The fracture-flip technique reveals new structural features of the Escherichia coli cell wall.

With few exceptions, all bacteria possess a wall which protects them and controls their communication with the environment. In Gram-negative bacteria the cell wall exhibits a complex and unique multilayered organization. We have applied a modification of the freeze-fracture technique known as 'fracture-flip' to visualize the real surfaces of the different wall layers in a Gram-negative bacterium, Escherichia coli. In combination with treatments to weaken the interlayer connections, this technique has provided new insights into the structure of the bacterial wall. Large areas of an intermediate layer (most probably the peptidoglycan-containing matrix) have been visualized for the first time between the plasma membrane and the outer membrane of the wall. Extensive regions corresponding to the cytoplasmic face of the plasma membrane have also been obtained. These images provide new three-dimensional views of the bacterial cell wall and provide the structural framework for the analysis of the molecular relationships between the different cell wall components.

Posttranscriptional regulation by Rev protein of human immunodeficiency virus type 1 results in nonrandom nuclear localization of gag mRNA.

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 beta-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.

Intracellular transport of the murine leukemia virus during acute infection of NIH 3T3 cells: nuclear import of nucleocapsid protein and integrase.

The entry and intracellular transport of Moloney-murine leukemia virions inside mouse NIH 3T3 cells have been followed by electron microscopy techniques. Five viral proteins--matrix (MA, p15), capsid (CA, p30), nucleocapsid (NC, p10), integrase (IN), and the envelope glycoprotein (SU, gp70)--were located by immunolabeling using gold probes. After entering the cells, viral particles were frequently detected inside cytoplasmic vesicles of variable size. Their viral envelope was apparently lost during intracytoplasmic transport. When the unenveloped viral cores reached the nuclear membrane or its vicinity, they were disrupted. Two of the immunolabeled proteins, NC and IN, were detected entering the nucleus of non-dividing cells, where both were targeted to the nucleolus. However, MA and CA were found only in the cytoplasm. NC is a nucleic acid-binding protein which contains potential nuclear localization signals. We suggest that NC could enter the nucleus as part of a nucleoprotein complex, associated with IN, and possibly, also with viral DNA.

Cellular functions during activation and damage by pathogens: immunogold studies of the interaction of bacterial endotoxins with target cells.

Bacterial endotoxins (lipopolysaccharides or LPS) are active components of Gram-negative 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.

Immunogold labeling of oncogenic and tumor related proteins.

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.

Protein synthesis is required for laminin-induced expression of the 67-kDa laminin receptor and its 37-kDa precursor.

The high affinity 67-kDa laminin receptor (67LR) is highly expressed in metastatically active human cancers. A 37-kDa polypeptide has been identified as its precursor (37LRP). Antibodies raised against 37LRP-derived synthetic peptides were used in immunogold electron microscopy and immunoblot studies to assess the effect of laminin on expression of the 67LR and the 37LRP. Laminin (15 micrograms/ml) treatment of suspended A2058 human melanoma cells doubled the expression of both 37LRP and the 67LR. Fibronectin had no effect. There was no effect of laminin on the expression of actin or galectin-3. Cycloheximide treatment of cells prior to laminin abrogated its inducible effect. The results suggest that binding of laminin by cell surface laminin receptors induces synthesis of the 37LRP and mature 67LR, with a consequent delivery to the cell surface of more laminin binding proteins for potentiated attachment of the melanoma cell to the basement membrane during invasion and metastasis.

Overexpression of mos oncogene product in Swiss 3T3 cells induces apoptosis preferentially during S-phase.

When Swiss 3T3 cells are acutely infected with Moloney murine sarcoma virus containing the v-mos oncogene, 90% of the cells round up and detach from the monolayer (floating cells) and express high levels of v-Mos. The majority of the floating cells are generated between 30 and 70 h post infection when the cellular level of Mos reaches approximately 0.1% of the total protein. Seventy percent of the floating cells exclude trypan blue but are growth arrested with 2C or 4C DNA content, whereas the remaining floating cells with < 2C DNA content, are dead or dying, and show characteristic apoptotic phenotypes. The apoptotic cells are most likely generated from cells in S-phase since these cells are absent from the viable floating cell population and the percentage of cells with < 2C DNA approximated the expected S-phase fraction of logarithmically growing cells. In addition, 5'-bromo-2'-deoxyuridine-labeling studies showed that approximately 50% of the floating cells with typical apoptotic phenotypes were metabolically-labelled with the drug. These analyses show that cell populations in different stages of the cell cycle are differently affected by high levels of v-Mos expression and cells in S-phase appear to be uniquely sensitive and undergo apoptosis.

Leishmania major: association of the differentially expressed gene B protein and the surface lipophosphoglycan as revealed by membrane capping.

The lipophosphoglycan (LPG) of Leishmania promastigotes forms a dense glycocalyx which effectively covers the entire surface of the cell, and which undergoes structural modifications during the differentiation of promastigotes to the infective or metacyclic stage. Recently, the first protein marker for metacyclic promastigotes of Leishmania major has been characterized. This protein, termed gene B protein, is located on the cell surface, yet it lacks any hydrophobic sequence for membrane attachment. It does contain an unusual amino acid repeat that is related to the peptidoglycan binding domain of protein A from Staphylococcus aureus, suggesting that the protein might interact with metacyclic LPG via this domain for attachment to the cell. We have studied the distribution of LPG, gene B protein, and the major surface protease, gp63, by labeling them with immunogold or immunofluorescence prior to and during capping events. Thin sections of double-labeled parasites revealed that the gene B protein-gold particles were colocalized with the LPG-gold particles in the LPG capping structures at the extremities of the cell. Cocapping of LPG and gene B protein was also observed with two-color fluorescence. No similar redistribution was seen in gp63 or with integral membrane proteins. In contrast to the gene B protein, gp63 could only be immunogold labeled on the metacyclic surface after capping and shedding of the LPG, providing further that it and other membrane-associated proteins are normally buried under the LPG coat. The unusual surface exposure of the gene B protein is consistent with its hydrophilic and LPG binding properties, which allow it to become incorporated into the cell coat and to localize to the most external aspects of the cell.

Cell localization and redistribution of the 67 kD laminin receptor and alpha 6 beta 1 integrin subunits in response to laminin stimulation: an immunogold electron microscopy study.

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)

Type II pneumocytes revisited: intracellular membranous systems, surface characteristics, and lamellar body secretion.

BACKGROUND: Type II pneumocytes, the producers of pulmonary surfactant, have been extensively studied during the last 20 years because of the importance of their metabolism in lung function and integrity. The ultrastructural studies of the 1970s and 1980s have shown that these cells present unique elements. EXPERIMENTAL DESIGN: In this work, we used thin-section, freeze-fracture, and fracture-flip electron microscopy techniques to obtain new information on the ultrastructural peculiarities of isolated rat type II pneumocytes, focusing our study on the intracellular membranous systems and their interrelationships and the microanatomy of their plasma membrane during secretory process. RESULTS: In thin-sections of pneumocytes postfixed with osmium tetroxide and potassium ferricyanide, we observed that lamellar bodies (LBs) are usually connected to membranes of the endoplasmic reticulum, and seem to emerge and grow from them. Unusual connections between the endoplasmic reticulum and mitochondria were detected, as well as numerous "bar-like structures" (BLSs), most of them in the early stages of development and often generating from the nuclear membrane. Membranes of the smooth endoplasmic reticulum that closely follow the outlines of mitochondria also appear to be the origin of some BLSs. Possible transition forms, BLS--LB, were also detected, although they were rare. New images of the surface of the pneumocytes and its changes during LB secretion showed a segregation and clearing of membrane particles at the areas of LB extrusion. CONCLUSIONS: We propose that LBs can originate directly from membranes of the endoplasmic reticulum or from BLSs. An indirect participation of mitochondria appears possible. The plasma membrane of pneumocytes displays structural changes associated with the secretion of LBs as visualized by a redistribution of intramembrane and surface particles.

The morphology of teleost meningocytes as revealed by freeze-fracture.

We describe the morphology of the cells in the three layers of the teleost endomeninx, as viewed by freeze-fracturing. The cells of the outer endomeningeal layer are fusiform and closely packed, with interdigitations that hold the cells together, gap junctions and a few strands of particles resembling tight junctions, but no desmosomes. The intermediate layer is formed by a single layer of flattened and elongated cells with rectangular shape, and well developed junctional complexes (gap junctions, tight junctions and desmosomes). These cells also show numerous plasmalemmal vesicles (6.5 +/- 1.3/microns 2) in the upper (in contact with the outer layer) and lower (in contact with the inner layer) membranes. Cross fracture of these cells shows many membrane-bound and free vesicles. The inner layer is formed by spindle shaped cells with wide intercellular spaces filled with a granular matrix and collagen fibers. The density of intramembrane particles is higher than that in the meningocytes of the outer and intermediate layers. The morphology of the teleost endomeninx appears similar to that of the leptomeninges of birds and mammals, but the outer endomeningeal layer of teleosts (which resembles the arachnoid of elasmobranchs and amphibians) appears different from any cell layer in the meninges of amniotic vertebrates.

Binding of bacterial endotoxins to the macrophage surface: visualization by fracture-flip and immunocytochemistry.

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.

Purification of immature cores of mouse mammary tumor virus and immunolocalization of protein domains.

The immature capsids of the mouse mammary tumor virus (MMTV), known as intracytoplasmic A particles, have been isolated from murine L1210 leukemia cells. The diameter of the isolated particles was 80 nm as determined by negative staining. Two polypeptides of 77 and 110 kDa were found to be their major polypeptide components, in agreement with the expected sizes of the Gag and Gag-Pro precursor polypeptides of the mature MMTV proteins. Both polypeptides were recognized by antibodies directed toward the matrix (p10) and capsid (p27) proteins of MMTV. Immunogold labeling of p10 on isolated A particles, visualized by negative staining, showed that this protein is located at the surface of the immature capsids, whereas p27 can be detected only in broken or disrupted particles, suggesting that it has an internal location. These observations were confirmed by immunolabeling of both proteins on thin sections of A particle-producing cells. In addition, the viral protease had a more internal position than p27. Since the sequential order of the viral proteins in the Gag precursor is p10-pp21-p27-p14 and that in Gag-Pro is p10-pp21-p27-p30-protease, our results demonstrate the radial organization of the polypeptide precursors forming the intracytoplasmic A particles.

Human meibomian glands: the ultrastructure of acinar cells as viewed by thin section and freeze-fracture transmission electron microscopies.

Heightened interest in meibomian glands dysfunction prompted the authors to examine the ultrastructure of the glandular epithelium in specimens of surgical origin, by thin section and freeze-fracture electron microscopies. In meibomian glands, the morphology and ultrastructure of acinar cells varies considerably according to their stage of holocrine differentiation. This study shows close interdependence between fat droplets and Golgi apparatus or endoplasmic reticulum. As the cells initiate their differentiation, the smooth endoplasmic reticulum and the Golgi apparatus become prominent and the first small lipid droplets appear in the cytoplasm. When fractured through a plane close to their surface, lipid droplets appear onion-like structured, ie made up of a variable number of irregular shaped concentric lamellae. This lamellar organization suggests that membranes are not only involved in synthesis, but also that some of their components are incorporated in the fat droplets. The authors conclude that human meibomian glands are a holocrine glandular complex that, despite great differences in type and location, present basic similarities with sebaceous glands.

Dynamics of transmembrane proteins during Sindbis virus budding.

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.

In vitro and in vivo characterization of pp39mos association with tubulin.

The product of protooncogene c-mos, pp39mos, is expressed and functions during oocyte maturation. We have previously found that pp39mos is complexed with and phosphorylates tubulin. In addition, part of pp39mos is localized on mitotic spindle and spindle pole regions in c-mosxe-transformed NIH/3T3 cells. Here, we further characterized the interaction between pp39mos and tubulin. We show that mos product synthesized in vitro appears in a 500 kD complex and can oligomerize with tubulin in vitro under tubulin polymerization conditions. Moreover, conditions which favor microtubule depolymerization facilitate pp39mos extraction from c-mosxe-transformed NIH/3T3 cells. We also show by immunofluorescence and immunoelectron microscopy that pp39mos is localized on microtubules. Thus, in vitro and in vivo the mos product is associated with tubulin and microtubules, respectively. Therefore, the mos product may be involved in the modification of microtubules and formation of the spindle.

Localization of wheat germ agglutinin and antibody binding sites on the plasma membranes of sea urchin sperm heads as revealed by label-fracture and fracture-flip.

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.

Giardia duodenalis: a freeze-fracture, fracture-flip and cytochemistry study.

The freeze-fracture technique was used to study the structural organization of the membranes of trophozoites of the protozoon Giardia duodenalis. No special array of intramembranous particles was observed in the membrane lining the protozoon body or the flagella. A large globular protuberance located in the ventral region displayed several small circular indentations similar to those seen in the dorsal region. These also occurred on the parasite surface as revealed in fracture-flip replicas. A large number of vesicles were observed below the plasma membrane; they corresponded to an acidic compartment as indicated by fluorescence microscopy of acridine orange-stained cells and contained acid phosphatase as indicated by cytochemistry. In addition, gold-labeled macromolecules (albumin, peroxidase, transferrin, and low-density lipoprotein) accumulated in the vesicles. These observations suggest that the peripheral vesicles of trophozoites are part of the endosomal-lysosomal system of G. duodenalis.

Simulcast: contiguous views of fracture faces and membrane surfaces in a single cell.

We introduce "simulcast", a new method that combines the advantages of freeze-fracture with those of "fracture-flip" (Anderson-Forsman, C., P. Pinto da Silva, J. Cell Sci. 90, 531-541 (1988)) to provide images of the fracture faces and membrane surfaces of the same membrane in a single cell. The method involves low-angle unidirectional shadowing, careful azimuthal reorientation of the replicas, flipping, and re-shadowing. Simulcast relates, in a single image, the freeze-fracture morphology of fracture faces, the nanoanatomy of membrane surfaces and the topochemistry of surface receptors/antigens.

High-resolution surface views of human lymphocytes during capping of CD4 and HLA antigens as revealed by immunogold fracture-flip.

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.