Distribution of Intramembrane Particles and Its Changes during the Acrosome Reaction in Spermatozoa of the Japanese Abalone

The distribution of intramembrane particles in the plasma and acrosomal membranes of sperm of the Japanese abalone, Haliotis discus , and its changes during the acrosome reaction were studied by the freeze-fracture replica technique. The P face of the plasma membrane covering the acrosome has sparse membrane particles except in the apical region, which includes the trigger and 'truncated cone' regions. Large particles with an average diameter of 10 nm are located in this apical region. The E face of the plasma membrane has only a few particles. On the outer acrosomal membrane, many particles are randomly distributed throughout the P face, but only a small number of particles are found on the E face. Numerous particles on the P face of the inner acrosomal membrane show a regular arrangement as a dense lattice or with a concentric circular pattern. The initial change in the acrosome reaction is clearance of membrane particles from both the P and E faces of the plasma and outer acrosomal membranes around the apical region, where fusion of the two membranes occurs. As the acrosomal process elongates, the dense arrangement of particles on the inner acrosomal membrane changes via a loose lattice arrangement to a patchy distribution with particle-free areas. Then the arrangement is further disorganized becoming a sparse, random distribution.     

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.     

Membrane assembly in retinal photoreceptors I. Freeze-fracture analysis of cytoplasmic vesicles in relationship to disc assembly

To study precursor-product relationships between cytoplasmic membranes of the inner segment of photoreceptors and the continually renewed outer disc membrane, we have compared the density and size distribution of intramembrane particles (IMP) in various membrane compartments of freeze-fractured photoreceptor inner and outer segments. Both rod and cone outer segments of Xenopus laevis are characterized by a relatively uniform distribution of approximately 4,400-4,700 IMP/micron2 in P-face (PF) leaflets of disc membranes. A similar distribution of IMP is found in the outer segment plasma membrane, the ciliary plasma membrane, and in the plasma membrane of the inner segment in the immediate periciliary region. In each case the size distribution of IMP can be characterized as unimodal with a mean diameter of approximately 10 nm. PF leaflets of endoplasmic reticulum, Golgi complex, and vesicles near the cilium have IMP with a size distribution like that in the cilium and outer segment, but with an average density of approximately 2,000/micron2. In contrast, IMP are smaller in average size (approximately 7.5 nm) in PF leaflets of inner segment plasma membrane, exclusive of the periciliary rgion. The similarity of size distribution of IMP in inner segment cytoplasmic membranes and those within the plasmalemma of the cilium and outer segment suggest a precursor-product relationship between the two systems. The structure of the vesicle-rich periciliary region and the segregation of IMP with different size distributions in this region suggest that components destined for incorporation into the outer segment exist as preformed membrane packages (vesicles) which fuse with the inner segment plasma membrane in the periciliary region. Subsequently, membrane components may be transferred to forming discs of the outer segment via the ciliary plasma membrane.     

Ultrastructure of ascospore delimitation in freeze substituted samples ofAscodesmis nigricans (Pezizales)

Summary Freeze substitution proved to be a valuable technique for studying the early stages of ascosporogenesis inAscodesmis nigricans. Our observations indicate that the ascus vesicle originated from the ascus plasma membrane. Invaginations of the plasma membrane produced ascus vesicle initials consisting of two closely spaced unit membranes. The appearance of the outer leaflet of each of these membranes was identical to that of the inner leaflet of the ascus plasma membrane. Apparent points of continuity between ascus vesicle initials and the plasma membrane were observed. Ascus vesicle initials accumulated in the ascus cytoplasm near the plasma membrane and then coalesced to form the ascus vesicle, a peripheral, cylinder-like structure consisting of two closely spaced unit membranes that extended from the ascus apex to the ascus base. The ascus vesicle then became invaginated in a number of regions and subsequently gave rise to eight sheet-like segments, or ascosporedelimiting membranes, that encircled uninucleate segments of cytoplasm forming ascospore initials. Like the ascus vesicle, each ascospore-delimiting membrane consisted of two closely spaced unit membranes, the inner of which became the ascospore plasma membrane. The ascospore wall then developed between the spore plasma membrane and the outer membrane. Many details of ascospore maturation were clearly visible in freeze substituted samples.     

REGULAR STRUCTURES IN MEMBRANES : I. Membranes in the Endocytic Complex of Ileal Epithelial Cells

An "apical endocytic complex" in the ileal lining cells of suckling rats is described. The complex consists of a continuous network of membrane-limited tubules which originate as invaginations of the apical plasma membrane at the base of the microvilli, some associated vesicles, and a giant vacuole. The lumenal surface of this tubular network of membranes and associated vesicles is covered with a regular repeating particulate structure. The repeating unit is an ~7.5-nm diameter particle which has a distinct subunit structure composed of possibly nine smaller particles each ~3 nm in diameter. The ~7.5-nm diameter particles are joined together with a center-to-center separation of ~15 nm to form long rows. These linear aggregates, when arranged laterally, give rise to several square and oblique two-dimensional lattice arrangements of the particles which cover the surface of the membrane. Whether a square or oblique lattice is generated depends on the center-to-center separation of the rows and on the relative displacement of the particles in adjacent rows. Four membrane faces are revealed by fracturing frozen membranes of the apical tubules and vesicles: two complementary inner membrane faces exposed by the fracturing process and the lumenal and cytoplasmic membrane surfaces revealed by etching. The outer membrane face reveals a distinct array of membrane particles. This array also sometimes can be seen on the outer (B) fracture face and is sometimes faintly visible on the inner (A) fracture face. Combined data from sectioned, negatively stained, and freeze-etched preparations indicate that this regular particulate structure is a specialization that is primarily localized in the outer half of the membrane mainly in the outer leaflet.     

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.     

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.     

Ultrastructural changes in the spermatid nuclear envelope of Periplaneta americana during spermiogenesis

The nuclear envelope in the earliest stage of spermatid development possesses double membranes with pores aggregated in certain areas and arranged in a hexagonal pattern. The convex face of the outer nuclear membrane is relatively smooth and the convex face of the inner nuclear membrane carries many particles which are arranged in net-like pattern. In the later developmental stages, nuclear pores were not observed, the convex face of the inner nuclear membrane being covered with densely packed particles and the convex face of the outer nuclear membrane having a rough appearance. During the final stage of spermatid development, the cross-fractured face of the nucleus reveals the profiles of the nuclear fibres, the granular appearance of the convex face of the outer nuclear membrane, and the convex face of the inner nuclear membrane carrying more densely packed particles.     

Freeze-fracture electron microscopic analysis of ultrarapidly frozen envelope membranes on intact chloroplasts and after purification

Summary Freeze-fracture electron microscopy of ultrarapidly frozen intact pea chloroplasts has been used to characterize the supramolecular architecture of their outer and inner envelope membranes, to follow changes in these membranes caused by experimental treatments, and to identify the composition of purified envelope membrane subfractions. Examination of intact chloroplasts revealed that the two membranes exhibit dramatically different densities of intramembrane particles, with the inner membrane particle density approximately fourfold that of the outer. Analysis of purified envelope membrane subfractions indicates that the low bouyant density fraction (1.08 g/cm³) corresponds to the outer envelope membrane, whereas the relatively higher bouyant density fraction (1.13 g/cm³) is predominantly inner membrane. From qualitative and quantitative morphological data we conclude that the outer membrane subfraction is pure whereas the inner membrane subfraction is significantly contaminated by outer membrane. These results confirm conclusions reached from biochemical analysis of these membranes.During the course of the studies on intact chloroplasts, sites were observed where the outer and inner envelope membranes appear to adhere to each other (contact sites). Some of the contact sites observed on intact chloroplasts survived the envelope purification procedures as evidenced by their presence on a small number of vesicles in inner membrane preparations. The practical significance of these putative contact sites is discussed.     

Fine structure of the chloroplast membranes ofEuglena gracilis as revealed by freeze-cleaving and deep-etching techniques

Summary The chloroplasts ofEuglena gracilis have been examined by freeze-cleaving and deep-etching techniques.The two chloroplast envelope membranes exhibit distinct fracture faces which do not resemble any of the thylakoid fracture faces.Freeze-cleaved thylakoid membranes reveal four split inner faces. Two of these faces correspond to stacked membrane regions, and two to unstacked regions. Analysis of particle sizes on the exposed faces has revealed certain differences from other chloroplast systems, which are discussed. Thylakoid membranes inEuglena are shown to reveal a constant number of particles per unit area (based on the total particle number for both complementary faces) whether they are stacked or unstacked.Deep-etchedEuglena thylakoid membranes show two additional faces, which correspond to true inner and outer thylakoid surfaces. Both of these surfaces carry very uniform populations of particles. Those on the external surface (the A surface) are round and possess a diameter of approximately 9.5 nm. Those on the inner surface (the D surface) appear rectangular (as paired subunits) and measure approximately 10 nm in width and 18 nm in length. Distribution counts of particles show that the number of particles per unit area revealed by freeze-cleaving within the thylakoid membrane approximates closely the number of particles exposed on the external thylakoid surface (the A surface) by deep-etching. The possible significance of this correlation is discussed. The distribution of rectangular particles on the inner surface of the thylakoid sac (D surface) seems to be the same in both stacked and unstacked membrane regions. We have found no correlation between the D surface particles and any clearly defined population of particles on internal, freeze-cleaved membrane faces. These and other observations suggest that stacked and unstacked membranes are similar, if not identical in internal structure.     

Topographical relations of intramembrane particle distribution patterns in human sperm membranes

The distribution of intramembrane particles in human sperm membranes has been explored with particular reference to the topographical region of the sperm cell and the membranes' fracture face. Conspicuous differences in the size, arrangement, density, and lateral mobility of intramembrane particles between some topographically distinct membrane domains are demonstrated. The greatest regionality is exhibited by the plasma membrane. In sperm head regions, it shows a significant variability and changes its particle distribution during culture in capacitating medium. In contrast, little variability and no changes during the incubation are seen in the acrosomal and nuclear membranes. Striking is the difference in particle distribution on the E face of the outer acrosomal membrane between the acrosomal and equatorial regions. It is suggested that the invariable regional difference in the organization of the outer acrosomal membrane may bear on the different behavior of its two main domains during sperm capacitation and acrosome reaction.     

Sulfonolipids are localized in the outer membrane of the gliding bacterium Cytophaga johnsonae

Earlier work in our laboratory demonstrated that gliding bacteria of the Cytophaga-Flexibacter group contain, in their cell envelopes, large quantities of unusual sulfonolipids (N-fatty acyl 2-amino-3-hydroxyisoheptadecane-1-sulfonic acids). Recently, it has been shown that these lipids are necessary for the gliding motility of C. johnsonae. As one approach to determining the role of the lipids in motility, methods have now been developed for separating the inner (cytoplasmic) and outer membranes of a strain (ATCC 43786) of this Gram-negative bacterium. Sulfonolipid is at least five times as abundant in the outer membrane as in the inner. The inner membrane has properties similar to those found for other Gram-negative bacteria; it has a buoyant density of 1.14 g/ml and is highly enriched in cytochromes and succinate dehydrogenase. The outer membrane (1.18 g/ml) is enriched in bound carbohydrate and sulfonolipid, but contains little or no 2-keto-3-deoxyoctonate (such as is found in the enterobacteria). The localization of the sulfonolipids in the outer membrane permits focus on the possible roles these unusual substances may play in gliding motility.Abbreviations used IM inner membrane - OM outer membrane - KDO 2-keto-3-deoxyoctonate - EDTA ethylenediaminetetraacetic acid - SDH succinate dehydrogenase     

Identification of the initial binding sites of alphas2-casein f(183-207) and effect on bacterial membranes and cell morphology

The aim of this work was to identify the initial binding sites to the bacterial membranes of the antimicrobial peptide alphas2-casein f(183-207) and also to acquire further insight into membrane permeabilization of this peptide. Furthermore, cell morphology was studied by transmission electron microscopy. In all the experiments, bovine LFcin was employed as a comparison. Results showed that initial binding sites of alphas2-casein f(183-207) peptide were lipoteichoic acid in Gram-positive bacteria and lipopolysaccharide in Gram-negative. The peptide was able to permeabilize the outer and inner membranes. Moreover, the alphas2-casein peptide f(183-207) generated pores in the outer membrane of Gram-negative bacteria and in the cell wall of Gram-positive bacteria. In the Gram-negative bacteria, f(183-207) originated cytoplasm condensation, and in the Gram-positive bacteria the cytoplasmic content leaked into the extracellular medium. Furthermore, the experiments of inner and outer membrane permeabilization performed with LFcin-B showed that this peptide also has the ability to permeabilize both the inner and outer membranes.     

FREEZE-FRACTURE STUDY OF THE SINGLE MEMBRANE BETWEEN HOST CELL AND ENDOCYTOBIONT IN THE DINOFLAGELLATES GLENODINIUM FOLIACEUM AND PERIDINIUM BALTICUM1

The dinoflagellates Glenodinium foliaceum Stein and Peridinium balticum (Levander) Lemmermann harbor a chrysophytic endocytobiont which is bounded by only a single membrane. This unique membrane is of particular interest because it could correspond to an intermediate stage in the evolution of “complex” plastids found in many Plastids of this type are surrounded by three or membranes instead of the usual two. With freeze-fracture techniques, we show that the single membrane in P. balticum has a pronounced polarity with respect to the distribution of intramembrane particles (IMPs) on the two corresponding fracture faces. The inner face exhibited more IMPs than the outer. We suggest that this stdedness identifies the separating membrane as the plasma membrane of the endocytobiont. A symbiontophoric vacuole with a separate membrane apparently is lacking. In the endocytobiosis of G. foliaccum, the single membrane separating host and endocylobiont exhibits a symmetrical particle partition. Nevertheless, from the size distribution of the IMPs it appears likely that this membrane, too, corresponds to the plasma membrane of the symbiont.     

Identification of the initial binding sites of αs2-casein f(183-207) and effect on bacterial membranes and cell morphology

The aim of this work was to identify the initial binding sites to the bacterial membranes of the antimicrobial peptide α_s2-casein f(183-207) and also to acquire further insight into membrane permeabilization of this peptide. Furthermore, cell morphology was studied by transmission electron microscopy. In all the experiments, bovine LFcin was employed as a comparison. Results showed that initial binding sites of α_s2-casein f(183-207) peptide were lipoteichoic acid in Gram-positive bacteria and lipopolysaccharide in Gram-negative. The peptide was able to permeabilize the outer and inner membranes. Moreover, the α_s2-casein peptide f(183-207) generated pores in the outer membrane of Gram-negative bacteria and in the cell wall of Gram-positive bacteria. In the Gram-negative bacteria, f(183-207) originated cytoplasm condensation, and in the Gram-positive bacteria the cytoplasmic content leaked into the extracellular medium. Furthermore, the experiments of inner and outer membrane permeabilization performed with LFcin-B showed that this peptide also has the ability to permeabilize both the inner and outer membranes.     

Structure of rough,smooth, stripped and reconstituted rough membranes derived from rat liver as visualized by the freeze fracture technique

The structure of purified fractions of rough, smooth, stripped rough and reconstituted rough membranes have been investigated by the freeze etching technique. Preparations of rough and reconstituted rough membranes, active in protein synthesis, show vesicles whose outer surface is covered with ribosome-like particles. The inner surface of these vesicles contains also numerous particles of the same size. The particles located on the outer surface are largely absent in the stripped rough membrane preparations which, however, retain the particles located on the inner face. Particles were not seen either on the outer nor on the inner face of the smooth membranes. The possibility is considered that the particles located on the inner face are specific to the rough membranes and might play a role in the specific binding of ribosomes to the membranes.     

Freeze fracture studies on the annelid septate junction

Summary Freeze-fractured preparations of septate junctions between epidermal cells of annelids (Lumbricus terrestris and Tubifex spec.) have been investigated. In Lumbricus the protoplasmic face (PF) of the plasma membrane is characterized by variously arranged rows of particles. Apically the rows take an undulating course and often are separated by wide distances. In the basal part of the junction the rows run closely together and more or less in parallel. The diameter of the particles measures 80–120 Å, the distance between two particles (centre to centre) is 150–250 Å. Additionally striking rows of large particles (long diameter 150–200 Å). Are to be observed mainly near the basal part of the junction. In Tubifex both faces of the plasma membrane could be studied in detail. The protoplasmic face (PF) contains rows of distinct individual particles (mean diameter 100–150 Å, centre to centre distance approx. 250 Å) whereas the particles of the extracellular face (EF, mean diameter 200–250 Å) usually form continuous strands in which the individual particles seem to fuse. The density of arrangement of the strands varies considerably. Additionally ladder-shaped membrane structures have been observed in plasma membranes of this species.     

The morphology and configurational states of isolated heavy beef heart mitochondria by the freeze fracture technique

Configurational changes of glutaraldehyde fixed heavy beef heart mitochondria are confirmed using the freeze fracture technique. Large amplitude swelling occurred after unfixed mitochondria were suspended in 30% glycerol. Fine structure of the outer and inner mitochondrial membranes is described using unfixed heavy beef heart mitochondria by the freeze fracture technique. The matrix side of the inner membrane appears to be covered with 90 Å particles while the opposite side (cytochromec side) is also particulate covered by a high density of lower profile particles with a smooth underlying mosaic layer beneath. The outer surface of the outer membrane is smooth with particles embedded within the membrane. Possible structure of the membrane is discussed.     

Structural Changes in Membranes of Synchronized Cells demonstrated by Freeze-cleavage

FREEZE-CLEAVAGE is a new technique for studying the ultra-structure· of biological membranes, which fractures cell membranes in half, exposing two intramembranous fracture faces^1–3: the outer fracture face (OFF) and the inner fracture face (IFF). These fracture faces are partially covered with 70 Å globular particles which are thought to be unique structural components of cell membranes, formed by the association of membrane glycoproteins and lipids⁴. The 70 Å particles are dynamic structures and rapidly increase in density in the membranes of lymphocytes following exposure to mitogenic plant proteins (Scott and Marchesi, unpublished work).     

Freeze-fracture studies of Cryptosporidium muris.

The attachment site of Cryptosporidium muris to host cells was investigated using the freeze-fracture method. Cryptosporidium muris was enveloped by a double membrane of host plasma membrane origin, which formed the parasitophorous vacuole. The outer membrane of the double membrane was continuous with the host plasma membrane at the dense band, while the inner membrane was connected with the anterior part of the parasite plasma membrane at the annular ring. The density of intramembranous particles (IMP) was dramatically altered at the above two junctures. The outer parasitophorous membrane showed low IMP-density as compared to the host plasma membrane, although both membranes were continuous. The inner parasitophorous membrane had few IMP, whereas the parasite plasma membrane showed numerous IMP. When the attachment sites of parasites and host cells were fractured, circular-shaped fractured faces were observed on both sites of the parasite and host cell. These exposed faces corresponded to the dense bands and were very similar in size in each parasite.