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     

The assembly of cellulose microfibrils in Valonia macrophysa Kütz

The assembly of cellulose microfibrils was investigated in artificially induced protoplasts of the alga, Valonia macrophysa (Siphonocladales). Primary-wall microfibrills, formed within 72 h of protoplast induction, are randomly oriented. Secondary-wall lamellae, which are produced within 96 h after protoplast induction, have more than three orientations of highly ordered microfibrils. The innermost, recently deposited micofibrils are not parallel with the cortical microtubules, thus indicating a more indirect role of microtubules in the orientation of microfibrils. Fine filamentous structures with a periodicity of 5.0–5.5 nm and the dimensions of actin were observed adjacent to the plasma membrane. Linear cellulose-terminal synthesizing complexes (TCs) consisting of three rows, each with 30–40 particles, were observed not only on the E fracture (EF) but also on P fracture (PF) faces of the plasma membrane. The TC appears to span both faces of the bimolecular leaflet. The average length of the TC is 350 nm, and the number of TCs per unit area during primary-wall synthesis is 1 per m². Neither paired TCs nor granule bands characteristic of Oocystis were observed. Changes in TC structure and distribution during the conversion from primary- to secondary-wall formation have been described. Cellulose microfibril assembly in Valonia is discussed in relation to the process among other eukaryotic systems.Abbreviations TC terminal complex - EF E (outer leaflet) fracture face of the plasma membrane - PF P (inner leaflet) fracture face of the plasma membrane - MT microtubule - PS protoplasmic surface of the membrane     

Structural differentiation of membranes involved in the secretion of polysaccharide slime by root cap cells of cress (Lepidium sativum L.)

The peripheral secretion tissue of the root cap of Lepidium sativum L. was investigated by electronmicroscopy and freeze-fracturing in order to study structural changes of membranes involved in the secretion process of polysaccharide slime. Exocytosis of slime-transporting vesicles occurs chiefly in the distal region of the anticlinal cell walls. The protoplasmic fracture face (PF) of the plasmalemma of this region is characterized by a high number of homogenously distributed intramembranous particles (IMPs) interrupted by areas nearly free of IMPs. Near such areas slime-transporting vesicles are found to be underlying the plasma membrane. It can be concluded that areas poor in particles are prospective sites for membrane fusion. During the formation of slime-transporting vesicles, the number of IMPs undergoes a striking change in the PF of dictyosome membranes and their derivatives. It is high in dictyosome cisternae and remarkably lower in the budding region at the periphery of the cisternae. Slime-transporting vesicles are as poor in IMPs as the areas of the plasmalemma. Microvesicles rich in IMPs are observed in the surroundings of dictyosomes. The results indicate that in the plasmalemma and in membranes of the Golgi apparatus special classes of proteins — recognizable as IMPs — are displaced laterally into adjacent membrane regions. Since the exoplasmic fracture face (EF) of these membranes is principally poor in particles, it can be concluded that membrane fusion occurs in areas characterized by a high quantity of lipid molecules. It is obvious that the Golgi apparatus regulates the molecular composition of the plasma membrane by selection of specific membrane components. The drastic membrane transformation during the formation of slime-transporting vesicles in the Golgi apparatus causes the enrichment of dictyosome membranes by IMPs, whereas the plasma membrane probably is enriched by lipids. The structural differentiations in both the plasma membrane and in Golgi membranes are discussed in relation to membrane transformation, membrane flow, membrane fusion, and recycling of membrane constituents.Abbreviations PF protoplasmic fracture face - EF exoplasmic fracture face - IMP intramembranous particle     

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.     

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-fracture studies on isolated sperm cells ofSpinacia oleracea L.

Summary With freeze-fracturing sperm cells appear to be fractured preferentially through the plasma membranes. Only few fracture planes through the cytoplasm are found. Both the PF as well as the EF side of the sperm cell plasma membranes show a slightly undulating surface and contain intramembrane particles. The particle distribution is irregular and does not show any clustering. The EF side of the plasmamembrane contains approximately 3 times more particles per m² than the PF side.Abbreviations EF extraplasmatic fracture face - IMP intramembrane particles - FDA fluorescein diacetate - PF plasmatic fracture face     

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.     

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     

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.     

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     

The surface membrane of Leishmania mexicana mexicana: comparison of amastigote and promastigote using freeze-fracture cytochemistry

The freeze fracture replica technique has been used to compare the plasma membranes of amastigote and promastigote stages of Leishmania mexicana mexicana with respect to intramembranous particle (integral protein) distribution and to beta-hydroxysterols content as revealed by the distribution of lesions induced by the polyene antibiotic filipin. Intramembranous particle (IMP) density was greater in promastigote than in amastigote plasma membranes. Intramembranous particles were more abundant in the protoplasmic face (PF) than in the exoplasmic face (EF) of promastigotes, but this situation was found to be reversed in amastigotes. Filipin-induced lesions in glutaraldehyde-fixed parasites indicated higher levels of beta-hydroxysterols in the amastigote than in the promastigote plasma membrane, and in the promastigote flagellar membrane than in the body membrane. Amphotericin B (a related polyene antibiotic used in chemotherapy of leishmaniasis) induced IMP aggregation in the PF of unfixed amastigotes but did not appear to influence sterol distribution as demonstrated by freeze-fracture of subsequently-fixed and filipin-treated organisms.     

Density changes of Phycomyces spores after reversible and irreversible activation

Spores harvested by conventional methods could be cleaned up considerably using discontinuous density gradients. This method yielded a population of almost entirely viable spores. Germination of the spores resulted in density changes probably due to spore swelling. These changes could be observed as early as 15 min after activation. The usefulness of this method in the study of spore activation was shown by its application to spores germinating after a heat treatment at various temperatures, or to spore reacquiring dormancy by incubation in water after activation at different temperatures. Dormant and activated spores can quickly and easily be separated from mixed populations.     

Exocytosis in non-plasmolyzed and plasmolyzed tobacco pollen tubes

Exocytosis occurring during deposition of secondary wall material was studied by freeze-fracturing ultrarapidly frozen non-plasmolyzed and plasmolyzed tobacco pollen tubes. The secondary wall of tobacco pollen tubes shows a random orientation of microfibrils. This was observed directly on fractures through the tube wall and indirectly as imprints of microfibrils on fracture faces of the plasma membrane of non-plasmolyzed tubes. About half of the plasmatic fracture faces from non-plasmolyzed and plasmolyzed pollen tubes carried hexagonal arrays of intramembraneous particles in between randomly distributed particles. Deposition of secondary wall material was often accompanied by an undulated plasma membrane and the presence of membrane-bound vesicles in invaginations of the plasma membrane, between the plasma membrane and secondary wall and-especially in plasmolyzed tubes-within the secondary wall of tube flanks and wall cap. The findings are discussed in connection with published schemes of membrane behaviour during exocytosis.Abbreviations EF extraplasmatic fracture face - IMP(s) intramembraneous particle(s) - PF plasmatic fracture face Extended version of a contribution (poster) presented at the 8th Int. Symp. on Sexual Reproduction in Seed Plants, Ferns and Mosses, Wageningen, The Netherlands, August 1984 Dedicated to Prof. Dr. H.F. Linskens (Nijmegen) on the occasion of his 65th birthday in 1986     

A freeze-etch study of the effects of extracellular freezing on cellular membranes of wheat

Seedlings of Triticum aestivum L. cv. Lennox were grown in different environments to obtain different hardiness. Pieces of laminae and leaf bases were slowly cooled to sub-zero temperatures and the damage caused was assessed by an ion-leakage method. Comparable pieces of tissue were slowly cooled to temperatures between 2° and-14°C and were then freeze-fixed and freeze-etched. Membranes generally retained their lamellar structures indicated by the abundance of typical membrane fracture faces in all treatments, and some membrane fracture faces had patches which lacked the usual scattering of intramembranous particles (IMP). These IMP-free areas were present in the plasma membrane of tissues given a damaging freezing treatment, but were absent from the plasma membrane of room-temperature controls, of supercooled tissues, and of tissues given a non-damaging freezing treatment. The frequency of IMP-free areas and the proportion of the plasma membrane affected increased with increasing damage. In the most damaged tissue (79% damage; leaf bases exposed to-8°C), 20% of the plasma membrane was IMP-free. The frequencies of IMP at a distance from the IMP-free areas were unaffected by freezing treatments. There was a patchy distribution of IMP in other membranes (nuclear envelope, tonoplast, thylakoids, chloroplast envelope), but only in the nuclear envelope did it appear possible that their occurrence coincided with damage. The IMP-free areas of several membranes were sometimes associated together in stacks. Such membranes lay both to the outside and inside of the plasma membrane, indicating that at least some of the adjacent membrane fragments arose as a result of membrane reorganization induced by the damaging treatment. Occasional views of folded IMP-free plasma membrane tended to confirm this conclusion. The following hypothesis is advanced to explain the damage induced by extracellular freezing. Areas of plasma membrane become free of IMP, probably as a result of the freezing-induced cellular dehydration. The lipids in these IMP-free patches may be in the fluid rather than the gel phase. The formation of these IMP-free patches, especially in the plasma membrane, initiates or involves proliferation and possibly fusion of membranes, and during or following this process, the cells become leaky.Abbreviations EF exoplasmatic fracture face - IMP intramembranous particles - PF protoplasmatic 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.     

Freeze fracture studies on the annelid septate junction

Summary Freeze-fractured preparations of septate junctions between epidermal cells of annelids (Lumbricus terrestris and Tubifex spec.) have been investigated. In Lumbricus the protoplasmic face (PF) of the plasma membrane is characterized by variously arranged rows of particles. Apically the rows take an undulating course and often are separated by wide distances. In the basal part of the junction the rows run closely together and more or less in parallel. The diameter of the particles measures 80–120 Å, the distance between two particles (centre to centre) is 150–250 Å. Additionally striking rows of large particles (long diameter 150–200 Å). Are to be observed mainly near the basal part of the junction. In Tubifex both faces of the plasma membrane could be studied in detail. The protoplasmic face (PF) contains rows of distinct individual particles (mean diameter 100–150 Å, centre to centre distance approx. 250 Å) whereas the particles of the extracellular face (EF, mean diameter 200–250 Å) usually form continuous strands in which the individual particles seem to fuse. The density of arrangement of the strands varies considerably. Additionally ladder-shaped membrane structures have been observed in plasma membranes of this species.     

Vertebrate-like tight junctions in the insect eye

Unequivocal vertebrate-like anastomosing tight junctions have been observed for the first time in insect tissues. In freeze-fractured replicas of dipteran compound eyes, the intercellular junctions between certain glial cells in regions distal to the optic neuropile display an extensive network of continuous intramembranous P face (PF) ridges. The intramembranous E face (EF) possesses a reticulum of grooves which occur in the depths of troughs and thereby produce a ‘quilted’ appearance. At PF/EF membrane face transitions, there is an obliteration of the intercellular space at points of membrane fusion; here the PF ridges and EF grooves appear in register and are therefore complementary. Although the septate junctions found here are patent, these tight junctions are occluding to lanthanum and appear to represent the blood-retinal barrier previously demonstrated electrophysiologically in insects. The existence and vertebrate-like structural complexity of these junctions in arthropods supports the concept of the universality of the membrane specializations that mediate cell-to-cell interactions.     

Treatment with oxidizing agents damages the inner membrane of spores of Bacillus subtilis and sensitizes spores to subsequent stress

AIMS: To determine if treatment of Bacillus subtilis spores with a variety of oxidizing agents causes damage to the spore's inner membrane. METHODS AND RESULTS: Spores of B. subtilis were killed 80-99% with wet heat or a variety of oxidizing agents, including betadine, chlorine dioxide, cumene hydroperoxide, hydrogen peroxide, Oxone, ozone, sodium hypochlorite and t-butylhydroperoxide, and the agents neutralized and/or removed. Survivors of spores pretreated with oxidizing agents exhibited increased sensitivity to killing by a normally minimal lethal heat treatment, while spores pretreated with wet heat did not. In addition, spores treated with wet heat or the oxidizing agents, except sodium hypochlorite, were more sensitive to high NaCl in plating media than were untreated spores. The core region of spores treated with at least two oxidizing agents was also penetrated much more readily by methylamine than was the core of untreated spores, and spores treated with oxidizing agents but not wet heat germinated faster with dodecylamine than did untreated spores. Spores of strains with very different levels of unsaturated fatty acids in their inner membrane exhibited essentially identical resistance to oxidizing agents. CONCLUSIONS: Treatment of spores with oxidizing agents has been suggested to cause damage to the spore's inner membrane, a membrane whose integrity is essential for spore viability. The sensitization of spores to killing by heat and to high salt after pretreatment with oxidizing agents is consistent with and supports this suggestion. Presumably mild pretreatment with oxidizing agents causes some damage to the spore's inner membrane. While this damage may not be lethal under normal conditions, the damaged inner membrane may be less able to maintain its integrity, when dormant spores are exposed to high temperature or when germinated spores are faced with osmotic stress. Triggering of spore germination by dodecylamine likely involves action by this agent on the spore's inner membrane allowing release of the spore core's depot of dipicolinic acid. Presumably dodecylamine more readily alters the permeability of a damaged inner membrane and thus more readily triggers germination of spores pretreated with oxidizing agents. Damage to the inner spore membrane by oxidizing agents is also consistent with the more rapid penetration of methylamine into the core of treated spores, as the inner membrane is likely the crucial permeability barrier to methylamine entry into the spore core. As spores of strains with very different levels of unsaturated fatty acids in their inner membrane exhibited essentially identical resistance to oxidizing agents, it is not through oxidation of unsaturated fatty acids that oxidizing agents kill and/or damage spores. Perhaps these agents work by causing oxidative damage to key proteins in the spore's inner membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: The more rapid heat killing and germination with dodecylamine, the greater permeability of the spore core and the osmotic stress sensitivity in outgrowth of spores pretreated with oxidizing agents is consistent with such agents causing damage to the spore's inner membrane, even if this damage is not lethal under normal conditions. It may be possible to take advantage of this phenomenon to devise improved, less costly regimens for spore inactivation.     

Plasma-membrane rosettes involved in localized wall thickening during xylem vessel formation of Lepidium sativum L.

Developing xylem vessel elements in roots of cress, Lepidium sativum L., were freeze-fractured after rapid freezing in nitrogen slush (without cryoprotection). With the double-replica technique, both the plasmatic fracture face (PF) and the extraplasmatic fracture face (EF) of the plasma membrane were exposed. The EF revealed abundant, but rather indistinct terminal globules; whereas the PF showed numerous rosettes. Terminal globules and rosettes were localized, restricted to regions of secondary wall thickening only, and showed comparale frequencies per m², supporting the assumption that they are part of the same synthase complex. The abundance of rosettes in regions of high cellulose production supports their postulated involvement in cellulose microfibril formation. With up to 191 rosettes per m², the rosettes appear to be too densely arranged to be directly aligned on individual microtubules. This favors the channelling hypothesis of synthase movement in the plasma membrane.Abbreviations EF extraplasmatic fracture face - PF plasmatic fracture face