Exoplasmic Fracture Face Research Articles

Efficient Light Harvesting by Photosystem II Requires an Optimized Protein Packing Density in Grana Thylakoids

A recently developed technique for dilution of the naturally high protein packing density in isolated grana membranes was applied to study the dependence of the light harvesting efficiency of photosystem (PS) II on macromolecular crowding. Slight dilution of the protein packing from 80% area fraction to the value found in intact grana thylakoids (70%) leads to an improved functionality of PSII (increased antenna size, enhanced connectivity between reaction centers). Further dilution induces a functional disconnection of light-harvesting complex (LHC) II from PSII. It is concluded that efficient light harvesting by PSII requires an optimal protein packing density in grana membranes that is close to 70%. We hypothesize that the decreased efficiency in overcrowded isolated grana thylakoids is caused by excited state quenching in LHCII, which has previously been correlated with neoxanthin distortion. Resonance Raman spectroscopy confirms this increase in neoxanthin distortion in overcrowded grana as compared with intact thylakoids. Furthermore, analysis of the changes in the antenna size in highly diluted membranes indicates a lipid-induced dissociation of up to two trimeric LHCII from PSII, leaving one trimer connected. This observation supports a hierarchy of LHCII-binding sites on PSII.

Low-Light-Induced Formation of Semicrystalline Photosystem II Arrays in Higher Plant Chloroplasts

Remodeling of photosynthetic machinery induced by growing spinach plants under low light intensities reveals an up-regulation of light-harvesting complexes and down-regulation of photosystem II and cytochrome b6f complexes in intact thylakoids and isolated grana membranes. The antenna size of PSII increased by 40-60% as estimated by fluorescence induction and LHCII/PSII stoichiometry. These low-light-induced changes in the protein composition were accompanied by the formation of ordered particle arrays in the exoplasmic fracture face in grana thylakoids detected by freeze-fracture electron microscopy. Most likely these highly ordered arrays consist of PSII complexes. A statistical analysis of the particles in these structures shows that the distance of neighboring complexes in the same row is 18.0 nm, the separation between two rows is 23.7 nm, and the angle between the particle axis and the row is 26 degrees . On the basis of structural information on the photosystem II supercomplex, a model on the supramolecular arrangement was generated predicting that two neighboring complexes share a trimeric light-harvesting complex. It was suggested that the supramolecular reorganization in ordered arrays in low-light grana thylakoids is a strategy to overcome potential diffusion problems in this crowded membrane. Furthermore, the occurrence of a hexagonal phase of the lipid monogalactosyldiacylglycerol in grana membranes of low-light-adapted plants could trigger the rearrangement by changing the lateral membrane pressure.

Ultrastructure of Acaryochloris marina, an oxyphotobacterium containing mainly chlorophyll d.

We present a detailed investigation of the ultrastructure of the chlorophyll a/d-containing unicellular oxyphotobacterium Acaryochloris marina, combining light and transmission electron microscopy and showing freeze fractures of this organism for the first time. The cells were 1.8-2.1 microm x 1.5-1.7 microm in size. The cell envelope consisted of a peptidoglycan layer of approximately 10 nm thickness combined with an outer membrane. Cell division was intermediate between the constrictive and the septum type. The nucleoplasm, which contained several carboxysomes, was surrounded by 7-11 concentrically arranged thylakoids, which were predominantly stacked, with the exception of distinct areas where phycobiliproteins were located. The thylakoids were perforated by channel-like structures connecting the central and peripheral portions of the cytoplasm and not yet observed in other organisms. In freeze fractures, the protoplasmic fracture faces of thylakoid membranes were densely covered with particles of inhomogenous size. The particle size histogram peaked at 10-11, 13 and 18 nm. The 18-nm particles are assumed to represent photosystem I trimers. The particles on exoplasmic fracture faces, proposed to represent photosystem II complexes, were significantly larger than the corresponding particles of cyanobacteria and clustered to form large aggregates. This kind of arrangement is unique among photosynthetic organisms.

Topographical distribution of phospholipids in boar sperm plasma and intracellular membranes as revealed by freeze-fracture cytochemistry.

We used fracture-label and label-fracture cytochemistry in conjunction with the phospholipase A2-colloidal gold (PLA2-CG) technique to study the distribution of phospholipids in ejaculated boar spermatozoa. These techniques provide visualization of the topographical distribution of phospholipids in freeze-fractured sperm membranes in a three-dimensional view. In various freeze-fractured boar sperm membranes and crossfractured cytoplasmic structures, quantitative analysis revealed that the nuclear envelope membranes and the nuclear content possessed the highest labeling density of PLA2-CG. Moderate labeling was detected over acrosomal membranes, especially the inner acrosomal membrane. Replicas of both protoplasmic and exoplasmic fracture faces of the plasma membrane of boar sperm head showed a relatively low density of PLA2-CG labeling. Moreover, a differential distribution of phospholipids was seen over the protoplasmic face of the plasma membrane domains of the sperm head, which showed the highest concentration of gold particles in the postacrosomal region, followed by the equatorial segment and the anterior acrosome region. The PLA2-CG labeling densities over the post-acrosomal region and the equatorial segment were significantly higher than that over the anterior acrosome region. In the flagellum, an intense labeling was also seen over crossfractured mitochondria, dense fibers, and fibrous sheath. The protoplasmic fracture face of the plasma membrane over the middle piece, the annulus, and the principal piece was moderately labeled by PLA2-CG. No significant difference in mean labeling density of PLA2-CG was detected among the three membrane domains. In label-fracture preparations, exoplasmic halves of the plasma membrane of the head and the middle piece of the tail were uniformly labeled with PLA2-CG. However, the annulus and principal piece were devoid of PLA2-CG binding sites. These results indicate that differential distribution of phospholipids associated with the boar sperm membranes may reflect phospholipid composition of membrane domains characteristic of special physiological functions.

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.

Particle regularity on thylakoid fracture faces is influenced by storage conditions

Specific temperature, storage times, and medium composition enable initiation of regular arrays of intramembranous particles on the exoplasmic fracture face during prolonged storage of isolated chloroplasts at 4 °C, producing about 2 – 10 regular arrays with 2 – 30 particles in each array, with a period of about 36 nm, oriented in 1 – 4 directions. The particle sizes do not change throughout the time of storage (1 – 4 weeks). The second type of particle regularity arises during prolonged storage of chloroplasts in greater than 1 M sucrose at −18 °C. Rounded areas of small particles tightly packed into paracrystalline arrays are found among less densely packed particles. The density of small particles is 4700 particles/μm2, and the mean size is 11 nm, whereas the particle density of the background is 1600 particles/μm2with a mean particle size of 13 nm compared with 1200 particles/μm2and mean size 16 nm in fresh chloroplasts. Based on the reduction of particle sizes and manner of packing on the fracture face, it is proposed that the small particles are a light-harvesting complex, separate from photosystem II and aggregated into paracrystalline arrays. The thylakoid lipids may participate in formation of particle regularity. Key words: thylakoid membrane, freeze fracture, particle regularity, low temperatures.

Ultrastructure of cell wall and photosynthetic apparatus of the phycobilisome-less Synechocystis sp. strain BO 8402 and phycobilisome-containing derivative strain BO 9201

The ultrastructures of two closely related strains of a novel diazotrophic cyanobacterium, Synechocystis sp. BO 8402 and BO 9201, were examined using ultrathin sections and freeze-fracture electron microscopy. Cells of both strains were surrounded by an unusual thick peptidoglycan layer. Substructures in the layer indicated the presence of microplasmodesmata aligned perpendicular to the free cell surface and in the septum of dividing cells. Synechocystis sp. strain BO 8402 contained lobed, electronopaque, highly fluorescent inclusion bodies consisting of phycocyanin-linker complexes. The thylakoids lacked phycobilisomes and accommodated, in addition to randomly distributed exoplasmic freeze-fracture particles, patches of two-dimensionally ordered arrays of dimeric photosystem II particles in the exoplasmic fracture face. Determination of photosystem I and photosystem II suggested an increase of photosystem II in strain BO 8402. Strain BO 9201 performed phycobilisome-supported photosynthesis and showed rows of dimeric photosystem II particles in the exoplasmic fracture face. Corresponding particle-free grooves in the protoplasmic fracture face were lined by a class of large particles tentatively assigned as trimers of photosystem I. The different lateral organization of protein complexes in the thylakoid membranes and the fine structure of the cell wall are discussed with respect to absorption cross-section of photosynthesis and nitrogen fixation.

TIP CELL GROWTH AND THE FREQUENCY AND DISTRIBUTION OF CELLULOSE MICROFIBRIL‐SYNTHESIZING COMPLEXES IN THE PLASMA MEMBRANE OF APICAL SHOOT CELLS OF THE RED ALGA PORPHYRA YEZOENSIS1

ABSTRACTThe supramolecular organization of the plasma membrane of apical cells in shoot filaments of the marine red alga Porphyra yezoensis Ueda (conchocelis stage) was studied in replicas of rapidly frozen and fractured cells. The protoplasmic fracture (PF) face of the plasma membrane exhibited both randomly distributed single particles (with a mean diameter of 9.2 ± 0.2 nm) and distinct linear cellulose microfibril‐synthesizing terminal complexes (TCs) consisting of two or three rows of linearly arranged particles (average diameter of TC particles 9.4 plusmn; 0.3 nm). The density of the single particles of the PF face of the plasma membrane was 3000 μm−2, whereas that of the exoplasmic fracture face was 325 μm−2. TCs were observed only on the PF face. The highest density of TCs was at the apex of the cell (mean density 23.0 plusmn; 7.4 TCs μm−2 within 5 μm from the tip) and decreased rapidly from the apex to the more basal regions of the cell, dropping to near zero at 20 μm. The number of particle subunits of TCs per μm2 of the plasma membrane also decreased from the tip to the basal regions following the same gradient as that of the TC density. The length of TCs increased gradually from the tip (mean length 46.0 plusmn; 1.4 nm in the area at 0–5 μm from the tip) to the cell base (mean length 60.0 plusmn; 7.0 μm in the area at 15–20 μm). In the very tip region (0–4 μm from the apex), randomly distributed TCs but no microfibril imprints were observed, while in the region 4–9 μm from the tip microfibril imprints and TCs, both randomly distributed, occurred. Many TCs involved in the synthesis of cellulose microfibrils were associated with the ends of microfibril imprints. Our results indicate that TCs are involved in the biosynthesis, assembly, and orientation of cellulose microfibrils and that the frequency and distribution of TCs reflect tip growth (polar growth) in the apical shoot cell of Porphyra yezoensis. Polar distribution of linear TCs as “cellulose synthase” complexes within the plasma membrane of a tip cell was recorded for the first time in plants.

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

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

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

The supramolecular organization of the vacuole membrane (or of the membranes of mucilage sacs) in 27 species of red algae is studied in replicas of rapidly frozen and fractured cells. Intramembranous particle complexes composed of four particles ('tetrads' with average diameters between 8·5 and 14·5 have been observed in the protoplasmic fracture (PF) face but most 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 or form clusters in several species and are randomly distributed. In the species Ceramium diaphanum var. strictum and Laurencia obtusa the intramembranous particle complexes ('tetrads') have been observed both in the EF and PF faces of the vacuole membrane; the 'membrane tetrads' at least as regards these two species seem to span both the outer and inner leaflets of the vacuole membrane ('transmembrane particles'). The occurrence of particle tetrads in the plasma membrane is probably due to exocytosis either of the Golgi vesicles or of the mucilage sacs. Tetrad frequency in the EF face of the vacuole membranes of the investigated red algae varies between 2 and 87 μm-2, while that of single particles varies between 102 and 695 μm-2. The PF face of the vacuole membrane is characterized by a higher particle density than the EF face. The particle densities of the PF and EF faces of the plasma membrane for a given species are higher than those of the corresponding fracture faces of the vacuole membrane. Some members of Bangiophycidae bear smaller protein particles (diameter between 8·5 and 10·5 nm) in comparison with those of Florideophycidae (diameter between 10·5 and 14·5 nm). It is suggested, based upon the particle tetrads lying in depressions of the vacuole membrane and the origin of vacuoles (mucilage sacs) from ER, that the particle tetrads originate from the ER or the Golgi complex. Since vacuoles (mucilage sacs) in red algae, along with the Golgi complex, are involved in the synthesis and export of cell surface polysaccharides, it could be assumed that the 'membrane-tetrads' within the vacuole membrane represent a membrane-bound multienzyme complex, participating in the synthesis of amorphous extracellular matrix polysaccharides.

Freeze‐fracturing for low‐temperature scanning electron microscopy of Hartig net in synthesized Picea abies–Hebeloma crustuliniforme and –Tricholoma vaccinum ectomycorrhizas *

SUMMARYRapidly frozen ectomycorrhizal roots were freeze‐fractured under high vacuum in a dedicated cryo‐preparation unit and investigated in the frozen, partially freeze‐dried, or fully hydrated state. Tangential, radial and occasional transverse surface views of Hartig net were obtained after freeze‐fracturing. Protoplasmic and exoplasmic fracture faces of the fungal plasmalemma und occasional fractures between the fungal cell wall and the extracellular matrix were seen. Primary pit‐fields with plasmodesmata occurred in non‐ectomycorrhizal parts of the root as well as in small gaps of Hartig net. Septa with dolipores were observed in the finger‐like complexes of the Hartig net.

Microtubules do not control orientation of secondary cell wall microfibril deposition inMicrasterias

The influence of the microtubule disorganizing substances amiprophos-methyl (APM) and colchicine on secondary wall formation inMicrasterias denticulata was investigated by the freezeetch technique. The results reveal that neither microtubule inhibitor changes the pattern of microfibril deposition. The application of APM or colchicine also does not cause any structural alterations of the microfibrils or of the protoplasmic (Pf) and the exoplasmic (Ef) fracture face of the plasma membrane, thus indicating that microtubules are not involved in secondary wall formation inM. denticulata. However, since areas of the plasma membrane which collapsed upon freeze-etching are restricted to the Pf-face of cells treated with microtubule inhibitors, cortical microtubules may function as mechanical support during secondary wall formation. In the cortical cytoplasm filamentous structures are found in close spatial relationship and an almost parallel alignment to rosettes of the plasma membrane.

A freeze fracture analysis of the surface of embryonic and non-embryogenic suspension cells of Daucus carota

Cell surface ultrastructure and exocytosis was studied during one batch cycle in cells of an embryogenic suspension culture of carrot, by means of freeze fracture electron microscopy of samples ultra-rapidly frozen by plunging in liquid propane. The culture of proliferating cells contains three cell types: (i) pro-embryogenic masses (PEMs) attached to (ii) clusters of isodiametric cells and (ii) elongated single cells, the latter deriving from the clusters, as long as the synthetic auxin 2,4-dichlorophenoxy acetic acid (2,4-D) is present in the culture medium. Differences between the three cell types occur in cell wall texture, number of plasmodesmata and number of exocytosis configurations, but not in the number of plasma membrane rosettes. Wall texture is ordered in embryogenic cells, but unordered in elongated, non-dividing cells. Embryogenic cells adhere more closely together and have a higher number of plasmodesmata. Exocytosis configurations occur as small pores, slit-shaped membrane fusion sites with a maximum length of 220 nm and, most abundantly, depressions (in the protoplasmic fracture face), bulges (in the exoplasmic fracture face) and horseshoe-shaped configurations (in both fracture faces), the latter three are 160 ± 20 nm in diameter. Morphology and size of exocytosis configurations are similar and do not change in time in the three cell types, but density differs in time and among cell types. From 1 to 14 days after subculturing the density of the 160 nm exocytosis configurations drops considerably in elongated cells, slightly but with statistical significance in cluster cells and hardly in PEMs.

Expression and organisation of antenna proteins in the light-and temperature-sensitive barley mutant chlorina-104

The nuclear gene mutant chlorina-(104) of barley (Hordeum vulgare L.) is chlorophyll-deficient when grown under high irradiance, particularly at low temperatures. Chlorina- (104) chloroplasts had fewer thylakoids than the wild type, and fewer appressed lamellae relative to non-appressed lamellae. The freeze-fracture ultrastructure showed a loss of particles from the protoplasmic fracture face of the stacked thylakoid region (PFs), consistent with the loss of most of the light-harvesting complex (LHC) II, and a loss of some of the large particles from the same face of the unstacked thylakoid region (PFu), indicating a loss of photosystem-I particles. The mutant is remarkable for the high density of particles on the exoplasmic fracture face of the unstacked thylakoid region (EFu), levels of which fell to normal after transfer to low light. The chlorophyll deficiency was shown to be primarily caused by the loss of LHCII and LHCI-680, with the consequent loss of much of the chlorophyll (Chl) b and the xanthophylls neoxanthin and lutein. The use of a monoclonal antibody which recognises the 23-kDa polypeptide of LHCI-680, confirmed that it was severely depleted in chloroplasts from chlorina-(104) grown under restrictive conditions. The 77 K fluorescence emission spectrum was characterised by a pronounced shoulder at 720 nm, arising from the photosystem-I reaction centre (CPI). Since fluorescence from CPI is normally quenched by LHCI-730, this indicates that LHCI-680 mediates excitation energy transfer between LHCI-730 and the reaction centre. After moving seedlings to permissive conditions, LHCII and LHCI-680 began to accumulate in the chlorotic leaves and the fluorescence emission spectrum resembled that of wild-type leaves. Measurement of the steady-state mRNA levels with specific Cab probes, showed no difference between wild type and mutant, indicating that control of LHCII and LHCI-680 accumulation was at a post-transcriptional level.

Temperature‐dependent changes in Photosystem II heterogeneity of attached leaves under high light

Attached leaves of pumpkin (Cucurbita pepo L. cv. Jattiläismeloni) were exposed to high light intensity at room temperature (ca 23°C) and at 1°C. Fluorescence parameters and electron transport activities measured from isolated thylakoids indicated faster photoinhibition of PSII at low temperature. Separation of the α and β components of the complementary area above the fluorescence induction curve of dichlorophenyl-dimethylurea-poisoned thylakoids revealed that at low temperature only the α-centers declined during exposure to high light intensity while the content of functional β-centers remained constant. Freeze-fracture electron microscopy showed no decrease in the density of particles on the appressed exoplasmic fracture face, indicating that the photoinhibited α-centers remained in the appressed membranes at 1°C. Because of the function of the repair and protective mechanisms of PSII, strong light induced less photoinhibition at room temperature, but more complicated changes occurred in the α/β-heterogeneity of PSII. During the first 30 min at high light intensity the decrease in α-centers was almost as large as at 1°C, but in contrast to the situation at low temperature the decrease in α-centers was compensated for by a significant increase in PSIIβ-centers. Changes in the density and size of freeze-fracture particles suggest that this increase in β-centers was due to migration of phosphorylated light-harvesting complex from appressed to non-appressed thylakoid membranes while the PSII core remained in the appressed membranes. This situation, however, was only transient and was followed by a rapid decrease in the functionalβ-centers.

Distribution of Chlorophyll-Protein Complexes during Chilling in the Light Compared with Heat-Induced Modifications

The effects of chilling in the light (4 days at 5 degrees C and 100-200 micromoles of photons per square meter per second) on the distribution of chlorophyll (Chl) protein complexes between appressed and nonappressed thylakoid regions of pumpkin (Cucurbita pepo L.) chloroplasts were studied and compared with the changes occurring during in vitro heat treatment (5 minutes at 40 degrees C) of isolated thylakoids. Both treatments induced an increase (18 and 65%, respectively) in the relative amount of the antenna Chl a protein complexes (CP47 + CP43) of photosystem II (PSII) in stroma lamellae vesicles. Freeze-fracture replicas of light-chilled material revealed an increase in the particle density on the exoplasmic fracture face of unstacked membrane regions. These two treatments differed markedly, however, in respect to comigration of the light-harvesting Chl a/b protein complex (LHCII) of PSII. The LHCII/PSII ratio in stroma lamellae vesicles remained fairly constant during chilling in the light, whereas it dropped during the heat treatment. Moreover, it was a minor light-harvesting Chl a/b protein complex of PSII, CP29, that increased most in stroma lamellae vesicles during light-chilling. Changes in the organization of LHCII during chilling were suggested by a shift to particles of smaller sizes on the protoplasmic fracture face of stacked membrane regions and a decrease in the amount of trans-Delta(3)-hexadecenoic acid in the phosphatidyldiacylglycerol fraction.

Morphological Detection of Filipin‐Sterol Complexes in the Cytoplasmic Membrane of Staphylococcal L‐Form

Filipin, a sterol-specific antibiotic, and freeze-fracture electron microscopy were used to study the presence and distribution of sterol in the cytoplasmic membrane of stable staphylococcal L-form cells. Fixed cells were treated with filipin, and then observed by freeze-fracture electron microscopy. Freeze-fractured profiles of the L-form cells treated with filipin demonstrated irregular distribution of protuberances or pits of 25-30 nm, representing filipin-sterol complexes, on the proto-plasmic fracture face (PF) and exoplasmic fracture face (EF) of the cytoplasmic membrane. In contrast, no such structure was detected in the filipin-treated parent cells or protoplasts. The results suggest that some sterol molecules, which are usually not found in staphylococcal or other bacterial cells, emerged on the cytoplasmic membrane after the cells were converted to the stable L-form.

Shape determinants of McLeod acanthocytes.

We have sought to elucidate the spiculated shape of McLeod erythrocytes. Red cells from a normal donor and from a McLeod patient were incubated in phosphate-buffered saline containing 0, 0.05, or 0.1 mM chlorpromazine at 0 degrees C for 5 min, then glutaraldehyde-fixed, and examined by scanning electron microscopy. The normal red cells were biconcave disks in which chlorpromazine induced inward (negative) curvature: deep cupping (stomatocytosis) and multiple invaginations. The McLeod cells were mostly spiculated. Chlorpromazine at lower concentration converted them into biconcave disks and, at higher concentration, into stomatocytes. These results support the hypothesis that the spiculation of McLeod cells is the result of an imbalance of surface area between the two lipid leaflets of the membrane; that is, a bilayer couple effect. We determined the numerical density of intramembrane particles (IMP) in replicas of both fracture faces of red cells subjected to freeze fracture and rotary shadowing. These values were as follows (expressed per microns 2 of membrane +/- SD): the normal protoplasmic fracture face had 2200 +/- 306 and the McLeod had 2300 +/- 250. The normal exoplasmic fracture face had 388 +/- 75 and the McLeod had 330 +/- 59. We conclude that there is no evidence for derangement of band 3, the principal protein in the IMP, in McLeod erythrocytes.

The ultrastructure of chemolithoautotrophic Gallionella ferruginea and Thiobacillus ferrooxidans as revealed by chemical fixation and freeze-etching

By using sodium thioglycolate to dissolve the high amount of excreted stalk material in axenic cultures of the chemolithoautotrophic iron bacterium Gallionella ferruginea, the ultrastructure of Gallionella cells from pure cell suspensions could be studied without any loss of viability or disturbance by dense ferric stalk fibers, and compared with Thiobacillus ferrooxidans, also grown chemolithoautotrophically with ferrous iron as energy source. Both organisms were chemically fixed or freeze-etched. Particular structural differences between these iron-bacteria could be ascertained. G. ferruginea possesses intracytoplasmic membranes and soluble d-ribulose-1,5-bisphosphate-carboxylase, whereas T. ferrooxidans contains carboxysomes but no intracytoplasmic membranes; Gallionella forms poly-β-hydroxybutyrate and glycogen as storage material; T. ferrooxidans produces only glycogen. Both organisms also differ from each other with respect to the freeze fracture behaviour of the cell envelope layers. Whereas the cells of T. ferrooxidans exhibit a characteristic double cleavage, exposing the plasmic fracture face and exoplasmic fracture face of the outer membrane and cytoplasmic membrane, the exceptionally thin multilayered cell envelope of G. ferruginea revealed a particularly intimate association between the layers, resulting in a visualisation of the supramolecular organisation of only the inner fracture face of the cytoplasmic membrane. The results are discussed predominantly in relation to the extremely distinct environments of both organisms.

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

ABSTRACTParamylon 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.