Freeze-fractured Cells Research Articles

Ultrastructure of pycnidiospore walls and hyphae of Septoria nodorum

Fine-structural features of pycnidiospore walls and hyphae of Septoria nodorum were studied using thin sections stained with tannic acid, freeze-fracturing and freeze-etching techniques. The wall was composed of two layers, an outer electron-dense layer and an inner electron-transparent layer. Fine-structural features of the walls of hyphae and pycnidiospores appeared to be similar. The fracture faces of the cytoplasmic membrane of pycnidiospores were seen in the freeze-fractured cells. Thin fibrillar and globular material of different sizes partly covered the etched surface of the cell. The fibrillar material was best visualized with the freeze-etching technique. The globular material was clearly revealed using the rotatory shadowing technique.

T-lymphocyte heterogeneity: wheat germ agglutinin labeling of transmembrane glycoproteins.

We have recently described "fracture-label" techniques that permit direct cytochemical labeling of freeze-fractured cells. We report here the use of fracture-labeling to investigate the distribution and partition of wheat germ agglutinin (WGA) receptor sites over the protoplasmic and exoplasmic plasma membrane faces of freeze-fractured human thymus-derived (T) lymphocytes. All exoplasmic faces are strongly labeled by WGA. In contrast, the protoplasmic faces exhibit remarkable variation, ranging from virtual absence of label in some faces to very high densities in other faces. We interpret the presence of WGA receptor sites over the protoplasmic faces to reflect the presence of transmembrane WGA-binding sialoglycoproteins that, during freeze-fracture, partition with the inner half of the plasma membrane. Our results, therefore, indicate heterogeneous expression of integral membrane proteins within populations of human T cells. Fracture-label techniques thus represent an additional tool in the definition of lymphocyte subpopulations.

Pigment production in chemostat cultures of Streptococcus bovis

Streptococcus bovis 2B formed bright red cell clumps in glucose-limited defined medium under certain nonsteady state growth conditions. Scanning electron micrographs showed that clumped cells were more rounded than those from complex medium. Clumped cells appeared to be bound to one another at the cell surface and there was no sign of a polysaccharide matrix. Freeze fractured cells showed that the inner membrane particle distribution was different in the clumped cells from fast growing cells in complex or defined medium. Clumping is potentially a mechanism to decrease transport by reducing the cell surface area. It appears to be a nonsteady state phenomenon, occurring in situations leading to unbalanced growth.

Rhizoplast-membrane associations in the flagellate Tetraselmis cordiformisStein ( Chlorophyceae) revealed by freeze-etching and thin sections

Summary The two rhizoplasts of Tetraselmis cordiformis are firmly attached to the nuclear envelope and the plasmalemma in addition to their connection with the flagellar apparatus. In the region of contact the freeze-fractured nuclear membrane is indented and partly devoid of nuclear pores. Links are probably mediated via the cross-bars of the rhizoplast at certain specific sites. At the plasma-lemma the contact zone in freeze-fractured cells displays a “ridge” of 5 × 0.4 μm. dimensions. Intramembraneous particle numbers on the E-fracture face are significantly higher in this region than in other parts of the plasmalemma. Variations in certain aspects of rhizoplast ultrastructure and its associations with membranes are discussed in accordance with the proposed contractile function of the rhizoplast. Evidence is presented that shortening preferentially occurs in the proximal region of the rhizoplast near the flagellar apparatus and that the “normal” image of an evenly spaced rhizoplast is probably a fixation artifact.

Electron Microscopy of a Non-pellicle-forming Strain of Acetobacter xylinum

A non-pellicle-forming culture of Acetobacter xylinum, isolated from a pellicle-forming culture by repeated transfer of the latter through agitated medium, has been examined. The cells of the non-pellicle-producing culture are often greatly elongated and possess a profusion of intra-cytoplasmic membranes. Freeze-fractured cells reveal extensive face views of both the outer and cytoplasmic membranes and show a cell coat composed of a large number of short fibrils arising from the outer membrane. X-ray diffraction of the cells in some cases shows the cellulose-I pattern but shadow-contrasted preparations do not show long microfibrils. It is suggested that the non-pellicle formers produce cellulose as only very short microfibrils which are synthesised all over the cell surface and that there are changes in the outer membrane compared to pellicle-producing cells. The possible significance of these differences between pellicle-forming and non-pellicle-forming A. xylinum strains is discussed.

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.

Bacteriophage-resistant mutants of Salmonella typhimurium deficient in two major outer membrane proteins.

Mutants resistant to bacteriophages (P221 and PH105 or PH51) were isolated from a rfa strain of Salmonella typhimurium. They were found deficient in separate 33,000- to 36,000-dalton band proteins (major band proteins). Double mutants derived from both types of mutants were deficient in both of the bands. The growth behavior of all the mutants was normal. The outer membrane of the mutants appeared to be more wrinkled than normal and formed vesicles in many of the mutants. In freeze-fractured cells, changes were seen in the outer membrane (particleless patches in the concave fracture face, the particles themselves being smaller than normal). These changes were more marked in the double mutants.

Ultrastructural properties of the extra membranes of Escherichia coli O111a as revealed by freeze-fracturing and negative-staining techniques.

Escherichia coli O111a is a thermosensitive strain which, when grown at 40 C, accumulates large quantities of intracellular membranes. The ultrastructure of these membranes in cells which have been chemically fixed, embedded, and examined as thin sections has been compared with that of membranes in cells negatively stained or freeze-fractured. Results indicate that the extra membranes are present in the three types of preparations examined and, therefore, clearly are not artifacts of chemical fixation. Negative staining has proved also to be a valuable tool as a rapid means of monitoring cells for the accumulation of large amounts of extra membranes. Also, examination of thin sections has shown that distinct continuities between the plasma membrane and the extra membranes exist. In general, membrane surfaces in freeze-fractured cells containing extra membranes appear smooth and lack the particles associated with the plasma membranes of many cells.

Ultrastructure of two species of halobacterium

The morphology of a halobacterium from the Dead Sea has been compared with Halobacterium halobium. A freeze-fracture study has revealed two hexagonal patterns with repeating distances of 155 A and 65 A within layers of the cell-envelope. The larger pattern is a characteristic of the genus Halobacterium. The smaller has been identified with a surface of the cell-membrane. By the same freeze-fracture technique, the membrane adjacent to the cell-envelope was shown to be characterized by two particulate fracture faces, presumably within the hydrophobic region of the membrane. The Dead Sea Halobacterium was shown to contain poly-β-hydroxybutyric acid (PHB). Freeze-fractured cells contained granules similar to PHB granules found in other bacterial species. Alterations in the physiological state of the Dead Sea Halobacterium were found to have little effect on cell structure which was also unaffected by depletion of cell K.

Structural properties and features of parasitic Bdellovibrio bacteriovorus.

The structure of five parasitic strains of Bdellovibrio bacteriovorus was studied by electron microscope after negative staining and in shadow-case and etched freeze-fractured preparations. Special attention was paid to the cell wall and the flagellar sheath which is continuous with the wall or part of it. These structural components reveal distinct features which are induced by certain staining substances; they are exceedingly susceptible to disruption by physical treatments, and in old cells often appear impaired. In freeze-fractured cells the wall shows characteristic fracturing tendencies not known in other microorganisms. These structural properties and features are distinct to Bdellovibrio wall and flagellar sheath, the structural integrity of which is a fundamental requirement for the infectivity and survival of this organism. The anterior end of Bdellovibrio is differentiated: 6 to 12 ring-like structures (9 to 12 nm, outer diameter) are built into its wall and several fibers (7 to 10 nm wide, up to 1.5 mum long) emerge from it. Intracellular structures, which are revealed as compact oval bodies bulging from the cell border and have internal laminated organization, are characteristic of Bdellovibrio after negative staining with certain compounds. These findings on the structure of parasitic Bdellovibrio substantiate previous observations indicating the uniqueness of this organism and add criteria for the identification of this genus.