Cytochemical demonstration of alkaline phosphatase in the contractile vacuole of Dictyostelium discoideum.
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Biomedical subjects
Publications and source records attributed to A Ryter.
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Electroplaques dissected from the electric organ of Electrophorus electricus are labeled by tritiated alpha1-isotoxin from Naja nigricollis, a highly selective reagent of the cholinergic (nicotinic) receptor site. Preincubation of the cell with an excess of unlabeled alpha-toxin and with a covalent affinity reagent or labeling in the presence of 10(-4) M decamethonium reduces the binding of [3H]alpha-toxin by at least 75%. Absolute surface densities of alpha-toxin sites are estimated by high-resolution autoradiography on the basis of silver grain distribution and taking into account the complex geopmetry of the cell surface. Binding of [3H]alpha-toxin on the noninnervated face does not differ from background. Labeled sites are observed on the innervated membrane both between the synapses and under the nerve terminals but the density of sites is approx. 100 times higher at the level of the synapses than in between. Analysis of the distance of silver grains from the innervated membrane shows a symmetrical distribution centered on the postsynaptic plasma membrane under the nerve terminal. In extrasynaptic areas, the barycenter of the distribution lies approximately 0.5 micrometer inside the cell, indicating that alpha-toxin sites are present on the membrane of microinvaginations, or caveolae, abundant in the extrajunctional areas. An absolute density of 49,600 +/- 16,000 sites/micrometer2 of postsynaptic membrane is calculated; it is in the range of that found at the crest of the folds at the neuromuscular junction and expected from a close packing of receptor molecules. Electric organs were denervated for periods up to 142 days. Nerve transmission fails after 2 days, and within a week all the nerve terminals disappear and are subsequently replaced by Schwann cell processes, whereas the morphology of the electroplaque remains unaffected. The denervated electroplaque develops some of the electrophysiological changes found with denervated muscles (increases of membrane resting resistance, decrease of electrical excitability) but does not become hypersensitive to cholinergic agonists. Autoradiography of electroplaques dissected from denervated electric organs reveals, after labeling with [3H]alpha-toxin, patches of silver grains with a surface density close to that found in the normal electroplaque. The density of alpha-toxin binding sites in extrasynaptic areas remains close to that observed on innervated cells, confirming that denervation does not cause an increase in the number of cholinergic receptor sites. The patches have the same distribution, shape,and dimensions as in subneural areas of the normal electroplaque, and remnants of nerve terminal or Schwann cells are often found at the level of the patches. They most likely correspond to subsynaptic areas which persist with the same density of [3H]alpha-toxin sites up to 52 days after denervation. In the adult synapse, therefore, the receptor protein exhibits little if any tendency for lateral diffusion.
The morphometric analysis of growing cells shows that the membranes of the digestive apparatus have a surface area equal to the cell surface area. After yeast phagocytosis, the surface area of the membrane surrounding the ingested yeast is equal to 40% of the surface area of the cell membrane. In spite of this internalization, the cell surface remains constant. Its renewal is insured by the translocation of the membrane of the digestive system, the surface area that concomitantly decreases by 40%. This means that the influx of plasma membrane is continually compensated for by the same outflow of internal membranes. During this turnover, the characteristic polysaccharide stainability (two different stains were used) of the plasma membrane is maintained after internalization, at the level of the digestive system, despite the presence of hydrolases in the digestive vacuoles. The cytochemical demonstration of acid phosphatase shows that this enzyme penetrates into phagosomes by fusion between phagosomes and vacloles of various sizes. The debris of digested yeast are released into the culture medium after 2 h. This process of defecation is accompanied by the appearance of new pinocytotic vacuoles, which indicates that the uptake of axenic medium has resumed. A model of membrane turnover is proposed to explain these observations.
The effects of starvation on the cell morphology of Dictyostelium discoideum were studied with different cytochemical techniques, and with a morphometric method by which the surface areas of the cell membrane and of the digestive system can be determined. During the first 2 h, the cell membrane becomes very wrinkled and many phagocytic cups and filopods are formed. These changes are in accord with the 40 percent increase in the cell surface area to cytoplasmic volume ratio observed, which is mainly due to a strong decrease in the cytoplasmic volume. At this time of starvation, cells are able to ingest twice as many yeast as during growth. Afterwards, while the phagocytic ability decreases, the phagocytic cups disappear, and all the cells become bristled with many thin filopods. In spite of these morphological changes, no quantitative or topological differences have been observed concerning the polysaccharide content of the plasma membrane, whether it was stained with phosphotungstic acid, silver proteinate, or ruthenium red. During this time, the digestive vacuoles imbricate one into the other. Part of the vacuoles are degraded by this process, thus leading to an atrophy of the digestive apparatus. The digestive apparatus is progressively replaced by an autophagic system. Polysaccharide stainings and morphological observations show that the cytosegresomes seem to originate from the food vacuoles which flatten and sequester portions of cytoplasm. After 5 h of starvation, the digestive system is entirely transformed into an autophagic apparatus. The cell population appears to be homogeneous with respect to these changes. Therefore, potential precursors of prestalk and prespore cells were not observed.
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Induction of the synthesis of the receptor for phage lambda is obtained by adding maltose and adenosine 3'-5'-cyclic monophosphate to glucose grown cells of Escherichia coli. Bacteria induced for a short period of time were infected with a high multiplicity of phage lambda , and examined under the electron microscope. Only a fraction of the bacteria were seen to have adsorbed a large number of phage particles. The majority of such bacteria had a constriction indicating formation of a septum, and, in this case, the density of adsorbed particles was highest in the vicinity of the constriction. When found on bacteria showing no sign of septum formation, the adsorbed particles were asymmetrically distributed, one pole of the bacteria being more heavily covered with phage particles than the other. Such asymetrically covered bacteria are believed to have originated from cells which divided during the induction period. The results suggest that the receptor for phage lambda, a protein of the outer membrane, is integrated in the cell envelope during the last quarter of each generation and that the integration process is initiated in the vicinity of the forming septum.
Growth of the cell wall of Bacillus megaterium was studied by pulse-labeling the cell wall of a DAP- Lys- mutant for a very short time with tritium-labeled diaminopimelic acid. The distribution of radioactivity along the cell wall was examined by high-resolution autoradiography on isolated cell walls and thin sections of bacteria. The results indicate that cell wall elongation occurs by diffuse intercalation of newly synthesized murein into the expanding cell wall during exponential growth, as well as during germination, and that the only zone of highly localized diaminopimelic acid incorporation is found at the cross wall during its synthesis. This zone contains about 30% of the radioactivity incorporated into the cell wall. Analysis of autoradiographs of thin sections of bacteria shows that the total radioactivity incorporated per bacterium doubles during the life cycle. This doubling occurs in the cylindrical part of the cell wall but not in the polar caps. This seems to indicate that elongation of the bacterium is not constant during the life cycle but increases with the length of the cell.
Study of the cell wall growth in Bacillus megaterium by pulse-labeling a DAP- Lys- mutant with tritiated diaminopimelic acid (DAP) had revealed the presence of intracytoplasmic radioactivity. The nature of this radioactivity was studied on one hand by autoradiographic analysis of bacteria treated in different ways and on the other hand by chromatography of the radioactive compounds extracted with boiling water. It is shown that cytoplasmic radioactivity corresponds neither to free DAP nor to DAP metabolized into lysine, but to murein precursors. Autoradiographic analysis of bacteria in which all murein precursors were removed gives exactly the same cell wall growth pattern as the one previously obtained for untreated bacteria. It can be concluded that, in B. megaterium, cell wall elongation occurs by diffuse intercalation of newly synthesized murein along the cylindrical part of the cell wall and that only cross wall formation occurs in a precise growth zone.
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