Organization of mesosomes in fixed and unfixed cells.

After the addition of glutaraldehyde (GA) to cells incubated at 3 or 37 degrees C, mesosomes were observed with increasing frequencies in freeze fractures of cells. These increases were related to the kinetics with which GA cross-linked adjacent amino acids. Upon the addition of GA, mesosomes were first observed in the periphery of freeze-fractured cells usually attached to septal membranes. However, the time, while the septal attachment sites were maintained, the "bodies" of the mesosomes were observed to move toward the center of the cytoplasm. This centralization process was much more rapid at 37 than at 3 degrees C. It is hypothesized that upon fixation, or receipt of some physical insult, mesosome precursors found in undisturbed cells undergo a change in state that results in their visibility in freeze fractures.     

CRYOPROTECTANT-INDUCED REDISTRIBUTION OF INTRAMEMBRANOUS PARTICLES IN MOUSE LYMPHOCYTES

This study demonstrates, by freeze fracture, clustering of intramembranous particles caused by cryoprotectant treatment of intact unfixed mouse lymphoid cells. Both T and B cells react in a similar fashion, while similar clustering of particles is not observed in some other cell types. The intramembranous particles can be aggregated by incubating unfixed cells in glycerol or dimethylsulfoxide (DMSO) before freezing. The aggregation phenomenon is dependent on the length of time of exposure and the concentration of the cryoprotectants. Further, the cells remain viable and the cryoprotectant-induced clustering is completely reversible. Prefixation of glycerol-treated cells with glutaraldehyde prevents the formation of these particle clusters, and unfixed nonglycerinated cells show no clusters. Thin sections of cells exposed to glycerol or DMSO without previous fixation exhibit bizarre membrane alterations and numerous other degenerative changes. These observations stress the importance of prefixation of lymphoid cells before exposure to glycerol or DMSO, as well as indicate that the membrane characteristics of certain cell types may be probed by glycerol treatment of unfixed cells.     

Morphokinetic Reaction of Cells of Streptococcus faecalis (ATCC 9790) to Specific Inhibition of Macromolecular Synthesis: Dependence of Mesosome Growth on Deoxyribonucleic Acid Synthesis

The application of quantitative electron microscopy to thin sections of cells of Streptococcus faecalis specifically inhibited for deoxyribonucleic acid (DNA), ribonucleic acid, and protein synthesis shows that septal mesosomes (i) increase in size when protein synthesis is inhibited by at least 80% while DNA synthesis proceeds at no less than 50% of the control rate and (ii) decrease in size when DNA synthesis is inhibited 50% or more during the initial 10 min of treatment. This indicates that fluctuations in mesosome size are dependent on the extent of DNA synthesis. The fluctuations in mesosome areas observed on treatment do not correlate with the kinetics of glycerol incorporation per milliliter of a culture. However, when glycerol incorporation is placed on a per cell basis, a strong correlation is observed between increases in (i) the thickness of the electron-transparent layer of the cytoplasmic membrane and (ii) the amount of glycerol incorporated per cell. It seems that the electron-transparent membrane layer may thicken to accommodate changes in lipid content when protein and lipid synthesis are uncoupled.     

Bacterial mesosomes: Real structures of artifacts?

The ultrastructural study of membrane organization in gram-positive bacteria related to the OsO₄ fixation conditions revealed that large, complex mesosomes are observed only when the bacteria are subjected to an initial fixation with 0.1% OsO₄ in the culture broth, as in the prefixation step of the Ryter-Kellenberger procedure. Evidence was obtained suggesting that the large mesosomes are produced by this prefixation. The kinetic study of the membrane morphological alterations occurring during the prefixation of Bacillus cereus with 0.1% OsO₄ in the culture broth showed that the amount of mesosome material increases linearly from zero to a maximum observed at 1.7 min of prefixation and that at about this time a maximum is reached for the number of mesosomes per unity of cell area and for the average individual mesosome area. The large mesosomes observed in gram-positives fixed by the complete Ryter-Kellenberger procedure would be the result of the membrane-damaging action of 0.1% OsO₄. Such damaging action was deduced from the observation that 0.1% OsO₄ quickly lyses protoplasts and induces a quick and extensive leakage of intracellular K⁺ from B. cereus and Streptococcus faeculis. In support of that interpretation is the observation that in bacteria subjected to several membrane-damaging treatments, mesosome-like structures are seen after three different fixation procedures. In bacteria initially fixed with 1% OsO₄, 4% OsO₄ or 2.5% glutaraldehyde, no large, complex mesosomes are observed, small and simple invaginations of the cytoplasmic membrane being present. The size of these minute mesosomes is inversely proportional that causes of fixation. Uranyl acetate was found among the studied fixatives the one to the rate the least damage to bacterial membranes. This fixative satisfactorily preserves protoplasts. In bacteria initially fixed with uranyl acetate no mesosomes were found. The results of the present work throw serious doubts on the existence of mesosomes, both large and small, as real structures of bacterial cells. It is proposed that a continuous cytoplasmic membrane without infoldings (mesosomes) would be the real pattern of membrane organization in gram-positives.     

Effect of chromosomal breaks induced by x-irradiation on the number of mesosomes and the cytoplasmic organization of Streptococcus faecalis

A model which explains mesosome formation via a contraction of the cytoplasm and nucleoid when bacteria are physiologically disturbed was tested by: (1) X-irradiation of unfixed cells of Streptococcus faecalis to produce chromosomal breaks and to remove DNA attached to the cell membrane; (2) subsequent determination of the number of irradiated cells in which mesosomes (using electron microscopy) and central density changes (using phase-contrast microscopy) could be visualized after fixative was added. Results obtained by exposure of cells to doses up to 1100 krads before fixation indicated that: (1) the number of cells with central mesosomes was reduced proportional to the decrease in the molecular weight of the DNA due to double-strand breaks: (2) the number of cells with total (central plus peripheral) mesosomes and the number of cells with peripheral mesosomes were both reduced proportional to the removal of DNA attached to the cell membrane (M band); (3) the nucleoid became more diffusely organized. Exposure of cells to doses greater than 1100 krads before fixation resulted in: (1) an increase in the number of cells with central and peripheral mesosomes (compared to cells exposed to lower dosages); (2) a return to the centralized, dense nucleoid seen in unirradiated cells.These results suggest that mesosomes are formed when localized sites on the cell membrane are pulled from close contact with the cell wall into the cytoplasm by the action of a cross-linking fixative via the aggregation of intracytoplasmic components such as DNA. This model considers the attachment of DNA and/or other cytoplasmic components to the membrane as an intrinsic part of its mechanism. The formation of central and peripheral mesosomes in unirradiated and X-irradiated cells are contrasted.     

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

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

The sulphur content of chondrocyte nuclei

The sulphur content as a measure of glycosaminoglycan content of growth plate cartilage was determined by energy dispersive x-ray analysis on fresh freeze dried unstained, unfixed ultra thin sections of rat growth plate. In the resting and proliferative zones, quantities of sulphur were found in the nuclei equal to that of the matrix. Less sulphur was present in the cytoplasm. In areas of cell degeneration nuclear and cytoplasmic content of sulphur fell to levels a fraction of that seen in the matrix. It was presumed that most of the sulphur was in glycosaminoglycans. Although glycosaminoglycans have been reported in small amounts in the nuclei of cells, no study of the glycosaminoglycan content of chondrocyte nuclei has been reported. The use of freeze dried unstained, unfixed sections presumably prevented the migration of sulphur and glycosaminoglycans from compartment to compartment.     

The sulphur content of chondrocyte nuclei

Summary The sulphur content as a measure of glycosaminoglycan content of growth plate cartilage was determined by energy dispersive x-ray analysis on fresh freeze dried unstained, unfixed ultra thin sections of rat growth plate. In the resting and proliferative zones, quantities of sulphur were found in the nuclei equal to that of the matrix. Less sulphur was present in the cytoplasm. In areas of cell degeneration, nuclear and cytoplasmic content of sulphur fell to levels a fraction of that seen in the matrix. It was presumed that most of the sulphur was in glycosaminoglycans. Although glycosaminoglycans have been reported in small amounts in the nuclei of cells, no study of the glycosaminoglycan content of chondrocyte nuclei has been reported. The use of freeze dried unstained, unfixed sections presumably prevented the migration of sulphur and glycosaminoglycans from compartment to compartment.Supported by grants from the Medical Research Council and the Shriners of North America     

THE FINE STRUCTURE OF CHONDROCOCCUS COLUMNARIS : I. Structure and Formation of Mesosomes

Cells of Chondrococcus columnaris were sectioned and examined in the electron microscope after fixation by two different methods. After fixation with osmium tetroxide alone, the surface layers of the cells consisted of a plasma membrane, a dense layer (mucopeptide layer), and an outer unit membrane. The outer membrane appeared distorted and was widely separated from the rest of the cell. The intracytoplasmic membranes (mesosomes) appeared as convoluted tubules packaged up within the cytoplasm by a unit membrane. The unit membrane surrounding the tubules was continuous with the plasma membrane. When the cells were fixed with glutaraldehyde prior to fixation with osmium tetroxide, the outer membrane was not distorted and separated from the rest of the cell, structural elements (peripheral fibrils) were seen situated between the outer membrane and dense layer, and the mesosomes appeared as highly organized structures produced by the invagination and proliferation of the plasma membrane. The mesosomes were made up of a series of compound membranes bounded by unit membranes. The compound membranes were formed by the union of two unit membranes along their cytoplasmic surfaces.     

Organization of the nucleoplasm in Escherichia coli visualized by phase-contrast light microscopy, freeze fracturing, and thin sectioning.

The organization of the nucleoplasm in Escherichia coli was studied by comparing the results obtained by freeze fracturing and thin sectioning. In addition to exponentially growing cells, we used chloramphenicol-treated cells which show a well-defined nucleoplasm, in the phase-contrast light microscope and can therefore function as a control for treatments necessary for electron microscopy. Two factors were found to determine the visibility of the nucleoplasm in freeze fractures: first, the state of lateral aggregation of deoxyribonucleic and fibrils, which is enhanced by postfixation with OsO4 according to the Ryter-Kellenberger technique; second, the presence of ice crystals. When their formation is prevented by the use of high concentration of freeze-protecting agents, the nucleoplasm appears as a smooth region in cells that have been prefixed. In unfixed cells, however, the freeze-protecting agent causes disappearance of the nucleoplasm by rearrangement of structures within the cell. This observation makes it hard to determine whether the deoxyribonucleic acid in vivo dispersed, as found after glutaraldehyde prefixation, or compact, as after OsO4 prefixation.     

PRESERVATION OF THE ULTRASTRUCTURE OF BACILLUS SUBTILIS BY CHEMICAL FIXATION AS VERIFIED BY FREEZE-ETCHING

The present study on the ultrastructure of Bacillus subtilis was undertaken in order to examine by means of the freeze-etching technique possible structural changes occurring during the chemical fixation procedure (Ryter-Kellenberger (R-K) fixation). Three stages were followed by freeze-etching, viz.: (a) fixation in osmium tetroxide, (b) fixation in osmium tetroxide and posttreatment with uranyl acetate, and (c) fixation in osmium tetroxide, posttreatment in uranyl acetate, and dehydration in a graded series of acetone. Preparations were made after each stage in the presence of 20% glycerol. Good preservation of ultrastructure was observed, after any of the three treatments, of the outer surface of the plasma membrane, and the inner surface of the plasma membrane. No alteration in fracturing properties could be observed. However, if we are to judge by the results of freeze-etching, any of the successive steps of the chemical fixation procedure achieve strong contrast between the nucleoplasmic region and the cytoplasm. Dependent on the quality of fixation, very delicately preserved DNA fibrils or strongly aggregated ones were seen. It appears that R-K fixation is capable of producing more or less distinctly visible changes in the native state of the nucleoplasm in young cells of B. subtilis.     

Visualization of the nuclear lamina in mouse anterior pituitary cells and immunocytochemical detection of lamin A/C by quick-freeze freeze-substitution electron microscopy.

We examined the nuclear lamina in the quickly frozen anterior pituitary cells by electron microscopic techniques combined with freeze substitution, deep etching, and immunocytochemistry and compared it with that in the chemically fixed cells. By quick-freeze freeze-substitution electron microscopy, an electron-lucent layer, as thick as 20 nm, was revealed just inside the inner nuclear membrane, whereas in the conventionally glutaraldehyde-fixed cells the layer was not seen. By quick-freeze deep-etch electron microscopy, we could not distinguish definitively the layer corresponding to the nuclear lamina in either fresh unfixed or glutaraldehyde-fixed cells. Immunofluorescence microscopy showed that lamin A/C in the nucleus was detected in the acetone-fixed cells and briefly in paraformaldehyde-fixed cells but not in the cells with prolonged paraformaldehyde fixation. Nuclear localization of lamin A/C was revealed by immunogold electron microscopy also in the quickly frozen and freeze-substituted cells, but not in the paraformaldehyde-fixed cells. Lamin A/C was localized mainly in the peripheral nucleoplasm within 60 nm from the inner nuclear membrane, which corresponded to the nuclear lamina. These results suggest that the nuclear lamina can be preserved both ultrastructurally and immunocytochemically by quick-freezing fixation, rather than by conventional chemical fixation.     

Electron Microscope and Autoradiographic Study of Ultrastructural Aspects of Competence and Deoxyribonucleic Acid Absorption in Bacillus subtilis: Localization of Uptake and of Transport of Transforming Deoxyribonucleic Acid in Competent Cells

With the aid of serial-section electron microscopy two types of mesosomes can be distinguished in cells of competent cultures of Bacillus subtilis: (i) mesosomes connected to the plasma membrane only (plasma membrane mesosomes) and (ii) mesosomes which extend from the plasma membrane into the nuclear bodies (nuclear mesosomes). Contrary to plasma membrane mesosomes, nuclear mesosomes are absent from the tip zones. Electron microscopic autoradiography of sections of Bacillus subtilis cells exposed to [(3)H]thymidine-labeled transforming deoxyribonucleic acid (DNA) for a short period of time shows that the DNA becomes associated with mesosomes. As a function of time the DNA migrates towards the nucleoids. Transport of DNA is completed within 15 to 60 min after termination of DNA uptake. During its migration the DNA continues to be associated with mesosomes, presumably with nuclear mesosomes. DNA initially associated with plasma membrane mesosomes of the tip zones is probably transported first towards the middle zones peripherally and from there towards the nucleoids.     

Changes in intramembranous particle topography and concanavalin A receptor mobility associated with myoblast differentiation.

These studies have examined the distribution of plasma membrane intramembranous particles (PMP) visualized by freeze fracture and concanavalin A receptors seen by ultrastructural cytochemistry of differentiated and undifferentiated L6 myoblasts. Undifferentiated mononucleated cells have a clustered distribution of PMP on the majority of the fracture faces. Associated with cell differentiation and cell fusion a more uniform distribution of PMP is observed. Changes also occur with myoblast differentiation in the topography and dynamics of receptors bound to concanavalin A. If undifferentiated or differentiated cells are fixed with glutaraldehyde and then reacted with con-A a uniform distribution of con-A is seen on the cell surfaces. In contrast to this if unfixed live cells are reacted at 37 degrees C with con-A a profound redistribution occurs on differentiated cells (greater than 99% showing redistribution) while receptors remain in a uniform array on undifferentiated cells (approximately 95% uniform distribution). In addition to the membrane binding, con-A is observed to bind to an extracellular filamentous matrix seen in high density undifferentiated cultures which then appears to be degraded with differentiation and myoblast fusion. These studies show that a number of membrane changes, both structural and dynamic occur with myoblast differentiation.     

Changes in Intramembranous Particle Topography and Concanavalin A Receptor Mobility Associated with Myoblast Differentiation

These studies have examined the distribution of plasma membrane intramembranous particles (PMP) visualized by freeze fracture and concanavalin A receptors seen by ultrastructural cytochemistry of differentiated and undifferentiated L6 myoblasts. Undifferentiated mononucleated cells have a clustered distribution of PMP on the majority of the fracture faces. Associated with cell differentiation and cell fusion a more uniform distribution of PMP is observed. Changes also occur with myoblast differentiation in the topography and dynamics of receptors bound to concanavalin A. If undifferentiated or differentiated cells are fixed with glutaraldehyde and then reacted with con-A a uniform distribution of con-A is seen on the cell surfaces. In contrast to this if unfixed live cells are reacted at 37° C with con-A a profound redistribution occurs on differentiated cells (greater than 99% showing redistribution) while receptors remain in a uniform array on undifferentiated cells (approximately 95% uniform distribution). In addition to the membrane binding, con-A is observed to bind to an extracellular filamentous matrix seen in high density undifferentiated cultures which then appears to be degraded with differentiation and myoblast fusion. These studies show that a number of membrane changes, both structural and dynamic occur with myoblast differentiation.     

Ultrastructural observations on tissues processed by a quick-freezing,rapid-drying method: Comparison with conventional specimen preparation

A quick-freeze, rapid-dry method for processing unfixed tissue for electron microscopy has been developed. The technique employs freezing on a cryogenchilled metal surface and drying in a cryosorption vacuum apparatus that allows osmium-vapor fixation and epoxy-resin embedment under high vacuum. Liver, kidney, bone marrow, and monolayer cultures of ventricular myocytes were selected as tissue specimens representing a wide range of physical properties, to demonstrate the practical aspects of achieving good ultrastructural morphology by freeze drying. A comparison was made between freeze drying and conventional processing using aldehyde fixation and alcohol dehydration. The preservation of cellular ultrastructure achieved by freeze drying allowed the identification of specific cell types within each specimen. Membranous organelles were well preserved, surrounded by cytoplasmic ground substance devoid of ice crystal damage. Electron-dense material was observed within the rough endoplasmic reticulum and Golgi cisternae and vesicles of frozen-dried, but not conventionally processed cells. This suggests the preservation by freeze drying of cytoplasmic components otherwise extracted from the cell by solvent exposure.     

STRUCTURAL FEATURES OF MESOSOMES (CHONDRIOIDS) OF BACILLUS SUBTILIS AFTER FREEZE-ETCHING

Freeze-etched cells of Bacillus subtilis have been studied with the electron microscope. The outer surface of the plasma membrane, i.e. the side facing the cell wall, is covered with numerous granules and short strands, each measuring approximately 50 A in diameter. These strands are occasionally seen to enter the cell wall. The inner surface of the plasma membrane, i.e. the side facing the cytoplasm, appears to be sparsely dotted with small particles measuring about 50 A. The envelope of mesosomes differs from the plasma membrane. Blunt protrusions arise from its outer surface; the inner surface appears smooth. Stalked particles, as described by other investigators after negative staining with phosphotungstic acid, were not observed on any membrane surface in our material. Preparations were also made of specimens prefixed in osmium tetroxide prior to freeze-etching. Under these conditions the bacterial membranes appeared to be surprisingly well preserved. In contrast to directly frozen, unfixed cells, some osmium tetroxide-fixed preparations showed a differentiation in cytoplasm and nucleoplasm, which made it possible to observe the close association of the mesosome with the latter.     

不同固定方法对植物细胞扫描电镜观察用冷冻断裂样品的影响

用冷冻断裂法在扫描电镜下研究了洋葱(Allium cepa)根端分生组织细胞内部的三维结构。采用了两种固定方法。冷冻断裂前只用1％锇酸固定的材料容易在细胞质和核之间断开,而用卡诺固定液(无水乙醇:冰醋酸3:1)前固定,然后再用1％锇酸固定的材料容易使细胞核断裂。前一固定方法适于研究细胞质的内部结构(细胞骨架的纤维、线粒体、内质网等及其三维分布关系):后一固定方法适于研究核内结构(染色质、核仁、核基质纤维)的三维形象,特别是核仁纤维中心染色质的三维结构。     

Nuclear and chromatin structure in rat spermatozoa

The nuclei of mature mammalian spermatozoa contain a highly ordered, lamellar substructure, presumably constituting the nucleoprotein of the haploid chromosomal complement. With a view toward constructing a plausible model of chromatin packing in sperm, we have determined some of the quantitative parameters associated with these “nuclear lamellae” in rat spermatozoa. Epididymal sperm from white, Sprague-Dawley rats were examined by conventional sectioning methods, freeze fracture of fixed and unfixed specimens, and by whole mount replica techniques. Fixation and glycerolation did not significantly alter nuclear structure as seen by freeze fracture. Numerical data obtained from cross fractures of sperm heads indicate that the number of lamellae are quite constant at 10.4 ± 1.8 and that the linear measure of the lamellae is 7.2 ± 2.3 μm per cross fracture. The total area of cross fracture, assuming an elliptical profile is 2.3 k 0.7 μm² and the thickness of the lamellae is 18.2 ± 3.5 nm with a range of 13.5 to 25.5 nm. An estimate of the total surface area of the nuclear lamellae could be made from measurements of projected nuclear area (from replicas and sections) as 173 ± 15 μm². From these data and the known amount of DNA in the rat sperm nucleus, a model can be proposed for the organization of the nucleoprotein in these lamellar sheets. It is suggested that the chromatin is arranged in a coiled-coil configuration closely associated together in a side-by-side fashion and continuous in extent. Approximate calculations based on this simple model are within a factor of 2 or 3 of predicting the correct amount of DNA in the sperm nucleus.