Localization of the binding sites for the Ricinus communis, Agaricus bisporus and wheat germ lectins on human erythrocyte membranes

Freeze-etch electron microscopy has been utilized to localize the binding sites for the Ricinus communis, Agaricus bisporus and wheat germ lectins on human erythrocyte membranes and to determine the relation of these different glycoprotein receptors to the intramembranous particles. A. bisporus lectin, which could be visualized directly on the surface of erythrocyte membranes, and ferritin conjugates of wheat germ agglutinin showed a distribution that correlates exactly with the intramembranous particles at all lectin concentrations tested. The binding sites for both of these lectins are located on the major sialoglycoprotein of the membrane. The R. communis agglutinin-ferritin conjugate which binds to receptors on membrane glycoproteins that are distinct from the major sialoglycoprotein showed a close correlation with the intramembranous particles at low lectin concentrations and a poor correlation at high lectin concentrations. High concentrations resulted in virtually complete coating of the surface of trypsinized ghosts which displayed marked aggregation of the intramembranous particles. We conclude that the intramembranous particles of erythrocyte membranes contain at least two glycoproteins and that some membrane lectin receptors are not associated with the intramembranous particles.     

Freeze-fracture evidence of gel-phase lipid in membranes of senescing cowpea cotyledons

The structural details of membrane organization in germinating and senescing cotyledons of cowpea (Vigna unguiculata (L.) Walp.) were studied by thin section and freeze-fracture electron microscopy. Germination- and senescence-related changes in the ultrastructure of parenchymal cells of cowpea cotyledons, as detected in thin sections, closely resemble those described for other leguminous seeds. Additionally, electron-dense deposits associated with the membranes, particularly the plasmalemma and endoplasmic reticulum, were seen to increase with advancing senescence. Freeze-fracture electron microscopy demonstrated that the membranes of cotyledons of 2-d-old seedings appear to be normal, with evenly dispersed intramembranous particles. However by 4 d, small areas or domains of the plasmalemma were free of intramembranous particles. These particle-free areas increased in both size and number as senescence progressed. We interpret these particle-free areas to be structural evidence for lateral phase separations of the membrane lipids into microdomains of gel-phase lipid from which intrinsic membrane proteins are excluded. Our results support wide-angle X-ray diffraction studies which have demonstrated the presence of gel-phase lipids in senescing bean cotyledons.Abbreviations EF exoplasmic fracture - ER endoplasmic reticulum - ESR electron-spin resonance - IMP(s) intramembranous particle(s) - PF protoplasmic fracture     

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 eggs of the freshwater fish Epiplatys dageti have tight plasma membranes without intramembranous particles

Summary The plasma membrane of fish (Epiplatys dageti) eggs, which are capable of developing in salt-free water despite intracellular osmolarity corresponding to a pressure gradient of 7 to 8 atmospheres, is almost devoid of intramembrane particles. The specific membrane resistance is at least 3 orders of magnitude larger than that of nerve or muscle cells of different species indicating that the membrane is tight. These findings support the view that intramembranous particles are involved in transmembrane transport of ions, and indicate that the ionic concentration gradient in this cell is maintained by a tight membrane rather than by active transport.     

Inhibition of membrane fusion by suppression of lateral movement of membrane proteins

Chicken erythrocytes were fused either by Sendai virus or by the combination of Ca²⁺ and ionophore A23187.Intramembrane particles and external anionic sites of cells undergoing fusion were found to acquire the ability to undergo a process of cold-induced clustering (thermotropic separation).Cationized ferritin (200 μg/ml 5% (v/v) cell suspension) inhibited both the fusion process and the thermotropic separation of intramembrane particles and external anionic sites. The correlation between the mobility of membrane proteins and the fusion process is discussed. It is suggested that an increase in the lateral mobility of membrane proteins is a prerequisite for initiation of membrane fusion.     

ANIONIC SITES OF HUMAN ERYTHROCYTE MEMBRANES : I. Effects of Trypsin, Phospholipase C, and pH on the Topography of Bound Positively Charged Colloidal Particles

The effects of pH, trypsin, and phospholipase C on the topographic distribution of acidic anionic residues on human erythrocytes was investigated using colloidal iron hydroxide labeling of mounted, fixed ghost membranes. After glutaraldehyde fixation at pH 6.5–7.5, the positively charged colloidal particles were bound to the membranes in small randomly distributed clusters. The clusters of anionic sites were reversibly aggregated by incubation at pH 5.5 before fixation at the same pH. These results correlate with the distribution of intramembranous particles found by Pinto da Silva (J. Cell Biol. 53:777), with the exception that the distribution of anionic sites on a majority of the fixed ghosts at pH 4.5 was aggregated instead of dispersed. The randomly distributed clusters could be nonreversibly aggregated by trypsin or phospholipase C treatment of intact ghosts before glutaraldehyde fixation. Previous glutaraldehyde fixation prevented trypsin and pH induced aggregation of the colloidal iron sites. Evidence that N-acetylneuraminic acid groups are the principal acidic residues binding colloidal iron was the elimination of greater than 85% of the colloidal iron labeling to neuraminidase-treated cell membranes. Colloidal iron binding N-acetylneuraminic acid residues may reside on membrane molecules such as glycophorin, a sialoglycoprotein which contains the majority of the N-acetylneuraminic acid found on the human erythrocyte membrane.     

Membranes and organelles of dehydratedSelaginella andTortula retain their normal configuration and structural integrity

Summary Dry (7–10% water content) leaves of the spikemossSelaginella lepidophylla (resurrection plant) and of the desiccationtolerant moss,Tortula ruralis were examined by freeze fracture electron microscopy. As has been described for dry seeds, the cells of these dehydrated leaves were shrunken, with highly convoluted walls and membranes. The membranes of all samples had a lipid bilayer organization with dispersed intramembranous particles (IMPs). Lipid droplets were very closely associated with the plasmamembrane. Chloroplasts were surrounded by a double membrane envelope and contained well-organized grana. Mitochondria were irregular in outline, and endoplasmic reticulum and cytoplasmic vesicles were present.Abbreviations ABA abscisic acid - EF exoplasmic fracture - FTIR Fourier transform infrared analysis - H_II hexagonal II - IMPs intramembranous particles - MGDG monogalactosyl diacylglycerol - NMR nuclear magnetic resonance - PE phosphatidylethanolamine - PF protoplasmic fracture - PS I photosystem I - PS II photosystem II     

Preparation-dependent distribution of intramembranous particles in freeze-fracture replicas of the neuromuscular junction of the locust Schistocerca gregaria

Summary Freeze-fracture replicas of the neuromuscular junction were prepared from untreated retractor unguis muscles of the locust Schistocerca gregaria that were rapidly frozen by contact with a copper block cooled by liquid helium. These replicas were compared with others prepared from tissue following fixation with glutaraldehyde and cryoprotection in glycerol. Freeze-fracture of rapidly frozen tissue produced replicas of high quality with little evidence of tissue damage by ice crystals in the superficial layers. The gross fracturing characteristics of the neuromuscular junction were consistent with replicas from fixed and cryoprotected tissue; all of the membrane specializations were recognisable but with some alterations in infrastructure. In tissue replicas prepared using either method intramembranous particles in the presynaptic membrane were arranged in a bar-like array. The intramembranous particles of this presynaptic bar array of the rapidly frozen material were large and found on the E-face of the cleaved membrane. This contrasts with the P-face distribution of the comparable particles in muscles fixed in glutaraldehyde and cryoprotected in glycerol, in which they are also smaller and more numerous. This difference in partitioning between rapidly frozen, and fixed, cryoprotected nerve terminals is not found at cholinergic synapses and thus may reflect functional differences between the two types of junction.Indentations of the nerve-terminal membrane occur in replicas from rapidly frozen muscle as well as fixed and cryoprotected muscle suggesting they are not fixation or glycerol-induced artifacts. It is suggested from their position and size that these indentations are more likely to be part of a membrane retrieval system than exocytotic figures.This work was supported by an S.E.R.C. project grant to I.R.D.     

Localization of the binding sites for the Ricinus communis, Agaricus bisporus and wheat germ lectins on human erythrocyte membranes.

Freeze-etch electron microscopy has been utilized to localize the binding sites for the Ricinus communis, Agaricus bisporus and wheat germ lectins on human erythrocyte membranes and to determine the relation of these different glycoprotein receptors to the intramembranous particles. A. bisporus lectin, which could be visualized directly on the surface of erythrocyte membranes, and ferritin conjugates of wheat germ agglutinin showed a distribution that correlates exactly with the intramembranous particles at all lectin concentrations tested. The binding sites for both of these lectins are located on the major sialoglycoprotein of the membrane. The R. communis agglutinin-ferritin conjugate which binds to receptors on membrane glycoproteins that are distinct from the major sialoglycoprotein showed a close correlation with the intramembranous particles at low lectin concentrations and a poor correlation at high lectin concentrations. High concentrations resulted in virtually complete coating of the surface of trypsinized ghosts which displayed marked aggregation of the intramembranous particles. We conclude that the intramembranous particles of erythrocyte membranes contain at least two glycoproteins and that some membrane lectin receptors are not associated with the intramembranous particles.     

Characteristics of the membranes of the pellicle of the ciliatePseudomicrothorax dubius

Summary The membranes of the pellicle of the ciliatePseudomicrothorax dubius are investigated using thin section electron microscopy and freeze-fracture replicas. The plasma membrane is covered by a surface coat and is connected to the outer alveolar membrane by short, sometimes branched, bridges. The inner alveolar membrane is coated on both sides. The epiplasm lies in intimate contact with the cytoplasmic surface of this membrane, and there is a corresponding deposit on the other surface. This deposit is regularly striated.The epiplasmic layer and the alveoli are interrupted at sites of cytotic activity,e.g., the attachment sites of trichocysts, the cytoproct, and the parasomal sacs. The striated deposit ends where the epiplasm ends, indicating a direct relationship between these two epimembranous layers.There is a deposit along the sides of the first part of the tip of the trichocysts, and in this region the trichocyst membrane is free of intramembranous particles.The membrane of the parasomal sacs has a coat on both surfaces. That on the extraplasmic surface is similar to the surface coat of the plasma membrane. The origin of the cytoplasmic coat is unknown. The cytotic activity of these sacs is indicated by their highly irregular profiles.     

Aggregates of particles on the plasmalemma of striated muscle from a spider.

Aggregates of large intramembranous particles are described in the surface membrane of muscle fibers of a spider. The particles are continuous with extensions into the extracellular space, forming large complexes. No intramembranous particles are visible at sites of Z line attachment to the plasma membrane.     

High resolution analysis of the formation of cellulose synthesizing complexes inVaucheria hamata

Summary Ultrastructure and assembly of cellulose terminal synthesizing complexes (terminal complexes, TCs) in the algaVaucheria hamata (Waltz) were investigated by high resolution analytical techniques for freeze-fracture replication.Vaucheria TCs consist of many diagonal rows of subunits located on the inner leaflet of the plasma membrane. Each row contains about 10–18 subunits. The subunits themselves are rectangular, approx. 7×3.5 nm, and each has a single elliptical hole which may be the site of a single glucan chain polymerization. The subunits are connected with extremely small filaments (0.3–0.5 nm). Connections are more extensive in a direction parallel to the subunit rows and less extensive perpendicular to them. Nascent TC subunits are found to be packed within globules (15–20 nm in diameter) which are larger than typical intramembranous particles (IMPS are 10–11 nm in diameter) distributed in the plasma membrane. The subunits in the globule, which may be a zymogenic precursor of the TC, are generally exhibited in the form of doublets. Approximately 6 doublets are connected to a center core with small filaments. The globules are inserted into the plasma membrane together with IMPS by the fusion of cytoplasmic (Golgi derived) vesicles. Two or three globules attach to each other, unfold, and expand to form the first subunit rows of the TC on the inner leaflet of the plasma membrane. More globules attach to the structure and unfold until the nascent TC consists of a few rows of subunits. These rows are arranged almost parallel to each other. Two formation centers of subunits appear at both ends of an elongating TC. New subunits carried by the globules are added at each of these centers to create new rows until the elongating TC structure is completed. On the basis of this study, a model of TC assembly and early initiation of microfibril formation inVaucheria is proposed.Abbreviations IMPS intramembranous particles - MF microfibril - TC terminal complex     

An electron-microscope and freeze-fracture study of the egg cortex of Brachydanio rerio

Summary We have examined the cortex of the teleost (Brachydanio rerio) egg before and during exocytosis of cortical granules by scanning, transmission, and freeze-fracture electron microscopy. In the unactivated egg, the P-face of the plasma membrane exhibits a random distribution of intramembranous particles, showing a density of 959/m² and an average diameter of 8 nm. Particles over P- and E-faces of the membranes of cortical granules are substantially larger and display a significantly lower density. An anastomosing cortical endoplasmic reticulum forms close associations with both the plasma membrane of the egg and the membranes of cortical granules. Exocytosis begins with cortical granules pushing up beneath the plasma membrane to form domeshaped swellings, coupled with an apparent clearing of particles from the site of contact between the apposed membranes. A depression in the particle-free plasma membrane appears to mark sites of fusion and pore formation between cortical granules and plasma membranes. Profiles of exocytotic vesicles undergo a predictable sequence of morphological change, but maintain their identity in the egg surface during this transformation. Coated vesicles form at sites of cortical granule breakdown. Differences in particle density between cortical granules and egg plasma membranes persist during transformation of the exocytotic profiles. This suggests that constituents of the 2 membrane domains remain segregated and do not intermix rapidly, lending support to the view that the process of membrane retrieval is selective (i.e., cortical granule membrane is removed).     

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     

Membrane differentiations in spermatozoa of the squid,Loligo pealeii

Regional differences in the structure of the plasma membrane and acrosome membrane of squid spermatozoa were studied by freeze-fracture and thin section electron microscopy. In regions of close apposition the plasma membrane and acrosome membrane are adjoined to one another by regularly spaced linkages. These linkage sites, overlie a set of fibers located at the inner face of the acrosomal membrane. The acrosomal fibers terminate in a layer of granular material located at the base of the acrosome. Detergent treatment of sperm releases the fibers and granular material as an interconnected complex. Freeze-fracture replicas reveal a random arrangement of intramembranous particles in the plasma membrane over the sperm head and linear aggregates of intramembranous particles in the acrosomal membrane. Several regional differences in the structure of the flagellar plasma membrane are present. The thickness of the glycocalyx is progressively reduced distally along the flagellum. Freeze-fracture replicas show evenly spaced linear arrays of intramembranous particles which extend parallel t o the flagellar long axis. Examination of spermatozoa extracted to disrupt flagellar geometry suggest that the dense fiber-doublet microtubule complexes are attached to the plasma membrane. The possible functional role of these membrane differentiations and their relationship t o membrane structures in mammalian spermatozoa are discussed.     

Pigment biogenesis in freshwater shrimp ventral nerve chord chromatophores

Summary The possible biogenesis of two pigment granule types present in the monochromatic, brown chromatosomes enveloping the ventral nerve chord of the freshwater palaemonid shrimps Macrobrachium acanthurus, M. heterochirus and M. olfersii is examined by transmission electron microscopy in thin section and freeze fracture replicas. Prominent, membrane limited granules are suggested to have their origin in a complex, juxtanuclear, smooth endoplasmic reticulum labyrinth, continuous with the nuclear envelope. Amembranous, lipocarotenoid granules possibly derive from the external surface of the smooth endoplasmic reticulum. Nuclear envelope and SER membranes contain numerous 11 nm diameter intramembranous particles while pigment granule membranes exhibit fewer particles. A dictyosomal origin for the lipocarotenoid granules is discounted. Granulogenesis is suggested to be a continuous process in crustacean chromatophores.     

Identification of tonoplast and plasma membrane in membrane fractions from garden cress (Lepidium sativum L.) with and without filipin treatment

Membranes from roots of Lepidium sativum L. were investigated in situ and after fractionation by applying morphological and biochemical methods. After freeze-fracture combined with filipin labelling the tonoplast and the plasma membrane could be easily characterized by the frequency of intramembranous particles and the arrangement of filipin-induced lesions. On tonoplast vesicles, the filipin-induced lesions were arranged in clusters of different size whereas they were evenly distributed on plasma membrane vesicles. Enrichment of tonoplast and plasma membrane in different fractions was documented by filipin labelling, phosphotungstic acid staining and by the profiles of marker enzyme activities and ATP-dependent H⁺-transport. Additionally, the presence of rightside-out and inside-out vesicles of both tonoplast and plasma membrane could be demonstrated. It was found that filipin labelling used in combination with freeze-fracturing is suitable for quantitative determinations of the percentages of tonoplast and plasma membrane in membrane fractions, which have been found to be more than 40% for the tonoplast and about 40% for plasma membrane in the respective enriched fractions.Abbreviations EF extraplasmatic fracture face - FIL filipin induced lesion - IMP intramembranous particle - PF plasmatic fracture face - PTA phosphotungstic acid-chromic acid stain - UDPG uridine 5-diphosphate glucose A preliminary report was presented at the joint Annual Meeting of the Belgian and German Societies for Cell Biology, Bonn, March 1985Dedicated to Professor Augustin Betz on the occasion of his 66th birthday     

Freeze-fracture observations of unfertilized and fertilized hamster oocytes with special reference to the use of lipid probes

Membrane and cytoplasmic changes were observed after in vitro fertilization of hamster oocytes by examining freeze-fracture replicas. The density of intramembranous particles on areas of membrane between microvilli increased following fertilization. Although the intramembranous particle density of microvilli is higher than that on the intermicrovillar membrane of unfertilized eggs, it did not change significantly after fertilization. Cytoplasmic changes in the Golgi complex and mitochondria upon fertilization indicate a change in cellular activity. Lipid binding probes were applied to the oocyte membranes in order to study the distribution of specific lipids before and after fertilization. Probes included the B-hydroxy-steroid complexing molecules, filipin and tomatin, and an anionic lipid binding antibiotic, polymyxin B. Both tomatin and filipin complex with steroids in the P and E faces of the plasma membrane (including the polar bodies), cortical granules and vesicles deeper in the cytoplasm, and the Golgi complex, leaving mitochondria, pronuclei, endoplasmic reticulum, and the majority of vesicles unlabeled. Polymyxin B binding is dependent on its application before or after fixation or in association with EGTA. With its application we detected both minor membrane perturbations of wrinkles and particle redistributions and major perturbations of vesicle fusions, the formation of blebs, and the loss of membrane morphology. Neither the distribution nor apparent quantity of these probes changed overall following fertilization, but this impression does not include specific sites of sperm-egg fusion.     

Localization and Characterization of Photosystem II in Grana and Stroma Lamellae

Attempts have been made to identify intramembranous particles observed in freeze-fracture electron microscopy as specific functional components of the membrane. The intramembranous particles of the exoplasmic fracture (EF) face of freeze-fractured pea (Pisum sativum) chloroplast lamellae are nonuniformly distributed along the membrane. Approximately 20% of the particles are in unpaired membrane regions whereas 80% are localized in regions of stacked lamellae (grana partitions). The EF particles within the grana regions of the chloroplast membrane are of a larger average size than those in stroma lamellae.     

Modification of protoplast cell wall regeneration by membrane perturbation

Summary Protoplasts derived from cells ofBoergesenia forbesii regenerated aberrant cell walls when treated with cholesteryl hemisuccinate (CHS). Protoplasts treated with CHS, for a short period during the initial stages of cell wall regeneration, developed a patchwork cell wall, possessing regions devoid of cell wall. This effect was reversible, and treated cells ultimately developed a normal, confluent cell wall when removed from the CHS. Freeze fracture studies revealed that for CHS-treated cells, regions without microfibril impressions did possess intramembranous particles (IMP's) but that these regions contained small domains free of IMP's suggestive of lateral phase separation. The data implies that the physical characteristics of the plasma membrane lipid are important to the deposition of cell wall microfibrils during cell wall regeneration. This effect may be attributed to altered lipid-protein interactions, modified membrane fusion characteristics, or altered membrane flow.