epithelial cells (1(ko)/2(kd)) in which the expression of ZO-1/ZO-2 was suppressed by homologous recombination and RNA interference, respectively. These cells were well polarized, except for a complete lack of TJs. When exogenously expressed in 1(ko)/2(kd) cells, ZO-1 and ZO-2 were recruited to junctional areas where claudins were polymerized, but truncated ZO-1 (NZO-1) containing only domains PDZ1-3 was not. When NZO-1 was forcibly recruited to lateral membranes and dimerized, claudins were dramatically polymerized. These findings indicate that ZO-1 and ZO-2 can independently determine whether and where claudins are polymerized.

Localisation of fucoxanthin chlorophyll a/c-binding polypeptides of the centric diatom Cyclotella cryptica by immuno-electron microscopy

Antisera were raised against the C termini of three fucoxanthin chlorophyll a/c-binding polypeptides, Fcp2, Fcp4, and Fcp6, of the centric diatom Cyclotella cryptica. Immunogold electron microscopy of ultrathin-sectioned cells indicated that Fcp2 and Fcp4 are present in almost the same amounts, whereas approximately 8- to 10-fold less gold label was registered for Fcp6. Immunogold electron microscopy of freeze-fracture replicas of thylakoid membranes showed that the C termini of at least Fcp2 and Fcp4 were located in the thylakoid lumen, thus demonstrating a 3-dimensional structure similar to that already described for the chlorophyll a/b-binding light-harvesting polypeptides of higher plants.

Compartmentalization established by claudin-11-based tight junctions in stria vascularis is required for hearing through generation of endocochlear potential.

Claudins are cell adhesion molecules working at tight junctions (TJs) that are directly involved in compartmentalization in multicellular organisms. The cochlea includes a rather peculiar compartment filled with endolymph. This compartment is characterized by high K+ concentration (approximately 150 mM) and a positive endocochlear potential (approximately 90 mV; EP), both indispensable conditions for cochlear hair cells to transduce acoustic stimuli to electrical signals. These conditions are thought to be generated by the stria vascularis, which is adjacent to the endolymph compartment. The stria vascularis itself constitutes an isolated compartment delineated by two epithelial barriers, marginal and basal cell layers. Because TJs of basal cells are primarily composed of claudin-11, claudin-11-deficient (Cld11-/-) mice were generated with an expectation that the compartmentalization in stria vascularis in these mice would be affected. Auditory brainstem response measurements revealed that Cld11-/- mice suffered from deafness; although no obvious gross morphological malformations were detected in Cld11-/- cochlea, freeze-fracture replica electron microscopy showed that TJs disappeared from basal cells of the stria vascularis. In good agreement with this, tracer experiments showed that the basal cell barrier was destroyed without affecting the marginal cell barrier. Importantly, in the endolymph compartment of Cld11-/- cochlea, the K+ concentration was maintained around the normal level (approximately 150 mM), whereas the EP was suppressed down to approximately 30 mV. These findings indicated that the establishment of the stria vascularis compartment, especially the basal cell barrier, is indispensable for hearing ability through the generation/maintenance of EP but not of a high K+ concentration in the endolymph.

Isoforms of caveolin-1 and caveolar structure.

The relationship between caveolin-1 isoforms alpha and beta and caveolar ultrastructure was studied. By immunofluorescence microscopy of human fibroblasts, caveolae were observed as dots positive for caveolin-1, but many dots labeled by an antibody recognizing both isoforms (anti-alphabeta) were not labeled by another antibody specific for the alpha isoform (anti-alpha). Immunogold electron microscopy of freeze-fracture replicas revealed caveolae of different depths, and indicated that anti-alpha labeled deep caveolae preferentially over shallow ones, whereas anti-alphabeta labeled both forms with an equivalent frequency and intensity. The presence of the beta isoform in deep caveolae was confirmed by labeling epitope-tagged beta-caveolin. When made to be expressed in HepG2 cells lacking endogenous caveolins, the alpha isoform formed caveolar depressions efficiently, but the beta isoform hardly did so. Caveolae were also formed in cells expressing the two isoforms, but their frequency was variable among cells of the same clone. Coexpression of caveolin-1 and caveolin-2 caused more efficient formation of deep caveolae than caveolin-1 alone. The result indicates that the two isoforms of caveolin-1 have a different potential for forming caveolae structure, and more importantly, that deep and shallow caveolae may be diversified in their molecular composition.

Seasonal Changes in the Freezing Behavior of Xylem Ray Parenchyma Cells in Four Boreal Hardwood Species

The freezing behavior of xylem ray parenchyma cells in several boreal hardwood species, namely,Betula platyphylla, Populus canadensis, P. sieboldii,andSalix sachalinensis,was examined by differential thermal analysis (DTA), cryo-scanning electron microscopy (Cryo-SEM), and freeze-fracture replica electron microscopy. Although DTA profiles of samples harvested in summer and in winter suggested that the xylem ray parenchyma cells in all four species responded to freezing stress by extracellular freezing, Cryo-SEM showed clearly that the xylem ray parenchyma cells in all these species responded to freezing stress by shallow supercooling in summer and by extracellular freezing in winter. It is suggested that DTA failed to reveal the true freezing behavior of xylem ray parenchyma cells because of an overlap of temperature ranges between the high-temperature exotherm and the low-temperature exotherm and/or because of the limited extent of the LTE. The seasonal changes in freezing behavior of xylem ray parenchyma cells in all these boreal species, which are results of seasonal cold acclimation, support the hypothesis that a gradual shift of freezing behavior in xylem ray parenchyma cells from shallow supercooling in hardwood species that grow in tropical zones to extracellular freezing in hardwood species that grow in cold areas might be a result of the evolutionary adaptation of hardwood species to cold climates.

Revaluation on the types and pattern of distribution of sinusoidal fenestrations in the lobule of normal rat liver.

There is a need to identify the existence of large fenestrations and to evaluate the type and pattern of the distribution of sinusoidal fenestrations in the hepatic lobule. The sinusoidal fenestrations in the rat liver lobule were investigated by scanning electron microscopy of perfusion-fixed specimens and by transmission electron microscopy of freeze-fracture replicas from immersion-fixed specimens. Ringer solution used for precleaning was equilibrated with 84%/16% O2/CO2 at 37 degrees C, and the perfusion pressure was maintained at 5-10 mmHg in the portal vein. The fenestrations were recognized on the luminal surface of sinusoids on the basis of regional difference in size and distribution within the lobule and divided into three types: small, medium-sized, and large. The small fenestrations were numerous in each region. The number of medium-sized fenestrations was large in the pericentral and intermediate regions, while that of large fenestrations was predominant in the intermediate region. Based on the occurrence of fenestrations, the sinusoids were also divided into three types. The type I sinusoid mainly showed small fenestrations and was predominant in the periportal region. The type II sinusoid showed small and medium-sized fenestrations and was predominant in the pericentral region. The type III sinusoid showed all the types of fenestrations including large ones and was predominant in the intermediate region. The images showing dumbbell-shaped or irregularly shaped margins were observed between small or medium-sized fenestrations on the freeze-fracture replica. 1) The fenestrations are divided into three types according to the size and pattern of distribution within the lobule. 2) The sinusoids are also divided into three types based on the distribution pattern of each type of fenestrations. 3) The small fenestrations are evenly distributed throughout the entire lobule, while the medium-sized and large fenestrations show the regional difference within the lobule. 4) The large fenestrations exist as real structures of the endothelial lining cells of sinusoids and may result from the fusion of contiguous smaller ones.

Seasonal changes in the low-temperature behaviour of xylem ray parenchyma cells in red osier dogwood ( Cornus sericea L.) with respect to extracellular freezing and supercooling

The low temperature behaviour of xylem ray parenchyma cells in red osier dogwood ( Cornus sericea L.) was examined by different thermal analysis (DTA), cryo-scanning electron microscopy, freeze-fracture replica electron microscopy and a survival assay (leakage of electrolytes). DTA provided a profile that is typical of extracellular freezing in xylem ray parenchyma cells in both winter and summer. Observations of xylem ray parenchyma cells by electron microscopy indicated, however, that low-temperature behaviour was different from that predicted by DTA. Electron microscopy revealed that, upon cooling at 0.1°C/min, the ray parenchyma cells in winter exhibited typical extracellular freezing, whereas cells in summer exhibited intracellular freezing below -15°C. Cooling at 1.25°C/day (<0.001°C/min) produced a slight collapse of the cell walls as a result of partial dehydration, but it did not inhibit the intracellular freezing in xylem ray parenchyma cells in summer. It is suggested that failure of DTA to reveal the low temperature exotherm (LTE) upon intracellular freezing was due to an overlap of temperature between the high temperature exotherm (HTE) and the LTE, in addition to a reduction in the LTE by the partial dehydration of cells. It is concluded that red osier dogwood has xylem ray parenchyma cells whose low-temperature behaviour changes from extracellular freezing in winter to supercooling in summer, possibly as a result of seasonal differences in permeability of the cell walls to water. This type of seasonal change in the low-temperature behaviour may produce a superior mechanism for the adaptation to freezing temperatures of cells of plants growing in cold regions, in which dehydration tolerance also changes seasonally.

Characterisation of clusters of alpha-tocopherol in gel and fluid phases of dipalmitoylglycerophosphocholine.

The effect of alpha-tocopherol on the phase behaviour of aqueous dispersions of dipalmitoylglycerophosphocholine has been examined by differential scanning calorimetry, freeze-fracture electron microscopy, and real-time X-ray diffraction methods. The presence of alpha-tocopherol in proportions 2.5, 5, 7.5, and 10 mol/100 mol results in a progressive decrease in the temperature of the gel to liquid-crystalline phase transition from 41.5 degrees C to 36 degrees C and a reduction in transition enthalpy from 35 kJ.mol-1 to 15 kJ.mol-1 phospholipid. The thermal data indicated that the pretransition of the phospholipid is eliminated even in mixtures containing 2.5 mol/100 mol alpha-tocopherol. Real-time X-ray diffraction measurements using synchrotron radiation performed under identical conditions to the thermal studies showed clear transition sequences of L beta-->P beta-->L alpha for all mixtures. The sequence was reversible with hysteresis of 2-3 degrees C on cooling. Low-angle X-ray scattering from mixtures in the gel phase showed three lamellar repeat spacings of 6.35, 7.5, and 8.4 nm. The spacing at 6.35 nm was assigned to pure phospholipid from which alpha-tocopherol has been phase separated into enriched domains giving lamellar repeat spacings of 7.5 nm and 8.4 nm. Low-angle diffraction patterns of mixtures in the fluid phase were characterised by two lamellar repeat spacings. The longer spacing of about 6.6 nm was assigned to pure phospholipid and the shorter spacing at about 6.1 nm to an alpha-tocopherol-enriched phase. Electron microscopy of freeze-fracture replicas of mixtures of phospholipid containing 10 mol/100 mol alpha-tocopherol thermally quenched from 10 degrees C and 60 degrees C, showed evidence of domain structures within the bilayer plane that appeared to be correlated between successive bilayers in multilamellar dispersions. Calculations of the stoichiometry of phospholipid: alpha-tocopherol in the alpha-tocopherol-enriched domains based on enthalpy data and integrated X-ray scattering intensity gave values of 9.6:1 for the fluid phase and 9.2:1 for the gel phase. This was consistent with a clustering of alpha-tocopherol molecules in both gel and liquid-crystal phases of dipalmitoyl-glycerophospholcholine in approximately the same stoichiometry.

Freeze-fracture replica electron microscopy combined with SDS digestion for cytochemical labeling of integral membrane proteins. Application to the immunogold labeling of intercellular junctional complexes.

We propose a new electron microscopic method, the sodium dodecylsulphate (SDS)-digested freeze-fracture replica labeling technique, to study the two-dimensional distribution of integral membrane proteins in cellular membranes. Unfixed tissue slices were frozen with liquid helium, freeze-fractured, and replicated in a platinum/carbon evaporator. They were digested with 2.5% SDS to solubilize unfractured membranes and cytoplasm. While the detergent dissolved unfractured membranes and cytoplasm, it did not extract fractured membrane halves. After SDS-digestion, the platinum/carbon replicas, along with attached cytoplasmic and exoplasmic membrane halves, were processed for cytochemical labeling, followed by electron microscopic observation. As an initial screening, we applied this technique to the immunogold labeling of intercellular junction proteins: connexins (gap junction proteins), occludin (tight junction protein), desmoglein (desmosome protein), and E-cadherin (adherens junction protein). The immunogold labeling was seen superimposed on the image of a fracture face visualized by platinum/carbon shadowing. The immunoreaction was specific, and only the structures where the proteins were expected were labeled. For instance, anti-occludin immunogold complexes were observed immediately adjacent to the tight junction strands on the protoplasmic and exoplasmic fracture faces. No significant levels of gold label were associated with non-tight-junctional regions of plasma membranes. The procedures of the SDS-digested freeze-fracture replica labeling and its potential significance are discussed.

Morphological alterations of gap junctions in phalloidin-treated rat livers.

Morphological alterations in the pattern of liver cell gap junctions were examined in phalloidin-treated rats to assess the role of gap junctions in experimental intrahepatic cholestasis. Double-labelled fluorescent staining of gap junctions and F-actin were performed using a monoclonal antibody against rat hepatocyte connexin 32 and rhodamine-phalloidin. Immunoelectron microscopy, using the anti-connexin 32 antibody, freeze-fracture replica electron microscopy, and conventional electron microscopy were also performed. In phalloidin-treated rat livers, the specific immunofluorescent staining of connexin 32 was markedly decreased in the pericentral area after 1 day of phalloidin treatment and, after 5 days of phalloidin treatment, there was a decrease in connexin 32 staining in the entire hepatic lobule. On the other hand, F-actin staining at the cell periphery and at the bile canaliculi was markedly increased in the pericentral area of the hepatic lobule after 1 day of phalloidin treatment and in the entire lobule after 5 days of treatment. Immunoelectron microscopy showed that both sides of the cytoplasmic domains of gap junctions were stained with anti-connexin 32 antibody in controls, whereas, in cholestatic rats, only one side of the cytoplasmic domain of some gap junctions was stained with anti-connexin 32 antibody after 1 or 3 days of phalloidin treatment. No gap junctions were observed after 5 days of phalloidin treatment either by freeze-fracture replica electron microscopy or by conventional electron microscopy. These results indicate that with phalloidin treatment, hepatocyte gap junctions decrease, first in the pericentral area, and finally throughout the entire lobule.

Identification of polypeptides associated with sarcolemmal vesicles enriched in orthogonal arrays

Electron microscopy of freeze-fracture replicas from the sarcolemmas of fast-twitch muscle fibers reveals orthogonal arrays of particles. The biochemical nature of macromolecules associated with the sarcolemmal orthogonal array was investigated using muscle fragments and isolated sarcolemmal vesicles. Muscle fragments incubated in vitro with the lectin concanavalin A exhibited a clustering of orthogonal arrays into local patches. Treatment with other lectins did not result in the clustering of arrays. Clustering was inhibited by the addition of α-methyl- d-mannoside, a ligand which also binds concanavalin A. These results suggest that the orthogonal arrays (or associated components) specifically bind concanavalin A. Sarcolemmal vesicles from rabbit sacrospinalis (SAC) and rat extensor digitorum longus (EDL) (both primarily fast-twitch) and rat soleus (SOL) (primarily slow-twitch) were obtained by a combination of low-salt fractionation and sucrose density gradient centrifugation. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of proteins and glycoproteins solubilized from these vesicles revealed several bands. Four of these bands were present in gels from both the rabbit and rat fast-twitch muscle sarcolemmal preparations (that contained arrays), yet were absent in gels from rat slow-twitch muscle sarcolemmal preparations (not bearing arrays). An enrichment in vesicles containing arrays was achieved by binding SAC sarcolemmal vesicles to Con A-Sepharose 4B beads. SDS-PAGE analysis of array-enriched vesicles from the concanavalin A beads revealed enrichment of three major bands at M r 93 000, 54 000 and 49 000. These enriched bands correlate with three of the four bands common to fast-twitch EDL and SAC, yet absent in slow-twitch SOL sarcolemmal preparations. We conclude that at least one macromolecular component associated with the sarcolemmal orthogonal array is a concanavalin A binding glycoprotein. We further conclude that three candidates for this component co-purify with the morphological array, and have approximate molecular weights of 93 000, 54 000 and 49 000.

Influence of the membrane undercoat on filipin perturbation of the red blood cell membrane

Filipin, a polyene antibiotic, interacts with β-hydroxy sterols such as cholesterol in most cell membranes, forming bumps and pits that are visible by electron microscopy of freeze-fracture replicas. The markedly reduced perturbability of the red blood cell (RBC) membrane, compared to other cells, has been attributed to the constraining influence of the red cell membrane skeleton, the undercoat composed of spectrin, actin, and protein 4.1. To test the influence of the membrane skeleton on filipin-induced perturbation of the RBC membrane, we studied the interaction of filipin with red cells that were inherently devoid of spectrin and RBC in which spectrin had been crosslinked or denatured. These spectrindeficient, crosslinked, and denatured cells have a fivefold increase in the number of filip-ininduced perturbations as compared to control cells, despite equivalent membrane cholesterol content. These findings confirm that the spectrin-based membrane skeleton strongly influences the organization of the membrane so as to limit perturbation by filipin:cholesterol interaction and that for membranes in which the cholesterol content is known, filipin is a useful probe for testing the avidity of spectrin-based cytoskeletal attachment.

Phase separation in frozen erythrocyte membrane preparations.

The reversible formation of a lipid-like phase in frozen preparations of erythrocyte membranes has been studied by X-ray diffraction and by electron microscopy of freeze-fracture replicas. The observations provide strong evidence for lateral migration or displacement at specific temperatures of intra-membrane particles. This creates large areas of particle-free membranes which fracture preferentially so as to dominate the freeze-fracture image.

The distribution of filipin-sterol complexes in photoreceptor synaptic membranes.

The polyene antibiotic filipin, which binds to membrane sterols, has been used to investigate the relative distribution of cholesterol at photoreceptor synaptic junctions in the chick retina. Following anesthesia and aldehyde perfusion fixation, the retina is removed and immersed in fixative solution containing the filipin for 36-48 hours. The retinas are then processed for freeze-fracture. Electron microscopy of freeze-fracture replicas demonstrates that the filipin-sterol complexes are not evident between intramembrane particles of the presynaptic and postsynaptic particle arrays that are present at ribbon and basal junctions. In contrast, the synaptic vesicle fusion zone of ribbon junctions contains large numbers of filipin-sterol complexes which are observed merging with the free margins of the presynaptic particle array. There is a scarcity of such complexes, however, around the free margins of basal junction presynaptic particle arrays. These latter sites do not contain a vesicle fusion zone. Particle-poor areas of membrane that surround postsynaptic particles arrays of ribbon and basal junctions also do not contain filipin binding sites. The nonsynaptic membrane of photoreceptor terminals contains large numbers of filipin-sterol complexes, less tightly packed than in the synaptic vesicle fusion zone. Coated vesicle fusion sites in the presynaptic membrane contain groups of intramembrane particles but the filipin-sterol complexes are excluded from these sites. The observations suggest that synaptic membrane domains which interact with cytoskeletal components, such as clathrin and pre- and postsynaptic densities contain less cholesterol than other domains such as the synaptic vesicle fusion zones.

ブドウ球菌L型菌のフリーズフラクチャー法による解析

L-form cells are generally pleomorphic and show variety in size ranging from 0.2 μm to more than 10 μm in diameter. We investigated the ultrastructure of the cell membrane of such variable L-form cells derived from Staphylococcas aureus 209P in terms of membrane particle distribution by freeze-fracture replica electron microscopy. The specimens were prepared by immediate freezing in liquid nitrogen without any chemical fixatives followed by freeze-fracture with Eiko FD-2. We focused on the structure of the P face only. The particles were densely dispersed on the cell membrane of mediumsized cells (0.4-4 μm). Large (>4 μm) and small (0.2-0.4 μm) cells showed very few particles. In conclusion, the medium-sized cells were growing actively, while large and small cells were not growing.