The Supramolecular Organization of Photosynthetic Membranes of the Thallus Stage of Porphyra leucosticta Thuret. (Bangiales,Rhodophyta) Visualized by Freeze-Fracture

The supramolecular organization of the thylakoid membranes of the thallus stage in the red alga Porphyra leucosticta is studied in replicas of rapidly frozen and fractured cells. Freeze-fractured thylakoid membranes exhibit only two types of fracture faces (EF and PF), because the lamellae in red algal chloroplasts are not stacked. The PF reveals numerous, tightly packed, but randomly distributed particles (density range from 2970 to 3550 particles/μm²). In contrast, the EF particles appear organized into parallel rows, the spacing of which is about 60–70 nm (about 8–9 particles occur along 100 nm of the line that is formed). Significant numbers of single EF particles are randomly distributed between the EF particle rows. The particles on both fracture faces (PF and EF) fall into two size classes: 10 to 11 nm (major size class) and 14 to 15 nm (minor size class).     

The eyespot of the flagellateTetraselmis cordiformis stein (Chlorophyceae): Structural spezialization of the outer chloroplast membrane and its possible significance in phototaxis of green algae

Summary The eyespot region of the flagellateTetraselmis cordiformis Stein (Chlorophyceae) was investigated with the freeze-fracture technique. The only fracture faces observed in this region were the two complementary fracture faces (PF and EF) of the outer chloroplast envelope membrane. Intramembranous particle numbers on both fracture faces of this membrane were significantly higher in the eyespot region as compared to regions outside the eye-spot. Higher numbers of particles on the PF face in the eyespot region were mainly caused by an increase in particle numbers of the size class 6–8 mm, while on the EF face particle size distribution was not significantly different between eyespot and other regions. Functional implications are discussed and evidence is presented that the outer chloroplast envelope membrane may be the site of photoreceptor location in green algal phototaxis.     

The Supramolecular Organization of Red Algal Vacuole Membrane Visualized by Freeze-fracture

The supramolecular organization of the vacuole membrane (orof the membranes of mucilage sacs) in 27 species of red algaeis studied in replicas of rapidly frozen and fractured cells.Intramembranous particle complexes composed of four particles('tetrads' with average diameters between 8·5 and 14·5have been observed in the protoplasmic fracture (PF) face butmost clearly and more frequently in the exoplasmic fracture(EF) face of the vacuole membrane of all red algae investigated.The tetrads lie individually within the vacuole membrane orform clusters in several species and are randomly distributed.In the species Ceramium diaphanum var. strictum and Laurenciaobtusa the intramembranous particle complexes ('tetrads') havebeen observed both in the EF and PF faces of the vacuole membrane;the 'membrane tetrads' at least as regards these two speciesseem to span both the outer and inner leaflets of the vacuolemembrane ('transmembrane particles'). The occurrence of particletetrads in the plasma membrane is probably due to exocytosiseither of the Golgi vesicles or of the mucilage sacs. Tetradfrequency in the EF face of the vacuole membranes of the investigatedred algae varies between 2 and 87 µm^-2, while that ofsingle particles varies between 102 and 695 µm^-2. ThePF face of the vacuole membrane is characterized by a higherparticle density than the EF face. The particle densities ofthe PF and EF faces of the plasma membrane for a given speciesare higher than those of the corresponding fracture faces ofthe vacuole membrane. Some members of Bangiophycidae bear smallerprotein particles (diameter between 8·5 and 10·5nm) in comparison with those of Florideophycidae (diameter between10·5 and 14·5 nm). It is suggested, based uponthe particle tetrads lying in depressions of the vacuole membraneand the origin of vacuoles (mucilage sacs) from ER, that theparticle tetrads originate from the ER or the Golgi complex.Since vacuoles (mucilage sacs) in red algae, along with theGolgi complex, are involved in the synthesis and export of cellsurface polysaccharides, it could be assumed that the 'membrane-tetrads'within the vacuole membrane represent a membrane-bound multienzymecomplex, participating in the synthesis of amorphous extracellularmatrix polysaccharides.Copyright 1993, 1999 Academic Press Red algae, freeze-fracture, vacuole membrane, mucilage sacs, membrane tetrads, supramolecular organization     

Evidence for a vertical displacement of intramembranous particles on the plasma membrane of Tetrahymena cells by a local anesthetic

We examined the effect of a local anesthetic, dibucaine, on the plasma membrane of Tetrahymena pyriformis strain NT-1 using freeze-fracture electron microscopy. Intramembranous particles (IMPs) were distributed homogeneously on the plasma membrane of untreated cells. But, when Tetrahymena cells had been treated with 1.3 mM dibucaine for 5 min at growth temperature, freeze-fracture micrographs of the plasma membrane showed marked alterations. Although IMPs showed an almost homogeneous distribution, their density was elevated markedly on the protoplasmic fracture (PF) face but greatly reduce on the exoplasmic fracture (EF) face. Areas around deciliated portions had a reverse IMP density distribution for the PF and EF faces. These results suggest that dibucaine induced vertical displacement of the IMPs in the plasma membrane.     

FREEZE-FRACTURED CHLOROPLAST MEMBRANES OF GONYAULAX POLYEDRA (PYRROPHYTA)1

The chloroplast membranes of Gonyaulax polyedra Stein were studied in replicas of rapidly frozen and fractured cells. The thylakoid EF_s face lacked the large 15–16 nm particles characteristic of plants with the light-harvesting chlorophyll a/b protein, presumably because the principal light-harvesting protein of Gonyaulax is the small water-soluble peridinin-chlorophyll-protein and the chlorophyll a/b protein is absent. As in other plants, the EF_s thylakoid fracture face carried more particles (4 ×) than EF_uface. The PF faces of the thylakoid showed twice as many particles as did the EF_s faces. No circadian differences in the number or size of thylakoid membrane particles could be detected. Three membranes comprise the chloroplast envelope in Gonyaulax. They could be clearly differentiated in freeze-fractured cells. The middle envelope membrane carried many fewer particles on both the EF and PF faces than did the other two envelope membranes. The PF faces of both the outer and inner envelope membranes showed more particles than the EF faces, as do many other membranes which have been examined.     

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     

Ultrastructure of thylakoid membranes in C. reinhardtii: Evidence for variations in the partition coefficient of the light-harvesting complex-containing particles upon membrane fracture

The ultrastructure of the thylakoid membranes of Chlamydomonas reinhardtii was investigated using cell cultures grown under light intensities of 200 and 4000 lx, respectively. A significant difference in the size distribution of the exoplasmic fracture face (EF) particles appears upon Mg²⁺ treatment of broken cell preparations from the two light growth conditions. Particles larger than 150 Å are seen at 4000 lx only. However neither the absorption spectra of chlorophyll at 77 °K, nor the chlorophyll a/chlorophyll b ratios differ in the two cell batches. In addition, the polypeptide composition of the thylakoid membranes and the Mg²⁺ effect (spillover) on the photochemical rate of Photosystem II are the same in both conditions. We conclude that the partition coefficient between the two fracture faces of light-harvesting complex-containing particles is variable. It depends on Mg²⁺ ion concentration in the incubating medium of the membranes and on the light growth conditions of the cell cultures. Our results suggest that 60- to 80-Å protoplasmic fracture face (PF) particles containing the light-harvesting complexes can aggregate either in larger PF particles (100–120 Å) or in EF particles larger than 120 Å which also contain the Photosystem II centers. That some light-harvesting complexes are located on the PF faces is confirmed by the analysis of the BF4 mutant of C. reinhardtii lacking in chlorophyll-protein complex II. The PF faces of the BF4 thylakoids display a reduced number of particles as compared to that in the wild type.     

Effect of hyperthermia on the plasma membrane structure of Chinese hamster V79 fibroblasts: a quantitative freeze-fracture study

Hyperthermia in the range 41-45 degrees C can induce wide biochemical, physiological, and morphological changes in mammalian cells both in vivo and in vitro. In general, its effects on membranes, particularly on the plasma membrane, are still poorly understood. To investigate the effects of heat on this cell structure, Chinese hamster V79 fibroblasts were exposed to 43 degrees C hyperthermia for 1 h, immediately fixed with glutaraldehyde after treatment, and freeze-fractured for electron microscopic examination. Particular attention was given to the density and size of intramembranous particles (IMPs) on both protoplasmic (PF) and external (EF) fracture faces of the plasma membrane. The quantitative study performed by an interactive image analyzer on the IMPs, generally reported as plasma membrane proteins, showed in heat-treated cells a statistically significant increase in their density and size on both fracture faces. The differences observed demonstrate that in our experimental conditions, hyperthermia in plasma membranes produces structural changes whose biological significance has to be clarified. Moreover, our findings seem to support recent data indicating an involvement of membrane proteins in the cell response to hyperthermia.     

Intercellular junctions of antennal gland epithelial cells in the crayfish,Orconectes virilis

Summary Labyrinth and nephridial canal cells of the crayfish (Orconectes virilis) antennal gland possess two types of intercellular junctions revealed by freeze-fracture studies. Apical margins of the cells are connected by long septate junctions. In replicas, these junctions consist of many parallel rows of 80–140 Å intramembrane particles situated on the PF membrane face (EF and PF fracture faces of Branton et al., 1975). Rows of pits are found on the EF fracture face and are deemed complementary to the rows of particles. Moreover, lateral margins of basal regions of the epithelial cells are attached by many intercellular junctions. These contacts are characterized in thin plastic sections by a narrow dense cytoplasmic plaque located subjacent to the plasma membrane at sites of adjoined cells, and 5 to 12 fine strands of dense material that extend across the intercellular gap between adjoined cells. In freeze-fracture replicas, EF intramembrane faces basal to the region of the plasma membrane containing septate junctions exhibit numerous discoid clusters of particles. The particle aggregates, assumed to represent freeze-cleave images of adhering junctions, range from 900 to 3,700 Å in diameter, with individual particles about 185 Å in diameter. These junctions appear to connect epithelial cell processes formed by basal infoldings of the plasma-lemma, and occur between adjacent cells as well as adjacent processes of a single cell. The discrete aggregates of particles resemble replicated desmosomes (Shienvold and Kelly, 1974) and hemi-desmosomes (Shivers, 1976); therefore, they probably do not constitute a basis for electrical coupling between antennal gland epithelial cells.Supported by the National Research Council of Canada     

Freeze-fracture analysis of phloem structure in plant tissue cultures. II. The sieve element plasma membrane

Sieve element plasma membranes reveal a unique distribution of intramembrane particles (IMPs) in tissue cultures fixed and cyroprotected prior to freeze-fracturing. Sieve element IMPs are smaller than those found in the plasma membranes of callus parenchyma cells from these same cultures. The PF/EF ratio of plasma membrane IMPs is 9.6 for parenchyma cells and 1.21 for sieve elements. The increased binding of IMPs to the sieve element E face may be related to the role of membrane proteins in the loading of sucrose and other molecules by these cells. The enlargement of the cell wall at the site of sieve area pores creates complementary ridges and depressions in the E and P fracture faces of sieve element plasma membranes. No alteration of IMP density is seen at the sieve area pore site.     

THE INTRAMEMBRANOUS PARTICLE PROFILE OF THE PARAMYLON MEMBRANE DURING PARAMYLON SYNTHESIS IN EUGLENA (EUGLENOPHYCEAE)1

Paramylon is the β-1, 3-glucan storage product in euglenoid algae. It is a fibrous crystal that occurs as membrane-bound granules in the cytosol. The role of the surrounding membrane in paramylon synthesis was investigated by the use of freeze-etch electron microscopy. When Euglena gracilis Klebs strain Z (Pringsheim) cells were frozen in supercooled liquid nitrogen, the fracture plane primarily was throuh the paramylon membrane. A large intramembranous particle (IMP, mean diam range 5.6-6.5 nm) and a small IMP (mean diam range 9.6-10.3 nm) were predominant in both PF (protoplasmic fracture) and EF (exoplasmic fracture) faces of the paramylon membrane. During paramylon synthesis induction, the ratio of small to large IMPs increased in both fracture faces. The IMP density decreased in both fracture faces concomitant to paramylon synthesis increase. These changes in IMP profile and density suggest that the paramylon membrane is involved in the synthesis of paramylon.     

Ultrastructure of cell wall and thylakoid membranes of the thermophilic cyanobacterium Synechococcus lividus under the influence of temperature shifts

The ultrastructure of the cell wall and the thylakoid membranes of the thermophilic cyanobacterium Synechococcus lividus was studied by freezefracture electron microscopy after temperature shifts. Different fracture faces of the outer, the cytoplasmic and the thylakoid membranes were demonstrated when the preparation-temperature was in the range of the optimal growth temperature at 52°C or after fixation at 52°C. In the outer membrane of the cell wall two fracture faces with holes and 7.5 nm intramembrane particles were detected. On both the outer (EF) and inner (PF) leaflet of the cytoplasmic membrane randomly distributed particles were demonstrated. The particle density on the PF-face was approx. three times that of the EF-face. The EF-face of the thylakoid membrane exposed rows of particles with an average diameter of 10 nm. The spacing between the particle rows was 35–50 nm. This regular particle arrangement on the EF-face was demonstrated only in a few cases. Mostly the intramembrane particles were distributed randomly on the thylakoid fracture faces. The particle density of thylakoids with a random distribution was approx. in the same range both on the EF-and PF-face. The EF-particles fall into four groups of 9,10,11, and 12.5 nm. The main particle class was the 10 nm class. The PF-face exposed smaller particles with two maxima at 8.5–9 nm and 10 nm. When Synechococcus lividus OH-53s was chilled to temperatures below 30–35°C before the freeze-etch preparation a phase transition took place after the temperature shift. On the fracture faces of the thylakoid and cytoplasmic membranes particle depleted areas occurred. The size of the areas were different in both membranes and dependent on the velocity of cooling. Contrary to Synechococcus lividus OH-53s in the mesophilic Synechococcus strain 6910 the phase transition point was 15°C. The lower phase transition point may be due to a higher content of unsaturated fatty acids.Dedicated to Prof. D. Peters (Hamburg) on the occasion of the 65th anniversary of his birthday     

Organization of the photosystem II centers and their associated antennae in the thylakoid membranes: a comparative ultrastructural,biochemical, and biophysical study of Chlamydomonas wild type and mutants lacking in photosystem II reaction centers

We investigated the ultrastructure of thylakoid membranes that lacked either some or all of their Photosystem II centers in the F34SU3 and F34 mutants of Chlamydomonas reinhardtii. We obtained the following results: (a) There are no particles of the 160-A size class on the EF faces of the thylakoids in the absence of Photosystem II centers (as in F34); the F34SU3 contains 50% of the wild-type number of PSII centers and EF particles. (b) The density of the particles on the PF faces of the thylakoids is higher in the mutants than in the wild type. (c) The fluorescence analysis shows that the organization of the pigments is the same regardless of whether 50% of the PSII centers are temporarily inactivated (by preilluminating the wild type) or are actually missing from the thylakoid membrane (F34SU3). Our results, therefore, support a model in which: (a) each 160-A EF particle has only one PSII center surrounded by light-harvesting complexes and (b) part of the PSH antenna is associated with 80-A PF particles in both of the mutants and the wild type.     

An in situ Freeze-Fracture Study of Spiroplasma citri and the Corn Stunt Spiroplasma

Spiroplasma citri and the corn stunt spiroplasma in sieve cells of Catharanthus roseus were investigated using freeze -fracture electron microscopy. Only the particle studded fracture faces of the plasmalemma could be exposed and not the surfaces of both the extraplasmatic and the plasmatic leaflet. The extraplasmatic fracture face (EF) shows a lower particle density than the plasmatic fracture face (PF). On the PF particle free areas could be observed, which are helically arranged in helical filaments. We suppose that the cytoplasmic fibrils, probably involved in motility processes and in maintaining the helical shape, underly the particle free area only.     

Ultrastructure and freeze-fracture studies of the thylakoids ofMantoniella squamata (Prasinophyceae)

Summary The ultrastructure and the supramolecular organization of the thylakoids of the small green flagellate,Mantoniella squamata, were examined in thin sections and freeze-fracture preparations. The whole chloroplast is tightly packed with thylakoids, which show a pattern of meandering, branching and/or anastomosing membranes. In freeze-fracture preparations only two fracture-faces can be distinguished: the PF- and the EF-face. The PF-face has a much higher particle density than the EF-face (PF: 4086 particles/m²; EF: 865 particles/m²). The EF-face is not as uniform as the PF-face. The areas which are packed with particles probably correspond to closely appressed thylakoid regions or adhesive patches, noticed in thin sections in some areas. The mean particle size on both faces is also different (EF: 10.5 nm; PF: 8.6 nm), but no information about the classification of the particles to special protein complexes is available at this time.Abbreviations chl chlorophyll - EF exoplasmic fracture face - ER endoplasmic reticulum - LHC light-harvesting chlorophyll-protein complex - PF protoplasmic fracture face - PS I photosystem I - PS II photosystem II     

Electron microscopic research on the mitochondria-rich bladder cells of the frog

The ultrastructural peculiarities of mitochondria-rich cells of the frog urinary bladder are analysed using three electron microscopic methods: ultrathin sections, scanning electron microscopy, freeze fracture. The mitochondria and tubular and vesicular structures are most abundant in the apical region of cytoplasm. The P-face (PF) of the apical plasma membrane is characterized by the presence of rod-shaped intramembrane particles (IMP), whereas the E-face (EF) possesses complementary pits. Depending on the distribution density of the rod-shaped IMP, three types of cells are described. The apical plasma membrane has an invert distribution of the globular IMP: a great quantity of IMP on the EF and a few particles on the PF. This structure of the apical plasma membrane is supposed to correlate with its very low water permeability. Using filipin as a marker of cholesterol localization, it has been shown that the mitochondria-rich cell apical membrane contains more cholesterol than that of the granular cells. The nature of the rod-shaped IMP and their role in the transmembrane ion transport have been discussed.     

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.     

Changes in plasmalemma organization in cowpea radicle during imbibition in water and NaCl solutions

Abstract Freeze-fracture electron microscopy of the plasmalemma of dry cowpea radicle cells disclosed a normal-appearing membrane with a high area density of intramembraneous particles (IMPs). Seeds imbibed in either water or salt solution exhibited decreased area density of IMPs, but water-imbibed tissue showed the greatest decline. Mean particle size increased with hydration but not enough to suggest aggregation as the cause for this density decrease. Calculations of plasmalemma area expansion during imbibition show that such expansion can account for the decrease in IMPs per unit area in the cytoplasmic side (PF) of the membrane in water-imbibed, but not in salt-imbibed, tissue. During imbibition, there is a change in the ratio of IMPs per unit area of the PF versus EF (external) membrane faces, suggesting a relative increase in the number of EF particles. These changes in membrane structure are probably not related to any decrease in membrane permeability during the early phases of imbibition.     

The plasma membrane of the Funaria caulonema tip cell: morphology and distribution of particle rosettes,and the kinetics of cellulose synthesis

Freeze-fracturing of Funaria hygrometrica caulonema cells leads to a cleavage within the plasma membrane. The extraplasmatic and the plasmatic fracture faces differ in their particle density. The plasmatic fracture face in caulonema tip cells or in tip cells of side branches, but never in other caulonema cells, is further characterized by the occurrence of particle rosettes. The highest density of rosettes is found at the cell apex but decreases steeply toward the cell base. The shape of the rosettes varies remarkably; 20% of them are found in an incomplete, presumably disintegrating or aggregating state. The complete rosette has a diameter of about 25 nm and consists of five to six particles. The size of the single particles varies between 4 nm to 10 nm. The rosettes are thought to posses cellulose-synthase activity. It is assumed that one rosette produces one elementary fibril; rough calculations, considering the number of rosettes and the estimated amount of cellulose produced in the tip region, indicate that an elementary fibrillar length of 900 nm is formed in 1 min by one rosette. The consequence of the kinetics on the life-time of the rosettes and the cellulose-synthase activity are discussed.Abbreviations EF extraplasmatic fracture face - PF plasmatic fracture face     

A freeze-fracture study on the differentiation of Golgi and plasma membranes in plant cells

Dictyosomes, Golgi vesicles, and plasma membranes were investigated after freeze-fracture in cells from growing root tips of cress (Lepidium sativum L.), that are distinguishable by different cellulose content of the cell wall, into (i) meristematic cells during early formation of the cell plate, (ii) statocytes of the root cap, and (iii) cortex cells of the differentiation zone. The results of this study show that the number of intramembrane particles (imps) is high in dictyosome cisternae, but low in membranes of budding or dictyosome-derived vesicles. Imps are disperse in the vesicle membranes of meristematic cells (i), but are often grouped into clusters in vesicle membranes of statocytes (ii), and of cortex cells (iii). For the number of particle aggregates in vesicle membranes, the following relation holds: (i) < (ii) < (iii). The number of particles on both fracture faces (PF and EF) of the plasma membrane differs widely between the cell types investigated. There are approximately 250, 1400, and 3100 imps microns-2 on the PF and 50, 500, and 300 on the EF of (i), (ii), and (iii), respectively. The structural complexity of the plasma membrane as judged by the degree of particle aggregations on the PF and the number of cellulose microfibrils in the cell wall show the same relationship: (i) < (ii) < (iii). Thus, the strong correlation between the distribution of imps in vesicle membranes, the structural complexity of the plasma membrane, and the content of cellulose microfibrils indicate that selection of imps during vesicle formation at dictyosome cisternae is an integral component of biogenesis and structural differentiation of plant plasma membranes.