Formation of B type gap junctions between PHA-stimulated rabbit lymphocytes.

Contact areas of PHA-stimulated and consequently agglutinated rabbit peripheral blood and spleen lymphocytes were studied with ultrathin-section and freeze-fracture techniques. Broad contact zones (BCZ) between adjacent cells were characterized in freeze-fracture replicas as plasma membrane areas in which at the protoplasmic fracture face (PF) a heterogeneous population of redistributed intramembranous particles (IMP) appear to assemble. In addition homogeneous particles of 11 nm diameter, found to be concentrated at the external fracture face (EF) at the site of the BCZ, aggregate to clusters and after longer culture periods appear to participate in the formation of gap junctional complexes. Evidence is provided that the BCZ—probably an area of concentrated PHA-binding sites—may well serve as a formation plaque for gap junction constitution in the system studied.     

Close-packing of plasma membrane particles during wall regeneration by isolated higher plant protoplasts—fact or artefact?

Summary Freeze-fracture preparations of protoplasts isolated from cell suspension cultures and leaf mesophyll tissue have been examined by transmission electron microscopy. During the first 72 hours of cell wall regeneration, the 8–10nm intramembraneous particles were randomly distributed on both the protoplasmic and extracellular fracture faces of the plasma membranes of protoplasts frozen and fractured in the culture medium without glutaraldehyde fixation or cryoprotection. Incubation of living protoplasts in culture medium containing 20% v/v glycerol as cryoprotectant prior to freezing without fixation caused deformation of the plasma membrane in the form of protrusions accompanied by particle aggregation on the protoplasmic fracture face of the membrane. Intramembraneous particle aggregation was not observed in protoplasts fixed in glutaraldehyde prior to incubation in medium containing glycerol. The aggregation of particles into hexagonal close packed arrays and elongate chains is discussed in relation to a previous report in the literature of the possible involvement of intramembraneous particle complexes in microfibril formation by isolated higher plant protoplasts.     

Molecular cytochemistry of CD3 and CD4 antigens in human lymphocytes as studied by label-fracture and by fracture-label

Label-fracture and fracture-label membrane immunocytochemistry are used to analyze the surface distribution, dynamics and partition on fracture of CD3 and CD4 antigens of human T lymphocytes. Redistribution of the antigens, induced by treatment at 37 degrees C with specific monoclonal antibodies, results in patching and capping of the labeling as observed in label-fractured specimens. Examination of platinum/carbon replicas of freeze-fractured plasma membranes of antibody-treated cells does not reveal recognizable domains of intramembrane particles. However, in cells where the aggregation of intramembrane particles is induced by incubation with glycerol, colloidal gold-labeled CD3 and CD4 molecules are seen confined to particulate domains of the membrane. Therefore, the lack of visible aggregation of intramembrane particles in patched or capped regions of the membrane implies that migration of CD3 and CD4 antigens with concentration in domains of the membrane is achieved contemporaneously with export of other non-capped integral membrane proteins from the same regions, in a process of diffusional equilibrium. Examination of fracture-labeled specimens shows that CD4 molecules partition on fracture with the inner protoplasmic face of the plasma membrane. This partition illustrates the transmembrane attitude of the antigen molecule and is a probable consequence of interaction of the protein with other components of the membrane or with the cytoskeleton.     

Particle Assemblies in the Plasma Membrane of Tetrahymena: Relationship to Cell Surface Topography and Cellular Morphogenesis1

During a freeze-fracture electron microscopical study of the plasma membrane of Tetrahymena, several different types of organized particle assemblies were observed. Three of these were found only on the protoplasmic face and were localized in the anterior-ventral region of the cell. These consisted of plate-like arrays composed of 4–25 triplet rows of small 3–4 nm particles; long, paired linear arrays localized at the tops of cortical ridges and composed of 7–8 nm particles; and elongated tetragonal arrays located in the grooves between ridges and composed of approximately 10 nm particles. The distribution of these arrays is consistent with roles in cellular morphogenesis, chemoreception, or cell-cell pairing during conjugation. In addition, a unique particle track associated with the cytoproct (anal pore) was observed in the external face of the plasma membrane. Furthermore, the protoplasmic face of the plasma membrane is characterized by a high density of particles organized into localized microarrays, consisting of small paracrystals or strings, which exhibit a loose higher-order patterning most evident toward the anterior end of the cell. Particle distributions on the protoplasmic face do not appear to be significantly altered by conditions that cause clumping of alveolar membrane particles. Taken together, these observations are consistent with the idea that the proteins of the plasma membrane are highly ordered and relatively immobile and that the structure of the plasma membrane is regionally differentiated.     

Tonoplast fine structure and osmotic regulation in Porphyra umbilicalis

In Porphyra, an intertidal red alga, the fine structure of the tonoplast was studied by freeze-fracture electron microscopy. It was shown that density and size of intramembraneous particles on the protoplasmic fracture face vary with external osmotic potential. The frequency of particles grouped in size classes (calculated per cell) increases with increasing osmotic stress and shows a maximum in 3 to 4 x artificial seawater medium ASP₁₂. It is concluded that the intensity of tonoplast transport, which probably is enhanced with increasing osmotic stress from 1 to 4 x media, is most likely correlated with a change in membrane fine structure of the tonoplast.     

Plasma-membrane rosettes in root hairs of Equisetum hyemale

Particle arrangement in the plasma membrane during cell wall formation was investigated by means of the double-replica technique in root hairs of Equisetum hyemale. Particle density in the protoplasmic fracture face of the plasma membrane was higher than in the extraplasmic fracture face. Apart from randomly distributed particles, particle rosettes were visible in the PF face of the plasma membrane. The rosettes consisted of six particles arranged in a circle and had an outer diameter of approx. 26 nm. No gradient in the number of rosettes was found, which agrees with micrifibril deposition taking place over the whole hair. The particle rosettes were found individually, which might indicate that they spin out thin microfibrils as found in higher-plant cell walls. Indeed microfibril width in these walls, measured in shadowed preparations, is 8.5±1.5 nm. It is suggested that the rosettes are involved in microfibril synthesis. Non-turgid cells lacked microfibril imprints in the plasma membrane and no particle rosettes were present on their PF face. Fixation with glutaraldehyde caused, probably as a result of plasmolysis, the microfibril imprints to disappear together with the particle rosettes. The PF face of the plasma membrane of non-turgid hairs sometimes showed domains in which the intramembrane particles were aggregated in a hexagonal pattern. Microfibril orientation during deposition will be discussed.Abbreviations EF extraplasmic fracture face - PF protoplasmic fracture face     

Asymmetric mass distribution of (Na+ + K+)-ATPase in membranes studied by freeze-fracture-etch electron microscopy

SDS-purified porcine kidney (Na+ + K+)-ATPase was studied by thin-section and freeze-etch electron microscopy. Freeze-fracturing of resealed membrane fragments shows no difference in the distribution of intramembranous particles of approx. 9.0 nm in diameter between convex and concave fracture faces. However, two types of convex face are found: FA, which shows a rather smooth background with many intramembranous particles, and FB, which shows a textured background with very few or no intramembranous particles. Etching the fractured samples further reveals that FA faces are covered with many intramembranous particles, while the etched external faces (EA) are either irregularly granulated or reveal many particles half the size of intramembranous particles. FB faces are covered with distinct pits of 9 nm or larger. The etched external surfaces (EB) are covered with many particles of intramembranous particle size. These results suggest that there are two vesicle orientations in our resealed purified membrane preparation: right-side-out, as in vivo, and inside-out. The majority of the protein mass is distributed only on one side of the membranes. Right-side-out resealed membrane vesicles after fracturing and etching show particulated FA convex fracture faces and irregularly granulated or smooth etched EA surfaces, indicating that the FA face is the protoplasmic fracture face and that the majority of the protein mass of the (Na+ + K+)-ATPase is located on the cytoplasmic half of the membrane.     

Rearrangements of integral membrane components during in vitro aging of sheep erythrocyte membranes

In vitro aged sheep erythrocytes and sheep erythrocyte ghosts spontaneously release vesicles that consist of long protrusions affixed to flattened headlike structures. The intramembranous particles seen on the protoplasmic face of freeze fracture electron micrographs of vesicle protrusions are arranged in paired particle rows. On the equivalent fracture face of headlike structures, the particle density is low; if particles are present, they are clustered along the rim of the flattened headlike structure and at the junction with the protrusion. The released vesicles are depleted of the intramembranous particles seen on the exoplasmic face of ghost but retain almost exclusively particles of the protoplasmic face. Correspondingly, the exoplasmic face of ghosts that have released vesicles reveals a 28 percent higher density of intramembranous particles than that of fresh ghosts. Purified vesicles are depleted of spectrin but retain integral membrane proteins, with one of an apparent mol wt of 160,000 accounting for nearly 50 percent of the total protein (Lutz, H.U.,R. Barber, and R.F. McGuire. 1976. J. Biol. Chem. 251:3500-3510). When vesicles are modified with the cleavable cross-linking reagent [(35)S]dithiobis (succinimidyl propionate)at 0 degrees C, the 160,000 mol wt protein is rapidly converted to disulfide-linked dimers and higher oligomers. Exposure of intact ghosts to the reagent in the same way fails to yield equivalent polymers. A comparison of the morphological and biochemical aspects of ghosts and vesicles suggest that a marked rearrangement of membrane proteins accompanies the supramolecular redistribution of intramembranous particles during spontaneous vesiculation. The results also suggest that the paired particles of the protoplasmic face of vesicle protrusions are arranged in paired helices and contain the 160,000 mol wt protein as dimers.     

Surface distribution and partition during freeze-fracture of CD8 antigens on human lymphocytes and on epithelial transfected cells

Freeze-fracture immunocytochemistry was used to analyse the surface distribution, redistribution induced by antibodies, and partition during freeze-fracture, of CD8 molecules on human T lymphocytes and rat epithelial transfected (FRT-U10) cells. Immunogold labelling of CD8 antigens was uniform over the unfractured cell surfaces of both lymphocytes and epithelial transfected cells. After freeze-fracture, the gold particles were associated with the exoplasmic outer leaflets of the plasma membranes in both cell types. In lymphocytes, incubation with antibodies at 37° C up to 20 min induced patching and capping of the antigens on the unfractured cell surface. After fracture, the patched molecules appeared associated with the protoplasmic inner leaflet of the plasma membranes. Parallel antibody-treatment at 37° C of FRT-U10 cells induced clustering of CD8 molecules but failed to cause further aggregation in larger patches or in caps. After freeze-fracture, the immunola-belling was clustered, but associated with the exoplasmic outer leaflet of the plasma membranes as in untreated cells. The different redistribution induced by antibodies and the different behaviour on fracture of the redistributed molecules in the two cell types may be regulated by CD8 interaction with the cytoskeleton.     

A multidisciplinary approach to the study of the plasma membrane of Zea mays pollen during controlled dehydration

A multidisciplinary approach (freeze-fracture, nuclear magnetic resonance, differential scanning calorimetry, isoelectric focusing and fluorochromatic reaction test) has been used to follow the behaviour of Zea mays pollen during dehydration - and to estimate its quality. At anthesis, the water content of maize pollen is 57–58% and the vegetative plasma membrane is continous and well structured with a very low density of intramembraneous particles on the extraplasmic fracture face. Maize pollen grains can withstand the drying process until a water content of 28% is reached, at which point 60–80% of the individuals show a negative reaction in the fluorochromatic test. At this water content, there is no more crystallizable water and thus metabolism decreases, leading to oxidative damage and the formation of gelphase microdomains in the plasma membrane. Consequently, the plasma-membrane permeability is modified. At 15–13% water content, all pollen grains show a negative fluorochromatic reaction, and gel-phase microdomains are more numerous but membranes still have a bilayer structure. Relaxation-time experiments indicate the occurrence of water replacement at the membrane level. Thus, sugar may stabilize the membrane structure at water contents as low as 3%. During the dehydration process, pollen walls act as elastic structures and remain closely applied to the protoplast. The combination of wall deformation and water replacement would permit pollen survival until oxidative damage occurs in the dehydrated grain.Abbreviations EF extraplasmic fracture face - FCR fluorochromatic reaction - IMP intramembraneous particle - NMR nuclear magnetic resonance - PF protoplasmic fracture face - T₂ relaxation time     

Surface domains in the pathogenic protozoan Tritrichomonas foetus.

The quick-freezing and freeze-etching techniques were used to analyze surface domains of Tritrichomonas foetus. The surface of the protozoan body was not smooth, presenting surface projections, except on the flagellar surface. Images of the actual surface of the anterior flagella revealed the presence of intramembranous particles that form rosettes, as observed on the protoplasmic fracture face. They may represent integral transmembrane proteins exposed at the cell surface. Surface specializations were also observed at the flagella base and where the recurrent flagellum attaches to the cell body.     

Surface Domains in the Pathogenic Protozoan Tritrichomonas foetus

ABSTRACT The quick-freezing and freeze-etching techniques were used to analyze surface domains of Tritrichomonas foetus . The surface of the protozoan body was not smooth, presenting surface projections, except on the flagellar surface. Images of the actual surface of the anterior flagella revealed the presence of intramembranous particles that form rosettes, as observed on the protoplasmic fracture face. They may represent integral transmembrane proteins exposed at the cell surface. Surface specializations were also observed at the flagella base and where the recurrent flagellum attaches to the cell body.     

Alteration of human erythrocyte plasma membranes by perfringolysin O as revealed by freeze-fracture electron microscopy. Studies on Clostridium perfringens exotoxins V.

When human erythrocyte membranes were treated with perfringolysin O (Clostridium perfringens theta-toxin) and examined by electron microscopy after freeze-fracture, two ultrastructural alterations were observed in fracture faces of membrane. (1) A random aggregation of intramembranous particles was seen in the fracture face of the protoplasmic half (PF face) of all membranes treated with the toxin, even if at a low concentration (40 hemolytic units/ml). On the other hand, the aggregation in the fracture face of the exoplasmic half (EF face) was observed only in membranes treated with a high concentration (3300 hemolytic units/ml) for 2 h. (2) Round protrusions and "cavities" with 30 nm in diameter were visible in EF and PF faces of membranes treated with a high concentration, respectively. These structures were always protruded toward cytoplasmic side, but did not appear to form holes through the membrane. Ring and arc shaped structures with a dark center of 26 nm and a distinct border of 5 nm in width were observed when the toxin alone was negatively stained at a very high concentration (170,000 hemolytic units/ml). These structures were also produced in the presence of cholesterol even if the toxin concentration was low.     

Alteration of human erythrocyte plasma membranes by perfringolysin O as revealed by freeze-fracture electron microscopy. Studies on Clostridium perfringens exotoxins V

When human erythrocyte membranes were treated with perfringolysin O (Clostridium perfringens θ-toxin) and examined by electron microscopy after freeze-fracture, two ultrastructural alterations were observed in fracture faces of membrane. (1) A random aggregation of intramembranous particles was seen in the fracture face of the protoplasmic half (PF face) of all membranes treated with the toxin, even if at a low concentration (40 hemolytic units/ml). On the other hand, the aggregation in the fracture face of the exoplasmic half (EF face) was observed only in membranes treated with a high concentration (3300 hemolytic units/ml) for 2 h. (2) Round protrusions and ‘cavities’ with 30 nm in diameter were visible in EF and PF faces of membranes treated with a high concentration, respectively. These structures were always protruded toward cytoplasmic side, but did not appear to form holes through the membrane.Ring and are shaped structures with a dark center of 26 nm and a distinct border of 5 nm in width were observed when the toxin alone was negatively stained at a very high concentration (170 000 hemolytic units/ml). These structures were also produced in the presence of cholesterol even if the toxin concentration was low.     

Ultrastructure of the vegetative gametophytic cells of Porphyra leucosticta (Rhodophyta) grown in red, blue and green light

The ultrastructure of the vegetative gametophytic cells of Porphyra leucosticta Thuret grown in red, blue and green light was studied both in ultrathin sections and in replicas of rapidly frozen cells. High activity of dictyosornes and mucilage sacs results in a dramatic decrease of the protoplasmic area and in thicker cell walls in red light in comparison with blue light and the control. There are numerous well‐formed phycobili‐somes in blue light, whereas not well‐formed ones are present in red and especially in green light. There are also many phycobilisomes in the intrapyrenoidal thylakoids in blue light, fewer in green light, but they are absent in red light and in the control. It seems that in red and especially in green light, the phycobilisomes have fewer rods than in blue light. In green light, chloroplasts bear numerous genophores in contrast to blue and red light. The spacings of neighboring parallel thylakoids are as follows: control 64.3 nm, blue light 90.6 nm, red light 41.3 nm, green light 43.7 nm. Due to the relatively small spacing of the neighboring parallel thylakoids in red (41.3 nm) and in green light (43.7 nm) and of the given height of phycobilisomes (35 nm), the alternate phycobilisomes attached to neighboring lamellae are forced to interdigitate. The density of phycobilisomes per square micrometer of thylakoid surface dramatically increases in blue light (800 μm^?2) in relation to red (250 μm^?2) and green light (180 μm^?2). The protoplasmic fracture face of the thylakoids reveals numerous, tightly packed, but randomly distributed particles. The particle size distribution is uniform in the two types of fracture faces, with an average diameter of about 11.5 nm. In blue light, both the phycobilisomes and exoplasmic face particles are organized into rows with a spacing of 60–70 nm. The results (changes: in the protoplasmic area; in the spacing of the thylakoids; in phycobilisome arrangement; in structure, shape and size of phycobilisomes; and in the accumulation of plastoglobuli), have shown that the monochromatic light (blue, red and green) brings about marked changes in the package effect and consequently in the efficiency of light absorption. In addition, the blue light contributes to the intense production of chlorophyll a, phycoerythrin, phycocyanin and soluble proteins, while intense production of polysaccharidic material is attributed to red light.     

Oligomerization state of MIP proteins expressed in Xenopus oocytes as revealed by freeze-fracture electron-microscopy analysis

The MIP (major intrinsic protein) family is a widespread family of membrane proteins exhibiting two major types of channel properties: aquaporins and solute facilitators. In the present study, freeze-fracture electron microscopy was used to investigate the oligomerization state of two MIP proteins heterologously expressed in the plasma membrane of Xenopus laevis oocytes: AQPcic, an aquaporin from the insect Cicadella viridis, and GlpF, a glycerol facilitator from Escherichia coli. Swelling assays performed on oocytes 48 and 72 h following cRNA microinjections showed that these proteins were functionally expressed. Particle density determinations indicated that expression of proteins is related to an increase in particle density on the P fracture face of oocyte plasma membranes. Statistical analysis of particle sizes was performed on protoplasmic fracture faces of the plasma membrane of oocytes expressing AQPcic and GlpF 72 h after cRNA microinjections. Compared to control oocytes, AQPcic-expressing oocytes exhibited a specific population of particles with a mean diameter of 8.7 +/- 0.1 nm. This value is consistent with the previously reported tetrameric organization of AQPcic. In addition, AQPcic particles aggregate and form orthogonal arrays similar to those observed in native membranes of C. viridis, consisting of homotetramers of AQPcic. On the protoplasmic fracture face of oocytes expressing GlpF, the particle density is increased by 4.1-fold and the mean diameter of specifically added particles is 5.8 +/- 0.1 nm. This value fits with a monomer of the 28-kDa GlpF protein plus the platinum-carbon layer. These results clearly demonstrate that GlpF is a monomer when functionally expressed in plasma membranes of Xenopus oocytes and therefore emphasize the key role of the oligomerization state of MIP proteins with respect to their function.     

Heliobacterium chlorum: cell organization and structure

The basic cellular organization of Heliobacterium chlorum is described using the freeze-etching technique. Internal cell membranes have not been observed in most cells, leading to the conclusion that the photosynthetic apparatus of these organisms must be localized in the cell membrane of the bacterium. The two fracture faces of the cell membrane are markedly different. The cytoplasmic (PF) face is covered with densely packed particles averaging 8 nm in diameter, while the exoplasmic (EF) face contains far fewer particles, averaging approximately 10 nm in diameter. Although a few differentiated regions were noted within these fracture faces, the overall appearance of the cell membrane was remarkably uniform. The Heliobacterium chlorum cell wall is a strikingly regular structure, composed of repeating subunits arranged in a rectangular pattern at a spacing of 11 nm in either direction. We have isolated cell wall fragments by brief sonication in distilled water, and visualized the cell wall structure by negative staining as well as deep-etching.Abbreviations PF protoplasmic fracture face - EF exoplasmic fracture face     

Protein translocation across the endoplasmic reticulum. I. Detection in the microsomal membrane of a receptor for the signal recognition particle

Thin-section and critical-point-dried fracture-labeled preparations are used to determine the distribution and partition of glycophorin- associated wheat germ agglutinin (WGA) binding sites over protoplasmic and exoplasmic faces of freeze-fractured human erythrocyte membranes. Most wheat germ agglutinin binding sites are found over exoplasmic faces. Label is sparse over the protoplasmic faces. These results contrast with previous observations of the partition of band 3 component where biochemical analysis and fracture-label of concanavalin A (Con A) binding sites show preferential partition of this transmembrane protein with the protoplasmic face. Presence of characteristic proportions of WGA and Con A binding sites over each fracture face is interpreted to indicate the operation of a stochastic process during freeze-fracture. This process appears modulated by the relative expression of each transmembrane protein at either surface as well as by their association to components of the erythrocyte membrane skeleton.     

Effects of amphotericin B and its methyl ester on plasma membranes of Candida albicans and erythrocytes as examined by freeze-fracture electron microscopy

Freeze-fracture electron microscopy of the plasma membranes of Candida albicans yeast cells and red blood cells treated with amphotericin methyl ester and amphotericin B showed that amphotericin B (50 micrograms ml-1) caused extreme aggregation of intramembranous particles on the protoplasmic fracture face of the C. albicans membrane, and a marked reduction of the density of intramembranous particles. On the other hand, the rearrangement of intramembranous particles induced by amphotericin methyl ester (50 micrograms ml-1) produced elevations of the particle-free membrane domains toward the outside of the cells, so that the particles were aggregated in linear furrows surrounding these elevations on the protoplasmic fracture face, and the corresponding ridges on the exoplasmic fracture face. The density of intramembranous particles was greatly reduced on the protoplasmic fracture face. Both polyenes produced only small changes in the erythrocyte membranes at the same concentration. These results suggest that amphotericin methyl ester affects the ergosterol-containing membranes more than amphotericin B, and that ergosterol has a higher sensitivity for these two polyene antibiotics than cholesterol.     

Freeze fracture studies of reaction centers from Rhodospirillum rubrum in chromatophores and liposomes

In freeze-fractures of chromatophores of Rhodospirillum rubrum the reaction centers are seen as hexagonal arranged particles of 13 nm diameter with a density of around 5,500 particles per m². Similar regions on the cytoplasmic membrane suggest that these parts are the prospective invagination sites.Isolated reaction centers are easily incorporated into liposomes. In freeze fractures of liposomes particles similar in shape and size, although less dense as in chromatophores are observed. In negative staining much smaller units of only 5 nm in diameter are found indicating that reaction centers occur in the membrane as tri- or tetramers. There is a strong correlation between particle density in chromatophores and titratable reaction centers remaining in these membranes after extraction of reaction centers by detergents; both values are in good agreement with the yield of reaction centers at a given detergent concentration.Abbreviations LDAO Lauryldimethylamine oxide - PF protoplasmic fracture face - EF exoplasmic fracture face