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A violência doméstica e de género é um fenómeno transversal à sociedade e afeta um número significativo de mulheres, mostrando-se como um grave problema. O impacto e as consequências que esta violência causa na mulher e na sua vida familiar são profundas, sendo uma prioridade alterar pensamentos pré-concebidos, que podem comprometer as respostas das mais variadas entidades públicas e privadas. A presente dissertação tem como principal objetivo compreender crenças, atitudes e valores dos/as militares da GNR (Guarda Nacional Republicana) em relação à violência de género e perceber de que modo estas determinam a atuação face ao fenómeno. Para atingir esse objetivo foi constituída uma amostra, a nível Nacional, de 1872 militares da GNR, pertença dos 20 Comandos Territoriais (Portugal Continental e Ilhas). Optamos por uma metodologia quantitativa, com a construção e aplicação de um questionário online, no qual se fez uma adaptação de duas escalas já aplicadas e testadas anteriormente, a Escala de Crenças sobre a Violência Conjugal (ECVC), de Machado, Matos & Gonçalves (2000), e a Escala de Atitudes Policiais (EAP), de Gracia, García & Lila (2008). A primeira escala permitiu avaliar as crenças dos/as militares da GNR quanto aos fatores legitimadores da violência contra a mulher e a segunda escala possibilitou aferir a atitude dos/as militares da GNR face a determinadas situações de intervenção ou não intervenção, bem como, saber qual o grau de responsabilidade pessoal por essa decisão e o grau de gravidade percebida perante as situações apresentadas. Em termos gerais, pode-se concluir que os/as militares da GNR, na sua maioria, não expressam crenças legitimadoras de violência de género. Todavia, os resultados mostram-nos que existem ainda elementos desta força de segurança que manifestam uma visão estereotipada e simplista do fenómeno, nomeadamente pela externalização da culpa, o que pode levar a inadequada atuação perante ocorrências. Domestic and gender-based violence is a phenomenon that transcends society and a certain number of women and is a serious problem for society. The impact and consequences of violence on women and their lives are profound, and it is a difficult case to overcome the preconceptions which can compromise the responses of the most varied public and private entities. The main objective of this dissertation is to understand the beliefs, attitudes and values of the military of the GNR (National Republican Guard) in relation to gender violence and to understand how these determine the action against the phenomenon. To achieve this objective, a national sample of 1872 GNR military personnel, belonging to the 20 Territorial Commands (Continental Portugal and Islands), was constituted. We chose a quantitative methodology, with the construction and application of an online questionnaire, in which we adapted two scales already applied and previously tested; the Beliefs Scale on Conjugal Violence (ECVC), by Machado, Matos & Gonçalves (2000), and the Police Attitude Scale (EAP), by Gracia, García & Lila (2008). The first scale allowed us to evaluate the GNR military's beliefs about the legitimating factors of violence against women, and the second scale allowed us to assess the attitude of the military of the GNR facing certain situations of intervention or non intervention, as well as to know the degree of personal responsibility for this decision and the degree of seriousness perceived in the situations presented. In general terms, it can be concluded that the GNR military, for the most part, do not express legitimizing beliefs about gender violence. However, the results show that still existing elements of this security force that manifest a stereotyped and simplistic view of the phenomenon, namely by the externalization of guilt, which can lead to inadequate action against occurrences.

Immunogold labeling electron microscopy technique has been used to study the ultrastructural localization of oncogenic proteins: Mos, Met, Ski, and the tumor-associated protein, Muc1, as well as their relationship with other tumor-related proteins. By pre- and postembedding immunogold labeling electron microscopy techniques, we showed that the Mos protein pp39mos colocalized with microtubule bundles, suggesting that microtubulin or microtubule-associated protein(s) may be the substrate of Mos. Met protein was labeled at the microvilli of the lumen that are formed in cultured T47D cells, implying its potential involvement in lumen formation. Ski localization experiments revealed a unique globular structure “Ski body” that is present inside the nucleus of interphase chicken embryo fibroblast infected with Ski cDNA FB29 and FB2–29. Ski bodies were also found scattered in the cytoplasm of metaphase FB29 and FB2–29 Ski expressing chicken embryo fibroblasts. In T47D cells, tumor-associated protein Muc1 was associated with both the plasma membrane and the membranes of secretory vesicles in the cytoplasm. In MUC1 infected NIH3T3 cells, however, labeling showed that in addition to the plasma membrane and the membranes of secretory vesicles, some Muc1 gold spheres were seen inside the secretory vesicles, suggesting that the subcellular localization of the protein may vary in different cell types. © 1995 Wiley-Liss, Inc.

The 67 kDa laminin receptor (67LR), one of several cell surface laminin-binding proteins, is involved in the interactions between cancer cells and laminin during tumor invasion and metastasis. A 37 kDa polypeptide (37LRP), previously identified as the 67LR precursor, is abundantly present in the cytoplasm and has been implicated in polysome formation. To better understand the cellular localization of the 67LR and its precursor, transmission electron microscopic studies of human melanoma A2058 cells were carried out using immunogold labeling and a variety of antibodies: (a) affinity purified antibodies directed against 37LRP cDNA-derived synthetic peptides; (b) anti-67LR monoclonal antibodies raised against intact human small cell lung carcinoma cells; and (c) monoclonal antibodies against the subunits of the integrin laminin receptor, alpha 6 beta 1. Double-labeling immunocyto-chemistry revealed that anti-67LR monoclonal antibodies as well as anti-37 LRP antibodies recognized antigens that were localized in the cytoplasm in electron dense structures. As expected, cell membrane labeling was also observed. Surprisingly, alpha 6 and beta 1 integrin subunits were detected in the same cytoplasmic structures positive for the 67LR and the 37LRP. After addition of soluble laminin to A2058 cells in suspension, the number of labeled cytoplasmic structures increased especially in the vicinity of the plasma membrane, and were exported onto the cell surface. Neither fibronectin nor BSA induced such an effect. The data demonstrate that the 67 LR and the 37 LRP antibodies detect colocalized antigens that are in cytoplasmic structures with alpha 6 beta 1 integrin.(ABSTRACT TRUNCATED AT 250 WORDS)

Endotoxins (lipopolysaccharides, LPS) are surface components of gram-negative bacteria that stimulate macrophage activation and cause endotoxic shock. How LPS is recognized by host cells is still an open question, but it is generally accepted that many effects of endotoxins follow the overproduction of cytokines by macrophages. In the present study, we used fracture-flip and immunolabeling to study the morphology of isolated commercial LPS (C-LPS), the endotoxin release from the bacterial wall in presence of serum (S-LPS), and the distribution of these two endotoxins on the macrophage surface. Cells treated with C-LPS exhibited large LPS aggregates bound to smooth and particulate areas of the membrane and to microvilli. In contrast, macrophages incubated with S-LPS showed a uniform monodispersed labeling over the free surface of the membrane. Our results show that fracture-flip provides high-resolution images of the binding of ligands to the cell surface. They also suggest the importance of using highly dispersed LPS suspensions when the mechanisms of cell activation and damage by endotoxins are studied.

To assess the density and distribution of native and recombinant GABAA receptors we used label-fracture and fracture-flip technologies combined with immunocytochemistry using monoclonal and polyclonal Abs directed against the extracellular domain of the GABAA receptor protein located in the freeze-fracture replicas. In cortical neurons there is a high density of GABAA receptors on both soma and dendrites with some areas were the density of receptors is higher, but there are no well defined clusters. In cerebellar granule cells most of the receptors are distributed in round clusters both in neurites and soma. In astroglial cells the receptor density is lower than in neurons and only occasionally they appear in clusters. In cells transfected with cDNAs encoding for various molecular forms of GABAA receptor subunits, the receptor density is moderate when cDNAs for alpha, beta and gamma subunits are cotransfected; however, on cells cotransfected with cDNAs for beta and gamma subunits the receptor density is significantly lower. Recombinant receptors appear randomly distributed and occasionally they aggregate in small groups.

We propose the use of fracture-flip combined with Triton X-100 extraction to visualize the cytoplasmic surface of plasma membranes. Unfixed human erythrocytes were freeze-fractured, carbon-cast, and thawed. The carbon casts, along with attached freeze-fractured erythrocytes, were treated with 2% Triton X- 100 to solubilize unfractured plasma membranes and to release haemoglobin. After repeated washing, the carbon-casts, along with attached protoplasmic and exoplasmic membrane halves, were picked on grids, flipped, and Pt-shadowed. Our method leads to extended views of the cytoplasmic surface revealing the fibrilar network that laminates the inner surface of the erythrocyte membrane. Spectrin immunogold labelling of fractured, carbon cast erythrocytes shows that the colloidal gold particles are associated with the fibrilar network at the cytoplasmic surface. Removal of membrane skeletal elements including spectrin by treatment with a low ionic strength buffer containing EDTA leads to loss of the network and reveals globular particles on the cytoplasmic surface of the membrane. These globular particles contained band 3, as shown by immunogold labelling. Our method can be extended to both the ultrastructural observation and the cytochemistry of the cytoplasmic surfaces of other biomembranes.

S05-01 Polycomb repressive complexes are required to maintain compact chromatin structure at Hox loci Ragnhild Eskeland, Duncan Sproul, Graeme Grimes, Shelagh Boyle, Martin Lieb, Clemence Kress, Nick Gilbert, Helle F. Jorgensen, Amanda G. Fisher, Arthur I. Skoultchi, Anton Wutz, Wendy A. Bickmore 1 University of Edinburgh, Edinburgh, United Kingdom 2 Research Institute of Molecular Pathology, Vienna, Austria 3 MRC Clinical Sciences Centre, London, United Kingdom 4 Albert Einstein College of Medicine, Bronx, United States

L. donovani promastigotes were subjected to heat treatment yielding an axenic amastigote stage which was long-term cultured at 37 °C. No differences were observed between the growth rates of axenic amastigotes and promastigotes. Flow cytometry-derived DNA histograms of axenic amastigotes and promastigotes were typical of exponentially growing cell populations. Moreover, axenic amastigotes were metabolically active as evidenced by the release of an immunoprecipitable extracellular acid phosphatase (SAcP) into their culture supernatant. Cell transformation was confirmed by transmission electronmicroscopic examination of thin sections and extended by fracture-flip survey which allowed differentiation of cell membranes. The ultrastructure and nanoanatomy of axenic amastigotes was identical to that of intracellular amastigotes. The production of large amounts of heat-shock axenic amastigotes suitable for biochemical and biological studies of differentiation in Leishmania donovani may have important implications in the development of prevention and/or treatment strategies.

Freeze-fracture electron microscopy reveals that intramembrane particles are concentrated in a band encircling the posterior portion of the acrosome of Strongylocentrotus purpuratus sperm. Two colloidal gold labeling methods, label-fracture and replica-staining fracture-flip, were employed to show that the plant lectin wheat germ agglutinin, which recognizes a 210 kDa sperm surface glycoprotein, binds to this localized band of intramembrane particles. Monoclonal antibody J18/2, which also recognizes the 210 kDa surface glycoprotein, shows this localized binding in approximately 20% of the sperm observed in this study. The majority of sperm displayed a uniform distribution of receptor sites for monoclonal antibody J18/2. Since wheat germ agglutinin and monoclonal antibody J18/2 are known to agglutinate Strongylocentrotus purpuratus sperm but not sperm of another sea urchin, Lytechinus pictus, similar determinations were made for the latter species. Lytechinus pictus sperm are not labeled with wheat germ agglutinin and are only sparsely labeled with monoclonal antibody J18/2. The acrosomal localizations of wheat germ agglutinin and monoclonal antibody J18/2 receptors in Strongylocentrotus purpuratus sperm are consistent with the involvement of the 210 kDa surface glycoprotein in an egg jelly-induced sperm acrosome reaction. Low-temperature post-embed labeling of thin sections with wheat germ agglutinin and monoclonal antibody J18/2 show concentrations of label within the acrosomal vesicle of Strongylocentrotus purpuratus sperm, suggesting the presence of an intracellular storage site for the 210 kDa glycoprotein.

The expression of the human immunodeficiency virus type 1 mRNAs containing the Rev-responsive element is regulated at the posttranscriptional level by the viral Rev protein. Rev increases the nucleocytoplasmic export of these mRNAs, leading to high expression. Using in situ hybridization and electron microscopy, we investigated the localization of a subgenomic gag mRNA in the absence and presence of Rev. In addition to the previously shown cytoplasmic accumulation of the Rev-dependent mRNA, we observed that in the presence of Rev the nuclear gag mRNA accumulates nonrandomly and forms specific localization patterns at the nuclear membrane and in the nucleoplasm. Cellular mRNAs for β-actin and glyceraldehyde-3-phosphate dehydrogenase were not found to form such patterns. These data suggest that Rev leads the gag mRNA to specific subnuclear locations, which further supports the transport function of Rev.

Bacterial endotoxins (lipopolysaccharides or LPS) are active components of Gramnegative bacteria that act on numerous cellular functions through the processes of cell activation and damage. The molecular mechanisms involved in the “endotoxic phenomenon” are not defined yet, although extensive studies have been carried out. Immunogold and electron microscopy (EM) have contributed to identify the primary target cells of endotoxins and the subcellular systems that receive the direct action of these bacterial agents. Here, we review our studies on immunogold detection of endotoxins in cellular and subcellular systems. The analysis of the interaction between endotoxins and cells was focussed on the following aspects: (1) morphological characteristics of the LPS aqueous suspensions used in experimental work; (2) binding of endotoxins to the plasma membrane of type II pneumocytes and alveolar macrophages (two of their cellular targets), and influence of the state of aggregation of the LPS; (3) movement and distribution of endotoxins inside the cell, from the plasma membrane to the nucleoplasm; and (4) interaction of LPS with microtubules and its effects on the integrity of the microtubular network. These approaches provide information at the molecular level as well as data for the establishment of physiological models of endotoxicity. © 1995 Wiley-Liss, Inc.

Coexpression of the human Met receptor and its ligand, hepatocyte growth factor/scatter factor (HGF/SF), in NIH 3T3 fibroblasts causes the cells to become tumorigenic in nude mice. The resultant tumors display lumen-like morphology, contain carcinoma-like focal areas with intercellular junctions resembling desmosomes, and coexpress epithelial (cytokeratin) and mesenchymal (vimentin) cytoskeletal markers. The tumor cells also display enhanced expression of desmosomal and tight-junction proteins. The apparent mesenchymal to epithelial conversion of the tumor cells mimics the conversion that occurs during embryonic kidney development, suggesting that Met-HGF/SF signaling plays a role in this process as well as in tumors that express both epithelial and mesenchymal markers.

Label-fracture and immunogold fracture-flip techniques are used to address at the ultrastructural level the dynamics of viral and cellular transmembrane proteins during the budding of Sindbis virus on the plasma membrane of infected cells. Immunolabeling with anti-Sindbis spike antibodies shows that the viral proteins are mostly in clusters, all associated with budding viruses. Ultrastructural observation of the unlabeled freeze-fractured plasma membranes shows that membrane particles aggregate over the budding viruses. These results indicate that the concentration of viral transmembrane proteins gives rise to a parallel concentration of membrane particles. Immunolabeling with anti-CD8 antibodies of cells expressing by transfection the CD8 transmembrane protein and infected with Sindbis virus shows absence of labeling on the particle aggregates over the forming virions. These findings indicate the exclusion of CD8 proteins from the portions of the membrane where budding occurs.

The surface ultrastructure of lymphocytes during capping of two transmembrane proteins is shown. As seen by fracture-flip the plasma membranes of human lymphocytes are covered by a high density of surface particles. Incubation in 30% glycerol leads to aggregation of these surface particles. Immunogold labelling shows that the transmembrane proteins bearing HLA class I and CD4 antigens are confined to the particle aggregates. These results indicate that surface particles revealed by fracture-flip represent surface protrusions of integral membrane proteins seen as intramembrane particles in freeze-fractured lymphocytes. During capping HLA or CD4 antigens aggregate into progressively larger patches and, finally, into single caps. As revealed by fracture-flip the patches/caps are seen as clearly differentiated raised platforms that are clearly and sharply demarcated relative to contiguous areas of the surface. In non-patched (non-capped) regions, the pattern of distribution and apparent density of surface particles remain unaltered. Immunogold labelling clearly demarcates patches and caps, and shows that virtually no antigen molecules remain dispersed over the non-patched (non-capped) regions. Estimates of the surface density of either HLA or CD4 antigens over the capped areas point to high planar concentrations of the transmembrane proteins that bear these antigens.

Cytochemical labeling of intracellular components presupposes direct access to the cell interior. Traditionally, this access is rendered possible through sectioning of tissues or of cell pellets. Sectioning can be performed in fresh tissues, in polymer-embedded specimens, as well as in frozen sections. Alternative, although less conventional, methods do not involve cutting of the tissue but, instead, cleavage or fracture of previously frozen tissues.

Membrane events during milk fat secretion were analyzed by freeze-fracture of the rat mammary gland. Two modes of milk fat secretion were observed: extrusion of fat droplets surrounded by a portion of the apical plasma membrane of the alveolar epithelial cells and, less frequently, release into the alveolar lumen of fat droplets contained in intracytoplasmic vacuoles. The extrusion process consists of two asynchronous events: clearing of membrane particles (probably including integral membrane proteins) and bulging of the apical plasma membrane. Most fat droplets are extruded with a bilayer membrane envelope (milk fat globule membrane) partially devoid of particles. The segregation of membrane particles may represent the onset of a process of structural degradation of the milk fat globule membrane.

Freeze-fracture techniques were used to study the ultrastructural distribution of plasma membrane particles in cultures of normal Balb/c and Swiss 3T3 and simian virus 40- or murine sarcoma virus-transformed fibroblasts. No apparent differences were observed. Cultures fixed in situ show a seemingly random distribution of membrane particles both in normal or in transformed cells. Treatment of cell cultures in situ with concanavalin A does not result in an altered pattern of particle distribution. EDTA-induced suspension of normal or transformed cells does not result, per se, in modification of the type of membrane particle distribution seen in cells fixed in situ. However, upon further incubation, a proportion of normal or transformed cells in suspension show a varying degree of particle aggregation following a network pattern. Concanavalin A treatment of normal and transformed cells in suspension does not result in a specific change of the pattern of particle distribution. Because it has been established that treatment with concanavalin A of simian virus 40-transformed Balb/c 3T3 fibroblasts causes pronounced clustering of the concanavalin A receptors at the outer-surface, our results probably imply independence of membrane particles and concanavalin A receptors of these transformed cells.

The freeze-fracture morphology of cellular membranes of HK9 Entamoeba histolytica trophozoites, a pathogenic strain, is reported. Structural complexity of the E. histolytica membranes is suggested by differences in size of membrane particles (particles and subparticles), the presence of short rodlike arrays, higher than average density of particles over certain regions of the plasma membrane and, possibly, composite particles. Incubation of live cells in 25% glycerol results in complete coaggregation of particles and subparticles into a single random network. The results are discussed in relation to current interpretations of the structure and dynamics of biological membranes.

We used fracture-label and surface labeling techniques to characterize the distribution and topology of wheat germ agglutinin (WGA) receptors in the plasma membrane of boar sperm heads. We show that freeze-fracture results in preferential, but not exclusive, partition of WGA-binding sites with the outer (exoplasmic) half of the plasma membrane. Labeling of the inner (protoplasmic) half of the membrane is significant, and is denser over the areas that overlie the acrosome. Exoplasmic membrane halves are uniformly labeled. Analysis of freeze-fracture replicas revealed that the distribution of intramembrane particles over protoplasmic faces parallels that of WGA-binding sites as observed by fracture-label. Coating of intact spermatozoa with cationized ferritin results in drastic reduction of the labeling of both protoplasmic and exoplasmic membrane halves. Labeling of sperm cells lysed by short hypotonic shock fails to reveal the presence of WGA-binding sites at the inner surface of the plasma membrane. We conclude that: (a) all WGA-binding glycoconjugates are exposed at the outer surface of the membrane; (b) some of these glycoconjugates correspond to transmembrane glycoproteins that, on fracture, partition with the inner half of the membrane; (c) these transmembrane proteins are accumulated in the region of the plasma membrane that overlies the acrosome; and (d) parallel distribution of intramembrane particles and WGA-binding glycoproteins provides renewed support for the view of particles as the morphological counterpart of integral membrane proteins.

We show that “fracture—permeation”—a method developed to assess the compactness of the cytoplasm—can establish the distribution of intermolecular spaces in the sarcomeres of glutaraldehyde-fixed striated muscle. As examples of striated muscle, we use sartorius muscle from toad (Bufo marinus) and papillary cardiac muscle from the left ventricle of Sprague—Dawley rats. Tissues were fixed in glutaraldehyde, frozen, cross-fractured in liquid nitrogen, and thawed. Tissue fragments were immersed in concentrated solutions of native ferritin (30% w/v). Random fractures gave ferritin direct access to all regions of the sarcomere. We show that a sequence of four qualitatively distinct patterns of ferritin distribution is associated to a progressive sequence of sarcomere lengths. The correspondence between sarcomere lengths and patterns of ferritin permeation permits the recognition of sarcomeres in rigor (no penetration of ferritin), stretched (penetration into the I band and H zone), or at rest length (penetration restricted to the I band). Similar patterns of ferritin permeation can also be recognized in oblique sections. In cardiac muscle, ferritin permeates into the A bands of contracted sarcomeres and shows an increase in disorder within the filament lattice during contraction. The patterns of ferritin permeation observed in sarcomeres in rigor, stretched, or at rest length are those predicted from changes in filament overlapping proposed by Huxley's “sliding filament theory of muscle contraction.” Our results illustrate the power of fracture-permeation to investigate intermolecular distances in cytoplasmic matrices.

We have developed a method—“fracture-permeation”—to assess the compactness of the cytoplasm in glutaraldehyde-fixed cells. Cells or tissues are fixed in glutaraldehyde, impregnated with glycerol, and frozen in liquid nitrogen. The cells are freeze-fractured, thawed, deglycerinated, and immersed in concentrated solutions of globular proteins. In initial experiments, we used native ferritin (NF) to permeate model matrices made of bovine serum albumin (BSA). We show that permeation depends on the concentraion of proteins within the cross-linked matrix: NF permeates matrices made from 10 or 15% (w/v) BSA solutions but do not permeate matrices made from solutions with 20% (w/v) protein or more. With freeze-fractured cells, ferritin molecules were unable to permeate the cross-linked cytoplasm of fungal zoospores and cysts, used here as examples of resting cells. In human lymphocytes from peripheral blood, permeation of ferritin was limited or absent, but it became massive in cells activated by phytohemagglutinin. Massive permeation of ferritin was also observed within the cytoplasmic matrix of other active cells (fungal sporangia, germinating cysts). In the examined resting cells, glutaraldehyde crosslinks the proteins into a dense matrix that effectively excludes ferritin (diameter 12 nm). These findings cannot be explained by models that envisage all cytoplasmic proteins congregated into a single-phase microtrabecular lattice with the nature and dimensions proposed by Porter and co-workers. We conclude that compactness of the cytoplasm matrix depends on the physiological state of the cell: It varies through differentiation and is related to the degree of cellular activity.

We report here rapid assembly of gap junctions in prostate epithelial cells in vitro. Assembly of gap junctions can be induced by incubation at 0 degrees C followed by incubation at 37 degrees C. Colchicine (10(-5) M, 10(-3) M) and cytochalasin B (25 micrograms/ml), 100 micrograms/ml) at room temperature or at 37 degrees C also induce assembly of gap junctions. Assembly of the junctions proceeds even in the presence of a metabolic inhibitor (dinitrophenol) or of an inhibitor of protein synthesis (cycloheximide). We conclude that assembly of gap junctions can proceed from a pool of pre-existing precursors. The experimental conditions that result in gap-junction assembly involve perturbation of the cytoskeleton. Therefore, we propose that the assembly of gap junctions requires convergent migration of precursor molecules whose positional control in the membrane is released by perturbation of the cytoskeleton. Aggregates of particles and rugosities, whose distribution size and shape is similar to that of gap junctions, may represent intermediate assembly stages. This would indicate that the final stages in the assembly take place only after convergence of the precursor molecules to the junctional site and involve profound conformational changes required for establishment of fully assembled connexons.

We have used fracture-flip to produce new, macromolecular-resolution images of the surface of boar spermatozoa. Over the head, acrosomal and postacrosomal regions display sharply demarcated, subtly different surface textures. The rim is particle-poor, as well as a region above the oblique cords over the posterior ring. The tail shows two morphologically distinct domains: (1) the principal piece is covered by a high density of parallel-helical strands and a high density of large globular particles; (2) the midpiece and the neck are covered by smaller particles with apparent random distribution. Rectangular surface specializations frequently seen near the annulus display a waffle-like texture. With the notable exception of the parallel-helical strands of the principal piece the fracture-flip images of the boar spermatozoon can be directly related to the freeze-fracture morphology of its plasma membrane.

Applications of the new fracture-labeling techniques for the observation of cytochemical labels on platinum-carbon replicas are described. Frozen cells, embedded in a cross-linked protein matrix, and frozen tissues are fractured with a scalpel under liquid nitrogen, thawed, labeled, dehydrated by the critical point drying method, and replicated. This method allows direct, high-resolution, two-dimensional chemical and immunological characterization of the cellular membranes in situ, as well as detection of sites within cross-fractured cytoplasm and extracellular matrix.

Interpretation of freeze-fracture and thin-section results shows that fusion of the peripheral vesicle with the plasmalemma of a Phytophthora palmivora zoospore occurs at several discrete sites and results in the formation and expansion of a particle-free bilayer membrane diaphragm and in the appearance of a polymorphic network of membrane-bounded tunnels, the lumina of which are continuous with the cytoplasm. The outer half of the bilayer membrane diaphragm appears continuous with the outer half of the plasma membrane; the inner half of the bilayer membrane diaphragm with the inner half of the peripheral vesicle membrane; and the inner half of the plasmalemma with the outer half of the peripheral vesicle membrane. Interpretation of our results leads us to formulate a hypothesis for a sequence of several intermediate stages involved in membrane fusion. The initial fusion event is viewed as a local catastrophe (Thom, R. 1972. Stabilité Structurelle et Morphogenèse. W. A. Benjamin Inc., Reading, Mass.) involving the sudden reorganization of apposed elements of the inner half of the plasmalemma and the outer half of the peripheral vesicle membrane. Fusion of apposed components at the rim of the perimeter of fusion results in the formation of a toroid hemi-micelle which provides continuity between the inner half of the plasmalemma and the outer half of the peripheral vesicle membrane. Simultaneously, apposed components at the site of fusion may reorganize into an inverted membrane micelle. A bilayer membrane diaphragm is then formed by apposition and flowing of components form the outer half of the plasmalemma and the inner (exoplasmic) half of the peripheral vesicle membrane. The existence of large areas of membrane contact before fusion may lead to several fusion events and the formation of a polymorphic network of membrane-bound tunnels.

The acrosome is a large secretory vesicle of the sperm head that carries enzymes responsible for the digestion of the oocyte's investments. The event leads to sperm penetration and allows fertilization to occur. Release of the acrosomal enzymes is mediated by the interaction between sperm acrosomal and plasma membranes (acrosome reaction). Biochemical characterization of the acrosomal membrane has been restrained by a lack of methods to isolate uncontaminated fractions of the membrane. Here, we use new methods to expose the membrane to in situ cytochemical labeling by lectin-gold complexes. We study the topology and relative density of glycoconjugates both across and along the plane of the acrosomal membrane of boar sperm. Detachment of the plasma membrane from glutaraldehyde-fixed cells exposed the cytoplasmic surface of the acrosome to the lectin markers; freeze-fractured halves of the acrosomal membrane were marked by "fracture-label" (Aguas, A. P., and P. Pinto da Silva, 1983, J. Cell Biol. 97:1356-1364). We show that the cytoplasmic surface of the intact acrosome is devoid of binding sites for both concanavalin A (Con A) and wheat germ agglutinin (WGA). By contrast, it contains a high density of neuraminidase-resistant anionic sites detected by cationic ferritin. On freeze-fractured sperm, the receptors for Con A partitioned with the exoplasmic membrane half of the acrosomal membrane. The Con A-binding glycoconjugates were accumulated on the equatorial segment of the membrane. A low density of WGA receptors, as well as of intramembrane particles, was found on the freeze-fracture halves of the acrosomal membrane. The plasma membrane displayed, in the same preparations, a high density of receptors for both Con A and WGA. We conclude that the acrosome is limited by a membrane poor in glycoconjugates, which are exclusively exposed on the exoplasmic side of the bilayer. Regionalization of Con A receptors on the acrosome shows that sperm intracellular membranes, like the sperm surface, express domain distribution of glycocomponents.

I will describe four approaches that combine cytochemistry with freeze-fracture: 1) FREEZE-ETCHING; 2) FRACTURE-LABEL; 3) FRACTURE-PERMEATION; and 4) LABEL-FRACTURE. These techniques, in particular fracture-label, involve delicate points of interpretation and numerous validating controls. In the publications listed at the end, these issues have been addressed in detail.1. FREEZE-ETCHING. I developed freeze-etching as a cytochemical approach to prove that membranes were split by freeze-fracture and to show that biological membranes were comprised of a bilayer membrane continuum interrupted by integral membrane proteins.1 - 4 In freeze-etching, the distribution of the marker over the membrane surface exposed by sublimation is compared to that of the intramembrane particles exposed by fracture. It is often required to aggregate the particles into domains larger than the labeling molecules (Fig. 1). This, and the need for freezing in distilled water, severely limits the application of freeze-etching.

We have analyzed previous thin-section and freeze-fracture observations of the tight junction. We propose that the tight-junction strands represent intramembranous, cylindrical, inverted micelles. At the junctional site, the exoplasmic halves of the plasma membranes are fused into a continuous leaflet. Therefore, topologically and structurally the tight junction is viewed as the outcome of a process of linear fusion between the plasma membranes of epithelial cells. The extracellular spaces delimited by the junction are separated by two distinct exoplasmic membrane halves and the cylindrical micelles. Junctional stability, fostered by the environmental symmetry of the cytoplasmic milieux of contiguous cells, may be maintained by transmembrane integral proteins at the junctional site, interacting at the cytoplasmic surface with cytoskeletal components.

Summary: Zoospores of Phytophthora palmivora were induced to encyst synchronously and changes in morphology, particularly in surface structure, were examined by freeze-fracture and freeze-etching techniques. A uniform ‘fuzzy coat’, 20 to 25 nm thick, covers the plasma membrane. This hitherto unknown structure surrounds the entire zoospore body and flagella. No structural correspondence between cyst wall microfibrils and plasma membrane particles was found. The pattern of distribution of plasma membrane particles remains unchanged during encystment.

We introduce replica-staining label-fracture, a method for the cytochemical mapping of membrane surfaces. This method is a corollary of the rationale of label-fracture (Pinto da Silva and Kan, 1984: J Cell Biol 99:1156). After freeze-fracture the exoplasmic halves of the membrane remain attached to the replica. We show that cytochemical labeling of cell surfaces can be performed by direct post-fracture staining of freeze-fracture replicas. This new variant of label-fracture leads to miniaturization of labeling procedures and allows standardization of labeling conditions and simultaneous processing of different specimens.

We used immunogold label-fracture to follow the migration of HLA I class and HLA II class antigens during capping as induced by specific monoclonal antibodies. Capping is achieved through a process of clustering and "consolidation" of clusters into larger patches and, finally, a single cap. All receptors appear to cluster from the very start, with no "stray" molecules joining already formed patches. Characterization of exoplasmic and protoplasmic fracture-faces of capping cells fails to reveal any corresponding accumulation of intramembrane particles and/or subtler rugosities. Our results are consistent with the concepts that view the migration of capping molecules as contemporaneous with the efflux of noncapping integral membrane proteins.

Membrane halves of boar sperm flagella were produced by freeze-fracture and labeled in situ with concanavalin A and wheat germ agglutinin; the lectins were visualized with protein-gold complexes. Concanavalin A and wheat germ agglutinin binding sites partition with both protoplasmic and exoplasmic halves of the membrane. A high density of lectin marking was found on protoplasmic membrane halves; we conclude that the label corresponds to transmembrane glycoproteins that, on freeze-fracture, are dragged across the outer (exoplasmic) half of the phospholipid bilayer. Our demonstration of numerous transmembrane proteins in sperm flagella offers the structural setting for previous models on flagellar surface motility that postulate accessibility of motile membrane components to the submembranous cytoskeleton.

This paper demonstrates the translational movement along the plane of the human erythrocyte ghost of the membrane particles exposed by freeze-fracture. The membrane particles can be aggregated by incubation of the ghosts in media with a pH in the vicinity of 5 5 or 3 5. The particles are disaggregated in neutral and alkaline media (pH 9 5) and also at pH 4.5 Aggregation of the particles at pH 5.5 is reversible, prevented by prefixation in glutaraldehyde and by media of high ionic strength. Particle aggregation occurs within 2–4 min. These results are consistent with the concept that the erythrocyte ghost membrane is a planar fluid domain formed by a bilayer membrane continuum which is interrupted by localized, yet mobile, proteic intercalations.

The freeze-etch technique was used to observe red blood cell ghosts labeled on both surfaces with covalently bound ferritin. Ferritin molecules were never observed on fracture faces, thus indicating that fracture does not show membrane-surface detail. Subliming away the surrounding ice did expose the ferritin on the membrane surface. These results were consistent with the concept that membranes split during the fracture process of freeze-etching.

Treatment of living entamoeba histolytica cells with low concentrations of concanavalin A (con A) and peroxidase results in redistribution of the plasma membrane con A receptors to one pole of the cell where a morphologically distinct region--the uroid--is formed. Capping of con A receptors is not accompanied by parallel accumulation of ruthenium red-stainable components. In capped cells, the pattern of distribution of acidic sites ionized at pH 1.8 (labeled by colloidal iron) at the outer surface and of membrane particles (integral membrane components revealed by freeze-fracture) is not altered over the uroid region. Cytochemistry of substrate-attached microexudate located in regions adjacent to E. histolytica cells demonstrates the presence of con A binding sites and ruthenium red- and alcian blue-stainable components and the absent of colloidal iron binding sites. In a previous report we demonstrated that glycerol-induced aggregation of the plasma membrane particles is accompanied by a discontinuous distribution of colloidal iron binding sites, while con A receptors and acidic sites ionized at pH 4.0 remain uniformly distributed over the cell surface. Taken together, our experiments show that, in E. histolytica cells, peripheral membrane components may move independently of integral components and, also, that certain surface determinants may redistribute independently of others. These results point to the complexity of the membrane structure-cell surface relationship in E. histolytica plasma membranes relative to the membrane of the erythrocyte ghost where integral components (the membrane-intercalated particles) contain all antigens, receptors, and anionic sites labeled so far. We conclude that fluidity of integral membrane components (integral membrane fluidity) cannot be inferred from the demonstration of the mobility of surface components nor, conversely, can the fluidity of peripheral membrane components (peripheral membrane fluidity) be assumed from demonstration of the mobility of integral membrane components.

THE extent to which the distribution of antigens and other receptors at the cell surface is determined by structural components within the plasma membrane is largely unknown. In erythrocyte ghost membranes, freeze-fracture and freezeetch studies have demonstrated that structural components, ‘membrane intercalated particles’, are the exclusive sites at the surface which bear A and B antigens1,2, wheat germ agglutinin3, influenza virus3, con A receptors (Pinto da Silva and Nicolson, unpublished), and anionic sites4. It is clear, however, that conclusions derived from the study of this membrane system cannot be freely extrapolated to living cells where the relationship cell surface-membrane structure may be more complex. We report a study of this relationship on the plasma membrane of living Entamoeba histolytica cells in conditions which induce a redistribution of membrane intercalated particles, anionic sites or con A receptors.

A method--"fracture label"--is described for the cytochemical labeling of the membrane faces produced by freeze-fracture. Human erythrocytes embedded in a crosslinked matrix are frozen, fractured in liquid nitrogen, thawed, labeled, and cut into thin sections. Electron microscope observation of the fracture faces shows preferential partition of concanavalin A binding sites with the inner half of the membrane. This signifies that, during freeze-fracture, binding sites are dragged from the outer surface across the outer ("exoplasmic") half of the membrane and retained on the protoplasmic fracture face (face P). The fracture process results in exposure of new anionic sites on face P. Fracture-label can be applied to the cytochemical characterization of the cellular components exposed by freeze-fracture of isolated cells and tissues.

We report here the macromolecular dynamics of the cell surface of rat alveolar macrophages during spreading on a substratum, a process that involves the formation of numerous coated pits. We used ‘fracture-flip’ to prepare high-resolution platinum-shadowed replicas of membrane surfaces. Our observations show the following sequence of events associated with coated pit formation: at 4 degree C the cell surface of macrophages is covered with a moderate density of particulate components, with most ranging from 10 to 25 nm in diameter. These particles appear to be randomly distributed over the cell surface. Incubation of adherent cells at 37 degree C for 15 min results in the formation of large loose clusters (area 0.5-4 micron2) of particles on the adherent surfaces. After incubation of macrophages for 30 min at 37 degree C, these clusters become tighter and eventually form circular depressions (200–300 nm in diameter), which we interpret as part of a process of invagination. After 60 min, the depressions become much steeper. At this time surface particles can be observed on the intervening non-invaginated regions, and the peripheral region of the adherent membrane, as well as the free membrane. Fracture-flip reveals the presence of structures undetected in previous electron-microscopic studies and provides ultrastructural evidence for the clustering of surface macromolecules that is involved in the formation of coated pits.

Publisher Summary The apical plasma membrane of secretory cells undergoes considerable transformations during release of the products of secretion. Secretory vesicles derived from Golgi cisternae contact and fuse with the apical plasma membrane with formation of a small bilayer diaphragm, which separates the vacuolar content from the acinar lumen of the gland. Rupture of this transient diaphragm results in the release of the secretory products and incorporation of the secretory vesicle membrane in the apical segment of the plasma membrane. This process has been investigated in different types of cells. In the lactating cells of the mammary alveolus, the spectrum of transformations observed at the apical surface is wider. In addition to the release of proteins and other secretory products by exocytosis, other events occur at the apical membrane during the secretion of fat droplets. Milk fat droplets are released into the alveolar lumen surrounded by a membrane envelope derived from the cell membrane. The process results in the exportation of plasma membrane, which can be easily isolated from milk. This membrane system represents a useful model to study the dynamics of the apical surface of a secretory cell, as well as other membrane events of wider biological significance. The observation of lactating tissues with freeze fracture techniques provides imaging of the process and allows the analysis of the structural rearrangements of these membranes.

We used thin-section fracture-label to determine the distribution of wheat-germ agglutinin binding sites in intracellular membranes of secretory and nonsecretory rat tissues as well as in human leukocytes. In all cases, analysis of the distribution of wheat germ agglutinin led to the definition of two endomembrane compartments: one, characterized by absence of the label, includes the membranes of mitochondria and peroxisomes as well as those of the endoplasmic reticulum and nuclear envelope; the other, strongly labeled, comprises the membrane of lysosomes, phagocytic vacuoles, and secretory granules, as well as the plasma membrane. The Golgi apparatus was weakly labeled in all studied tissues.

The organization of the secretory epithelium of the rat ventral prostate as seen by freeze-fracture is analyzed. Ultrastructural interpretation of the tall columnar epithelial cell is facilitated by the axial regionalization of the cytoplasm. The endoplasmic reticulum, which exists in all the regions of the cell, appears to have a role in the maintenance of cellular organization. Its cisternae delimit the spaces for the nucleus, the Golgi, and the apical secretory apparatus. Elements involved in the formation of secretory products for export are located within the apical region of the cell and are regionally separated from the Golgi complex. Structural and functional specializations of the plasma membrane in the apical, lateral, and basal areas are illustrated. Artifacts related to specimen preparation procedures are reported. Displacement of membrane particles, which we observed in the fracture faces of plasma and intracellular membranes, appears related to fixation at low temperature. Fixation at 37 degrees C, which does not induce membrane-particle displacement, results in proliferation of tight-junctional elements.