A Freeze-Fracture Study of the Cortex of Xenopus laevis Eggs

The organization of the cortex of Xenopus laevis eggs was investigated by freeze-fracture electron microscopy. The cortical endoplasmic reticulum (CER) formed a network surrounding and interconnecting the cortical granules. It formed junctions with the plasma membrane and was confluent with the ER in subcortical regions. Intramembranous particles (IMP₁) were only present in the P face of the CER, the E face being apparently devoid of pits and particles. Arrays of densely packed IMP₁, having a mean diameter of 17 nm, were restricted to the microvillar region of the plasma membrane. The cortical granule membrane also contained IMP₁ (mean diameter, 21 nm) that were sparsely and randomly distributed. Several types of cortical granule seemed to exist based on an analysis of the distribution of the different IMP sizes.     

Inhibition of Pair Formation by Concanavalin A and Concanavalin A-Binding Glycoconjugates in Euplotes octocarinatus

ABSTRACT. Concanavalin A (≥ 50 μg/ml) inhibits pair formation in both of the two complementary mating types of Euplotes octocarinatus studied in this investigation. This effect can be reversed by methyl-α- d -mannose. Concanavalin A is accessible for methyl-α- d -mannose until pairs are formed. Methyl-α- d -mannose as well as methyl-α- d -glucose and 2-acetamido-2-deoxy- d -glucose alone do not inhibit pair formation unless applied in concentrations ≥ 60 mM. The Concanavalin A-sensitive phase of preconjugant interaction starts 2 h after cells are induced to conjugate. Based on these observations we suggest that Concanavalin A might exhibit its action by binding to carbohydrate moieties of preconjugation-specific adhesion molecules and thereby might allosterically block interactions with their counterparts. To identify preconjugation-specific alterations in number or localization of Concanavalin A-binding glycoconjugates, we probed western blots of total cell proteins or fixed cells, respectively, with digoxigenin-labeled Concanavalin A. On Concanavalin A blots 20 different Concanavalin A-binding glycoconjugates were identified in mating-competent cells. Localization of Concanavalin A-binding sites on mating-competent cells by light microscopy resulted in predominant labeling of a comma-shaped structure near the paroral membranelle. During the preconjugation period no changes in number or localization of Con A-binding glycoconjugates were detected. Possible reasons are discussed.     

Affinity-Purification of Concanavalin A-Binding Ciliary Glycoconjugates of Starved and Feeding Tetrahymena thermophila

Development of mating competency in Tetrahymena thermophila requires starvation for at least 70 min in low ionic strength buffer. Pair formation between conjugating cells is blocked at early stages by the lectin Concanavalin A (Con A). To investigate the role of Con A-binding proteins in this induced cellular change and pairing, and to confirm and extend an earlier study from our laboratory, a method was developed for preparation of Con A-binding proteins from ciliary membrane-rich fractions of T. thermophila. Con A-binding ciliary proteins were prepared from non-starved and starved cells from two wild type strains and a mating mutant, RH179E1. Comparison of these proteins by SDS-PAGE revealed on overall reduction in number of wild-type bands after starvation. In particular, a major band at 28 kDa was present in non-starved cells and absent in starved cells. However, in the mating mutant, no change in banding profile was seen after starvation: the 28 kDa band was present in both non-starved and starved cells. This, Con A-binding ciliary membrane proteins undergo a major change during starvation-induced development of mating competency in wild-type T. thermophila. In contrast, the mutant differed from wild-type in overall composition of its ciliary Con A-binding glycoproteins and in the response of these proteins to starvation, suggesting that it may be deficient in its ability to be initiated by starvation. Our results are consistent with the hypothesis that a change affecting ciliary membrane Con A-binding proteins is essential for the cellular response to mating signals.     

Development of Cleaving Mouse Embryos under Pressure

A chamber for applying mechanical pressure to developing mouse eggs is described.
Morphologically normal blastocysts developed from 4-cell eggs cultured under pressure initially near to the point of cell rupture. Four-cell, 8-cell eggs and late morulae, developing with the pressure regularly adjusted to this point, formed trophoblast-like cells internally and peripherally to the cell mass. These observations are consistent with the idea that cells will become trophoblast unless subjected to a specific intercellular environment. A glass plate closely apposed to the cells cannot substitute for this environment. Total enclosure by other cells seems necessary for inner cell mass differentiation.     

Freeze-Fracture Study of Malaria Sporozoites: Antibody-Induced Changes of the Pellicular Membrane*

Plasmodium cynomolgi, Plasmodium knowlesi, and Plasmodium berghei sporozoites, before and after incubation with immune serum, were studied after freeze-fracture by electron microscopy. There were evenly distributed numerous intramembranous particles (IMP) on the P face of the outer membrane. The E face of the plasma membrane had fewer IMP than its P face. The E face of the intermediate membrane had few IMP and also linear arrays of slightly raised ridges running the length of the parasite. The P face of the intermediate membrane had many IMP aligned along the long axis of the sporozoite. On the P face of the inner membrane. IMP were arranged in very distinct rows conforming to the long axis of the parasite; the E face of this membrane had a few randomly distributed IMP. A prominent change in the sporozoite incubated in immune serum was the appearance of a layer of aggregated particles around the parasite. The P face of the plasma membrane had several clear areas devoid of IMP and IMP aggregates. No changes were seen in the other fractured faces of the pellicle. These observations suggest that immune serum acts only on the P face of the plasma membrane.     

A Freeze-Fracture Study of the Microcyst Wall,Plasma Membrane,and Intramembrane Components of Physarum polycephalum1

ABSTRACT. Freeze-cleave replicas of encysted cells of the myxamoebae of Physarum polycephalum were examined to determine the intrawall substructure and to compare the intramembrane structural components. Cleaved areas of the cyst wall revealed a laminated substructure devoid of the macromolecular interruptions (intramembrane particles or IMP) visible in the cleaved cell membranes. The cyst wall adjacent to the cell membrane appears laminated, and a loose branching network of fibers and fibrils occurs at the wall periphery. The absence of intrawall particles is interpreted as a lack of protein or polypeptide components, thus suggesting additional support for the conclusion that polysaccharides are the major wall constituents. When cleaved cell membranes of encysted cells were examined, more intramembrane particles per unit area were observed on the extracellular membrane leaflet than on the protoplasmic membrane leaflet. In addition, homogeneous as well as aggregated particle distributions were visible on cleaved membrane leaflets. Moreover, the presence of aggregated and unaggregated particles on the same membrane leaflets similarly suggests asynchrony of the cell population. This paper examines and compares biological processes involving the cell membrane that may be related to different stages in the cell cycle or to periods of temporary stasis during the cell cycle.     

Topography of Membrane Concanavalin A Sites modified by Proteolysis

The topographic distribution of membrane Con A sites becomes more clustered after treatment with proteolytic enzymes. The clustered sites appear to be involved in the formation of intercellular Con A cross-bridges between agglutinated cells.     

Freeze-Fracture Study of the Endosymbiont of Blastocrithidia culicis1

The two unit membranes which envelope the endosymbiont of the trypanosomatid protozoon, Blastocrithidia culicis, were studied using the freeze-fracture technique. The distribution of the intramembranous particles on both fracture faces of the inner and outer membrane of the endosymbiont was analyzed in the replicas. The protoplasmic face of the inner membrane (PFi) had a higher density of membrane particles than that observed on the extracellular face (EFi), a pattern typical of plasma membranes. The extracellular face of the outer membrane (EFo) presented a density of membrane particles much higher than that observed on the P face of the outer membrane (PFo) a distribution significantly different from that found in the inner membrane of the endosymbiont and in the plasma membrane of the protozoon, but similar to that observed in Gram-negative bacteria. The data obtained support the idea that the endosymbiont of trypanosomatids represents a Gram-negative bacterium-like microorganism enveloped by two unit membranes and lacking a peptidoglycan layer and which lives in direct contact with the cytoplasm of the protozoon.     

Transient Concanavalin A-Binding Glycoproteins of the Parallel Fibres of the Developing Rat Cerebellum: Evidence for the Destruction of Their Glycans

Abstract: The lifetime of the glycoprotein glycans of the rat cerebellum was followed in the 2nd and 3rd weeks of postnatal age, after injection of labelled glucosamine. It appears that a particular class of glycans binding to Concanavalin A synthesized at an early age has a short lifetime. These results indicate that the glycans of the Concanavalin A-binding glycoproteins abundant on the newly formed parallel fibres are rapidly degraded between the 14th and the 18th postnatal day. Reeber A. et al. Transient Concanavalin A-binding glycoproteins of the parallel fibres of the developing rat cerebellum: Evidence for the destruction of their glycans. J. Neurochem. 35 , 1273–1277 (1980).     

Membrane mobility and the Concanavalin A binding system of the plasmalemma of higher plant protoplasts

The binding of a colloidal gold-Concanavalin A (ConA) complex to the plasmalemma of tobacco leaf protoplasts has been investigated using scanning electron microscopy. At 5° C the particles of gold-ConA appear to be randomly distributed over the surface of the protoplast. If the temperature is raised, the particles associate into clusters. Saturation of the membrane with particles can only occur when the weight of ConA in solution exceeds 1 g/10⁴ protoplasts in suspension, and when its concentration exceeds 15 g/ml. These results are discussed in terms of the properties of the ConA binding site and the mobility of such sites within the membrane surface.Abbreviations ConA Concanavalin A - AuConA Colloidal gold-Concanavalin A complex     

Xenopus Embryos as a Model to Study the Genetic Mechanisms of Brain Development

The review considers the advantages of Xenopus embryos as an experimental model to study the molecular-genetic mechanisms of embryo development. The results are described that were obtained with this model in studies on the early brain development within the framework of the Russian program Human Genome.     

Ultrastructure of the Squid Axon Membrane as Revealed by Freeze-Fracture Electron Microscopy

The structure of the axolemma of the squid giant axon was studied by freeze-fracture electron microscopy. Three types of preparations were examined: intact axons, axons with their Schwann cell sheaths stripped off prior to freezing, and axons with their Schwann cell sheaths chemically detached but not mechanically removed. Because of a problem of cross-fracturing, the first two types of preparations revealed very few membrane faces of the axolemma. This cross-fracturing problem, however, was eliminated when we used a complementary replication method to fracture the third type of preparation. We found that the E-face of the axon membrane was smooth relative to the P-face, which showed many prominent intramembrane particles (IMP). The diameters of the typical IMP range from 6 to 15 nm. The P-face of the adjacent Schwann cells also showed many large IMP. The sizes and heights of the Schwann-cell IMP, however, appear to be more homogeneous than the P-face axolemma.     

Freeze-Fracture Study of Blastocystis hominis1

The ultrastructure of Blastocystis hominis was investigated by the freeze-fracture method. Freeze-fracture replicas of the membranes of B. hominis and its organelles were studied with special regard to the density and distribution of the intramembranous particles (IMF's). On all membrane replicas, the concentration of IMF's on the protoplasmic face (P face) invariably was greater than on the exoplasmic face (E face). On the P face, IMP's were heterogeneously distributed in dense aggregates, alternating with particle-free, smooth surface areas. Occasionally, small depressions and protrusions were observed in these areas. On the membrane of the central vacuole, invaginations into the vacuole were frequently observed within the smooth surface regions. Since most of the granules in the central vacuoles had no IMF's, it seems likely that the intervacuolar granules were formed from these invaginations of the vacuole membrane. The width of the intermembrane space between the inner and outer membranes of the nuclear envelope was uneven, with regions of relative narrowness interspersed with regions of expansion. Nuclear pores were localized within the narrow portions of this space. A nucleus, apparently in the process of dividing, was observed enclosed within an intact outer membrane. Division of the outer membrane would then result in the formation of two discrete nuclei.     

Interaction of Concanavalin A and a Divalent Derivative with Lymphocytes and Reconstituted Lymphocyte Membrane Glycoproteins

Both concanavalin A (con A) and its divalent derivative, succinyl-concanavalin A (S-con A) are mitogenic for porcine lymph node lymphocytes. We have compared the binding of these two lectins to intact porcine lymphocytes and phospholipid vesicles containing reconstituted lymphocyte membrane glycoproteins. Both con A and S-con A showed high- and low-affinity binding to intact cells, as indicated by LIGAND analysis of Scatchard plots of binding data. Despite the apparently identical saccharide specificities of the two lectins, high-affinity binding sites for S-con A were only one-third as numerous as high-affinity sites for the parent lectin. Large numbers of low-affinity binding sites existed for con A, while many fewer were present for S-con A. It is suggested that these sites result from hydrophobic association. Con A bound to lymphocytes in a positively cooperative fashion, while S-con A showed non-cooperative behavior. Lectin binding to large unilamellar phospholipid vesicles containing reconstituted lymphocyte membrane glycoproteins was measured using a rapid filtration assay, and was linear with the glycoprotein content of the vesicles. Almost all of the outward-facing glycoprotein was functional in terms of lectin binding. Reconstituted glycoproteins showed only a single class of high-affinity binding sites for both con A and S-con A, with association constants similar to those measured for intact cells. Con A, but not S-con A, showed positively cooperative binding to reconstituted vesicles. Cooperativity was observed in both gel phase and liquid crystalline phase lipid, and was thus not dependent on long-range lateral rearrangement of glycoprotein receptors. Results suggested that con A induces a microre-distribution of receptors on the lymphocyte membrane surface, leading to the exposure of glycoproteins that were previously inaccessible to the lectin. S-Con A does not cause glycoprotein redistribution, and a large fraction of the receptors remain cryptic.     

Freeze-Fracture Study of the Bloodstream Form of Trypanosoma brucei gambiense

The ultrastructure of Trypanosoma brucei gambiense was investigated by the freeze-fracture method. Three different regions of the continuous plasma membrane; cell body proper, flagellar pocket, and flagellum were compared in density and distribution of the intramembranous particles (IMP's). The IMP-density was highest in the flagellar pocket membrane and lowest in flagellum. Intra membranous particles of the cell body membrane were distributed uniformly on both the protoplasmic (P) and exoplasmic (E) faces. On the P face of the flagellar membrane, a single row of IMP-clusters was seen along the juncture of the flagllum to the cell body. Since the spacing of the IMP-clusters was almost equal to the spacing of the paired rivet structures observed in thin section, these clusters likely are related to the junction of flagellum and cell body. At the neck of the flagellar pocket, several linear arrays of IMP's were found on the P face of the flagellar membrane, while on the E face rows of depressions were seen. At the flagellar base, the clusters of IMP's were only seen on the P face. On the flagellar pocket membrane, particle-rich depressions and linear particle arrays were also found on the P face, while on the E face such special particle arrangements were not recognized. These particle-rich depressions may correspond to the sites of pinocytosis of the bloodstream forms which have been demonstrated in thin sections.     

Freeze-Fracture Study of the Trophozoite and the Cyst of Entamoeba histolytica

ABSTRACT The fine structure of the trophozoite and cyst of Entamoeba histolytica from the stool of a patient was compared using the freeze-fracture method. The intramembranous particles (IMP's) were heterogeneously distributed on the plasma membrane of the trophozoite and their density was 1139 ± 105/μm² on the P face and 27 ± 9/μm² on the E face. Particle-rich depressions and linear particle arrays, reported by other investigators on cultured trophozoites, were also observed on the P face while on the E face such special particle arrangement was not recognized. Particle-free, small protrusions were frequently observed on the P face of the trophozoite membrane. The existence of these protrusions is a new finding. In the cyst, the IMP's were also distributed heterogeneously on both the P and E faces of the plasma membrane. The density of the IMP's, however, was much lower than in the trophozoite: 6 ± 2/μm² on the P face and averaging less than 1/μm² on the E face. In freeze-fracture images, the plasma membrane of the cyst showed a variety of configurations from smooth to uneven or ridged surfaces. These morphological alterations of the plasma membrane may be attributed to the aging of the cyst. The thick wall of the cyst had a filamentous tri- or tetra-lamellar structure. The cytoplasm of the cyst was similar in structure to that of the trophozoite and the diameter of the nuclear pores was equal in both trophozoites and cysts.     

A Freeze-Fracture Study of Membranes of Rapidly Drought-Stressed Leaf Bases of Wheat

Pearce, R. S. 1985. A frceze-fracture study of membranes ofrapidly drought-stressed leaf bases of wheat.—J. exp.Bol. 36: 1209-1221. Bases of expanding leaves were taken from well-watered or drought-hardenedwheat seedlings, and were progressively dehydrated (over ?–9h or, more slowly, for 24 h or 36 h) to between 76% and 5% ofthe water content of the turgid tissue. Damage was assessedby an ion-leakage test. The dehydrated tissues were freeze-fixedwithout rehydration. Patches free from intramembraneous particles(IMP) occurred in the plasma membrane, tonoplast and chloroplastenvelope of all the damaged leaf bases, and were mostly absentfrom undamaged tissues and controls. 15% of these patches appearedto have an ordered sub-structure. Lamellae with few or no IMP,were associated with some IMP-free patches of plasma membrane.Sometimes IMP-free patches and lamellae were associated withIMP-free folds. Groups of IMP-free lamellae occurred in thecytoplasm of the most severely stressed material. Vesicles andmembraneous sacs accumulated just below the plasma membranein some cells from stressed drought-hardened leaf bases. Depressions,‘lesions’ (mainly unusual circular discontinuities),and associated IMP-free patches, occurred in some plasma membranes,mostly in the stressed hardened tissues, including in non-damagedtissue. The results are related to an hypothesis previouslysuggested to explain damage due to extracellular freezing inwheat tissues: the stress causes cell dehydration and this inducesIM P-free patches leading to membrane reorganization (here expressedas IMP-free lamellae and folds) which results in leakage. Thepresent results confirm the role of cytoplasmic dehydrationin the formation of IMP-free patches and in other membrane changes. Key words: Drought stress, freezing stress, plasma membrane