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B Bowers

Publications and source records attributed to B Bowers.

At least 73 records · Page 4Linked to original sources
PubMed1984

Hydrolase secretion is a consequence of membrane recycling.

Acanthamoeba releases lysosomal hydrolases continuously into the culture medium. This release is specific for lysosomal hydrolases, but not other cellular proteins, and is energy dependent. The secreted hydrolases can be separated into two groups on the basis of their secretion kinetics: one is secreted at approximately 15% of the cellular activity per hour and the other at approximately 5%. Intracellularly the lysosomal hydrolases are restricted almost exclusively to secondary lysosomes where the hydrolases demonstrate a differential pH-dependent binding to membrane. Hydrolase secretion is not the result of secondary lysosomes' fusing with the plasma membrane since soluble and particulate lysosomal contents are not released at the same rate. Together the data suggest that the secreted hydrolases are trapped in shuttle vesicles that cycle membrane from secondary lysosomes to the cell surface. The inner membrane and content of these vesicles undergo a marked pH shift when, following fragmentation from lysosomes, these vesicles fuse with plasma membrane. This rapid pH shift and the differential pH-dependent membrane binding of hydrolases appear to account for the heterogeneous hydrolase secretion kinetics.

Amoeba↗
PubMed1984

Saccharomyces cerevisiae mannoproteins form an external cell wall layer that determines wall porosity.

A beta-glucanase (Z-glucanase) from Zymolyase was freed from a protease (Z-protease) by affinity chromatography on alpha 2-macroglobulin-Sepharose columns and used to solubilize proteins from isolated cell walls of Saccharomyces cerevisiae. The cell wall proteins were labeled with 125I and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The bulk of the labeled material had very low mobility. Its mannoprotein nature was demonstrated by precipitation with specific antibodies and by conversion to a band with an average molecular weight of 94,000 after incubation with endo-beta-N-acetylglucosaminidase. The intact mannoproteins were hydrolyzed by Z-protease, but were resistant to the enzyme when the carbohydrate was first removed by endo-beta-N-acetylglucosaminidase. In intact cells, lysis of cell walls by Z-glucanase required a previous incubation with z-protease, which led to solubilization of most of the 125I-labeled proteins. Other proteases that did not attack the cell wall mannoproteins were unable to substitute for Z-protease. The specific effect of Z-protease is consistent with the notion that mannoproteins form a surface layer of the cell wall that penetrates the wall to some depth and shields glucans from attack by Z-glucanase. Mannoproteins, however, do not appear to cover the inner face of the cell wall, because isolated cell walls, in contrast to intact cells, were completely solubilized by Z-glucanase in the absence of protease. The function of mannoproteins in determining cell wall porosity was highlighted by the finding that horseradish peroxidase (Mr, 40,000) causes lysis of cells that had been treated with Z-protease. Depletion of mannoproteins by Z-protease also resulted in the disappearance of a darkly stained surface layer of the cell wall, as observed by electron microscopy. Other agents that facilitate cell lysis by Z-glucanase, such as 2-mercaptoethanol, digitonin, and high concentrations of salts, caused little or no solubilization of mannoprotein. We assume that they perturb and loosen the structure of the mannoprotein network, thereby increasing its porosity. The implications of our results for the construction of the yeast cell wall and the anchoring of mannoprotein to the cell are discussed.

Cell Wall↗
PubMed1983

Vectorial synthesis of a polysaccharide by isolated plasma membranes.

To ascertain the directionality of chitin synthesis by yeast plasma membranes, the external surface of Saccharomyces cerevisiae protoplasts was labeled with ferritin--concanavalin A. After protoplast lysis, plasma membranes were isolated and treated with trypsin to activate chitin synthase (UDP-2-acetamido-2-deoxy-D-glucose:chitin 4-beta-acetamidodeoxy-D-glucosyl-transferase, EC 2.4.1.16). The membranes were then enrobed in agar and allowed to synthesize chitin from UDP-N-acetylglucosamine. After fixation and embedding in Epon, thin sections were stained for chitin with wheat germ agglutinin--colloidal gold complexes. The chitin marker was found near the ferritin-labeled external face of the membrane--i.e., the polysaccharide was located on the outside of the membrane, as it is in the intact cell. Chitin synthase activity was not detected in intact protoplasts before or after treatment with trypsin. The enzyme became available to trypsin activation after lysis of the protoplasts. Together with similar, previously reported experiments on the inactivation of chitin synthase by glutaraldehyde, these results indicate that the enzyme faces the interior of the cell. We conclude that, both in vivo and in vitro, the synthase receives N-acetylglucosamine residues from UDP-N-acetylglucosamine at the cytoplasmic face of the membrane and transfers them vectorially to a growing chain of chitin that is concomitantly extruded to the outside.

Cell Membrane↗
PubMed1983

Acanthamoeba discriminates internally between digestible and indigestible particles.

The capacity of Acanthamoeba to distinguish nutritive yeast particles from non-nutritive plastic beads during phagocytosis was investigated. When cells were allowed to phagocytose yeast to capacity, endocytosis stopped and subsequent presentation of particles (either yeast or beads) did not result in further uptake. By contrast, when cells were allowed to phagocytose plastic beads to capacity and a second dose of particles was presented (either yeast or beads), the cells exocytosed the internal particles and took up new ones. Yeast rendered indigestible by extensive chemical cross-linking were taken up at rates similar to those of untreated yeast, but, like beads, they were exocytosed when a second dose of particles was presented. The results show that an internal distinction is made between vacuoles containing yeast and vacuoles containing plastic beads, and they are consistent with the hypothesis that the presence within the vacuoles of material capable of being digested prevents exocytosis.

Amoeba↗
PubMed1983

Chitin synthesis and localization in cell division cycle mutants of Saccharomyces cerevisiae.

Growth of Saccharomyces cerevisiae cell cycle mutants cdc3, cdc4, cdc7, cdc24, and cdc28 at a nonpermissive temperature (37 degrees C) resulted in increased accumulation of chitin relative to other cell wall components, as compared with that observed at a permissive temperature (25 degrees C). Wild-type cells showed the same chitin/carbohydrate ratio at both temperatures, whereas mutants cdc13 and cdc21 yielded only a small increase in the ratio at 37 degrees C. These results confirm and extend those reported by B. F. Sloat and J. R. Pringle (Science 200:1171-1173, 1978) for mutant cdc24. The distribution of chitin in the cell wall was studied by electron microscopy, by specific staining with wheat germ agglutinin-colloidal gold complexes. At the permissive temperature, chitin was restricted to the septal region in all strains, whereas at 37 degrees C a generalized distribution of chitin in the cell wall was observed in all mutants. These results do not support a unique interdependence between the product of the cdc24 gene and localization of chitin deposition; they suggest that unbalanced conditions created in the cell by arresting the cycle at different stages result in generalized activation of the chitin synthetase zymogen. Thus, blockage of an event in the cell cycle may lead to consequences that are not functionally related to that event under normal conditions.

Cell Cycle↗
PubMed1981

Morphometric analysis of volumes and surface areas in membrane compartments during endocytosis in Acanthamoeba.

Stereologic analysis was made of cell surface membrane (PM) and two interrelated cytoplasmic membrane systems, the vacuole membranes (VM) and small vesicle membranes (SVM). Volumes and surface areas of the three membrane compartments were measured during steady-state pinocytosis, when membrane recycling is rapid, and during phagocytosis, when a shift to a lower rate of membrane uptake by endocytosis occurs (B. Bowers, 1977, Exp. Cell Res. 110:409). Total membrane area in the three compartments was 3.2 micrometers 2/micrometers 3 of protoplasmic volume and was constant throughout the experiments. In pinocytosing cells, 32% of the membrane was in the PM, 25% in the vM, and 43% in the SVM. The vacuole compartment occupies approximately 20% of the total cell volume, and the small vesicle, approximately 3%. As the endocytic uptake of membrane from the surface decreased, there was an increase in PM area and a marked decrease in SVM area. The VM area remained constant even though "empty" vacuoles were almost completely replaced by newly formed phagosomes within 45 min. This demonstrates directly a rapid flux of membrane though this compartment. A model, taking into consideration these and other data on Acanthamoeba, is proposed to account for the observed membrane shifts. The data suggest that the vacuolar (digestive) system of Acanthamoeba is central to cellular control of endocytosis and membrane recycling.

Amoeba↗
PubMed1981

Localization of lipophosphonoglycan in membranes of Acanthamoeba by using specific antibodies.

Antibodies against two electrophoretically distinct forms of lipophosphonoglycan (LPG) were produced in rabbits. Antibody specificity was demonstrated by the coupled antibody 125I-protein A assay (Adair et al., J. Cell Biol. 79:281-285, 1978). Indirect immunofluorescent labeling of intact Acanthamoeba showed that antibodies to both LPG components had the same uniform distribution on the cell surface. Both antibodies also bound to the cytoplasmic surface of isolated phagosomes. The location of LPG in other membranes of the amoeba was demonstrated on sections by the unlabeled antibody method. Although LPG was absent from the nuclear membrane, virtually all of the internal vacuole membranes were labeled, including the contractile vacuole. Antibodies directed against LPG were utilized to label lipophosphonoglycan in the plasma membrane of living amoebae. Labeled membrane was internalized and then localized by immunofluorescence in cytoplasmic vacuoles within 10 min of incubation. Although these results are evidence for exchange between plasma and cytoplasmic vacuolar membranes, the contractile vacuole remained unlabeled and can be considered, therefore, a separate membrane compartment. Concanavalin A also was bound and internalized by the amoeba, but electron microscopy showed that this label caused pronounced membrane perturbation, limiting its usefulness as a membrane marker in this system.

Acanthamoeba↗
PubMed1980

A morphological study of plasma and phagosome membranes during endocytosis in Acanthamoeba.

Particle ingestion by Acanthamoeba is rapid. Within 40 s bound particles can be surrounded by pseudopods, brought into the cytoplasm, and released as phagosomes into the cytoplasmic stream. In electron micrographs the phagosome appears as a flasklike invagination of the surface. Separation from the surface occurs by fragmentation of the attenuated "neck+ of the invagination. The separated phagosome membrane has a three- to fourfold greater density of intramembrane particles than the plasma membrane from which it derives. This change is evident within 15 min of ingestion and is detectable while the membrane is still tightly apposed to the particle. There is no direct evidence for the mechanism of this increase; no increase in particle density was seen in the membrane at an early stage in the forming phagosomes still connected to the surface. These morphological observations are consistent with chemical analyses, to be reported in a separate communication, that show that the phagosome membrane has a higher protein to phospholipid ratio and a higher glycosphingolipid content than the plasma membrane. Enlarged phagosomes (presumptive phagolysosomes) show multiple small vesiculations of characteristic morphology. The small vesicles are postulated to be the major route of membrane return to the cell surface.

Amoeba↗
PubMed1980

Distribution of chitin in the yeast cell wall. An ultrastructural and chemical study.

The distribution of chitin in Saccharomyces cervisiae primary septa and cell walls was studied with three methods: electron microscopy of colloidal gold particles coated either with wheat germ agglutinin or with one of two different chitinases, fluorescence microscopy with fluorescein isothiocyanate derivatives of the same markers, and enzymatic treatments of [14C]glucosamine-labeled cells. The septa were uniformly and heavily labeled with the gold-attached markers, an indication that chitin was evenly distributed throughout. To study the localization of chitin in lateral walls, alkali-extracted cell ghosts were used. Observations by electron and fluorescence microscopy suggest that lectin-binding material is uniformly distributed over the whole cell ghost wall. This material also appears to be chitin, on the basis of the analysis of the products obtained after treatment of 14C-labeled cell ghosts with lytic enzymes. The chitin of lateral walls can be specifically removed by treatment with beta-(1 leads to 6)-glucanase containing a slight amount of chitinase. During this incubation approximately 7% of the total radioactivity is solubilized, about the same amount liberated when lateral walls of cell ghosts are completely digested with snail glucanase yield primary septa. It is concluded that the remaining chitin, i.e., greater than 90% of the total, is in the septa. The facilitation of chitin removal from the cell wall by beta-(1 leads to 6)-glucanase indicates a strong association between chitin and beta-(1 leads to 6)-glucan. Covalent linkages between the two polysaccharides were not detected but cannot be excluded.

Cell Wall↗
PubMed1979

Chitin synthetase distribution on the yeast plasma membrane.

Purified, intact yeast plasma membranes were allowed to synthesize chitin, and the nascent chains of polysaccharide were observed either by the fluorescence produced with a brightener or by autoradiography. By both methods, it was concluded that the newly formed chitin emerged at many sites on each membrane. Thus, the synthetase that catalyzes chitin formation has a similar distribution. Since chitin synthetase is found mainly in a zymogen form, these results confirm the hypothesis that initiation of the chitinous primary septum of Saccharomyces occurs by localized activation of the uniformly distributed zymogen.

Cell Membrane↗
PubMed1977

Methanococcus vannielii: ultrastructure and sensitivity to detergents and antibiotics.

Methanococcus vannielii is a strictly anaerobic motile coccus that possesses a tuft of flagellae. The cells are markedly sensitive to mechanical stress and are readily lysed by detergents, but the organism grows normally in media of low ionic strength. The absence of a typical cell wall, further suggested by resistance of M. vannielii to penicillin, cycloserine, and vancomycin, was confirmed by ultrastructural studies. Electron micrographs showed that the cell envelope lacks a peptidoglycan layer. On the outer surface there is a regular array of subunits similar to those of the glycoprotein envelopes of the halobacteria. However, the M. vannielii cell envelope, unlike those of the holobacteria, is unable to maintain a definite shape, and a high salt concentration is not required for its integrity.

Anti-Bacterial Agents↗
PubMed1975

Chitin synthetase zymogen is attached to the yeast plasma membrane.

Pretreatment of yeast protoplasts with concanavalin A, according to the method used by G. A. Scarborough for Neurospora (J. Biol. Chem. 250, 1106-1111, 1975), reinforced the plasma membranes, and helped to maintain their integrity during subsequent lysis of the protoplasts. After purification by centrifuging on a Renografin density gradient, practically intact membranes were obtained. Previous labeling of the protoplasts with 125I or with [3H]concanavalin A resulted in recovery of the radioactivity in the membrane fraction. The bulk of the chitin synthetase (chitin synthase; UDP-2-acetamido-2-deoxy-D-glucose:chitin 4-beta-acetamidodeoxyglucosyltransferase; EC 2.4.1.16) recovered in the gradient was also found In this fraction; in the zymogen form. About 20% of the activity sedimented in a plasma-membrane-free fraction at lower density. Glutaraldehyde inactivated chitin synthetase when it was added to a lysate, but not when applied to intact protoplasts. It is concluded that chitin synthetase is so oriented in the membrane that it is only accessible from the inside of the cell. These results confirm our previous hypothesis that the chitin synthetase zymogen is associated with the plasma membrane, a basic assumption for the explanation of localized activation of the enzyme and initiation of septum formation.

Cell Fractionation↗