The ultrastructural basis of endothelial cell surface functions

Several enzymes, enzyme inhibitors, receptors and transport structures are situated on the luminal surface of endothelium. Some enzymes and transport systems are continuously active and, in effect, regulate the composition of blood moving downstream. Other components are latent. Their activities are not expressed in the absence of stimulus. Thus, endothelial cells injured by granulocytes or viral infection possess receptors for the Fc segment of IgG and for C3b, whereas normal endothelial cells do not. Both normal and injured endothelial cells express receptors for Clq. To visualize surface enzymes, inhibitors, receptors and transport structures, we have prepared surface replicas suitable for high resolution EM. The surface replication technique, coupled with immunocytochemical procedures, facilitates studies of the topographies of surface enzymes such as angiotensin converting enzyme (ACE) and carboxypeptidase N (CPN). In addition, the replicas provide unique views of the glycocalyx, a cell coating previously believed to be amorphous but now seen to be a highly organized carpet-work under which surface enzymes, receptors, etc are embedded. Given its content of fibronectin, the glycocalyx may be the Clq receptor. Cells treated with antibodies to ACE or CPN show disarrayed glycocalyces and bind Fc and C3b. Latency of the Fc and C3b receptors may be owing to the physical barrier provided by the glycocalyx. Apparently, damage to the glycocalyx creates conditions favoring binding of immune complexes, complement activation and intravascular coagulation with loss of gradients between blood and parenchyma. Whether some non-thrombogenic properties of endothelium require an intact glycocalyx deserves consideration as does the role of the glycocalyx in regulating microvascular permeability.     

Cell surface labelling of mononuclear cells with antisera associated to turnip yellow mosaic virus or alphalpha mosaic virus particles. A freeze-etch study

Synopsis Turnip yellow mosaic virus (TYMV) and alphalpha mosaic virus (AMV) were used as immuno-electron microscopical markers to detect cell surface receptors on mononuclear cells in freeze-etch replicas. TYMV particles were conjugated with vacuum-distilled glutaraldehyde to rabbit IgG anti-mouse immunoglobulins (TYMV-RAMIg conjugate) or to rabbit IgG anti-mouse antigen (TYMV-RAMTh conjugate). B-lymphocytes incubated with TYMV-RAMIg conjugate showed either randomly distributed particles or patches of virus particles on the etched surface of the cell membrane. Mouse thymocytes incubated with TYMV-RAMTh conjugate, however, showed only a random distribution of the virus particles. Human mononuclear cells incubated with rabbit IgG anti-AMV and AMV for the demonstration of the receptors for the Fc fragment of IgG showed the oblong shape of the AMV particles on the etched cell membrane. Fc receptors were either randomly distributed or aggregrated into patches. It is concluded that both types of virus particles are useful markers for the demonstration of membrane receptors in freeze-etch replicas of labelled cells.     

Demonstration of the fuzzy surface coat of rat intestinal microvilli by freeze-etching

In freeze-etch replicas of epithelial cells of rat rectum, the fuzzy surface coat is composed of discrete filaments which are aligned in parallel with each other and with the long axes of the microvilli.     

The dynamics of exoskeletal-epidermal structure during molt in juvenile lobster by electron microscopy and electron spectroscopic imaging

The exoskeletal-epidermal complex of juvenile lobsters at various stages throughout the molt cycle was examined by conventional electron microscopy, freeze-etch replicas, and electron spectroscopic imaging. This latter technique which enables the direct localization of atomic elements superimposed over morphological fine structure has been applied to this tissue complex to determine the spatial distributions and interrelationships of calcium, phosphorus, and sulphur. Chitin microfibril assembly is visualized in thin sections as occurring at the surface of apical membrane plaques which in freeze-etch replicas invariably possess a rich distribution of intramembrane particles on both P and E faces. In early stages of mineralization the exo- and endocuticular zones of the exoskeleton possess a dense Ca-containing lamellar repeat. These bands are unrelated to the helicoidal arrangement of chitin microfibrils. At later stages of development mineral deposits occur within the exocuticle and advance through to the endocuticle. These deposits align with chitin microfibrils and exhibit a helicoidal pattern. Morphological and chemical alterations associated with mineralization and demineralization of the exoskeleton are discussed.     

Virus-Induced Cytoplasmic Membrane Structures Associated with Semliki Forest Virus Infection Studied by the Freeze-Etching Method

Intracellular membrane structures associated with the Semliki Forest virus replication process were studied from freeze-etch replicas. Cleaved membrane structures inside the CPV I type vacuoles lacked the typical membrane particles present on most other fractured membranes. CPV II type vacuoles present in thin sections were obscured in the freeze-etch replicas by the cytoplasmic ground substance.     

DEVELOPMENT AND EVALUATION OF A FREEZE-ETCH,FREEZE-FRACTURE TECHNIQUE APPLIED TO DEVELOPING WHEAT ENDOSPERM

A technique was developed and evaluated whereby differentiating wheat endosperm tissue could be processed for the freeze-etch, freeze-fracture technique without the use of chemical fixatives. Field grown, developing hard red winter wheat (cv. Newton) caryopses were infiltrated with glycerol prior to freezing in liquid nitrogen-cooled Freon-22. Frozen samples were placed in cryogenic vials and stored in liquid nitrogen until needed. Freeze-fracture was carried out under standard conditions. Evaluation of replicas from glycerol-imbibed endosperm was made by comparing them to replicas obtained from freshly frozen untreated endosperm, and endosperm that had been prefixed in glutaraldehyde and paraformaldehyde. Other evaluations were made by comparing replicas of glycerol-treated endosperm to thin sections obtained from wheat that had been imbibed with glycerol, fixed, dehydrated, and infiltrated and embedded in epoxy resin for routine electron microscopy. The results indicate that if the unfixed glycerol-treated wheat endosperm is handled carefully, replicas can be obtained which show few artifacts due to glycerol and freezing. Typical artifacts such as vesiculation of RER, ice crystal damage, production of fusion intermediates, and membrane particle segregation can be nearly eliminated when this technique is applied to developing wheat endosperm.     

Unit Cell Hypothesis for Streptococcus faecalis

The mass doubling times of exponential-phase cultures of Streptococcus faecalis were varied from 30 to 110 min by omitting glutamine from a defined growth medium and providing different concentrations of glutamate (ranging from 300 to 14 μg/ml). After Formalin fixation, cells were dried by the critical point method, and carbon-platinum replicas were prepared. The surface area and volume of cell poles seen in these replicas were estimated by a computer-assisted, three-dimensional reconstruction technique. It was found that the amount of surface area and volume of poles seen in these replicas were independent of the growth rate of culture from which the samples were taken. These observations were consistent with the unit cell model hypothesis of Donachie and Begg, in which a small number of surface sites would produce a constant amount of new cell surface regardless of the mass doubling time of the culture. However, measurements of the thickness of the cell wall taken from thin sections of the same cells showed that the cell wall increased in thickness as a function of the increase in cellular peptidoglycan content which occurs when the growth rate of this organism is slowed down by a decrease in glutamate concentration. Thus, it would seem that although the size of polar shells made by S. faecalis is invariant with growth rate, the amount of wall precursors used to construct these shells is not.     

Freeze-fracturing in ultrahigh vacuum at -196 degrees C

Conventional freeze-etching is carried out in a vacuum of approximately 10(-6) torr and at a specimen temperature of -100 degrees C. The relatively poor topographic resolution of most freeze-etch replicas, and the lack of complementarity of morphological details in double replicas have been thought to be caused by structural distortions during fracturing, and radiation damage during replication. Both phenomena can be reduced by lowering the specimen temperature. To prevent condensation of residual gases (especially H2O) on the fracture faces at lower specimen temperature, an improved vacuum is required. Therefore, an ultrahigh vacuum freeze-fracture apparatus has been developed which allows fracturing and Pt/C-shadowing of specimens at -196 degrees C while maintaining a vacuum of 10(-9) torr. It consists of a modified Balzers BA 350 ultrahigh vacuum (UHV) unit, equipped with an airlock which enables the input of nonhoar-frosted specimens directly into the evacuated bell jar. A comparison of the paracrystalline plasmalemma structure in yeast cells portrayed by the conventional technique and by UHV-freeze-fracturing at -196 degrees C shows the improved topographic resolution which has been achieved with the new technique. The improvement is explained by less structural distortions during fracturing at lower temperatures. The particles of the paracrystalline regions on the P face are more regularly arranged and exhibit a craterlike substructure which corresponds with a ringlike depression in the E face. The optical diffraction patterns of these paracrystalline regions demonstrate the improvement of the structural record by showing well-defined third- and fourth-order spots.     

Freeze-fracture investigation of the red pulp of human spleen

This study concerns the morphology of the human spleen in freeze-fracture replicas and compares this with the findings in ultrathin sections. The material investigated consisted of two spleens resected at gastrectomy and one resected because of splenomegaly in a case of hairy cell leukaemia. The current concepts concerning the ultrastructure of the spleen were generally confirmed with the freeze-fracture technique. The sinus walls were found, as expected, to consist of closely fitting endothelial cells, which were identifiable in freeze-fracture replicas by numerous caveolae of the cell membrane. Contrary to the opinion upheld in the literature, the sinus endothelial cells were occasionally found to be connected by desmosomes or maculae adhaerentes. Corresponding to the finding of desmosomes in ultrathin sections, focal collections of intramembranous particles were observed in freeze-fracture replicas and a positive immunohistochemical reaction for desmoplakin in the sinuses was found at the light microscopic level. The view generally held in the literature that sinus endothelial cells can exhibit tight junctions was not confirmed. However, such junctions were found between vascular endothelial cells. The ring fibres of the sinuses, which are closely connected to the sinus endothelial cells through contractile fibres, apparently have various functions. Firstly, they contribute towards maintaining mechanical stability. Secondly, they represent basement membranes through which exchange occurs between the sinus endothelial cells and their surroundings. This is indicated by the caveolae and vesicles that are often found here in large numbers and in focal collections. Hairy cells exhibit no features in freeze-fracture replicas to suggest a cytogenetic relationship to interdigitating reticulum cells.     

Fine structure of Sertoli cells in the rat testis after hypophysectomy, testosterone treatment and re-involution

Using thin sections and freeze-etch replicas the fine structure of the Sertoli cells of the rat testis was investigated after hypophysectomy, testosterone treatment and re-involution. 41 days after hypophysectomy the Sertoli cells contain numerous dense bodies and remnants of degenerating spermatocytes and spermatids. The Sertoli cell junctions are most prominent. The membranes of neighbouring cells are folded into several layers. Freeze-fracture replicas reveal a normal arrangement of Sertoli cell tight junctions with linear array of membrane particles preferentially on the B-face and complementary grooves on the A-face. The geometric pattern of the ridges is varying with respect to the basal, intermediate and apical portions of the lateral Sertoli cell membranes. Since no major changes of the size, distribution and localization of the Sertoli cell junctions were observed in the different experimental groups these junctions, once formed, are inferred to be independent from hypophyseal hormones.     

The structure of cellulose-producing bacteria, Acetobacter xylinum and Acetobacter acetigenus.

The structure of the pellicles and cells of the cellulose-producing bacteria, Acetobacter xylinum and Acetobacter acetigenus, was studied by transmission electron microscopy of thin sections and freeze-etch replicas of glucose-stimulated cell suspensions, quiescent cell suspensions, and discrete pellicles. These bacteria have a relatively thin cell wall in section, with several irregular features superimposed on an otherwise simple, Gram-negative morphology. There are no flagella or pili. Unfixed, unextracted cells, viewed as whole mounts, show spherical or ellipsoidal bodies of undetermined composition which disappear after extraction with water or ethanol and propylene oxide. For both species, there are several kinds of cell surface irregularities, some of which are localized protrusions of the cell envelope. A variety of irregularities is seen frequently on cells in the first minutes of glucose incubation, on cells in a discrete pellicle, on quiescent cells, and on starved cells. Immediately after the addition of glucose to cellulose-free cells in suspension culture, fine fibrils appear on and (or) near the cell envelope. The fine fibrils are frequently as small as 3 nm in diameter in both freeze-etch and thin-section preparations and are frequently associated with freshly synthesized cellulose fibrils. Starved cells in suspensions free of (classical) microfibrils sometimes reveal stubs of an extracellular structure whose morphology resembles that of a nascent cellulose fibril.     

Multilamellar surface layer of the cell wall of Albugo candida and Phycomyces blakesleeanus.

The surface layer of the cell wall of the sporangia of Albugo candida and of the sporangiophores of Phycomyces blakesleeanus was composed of a series of lamellae. The evidence from freeze-fracture, freeze-etch, and single-stage replicas indicated that the lamellae are organized as bilayers, an organization associated with the presence of lipids. The role of these lamellae in dispersibility and resistance is discussed.     

Electron microscopy of liquid crystalline DNA: direct evidence for cholesteric-like organization of DNA in dinoflagellate chromosomes

Freeze-fracture-etch replicas of concentrated DNA solutions which appeared, by polarized light microscopy, to be in a cholesteric-like liquid crystalline state were examined by high resolution transmission electron microscopy (TEM). Individual DNA molecules were resolvable, and the microscopic morphologies observed for such replicas confirmed the cholesteric organization of DNA molecules in this liquid crystalline state. Furthermore, replica morphologies were strikingly similar to TEM images of dinoflagellate chromosomes in both thin section and freeze-etch replicas, providing strong support for the cholesteric DNA packing model proposed for the organization of DNA in these chromosomes by Bouligand and Livolant.     

FREEZE-ETCH STUDIES OF THE PLASMA MEMBRANE OF PULMONARY ENDOTHELIAL CELLS

Pulmonary endothelial cells are capable of metabolizing a variety of circulating hormonal substances. Indirect evidence indicates that some of the relevant enzymes are located on the plasma membrane. The associated caveolae are of special interest as globular subunits, possibly enzyme clusters, are evident in their membranes. In the present study, freeze-etch techniques were used to improve understanding of the fine structure of endothelial cells and to extend our investigations of possible sites of enzymes capable of metabolizing circulating vasoactive agents. As in other cells studied by freeze-etching, intramembranous particles are found on both inner aspects of the plasma membrane. In undifferentiated areas of plasma membrane, the particles appear to have a random distribution. These areas fracture such that approximately equal proportions of the particles adhere to the cytoplasmic aspect of the outer leaflet and the extracellular aspect of the inner leaflet. However, the particles organize into rosettes and plaques at the base of caveolae, and, after fracture, the rosettes and plaques adhere predominantly to the cytoplasmic aspect of the outer leaflet. The peculiar organization of particles in association with caveolae supports the concept that caveolae have a stomal skeletal structure and raises the possibility that the organization may be in some way related to pinocytosis.     

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

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

Freezing of tissue-limits for the autoradiographic localization of diffusible substances

Frozen thin sections and sections from freeze-dried and embedded tissue are used for the autoradiographic localization of diffusible substances at the electron microscope level. The presence of ice crystals in such sections may limit the autoradiographic resolution. Ice crystals are formed during freezing and may grow during subsequent processing of tissue. The contribution of ice crystal growth to the final image was estimated by measuring the distribution of the ice crystal sizes in freeze-etch replicas and in sections from freeze-dried and embedded tissues. A surface layer (10-15 mu) without visible ice crystals was present in both preparations. Beneath this surface layer the diameter of ice crystals increased towards the interior with the same relationship between crystal size and distance from the surface in the freeze-etch preparation as in the freeze-dry preparation. Ice crystal growth occurring during a much longer time during freeze-drying compared to freeze-etching does not significantly contribute to the final image in the electron microscope. The formation of ice crystals during freezing determines to a large extent the image (and therefore the autoradiographic resolution) of freeze-dry preparations and this probably holds also for thin cryosections of which examples are given.     

Prefracture and cold-fracture images of yeast plasma membranes

Fracture-temperature related differences in the ultrastructure of plasmalemma P faces of freeze-fractured baker's yeast (Saccharomyces cerevisiae) have been observed in high-resolution replicas prepared in freeze-etch systems pumped to 2 X 10(-7) torr in which the specimens were protected from contamination by use of liquid nitrogen-cooled shrouds. Two major P-face images were observed regardless of the source of the yeast, the age of the culture, the growth temperature, the physiological condition, or the suspending medium used: (a) a "cold- fracture image" with many strands closely associuated with tubelike particles (essentially the same image as those previously published for yeast freeze-fractured at 77 degrees K), and (b) a "prefracture image" characterized by the presence of more distinct tubelike particles with few or no associated strands (for aging cultures, the image recently referred to as "paracrystalline arrays" of "craterlike particles"). Both types of P-face image can be found in separate areas of single replicas and occasionally even within a single plasma membrane. Whereas portions of replicas known to be fractured at any temperature colder than 218 degrees K reveal only the cold-fracture image, prefracture images are found in cells intentionally fractured at 243 degrees K and in cracks or fissures which develop during the freezing of other specimens. These findings demonstrate that the prefracture image results from the fracturing of specimens at some temperature above 230 degrees K, no t from fracturing specimens at some temperature between 173 degrees and 77 degrees K, and not from the use of "starved" yeast cells.     

Surface Rodlets of Tilletia indica Teliospores

Tilletia indica teliospores were studied by use of thin sections and freeze-etch replicas. Surfaces of these spores have rodlet patterns which differ from those previously reported for spores of other fungi. The rodlets on T. indica teliospores average 240 nm in length and are not grouped into fascicles.     

Structural and biochemical examination of ghosts derived from a deep rough (heptose-deficient lipopolysaccharide) strain and a smooth strain of Escherichia coli.

Outer membrane derived 'ghosts' can be readily generated from both smooth and deep rough (heptose-deficient LPS) strains of Escherichia coli 08. MORPHOlogical and biochemical studies confirmed that 'ghosts' of both strains are composed of protein (four major proteins), LPS, and phospholipid (cardiolipin and phosphatidylethanolamine) in the form of a single membrane of roughly the same shape as intact normal cells. The ghost membrane cleaves only slightly in freeze-etch preparations of ghosts derived from the smooth strain as compared to the extensive cleavage plane of ghosts derived from the rough strain. The asymmetrical distribution of ghost proteins was visualized, by critical point drying and shadowing with platinum, as a relatively smooth outer surface with some discernible particles (10-15 nm) and an extremely particulate inner surface (10-15-mm particles. Ghosts derived from the smooth strain retained their structure following chloroform-methanol extraction, while ghosts derived from the rough strain fragmented with chloroform-methanol extraction. Evidence is presented that LPS-protein interactions as well as protein-protein interactions are significant in maintaining the ghost structure.     

Multiple forms of acid phosphatase in rat liver parenchymal,endothelial, and kupffer cells

Purified suspensions of highly viable parenchymal, endothelial, and Kupffer cells were prepared from rat liver. In the liver cell classes, total activities of acid phosphatase were determined with 4-methylumbelliferylphosphate, 1-naphthylphosphate, and p-nitrophenylphosphate. The specific enzyme activities were different for each type of cell and, even within one cell class, the enzymes showed different conversion rates for the three substrates. These results indicate the presence of multiple forms of acid phosphatase enzymes in each cell class. The inhibiting effects of tartrate, fluoride, and alloxan on the acid phosphatase activities were investigated. Depending on the substrate used, the inhibitors inactivated the enzymes at different rates, which also indicates the presence of multiple forms of acid phosphatase enzymes in the liver cell classes. By means of an isoelectric focusing technique, acid phosphatase enzymes could be separated on the basis of their differences in isoelectric points. One form with an isoelectric point around 4 is found in Kupffer cells, whereas another form with an isoelectric point of about 7 is found in parenchymal cells. Endothelial cells possess both forms. These findings suggest a specificity in the function of this lysosomal enzyme in each cell class.