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.     

Freeze fracture studies on the interaction between the malaria parasite and the host erythrocyte in Plasmodium knowlesi infections.

The freeze fracture technique has been used to study the internal cyto-architecture of the surface membranes of the parasite and erythrocyte in Plasmodium knowlesi infections. Six fracture faces, derived from the plasma membrane and 2 pellicular membranes, have been identified at the surface of the free merozoite. The apposed leaflets of the 2 pellicular membranes show the characteristic features of E fracture faces, a result compatible with the view that the pellicular membranes line a potential cisterna. There is evidence to suggest that there may be changes in the distribution and density of the integral proteins in the merozoite plasma membrane at invasion. Furthermore, vesicles consisting of stacked membranes occur within and around the erythrocyte invagination at invasion; it is suggested that these vesicles are released from the merozoite rhoptries. Formation of the parasitophorous vacuole is accompanied by dramatic changes in the density and distribution of intra-membraneous particles (IMP) in the vacuolar membrane. Initially there is a great reduction in particle numbers, but subsequently the particles reappear and show reversed polarity. The possible causes and implications of these changes are discussed. The intra-erythrocytic parasite synthesizes new transmembrane proteins as development proceeds, and the trophozoite and schizont stages of development are characterized by the appearance of circular, particle-free regions in the parasite plasmalemma. There is a decrease in the density of transmembrane proteins in the erythrocyte plasma membrane during parasite maturation, and the P face IMP show the characteristic features of aggregation.     

Corbular sarcoplasmic reticulum of rabbit cardiac muscle

The structure of corbular sarcoplasmic reticulum as part of the sarcoplasmic reticulum (SR) in perfusion-fixed rabbit cardiac muscle was studied by thin sections and freeze fracture. In thin sections, processes on the surface of corbular SR have all the anatomical features of junctional processes of junctional SR. By freeze fracture, the E face of corbular SR was particle poor and showed deep pits; the P face was particle rich. The demonstrated structural homology of corbular SR to all forms of junctional SR justifies its inclusion in that group.     

Intramembrane particles and the organization of lymphocyte membrane proteins

An experimental system was developed in which the majority of all lymphocyte cell-surface proteins, regardless of antigenic specificity, could be cross-linked and redistributed in the membrane to determine whether this would induce a corresponding redistribution of intramembrane particles (IMP). Mouse spleen cells were treated with P-diazoniumphenyl- β-D-lactoside (lac) to modify all exposed cell-surface proteins. Extensive azo- coupling was achieved without significantly reducing cell viability or compromising cellular function in mitogen- or antigen-stimulated cultures. When the lac-modified cell- surface proteins were capped with a sandwich of rabbit antilactoside antibody and fluorescein-goat anti-rabbit Ig, freeze-fracture preparations obtained from these cells revealed no obvious redistribution of IMP on the majority of fracture faces. However, detailed analysis showed a statistically significant 35 percent decrease (P less than 0.01) in average IMP density in the E face of the lac-capped spleen cells compared with control cells, whereas a few E-face micrographs showed intense IMP aggregation. In contrast, there was no significant alteration of P-face IMP densities or distribution. Apparently, the majority of E-face IMP and virtually all P-face IMP densities or distribution. Apparently, the majority of E-face IMP and virtually all P-face IMP do not present accessible antigenic sites on the lymphocyte surface and do not associate in a stable manner with surface protein antigens. This finding suggests that IMP, as observed in freeze-fracture analysis, may not comprise a representative reflection of lymphocyte transmembrane protein molecules and complexes because other evidence establishes: (a) that at least some common lymphocyte surface antigens are indeed exposed portions of transmembrane proteins and (b) that the aggregation of molecules of any surface antigen results in altered organization of contractile proteins at the cytoplasmic face of the membrane.     

大豆根瘤细菌周膜的冷冻复型研究

用冷冻复型电镜技术研究了中国丰收１１号大豆根瘤中的细菌周膜。细菌周膜的断裂面上有颗粒状物质,但Ｐ面和Ｅ面有所不同,前者颗粒密度较大。即使都在Ｐ面或Ｅ面上,不同的细菌或同一细菌不同部位的颗粒密度也不一样。在细菌周膜与细菌细胞壁之间有一个环形腔隙,腔的大小随细菌和细菌部位不同而异。腔中不仅有泡状和管状结构,有时也有类寄主细胞质物质。细菌周膜表面有近似半球形或嵴形隆起,它们可能是腔中管泡状结构压迫细菌周     

Colicin K decreases the density of intramembrane particles (IMP) in the cell membrane of Escherichia coli

Toxic effects of both main colicin types, i.e. of porin and nuclease types, involve the direct contact of their molecules with the plasma membrane of sensitive cells. In the present study, it was tested whether this contact provokes a lateral or vertical movement of intramembrane protein particles (IMP) or a direct cleavage of the proteins. IMP were visualized by freeze-fracturing and electron microscopy on the protoplasmic fracture face (PF) of colicin-treated cells of Escherichia coli. Possible changes in distribution and in density of IMP due to treatment with colicins E1-E7 and K were followed. As a control, the bacteria were equilibrated at 0 degrees C before quenching, which caused a reversible formation of smooth areas and a decrease in the mean density of IMP on the PF. Colicins E1-E7 had no clear-cut effect on the disposition of IMP. Only colicin K decreased the IMP density, by 10% in E. coli strain 58-161 and by 17% in strain C6; the distribution of IMP remained homogeneous. Trypsin reactivation of colicin-K-inactivated bacteria was not reflected by restoration of the original density of IMP; on the contrary, it led to a further decrease, of 1-13%, in IMP density, presumably by proteolytic cleavage. Varying densities of IMP in different strains of the same bacterial species (under standard conditions) were confirmed.     

Reduction of intramembranous particles in the periacrosomal plasma membrane of boar spermatozoa during in vitro capacitation: a statistical study

Membrane remodeling in the periacrosomal plasma membrane (PAPM) of boar spermatozoa during incubation in capacitation medium was examined by the freeze-fracture technique. In the preservation medium (PM) group, the major small (about 8 nm) intramembranous particles (IMP) and the minor large (> 10 nm) IMP were distributed evenly in the PAPM. The IMP-free area increased during capacitation. To correct the IMP-free area, arithmetically redistributed (ARD)-IMP density was used for statistical analysis. In the PM group, the mean density +/- SD of large IMP was 379 +/- 64 and 266 +/- 58/microm2, and that of small IMP was 1450 +/- 155 and 672 +/- 252/microm2 in protoplasmic (P) and external (E) faces, respectively. During capacitation, the significant (P < 0.01) reduction of large IMP density was encountered only in the E face of a few incubation groups, while that of the small IMP density occurred in the P face by 2 h. Consequently, reduction of the total IMP density of both faces was not significant in the large IMP, but it was significant (P < 0.01) in the small IMP. One-fifth of the total small IMP density reduced by 2 h. Filipin-sterol complexes (FSC) were numerous in the PAPM, and FSC-free areas also increased during capacitation. The mechanism of IMP-free area formation and the behavior of the small IMP in the PAPM during capacitation were discussed in relation to membrane stability.     

Freeze-fracture studies of the developing cell surface. I. The plasmalemma of the corneal fibroblast

The freeze-fracture technique was used to study changes in the corneal fibroblast cell membrane during morphogenesis in chick embryos. Fibroblasts migrate into the acellular primary corneal stroma on day 6 of embryogenesis, moving between the orthogonal layers of collagen fibrils which serve as their substratum. Morphometric analysis of the intramembrane particles (IMP) reveals their concentration on the P face to decrease from 756 to 534/mum2 from day 6 to day 14. After day 14, fibroblast migration and cell division cease and the stroma condenses due to dehydration, so that by day 18 all of the layers of fibroblasts are extremely flattened and the cornea has taken on its mature, transparent form. The cell membranes of the terminally differentiated, highly compacted fibroblasts are rich in IMP (1,300/MUM2, P face). In seeking to relate the particle increase to cell differentiation, we analyzed synthetic events taking place at this time, but no correlation, we analyzed synthetic events taking place at this time, but no correlation with 25SO4 or proline-3H incorporation was found. The event which seems best correlated with the doubling of P face particles between days 15 and 18 is the dehydration and condensation of the stroma, an event which is associated with cessation of both cell division and migration. Thyroxine stimulates premature condensation of the stroma, whereas thiouracil delays condensation, but neither of these treatments affects IMP concentration. Interestingly, IMP concentration on the filopodia of migrating fibroblasts is similar to that on the cell bodies, suggesting that the new membrane has the same composition as the pre-existing membrane. Observations are also presented on tight and gap junctions between fibroblasts and on the relation of extracellular matrix to the outer etched surface of the fibroblast plasmalemma.     

Animal-vegetal polarity in the plasma membrane of a molluscan egg: a quantitative freeze-fracture study

Using freeze-fracture electron microscopy, the numerical particle distribution in the fertilized Nassarius egg plasma membrane has been analyzed in four areas at different positions along the animal-vegetal axis of the egg. These areas can be distinguished by distinct microvilli patterns and differences in microvilli densities. In all areas, more IMPs (intramembrane particles) are present on the P face than on the corresponding E face. The ratio of the number of IMPs present on E and P face is similar in all areas (0.48-0.55) except for the most animal part of the vegetal hemisphere, where relatively more IMPs remain attached to the exterior half of the fractured membrane (E/P ratio = 0.88). The IMP density at the vegetal pole of the egg is considerably higher than in the animal hemisphere and in the animal part of the vegetal hemisphere. This difference is due to an increased number of IMPs in all size classes (4-18 nm). In the area adjacent to the vegetal pole the density of particles is also higher than in the two more animal areas, but here the difference is exclusively due to the smaller IMP size classes (4-8 nm). Statistical analysis of our data reveals that the area adjacent to the vegetal pole patch is significantly different from the other areas with respect to the distribution of the IMPs over the different IMP size classes. These results demonstrate the polar organization of the Nassarius egg plasma membrane. The possible role of this surface heterogeneity in the spatial organization of the egg cell and the later embryo is discussed.     

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

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

Fine structure and freeze-etch study of the protoscolex tegument of Echinococcus multilocularis (cestoda)

Freeze-etch replicas of the protoscolex tegument of Echinococcus multilocularis were examined and compared with conventional thin sections by TEM. The microtopography of the protoscolex tegument was also examined by SEM. The protoscolex consisted of morphologically-distinct, apical and basal tegumentary regions, the latter of which lacked microtriches. The hook area of the apical region contained long, slender, filamentous microtriches that obscured the hook arrangement. These microtriches were structurally different from those found on the suckers and rostellum of the protoscolex. Freeze-etch replicas of the tegumental membrane of the sucker and rostellar microtriches showed that the protoplasmic (P) and exoplasmic (E) faces of the microthrix base and tip contained numerous intramembranous particles (IMP). The densities of the IMP on both the P and E faces of the microthrix tip were approximately twice the number of the larger diameter IMP found on the P and E faces of the microthrix base. No freeze-etch replicas of the microtriches from the hook area were obtained. The basal tegumentary region of the protoscolex consisted of irregularly-distributed, knoblike processes that were variable in size and shape, and contained an electron-dense cap. The IMP on the P face of the knoblike processes measured approximately the same diameter as those on the P face of the microthrix base. However, their density was about half that of the latter. The density of IMP on the E face of the knoblike processes could not be determined from the freeze-etch replicas.     

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.     

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.     

Ultrastructure of the Mesocarp of Mature Avocado Fruit and Changes Associated with Ripening

The mesocarp tissue of ripening avocado fruits was studied byfreeze fracture, thin section and scanning electron microscopy.Carbon dioxide and ethylene production by individual fruit weremonitored, and samples were analysed at several stages of theripening process. The tissue is composed primarily of large, isodiametric, lipid-containingparenchyma cells. At maturity these cells contain the normalcomplement of cell organelles, and all membranes appear intact.When ripening begins, several changes in the ultrastructureoccur. The most obvious changes are a loosening and eventualbreakdown of the cell wall, and swelling and vesiculation ofthe rough endoplasmic reticulum. In freeze fracture replicasa significant increase in the number of intramembranous particlesin the EF face of the plasmamembrane was observed at the climactericpeak. In post-climacteric, soft fruit the particle density of theEF face of the plasmamembrane decreased to the density observedin the membrane of pre-climacteric cells. All of the organellesand membranes appear whole and intact whether examined by thinsection, freeze fracture or scanning electron microscopy. However,the cell walls in post-climacteric fruit have almost completelydisappeared. These results indicated that the ripening process per se inavocados does not involve a complete loss of compartmentationnor a breakdown of organelle and membrane integrity. It may,however, lead to these or similar senescence changes as a resultof the loss of the cell walls. The variations in particle densityof the plasmamembrane during ripening may reflect one or moreof several structural, compositional, or functional membranephenomena, and this aspect of ripening warrants further study. Persea americana Mill., avocado pear, freeze fracture, fruit ripening, scanning electron microscopy, senescence, ultrastructure     

Concomitant immunity in a rodent model of filariasis: the infection of Meriones unguiculatus with Acanthocheilonema viteae

In an attempt to study the occurrence of concomitant immunity in filarial infections, jirds (Meriones unguiculatus) were experimentally infected with Acanthocheilonema viteae, and patent animals were superinfected with a defined dose of A. viteae stage 3 larvae (L3). Infected animals harbored significantly less worms deriving from the superinfection than the control group (P < 0.05, 56.2%, and 63.4% protection), as shown by analysis of female worms 6 wk after superinfection on the basis of their developmental status and their length. This protection was not due to contact with L3 antigens because a significant reduction of worm burdens deriving of a superinfection was also observed after subcutaneous implantation of a single female worm (P < 0.05, 40.2% and 64.9% protection). The induced protective responses target L3 and restrict their migration because an established infection resulted in a reduction of L3 recovery (95.6% and 94.3%, P < 0.001) from tissues of jirds at day 5 after superinfection. Other data show that L3 from a superinfection are trapped within eosinophil-rich granulomas, which is likely to create unfavorable conditions for the worms and to lead to later death. Taken together, established A. viteae-infections partially protect hosts against homologous superinfection by an immune-mediated mechanism and, thus, regulate the population density of the parasites within the host by concomitant immunity.     

Freeze-fracture studies on Pneumocystis carinii. I. Structural alteration of the pellicle during the development from trophozoite to cyst

Pneumocystis carinii has generally been distinguished in three developmental stages, namely, trophozoite, precyst and cyst. The fine structure of the pellicle--the plasma membrane and the outer layer existing outside this plasma membrane--of each stage was studied by freeze-fracture technique. By this technique, P. carinii was cleaved through the cytoplasm or through the hydrophobic region of the plasma membrane, and the cross-fractured face of the outer layer was revealed on the replicas. The outer layer, which is electron-dense in the thin section, consisted of numerous fine granules about 15 nm in diameter in freeze-fracture images, whereas the electron-lucent middle layer which appeared in the precyst and cyst was less granular. Measurement of the intramembranous particles (IMP) also was carried out. The number of IMP per square micrometer of the plasma membrane of the trophozoite was 1,512 +/- 125 on the P face and 417 +/- 44 on the E face. In the precyst, the IMP density decreased, and 1,037 +/- 56 on the P face and 262 +/- 22 on the E face. In the cyst, it further decreased, nd 875 +/- 59 and 150 +/- 20 respectively. It is generally assumed that the density of IMP is related to the physiological activity of the cell membrane, so that the present results obtained in P. carinii suggest that the trophozoite is the most active stage, and that metabolic activity of the pellicle gradually decreases with the progress of development to the precyst then to the cyst.     

Freeze-fracture study of the trophozoite and the cyst of Entamoeba histolytica

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/micron 2 on the P 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/micron 2 on the P face and averaging less than 1/micron 2 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.     

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.     

Interdigitating reticulum cells in dermatopathic lymphadenitis: freeze-fracture and ultrathin-section morphology

Dermatopathic lymphadenitis is a non-neoplastic lesion found with various chronic skin lesions and associated with hyperplasia of the thymus-dependent (T) areas. These areas consist chiefly of interdigitating reticulum cells (IDC's), which are known to be accessory cells for T-cell-dependent immune reactions. The most characteristic features of IDC's are the bizarre-shaped cell nucleus, numerous cytoplasmic processes, deep cytoplasmic invaginations, and a close topographical relationship to surrounding (T) lymphocytes. The cytoplasmic processes of IDC's do not interdigitate with adjacent lymphocytes, as previously reported in the literature, but show close interdigitations with the processes of neighboring IDC's. With the freeze-fracture technique it can be seen that IDC's exhibit a characteristic distribution of intramembranous particles (IMP). While, for example, macrophages, epithelioid cells and lymphocytes display a clearly greater number of IMP on the P face than on the E face, IDC's show an equally high particle density on both the P face and the E face. The organelle content of IDC's in dermatopathic lymphadenitis varies considerably. Tubular profiles, the Golgi apparatus and vesicles may be increased in number. Birbeck granules are also found in IDC's, but only rarely. Variations in the numbers of the different cytoplasmic organelles may be a reflection of varying degrees of metabolic activity of IDC's.     

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

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