Freeze-fracture study of Blastocystis hominis

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 (IMP's). On all membrane replicas, the concentration of IMP'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 IMP'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.     

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.     

William Trager: An Appreciation

SYNOPSIS. Additional information on host interactions with trypanosomatid membranes was obtained from studies of a monomorphic strain of Trypanosoma brucei harvested at peak parasitemia from intact and lethally irradiated rats. Pellets of trypanosomes were fixed briefly in glutaraldehyde and processed for thin section electron microscopy or freeze-cleave replicas. Observations of sectioned material facilitated orientation and comparison of details seen in replicas. Fracture faces of cell body and flagellar membranes as well as 3-dimensional views of the nuclear membrane were studied. Cell body membranes of 80% of the organisms from intact rats contained random arrays of intramembranous particles (IMP). Aggregated clusters of particles appeared on the fracture faces of 20% of the trypanosomes. Some of these membranes had nonrandomly distributed particles aligned in distinct rows on the outer fracture face of both cell body and flagellum. Many inner face fractures of the cell body membranes had a particle arrangement similar to the longitudinal alignment of cytoskeletal microtubules. No aggregated particle distribution was seen in membranes of trypanosomes harvested from lethally irradiated rats. Replicas of trypanosome pellets also had plasmanemes as a series of attached, empty, coated membrane vesicles. These structures were found in close association with, as well as widely separated from the parasites. The shedding of these vesicles and the variation of particles in cell body membranes are discussed in light of antibody-induced architectural and antigenic changes in surface properties of trypanosomatids. The convex face of the inner membrane of the nucleus also is covered with randomly arrayed particles. More IMP were observed on the inner than on the outer nuclear membranes. Images of nuclear pores were also seen. The importance of these structures in drug and developmental studies of trypanosomes is discussed. On fracture faces of the flagellar membrane there were miniature maculae adherentes, unique to the inner fracture face and occurring only at regions of membrane apposition between cell body and flagellum. Each cluster of particles exposed by the freeze-cleave method corresponds to an electron-dense plaque seen in thin section images. However, because of a unique fracture pattern, these plaques were not revealed on the apposing body membranes, as illustrated in thin sectioned organisms.     

Phagosome fusion vesicles of Paramecium. II. Freeze-fracture evidence for membrane replacement

Phagosome fusion vesicles (PFVs), a new population of relatively large granules in Paramecium caudatum which fuse with the first stage of digestive vacuoles (DV-I) shortly after these vacuoles are released from the cytopharynx (their site of formation), have been studied by using the freeze-fracture technique. Identification of PFVs is possible in the resulting replicas at all sites where they are commonly found in thin sections, at the cytopharynx, bound but not fused with nascent digestive vacuoles and fused with released vacuoles in the cell's posterior end. These PFVs have membranes which do not resemble the membranes of the forming digestive vacuole membrane or the discoidal vesicle membranes from which vacuole membrane is derived. Their smooth E-fracture face with only 50 to 100 intramembrane particles (IMPs) per micrometers 2 and particulate P-face (approximately 2500 IMPs/micrometers) do resemble the second vacuole stage (DV-II) which is characterized by a smaller diameter and acid pH. Evidence is presented for PFV fusion with the DV-I and for membrane replacement, at least in part, as the DV-I becomes a DV-II. Membrane replacement entails first adding PFVs to the DV-I and then removing the original discoidal vesicle-derived membrane as tubules as the vacuole condenses. Implications of the possible role of PFVs in forming intravacuolar symbiotic relationships are also discussed.     

The structure and function of the smooth septate junction in a transporting epithelium: The malpighian tubules of the new zealand glow-worm Arachnocampa luminosa

Junctional complexes between the epithelial cells in the four distinct regions of the glow-worm Malpighian tubule were investigated by electron microscopy using thin sectioning, freeze-fracturing, osmotic disruption and tracer techniques. The lateral plasma membranes of all four cell types are joined by smooth septate junctions but the extent of the complex across the cell depth varies in the four different regions. The width of the septa, the interseptal spacing and the separation between the outer leaflets of the adjacent plasma membranes are different for each cell type. Gap junctions were identified only in the junctional complex between Type IV cells and were intercalated amongst large lateral sinuses. In oblique sections of lanthanum infiltrated tissue, the electron-lucent septa at the basal side of the junction are outlined by the tracer as it penetrates. In the Junctional complexes of all four regions the septa appear as short, distinct, linear bars. In tangential sections of gap junctions between Type IV cells, the junctions appear as a hexagonal array of intermembrane particles with a centre to centre spacing of 18 nm. Horseradish peroxidase did not penetrate the junctional complexes very far but readily passed through the basal lamina into the spaces between extracellular invaginations of the basement membrane of the cells. Junctional complexes in all four areas of the tubule have similar freeze-fracture faces. In freeze-fracture replicas of fixed tissue continuous ridges of fused particles are seen on the P face and complementary furrows are found on the E face. Junctional response to osmotically adjusted Ringer solutions was similar in all four cell types. Distortion or ‘blistering’ of the intercellular space between the septa of the junction occurred when the tissue was bathed in or injected with a hypertonic Ringer solution. The structure of these junctions, visualized by the different techniques, and the role of the septate junction in a transporting epithelium, are discussed.     

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

The supramolecular organization of the vacuole membrane (orof the membranes of mucilage sacs) in 27 species of red algaeis studied in replicas of rapidly frozen and fractured cells.Intramembranous particle complexes composed of four particles('tetrads' with average diameters between 8·5 and 14·5have been observed in the protoplasmic fracture (PF) face butmost 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 orform clusters in several species and are randomly distributed.In the species Ceramium diaphanum var. strictum and Laurenciaobtusa the intramembranous particle complexes ('tetrads') havebeen observed both in the EF and PF faces of the vacuole membrane;the 'membrane tetrads' at least as regards these two speciesseem to span both the outer and inner leaflets of the vacuolemembrane ('transmembrane particles'). The occurrence of particletetrads in the plasma membrane is probably due to exocytosiseither of the Golgi vesicles or of the mucilage sacs. Tetradfrequency in the EF face of the vacuole membranes of the investigatedred algae varies between 2 and 87 µm^-2, while that ofsingle particles varies between 102 and 695 µm^-2. ThePF face of the vacuole membrane is characterized by a higherparticle density than the EF face. The particle densities ofthe PF and EF faces of the plasma membrane for a given speciesare higher than those of the corresponding fracture faces ofthe vacuole membrane. Some members of Bangiophycidae bear smallerprotein particles (diameter between 8·5 and 10·5nm) in comparison with those of Florideophycidae (diameter between10·5 and 14·5 nm). It is suggested, based uponthe particle tetrads lying in depressions of the vacuole membraneand the origin of vacuoles (mucilage sacs) from ER, that theparticle tetrads originate from the ER or the Golgi complex.Since vacuoles (mucilage sacs) in red algae, along with theGolgi complex, are involved in the synthesis and export of cellsurface polysaccharides, it could be assumed that the 'membrane-tetrads'within the vacuole membrane represent a membrane-bound multienzymecomplex, participating in the synthesis of amorphous extracellularmatrix polysaccharides.Copyright 1993, 1999 Academic Press Red algae, freeze-fracture, vacuole membrane, mucilage sacs, membrane tetrads, supramolecular organization     

Fine Structure of Hepatozoon mocassini (Apicomplexa,Eucoccidiorida) Gamonts and Modifications of the Infected Erythrocyte Plasmalemma1

Transmission electron microscopy and scanning electron microscopy were used to investigate the fine structure of Hepatozoon mocassini gamonts and modifications of the infected erythrocyte plasmalemma. Intraerythrocytic gamonts were contained within a parasitophorous vacuole. An electron-lucid space observed between the gamont pellicle and the membrane of the vacuole corresponded to the unstained space described in light microscopy studies. Gamonts possessed a conoid, polar ring, subpellicular microtubules, four pairs of rhoptries, micronemes, ovoid granules, mitochondria with tubular cristae, and a pellicle composed of three individual unit membranes. The conoid had an anterior diameter of 320 nm, a posterior diameter of 360 nm, and a length of 150 nm. In contrast to a report on Hepatozoon aegypti, no micropore or “canopy-like structure” was observed. The plasmalemma of infected erythrocytes exhibited two types of modifications: gross membrane deformations and knobs with an electron-dense central mass. These knobs are structurally distinct from previously described membrane excrescences.     

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.     

Morphological features of the surface of the sea urchin (Arbacia punctulata) egg: Oolemma-cortical granule association

Scanning microscopy and transmission electron microscopy of sectioned specimens and freeze-fracture replicas revealed the presence of slightly elevated regions, approximately one-fourth to one-half the diameter of microvilli, which were situated along the surface of unfertilized Arbacia eggs. These modifications of the surface of the egg were observed in areas occupied by cortical granules and were greatly reduced in number following the cortical granule reaction. Few such modifications were present in immature and urethane-treated ova, in which cortical granules were located in regions of the egg other than the cortex. Freeze-fracture replicas of unfertilized eggs revealed a significantly higher density of intramembranous particles within the plasmalemma when compared to replicas of the membrane surrounding cortical granules. Areas characteristic of the cortical granule membrane, i.e., sparsely laden with particles, were not observed within the plasmalemma of the fertilized egg. Hence, following its fusion with the egg plasma membrane there is a dramatic reorganization in particle distribution of the membrane derived from cortical granules.     

Phagocytosis of bacteria by polymorphonuclear leukocytes: a freeze-fracture, scanning electron microscope, and thin-section investigation of membrane structure

The changes in membrane structure of rabbit polymorphonuclear (PMN) leukocytes during bacterial phagocytosis was investigated with scanning electron microscope (SEM), thin-section, and freeze-fracture techniques. SEM observations of bacterial attachment sites showed the involvement of limited areas of PMN membrane surface (0.01-0.25μm(2)). Frequently, these areas of attachment were located on membrane extensions. The membrane extensions were present before, during, and after the engulfment of bacteria, but were diminished in size after bacterial engulfment. In general, the results obtained with SEM and thin-section techniques aided in the interpretation of the three-dimensional freeze-fracture replicas. Freeze-fracture results revealed the PMN leukocytes had two fracture faces as determined by the relative density of intramembranous particles (IMP). Membranous extensions of the plasma membrane, lysosomes, and phagocytic vacuoles contained IMP's with a distribution and density similar to those of the plasma membrane. During phagocytosis, IMPs within the plasma membrane did not undergo a massive aggregation. In fact, structural changes within the membranes were infrequent and localized to regions such as the attachment sites of bacteria, the fusion sites on the plasma membrane, and small scale changes in the phagocytic vacuole membrane during membrane fusion. During the formation of the phagocytic vacuole, the IMPs of the plasma membrane appeared to move in with the lipid bilayer while maintaining a distribution and density of IMPs similar to those of the plasma membranes. Occasionally, IMPs were aligned to linear arrays within phagocytic vacuole membranes. This alignment might be due to an interaction with linearly arranged motile structures on the side of the phagocytic vacuole membranes. IMP-free regions were observed after fusion of lysosomes with the phagocytic vacuoles or plasma membrane. These IMP-free areas probably represent sites where membrane fusion occurred between lysosomal membrane and phagocytic vacuole membrane or plasma membrane. Highly symmetrical patterns of IMPs were not observed during lysosomal membrane fusion.     

Membrane interactions between adjacent mucols secretion granules

In primate goblet cells, the membranes of adjacent mucous granules from contact areas which appear as extensive pentalaminar fusion sites in thin sections. In freeze-fracture replicas, the same membrane areas are smooth, except for a few 6-8-nm particles which adhere to the E face. These protein-poor membrane interaction sites are relatively long-lived, and it is proposed that further stimulus may be required to trigger membrane fission.     

Studies of Blastocystis hominis

Blastocystis hominis, grown in Boeck-Drbohlav culture medium, modified by the omission of rice starch and the addition of 20% human serum and mineral oil cover to the Locke's solution overlay, can assume 3 morphologic forms. In the absence of human serum the vacuolated form, which divides by binary fission predominates. In medium with high serum content the granular form appears, with 3 types of granules. Spheroid or more elongate cytoplasmic granules predominate. In older organisms, lipid granules are found either in the peripheral cytoplasm or in the central vacuolar space. In occasional cells, variable numbers of reproductive granules develop in the central vacuolar space. These latter granules are released from the organism and give rise to typical B. hominis cells. The 3rd form, the ameba form, appears in small numbers in older cultures and in those treated with antibiotics. Ameba forms feed on bacteria and have slow pseudopodial activity. Exposure to oxygen causes rapid damage to cell membrane, with resulting leakage and collapse.     

Membrane particle changes attending the acrosome reaction in guinea pig spermatozoa

To examine the freeze-fracture appearance of membrane alterations accompanying the preparation of sperm membranes for fusions-the first preparatory stage occurring before physiological release of the acrosomal content, the second afterward-we induced the acrosome reaction in capacitated guinea pig spermatozoa by adding calcium to the mixture. The most common features observed before fusion of the acrosomal and plasma membranes were the deletion of fibrillar intramembranous particles from the E-fracture faces of both membranes, and the clearance of globular particles from the P face of the plasma membrane-events taking place near the terminus of the equatorial segment. Large particles, >12nm, remained not far from the cleared E-face patches. The P face of the outer acrosomal membrane is virtually clear from the outset. In addition, when fusion was completed, occasional double lines of large particles transiently embossed the P face of the plasma membrane (postacrosomal) side of the fusion zone. Behind the line of fusion, another series of particle-cleared foci emerged. We interpreted these postfusion membrane clearances as a second adaptation for sperm-egg interaction. Induction of the acrosome reaction in media containing phosphatidylcholine liposomes resulted in their apparent attachment, incorporation, or exchange in both the originally and secondarily cleared regions. Our observations support the concepts that membranes become receptive to union at particle- deficient interfaces, and that the physiologically created barren areas in freeze-fracture replicas may herald incipient membrane fusion.     

Histochemical Detection of Carbohydrates of Blastocystis hominis

The carbohydrates of Blastocystis hominis were detected by histochemical techniques using light and electron microscopy. B. hominis, fixed with various fixatives, followed by treatment with detergents, were stained with periodic acid-Schiff (PAS) or alcian blue (AB). Intense PAS reactions were observed in cells fixed with glutaraldehyde or 1/2 Karnovsky fixative. The cells fixed with other fixatives showed weak or no reactions with PAS staining. Similar results were seen in the case of AB stain. These results indicated that, depending on the fixative used, B. hominis contained PAS- or AB-reactive carbohydrates. At the electron microscopic level, ultrathin sections of B. hominis were stained with periodic acid methenamine silver (PA-MS) or periodic acid thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining techniques. Intense, positive reactions with PA-MS or PA-TCH-SP were observed on the central vacuole, Golgi apparatus, and cytoplasmic vesicles. The filamentous layer showed moderate reactions with PA-MS, whereas in PA-TCH-SP stain, it was stained more densely. The staining intensity of the central vacuole varied from cell to cell. The presence of membrane fusions of the cytoplasmic vesicles with the central vacuole indicated the accumulation of carbohydrates in the central vacuole.     

Fine Structures Associated with Nutrition of the Intracellular Parasite Eimeria auburnensis*

SYNOPSIS. In the nearly mature macrogametes of Eimeria auburnensis, the cell membrane is a unit membrane, with underlying and overlying osmiophilic layers usually present. Cup-shaped micropores were occasionally seen. Smaller, V-shaped invaginations were also found in considerable numbers at the surface. At the deepest point, these invaginations were bounded only by a unit membrane. Immediately adjacent to this point, vesicles with homogenous electron-pale contents bounded by a similar unit membrane, were frequently seen. Pinocytosis evidently occurs at the site of these invaginations. Numerous folds of the host cell membrane bordering the vacuole in which the parasite lay extended about 0.1–0.7 μ into the vacuole. These “intravacuolar folds” varied in depth and number in different specimens. In some, the majority of folds had apparently become disconnected from the host cell membrane. A highly developed smooth endoplasmic reticulum occurred in the adjacent host cell cytoplasm. The intravacuolar folds may assist in transfer of nutrients, including membrane material, from the host cell to the parasite. The evidence indicates that in this species of Eimeria nutrients are taken into the parasite primarily as fluids by pinocytosis and possibly other processes.     

Electron-microscopic study of the nuclear membrane of the PKEV cells during mitosis. 1. The breakdown of the nuclear membrane (prophase, prometaphase, metaphase)]

An ultrastructural study on dividing PKEV cells provided a possibility to distinguish between certain stages of their desintegration. The changes preceding fragmentation of the nuclear envelope commence with desorganization of its structural components: vanishing of granular peripherial chromatin layer; appearance of the pores without central granules; formation of deep invaginations of the nuclear membranes. The desintegration of the nuclear envelope starts from the disapearance of many pores and the appearance of perforations almost of the same size. Simultaneously, the number of polysomes is reduced on the outer membrane of the nuclear envelope and in the cytoplasm. Specific features of the nuclear envelope being lost it becomes undistinguishable from the reticulum elements. On serial sections, no contacts were observed between chromosomes and membranous elements.     

The morphological features of Aegyptianella bacterifera: an intraerythrocytic rickettsia of frogs from Corsica

Study of ultrathin sections and freeze-fracture replicas of erythrocytes containing Aegyptianella bacterifera (Rickettsiales; Anaplasmataceae) revealed that the organisms develop within a membrane bound vacuole in the erythrocyte cytoplasm. The organisms divide by binary fission to produce about 12 rickettsiae in a mature inclusion. The organisms have gram-negative cell envelopes. The distribution of intramembranous particles within the outer and plasma membranes of A. bacterifera is similar to that of other rickettsiae and gram-negative bacteria such as Escherichia coli. The definition of the genus Aegyptianella should be broadened to include rickettsiae measuring up to 5 microns in length prior to division which develop within membrane bound inclusions in erythrocytes of their hosts.     

A novel model for fluid secretion by the trypanosomatid contractile vacuole apparatus

We have studied fluid secretion by the contractile vacuole apparatuss of the trypanosomatid flagellate Leptomonas collosoma with thin sections and freeze-fracture replicas of cells stabilized by ultrarapid freezing without prior fixation or cryoprotection. The ultrarapid freezing has revealed membrane specializations related to fluid segregation and transport as well as membrane rearrangements which may accompany water expulsion at systole. This osmoregulatory apparatu consists of the spongiome, the contractile vacuole, and the fluid discharge site. The coated tubules of the spongiome converge on the contractile vacuole from all directions. These 60- to 70-nm tubules contain characteristic double rows of 11-nm intramembrane particles in a helical configuration which fracture predominantly with the E face. Short double rows of similar particles are also frequently found on both faces of the contractile vacuole itself, in addition to many smaller particles on the P face. The spongiome tubules fuse with the vacuole during the filling stage of each cycle and then detach before secretion. The contractile vacuole membrane is permanently attached to the plasma membrane of the flagellar pocket by a dense adhesion plaque. In some ultrarapidly frozen cells, 20- to 40-nm perforations can be visualized within the plaque and the adjacent membranes during the presumptive time of discharge. The formation of the plaque perforations and the membrane channels occurs without fusion of the vacuole and the plasma membrane and does not require extracellular calcium. On the basis of our results, we have developed a model for water secretion which suggests that the adhesion plaque may induce pore formation in the adjoining lipid bilayers, thereby allowing bulk expulsion of the fluid.     

Alignment of cholesterol in the membrane bilayer

Freeze-fracture replicas of filipin-treated samples of guinea pig colon mucosa reveal areas in the membrane of the goblet cell granules labeled by filipin-cholesterol complexes (FCC) intermingled with regions patterned by "lines." The FCC and "lines" are arranged in an approximately rhombic pattern. Other membranes of the same cell or of other cells display either FCC only, aligned and occasionally ordered in "rhombs," "lines" only, with a similar pattern, or randomly distributed FCC. Optical diffraction was used to analyze and compare replicas of membranes with ordered FCC and "lines", as well as randomly distributed FCC. The results demonstrate that all these structures are reciprocally related through a common distribution pattern in the membrane. This observation supports the assumption that cholesterol has a preferential ordered distribution within the membrane bilayer.     

Strategies for maximizing ATP supply in the microsporidian Encephalitozoon cuniculi: direct binding of mitochondria to the parasitophorous vacuole and clustering of the mitochondrial porin VDAC

Microsporidia are obligate intracellular parasites with extremely reduced genomes and a dependence on host‐derived ATP. The microsporidium Encephalitozoon cuniculi proliferates within a membranous vacuole and we investigated how the ATP supply is optimized at the vacuole–host interface. Using spatial EM quantification (stereology), we found a single layer of mitochondria coating substantial proportions of the parasitophorous vacuole. Mitochondrial binding occurred preferentially over the vegetative ‘meront’ stages of the parasite, which bulged into the cytoplasm, thereby increasing the membrane surface available for mitochondrial interaction. In a broken cell system mitochondrial binding was maintained and was typified by electron dense structures (< 10 nm long) bridging between outer mitochondrial and vacuole membranes. In broken cells mitochondrial binding was sensitive to a range of protease treatments. The function of directly bound mitochondria, as measured by the membrane potential sensitive dye JC‐1, was indistinguishable from other mitochondria in the cell although there was a generalized depression of the membrane potential in infected cells. Finally, quantitative immuno‐EM revealed that the ATP‐delivering mitochondrial porin, VDAC, was concentrated atthe mitochondria‐vacuole interaction site. Thus E. cuniculi appears to maximize ATP supply by direct binding of mitochondria to the parasitophorous vacuole bringing this organelle within 0.020 microns of the growing vegetative form of the parasite. ATP‐delivery is further enhanced by clustering of ATP transporting porins in those regions of the outer mitochondrial membrane lying closest to the parasite.