Poliovirus RNA replicative complexes are associated with cytoplasmic membranous structures that accumulate during viral infection. These membranes were immunoisolated by using a monoclonal antibody against the viral nonstructural protein 2C. Biochemical analysis of the isolated membranes revealed that several organelles of the host cell (lysosomes, trans-Golgi stack and trans-Golgi network, and endoplasmic reticulum) contributed to the virus-induced membranous structures. Electron microscopy of infected cells preserved by high-pressure freezing revealed that the virus-induced membranes contain double lipid bilayers that surround apparently cytosolic material. Immunolabeling experiments showed that poliovirus proteins 2C and 3D were localized to the same membranes as the cellular markers tested. The morphological and biochemical data are consistent with the hypothesis that autophagy or a similar host process is involved in the formation of the poliovirus-induced membranes.
Bac8c (RIWVIWRR-NH 2 ) is an 8-amino-acid peptide derived from Bac2A (RLARIVVIRVAR-NH 2 ), a C3A/ C11A variant of the naturally occurring bovine peptide, bactenecin (also known as bovine dodecapeptide), the smallest peptide with activity against a range of pathogenic Gram-positive and Gram-negative bacteria, as well as yeast. The effects of Bac8c on Escherichia coli were examined by studying its bacteriostatic and bactericidal properties, demonstrating its effects on proton motive force generation, and visually analyzing (via transmission electron microscopy) its effects on cells at different concentrations, in order to probe the complexities of the mechanism of action of Bac8c. Results were consistent with a two-stage model for the Bac8c mode of action. At sublethal concentrations (3 g/ml), Bac8c addition resulted in transient membrane destabilization and metabolic imbalances, which appeared to be linked to inhibition of respiratory function. Although sublethal concentrations resulted in deleterious downstream events, such as methylglyoxal formation and free radical generation, native E. coli defense systems were sufficient for full recovery within 2 h. In contrast, at the minimal bactericidal concentration (6 g/ml), Bac8c substantially but incompletely depolarized the cytoplasmic membrane within 5 min and disrupted electron transport, which in turn resulted in partial membrane permeabilization and cell death.
To circumvent the limitations of chemical fixation (CF) and to gain more reliable structural information about higher plant tissues, we have cryofixed root tips of Nicotiana and Arabidopsis by high pressure freezing (HPF). Whereas other freezing techniques preserve tissue to a relatively shallow depth, HPF in conjunction with freeze substitution (FS) resulted in excellent preservation of entire root tips. Compared to CF, in tissue prepared by HPF/FS: (1) the plasmalemma and all internal membranes were much smoother and often coated on the cytoplasmic side by a thin layer of stained material, (2) the plasmalemma was appressed to the cell wall, (3) organelle profiles were rounder, (4) the cytoplasmic, mitochondrial, and amyloplast matrices were denser, (5) vacuoles contained electron dense material, (6) microtubules appeared to be more numerous and straighter, with crossbridges observed between them, (7) cisternae of endoplasmic reticulum (ER) were wider and filled with material, (8) Golgi intercisternal elements were more clearly resolved and were observed between both Golgi vesicles and cisternae, and (9) larger vesicles were associated with Golgi stacks. This study demonstrates that HPF/FS can be used to successfully preserve the ultrastructure of relatively large plant tissues without the use of intracellular cryoprotectants.
We have expressed a fusion protein formed between the avian infectious bronchitis virus M protein and the bacterial enzyme f-glucuronidase in transgenic tobacco cells. Electron microscope images of such cells demonstrate that overexpression of this fusion protein gives rise to a type of endoplasmic reticulum membrane domain in which adjacent membranes become zippered together apparently as a consequence of the oligomerizing action of j3-glucuronidase. These zippered (Z-) membranes lack markers of the endoplasmic reticulum (NADH cytochrome c reductase and ribosomes) and accumulate in the cells in the form of multilayered scroll-like structures (up to 2 ,um in diameter; 20-50 per cell) without affecting plant growth. The discovery of Zmembranes has broad implications for biology and biotechnology in that they provide a means for accumulating large quantities of recombinant membrane proteins within discrete domains of native membranes.
The accompanying paper describes the effects of applied electric fields on the morphogenesis and patterns of wall deposition of growing cells of Micrasterias denticulata. This paper details the effects of electric fields (approximately 14 V cm-1) on the subcellular components of Micrasterias, including a description of the plasma membrane of growing semi-cells as visualized by freeze-fracturing. There are no gross cytoplasmic abnormalities or asymmetrics in the distributions of cytoplasmic organelles caused by the fields. In particular, neither the Large Vesicles nor Dark Vesicles are concentrated in the cathode-facing (CF) halves of lobes oriented perpendicular to the fields, where extra deposition of wall material has been shown to occur. In freeze-fracture replicas, there are about twice as many plasma membrane particles near the tips of growing lobes as there are in proximal regions of the lobes. Additionally, rosettes, consisting of 6 membrane particles, are seen predominantly in the distal parts of the lobes, and these rosettes are believed to be important in the synthesis of cell wall microfibrils. The applied fields cause a large asymmetry in the distributions of membrane particles, with larger numbers being found on the CF sides of lobes oriented perpendicular to the fields. We were not able to detect a specific effect on any class of particles. Taken all together, the data support the hypothesis that some of the factors responsible for growth localization in Micrasterias reside in the plasma membrane.
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