Effect of 2-deoxyglucose on cell wall formation in Saccharomyces cerevisiae and its relation to cell growth inhibition

The growth inhibition and the lysis of Saccharomyces cerevisiae caused by 2-deoxy-d-glucose (2-DG) were shown to be a consequence of unbalanced cellular growth and division. The lysis, but not the repression of growth and osmotic fragility of cells, could be suppressed by the addition of mannitol as an osmotic stabilizer. This result, as well as the morphological changes observed in the cells and changes in the chemical composition of the cell walls, showed that S. cerevisiae grown in the presence of 2-DG formed weakened cell walls responsible for the osmotic fragility. Evidence is presented for the first time demonstrating the incorporation of 2-DG into yeast cell wall material. Other data suggest that the inhibition of yeast growth by 2-DG results from an interference of phosphorylated metabolites of 2-DG with metabolic processes of glucose and mannose involved in the synthesis of structural cell wall polysaccharides.     

Electron Microscopy and Viability of Lysostaphin-induced Staphylococcal Spheroplasts, Protoplast-like Bodies, and Protoplasts

The cell walls of a selected isolate of Staphylococcus aureus FDA 209P were observed undergoing progressive disintegration when exposed to lysostaphin (1 unit/ml) in 24% NaCl solution. Electron micrographs of ultrathin sections of test cells after exposure to lysostaphin for 2 min showed only superficial evidence of lytic damage. However, an average of 89% of these cells were osmotically fragile, and 21% were damaged beyond their capacity to regenerate cell walls and to grow as normal staphylococci. The 68% (average) of the osmotically fragile cells which retained the capacity to revert to normal staphylococci were designated spheroplasts. Neither perforations of the cell walls nor separation of the cell walls from the plasma membranes were observed in the micrographs of these 2-min spheroplasts. Thus, it appears that the osmotic fragility of these and possibly all lysostaphin-induced staphylococcal spheroplasts results from the hydrolysis of a critical number of the pentapeptide cross-linkages of the murein of the cell wall. Electron micrographs of cells exposed to lysostaphin for 5 to 10 min showed perforations and more extensive damage, including the separation of walls from the plasma membranes and the disintegration of large sections of the walls. Smaller numbers of spheroplasts (21 and 8%) were recovered from these 5- and 10-min preparations; those recovered probably represent cells which were attacked more slowly than the majority by the lytic enzyme. The nonrevertible, osmotically fragile cells that retained segments of cell wall were designated protoplast-like bodies. After 20-min exposure to lysostaphin, all of the cell wall was digested away from most of the cells, and true staphylococcal protoplasts were produced. These lysostaphin-induced, osmotically fragile forms appear to have different osmotic properties from the staphylococcal "protoplasts" reported by other investigators and should serve as the basis for a variety of fundamental investigations.     

Genetic alteration of pore size and other properties of the Neurospora cell wall

Trevithick, John R. (University of Wisconsin Medical School, Madison), Robert L. Metzenberg and Donald F. Costello. Genetic alteration of pore size and other properties of the Neurospora cell wall. J. Bacteriol. 92:1016-1020. 1966.-Several properties of the cell walls of wild type and the osmotic mutant of Neurospora crassa have been examined. The peameability of the isolated cell walls to polyethylene glycol and dextran polymers of different molecular weights was investigated by the volume of distribution technique. The exclusion thresholds were evaluated by a statistical treatment. The molecular weights corresponding to these thresholds for wild type and osmotic were approximately 4,750 and 18,500, respectively; these values are significantly different. The cell walls of osmotic appeared to be thinner, more easily broken, and more easily compressed to ribbonlike shapes, whereas those of wild type were tubular and strong. Chemical analysis showed that osmotic walls had roughly a 30-fold higher galactosamine-glucosamine ratio than did wild type. It is proposed that the osmotic mutant has a cell wall with abnormally large pores, and that this may account for the increased rate of egress of invertase and the decreased fractionation of light from heavy invertase in this strain.     

The relationship between the osmotic fragility of human erythrocytes and cell age

Erythrocytes in a normal blood sample are hemolyzed over a range of hypotonic salt concentrations. In order to investigate the relationship between the distribution of osmotic fragilities and the distribution of cellular ages, the osmotic fragility has been compared with three indices of cellular age. The activity of glutamic-oxalacetic transaminase (GOT) and the percentage hemoglobin A_1C were measured in samples hemolyzed in different hypotonic salt concentrations. The osmotic fragility curve was also obtained for cells of different density separated by centrifugation. These experiments indicate that the fragility distribution is not an accurate reflection of the distribution of cellular ages. The mean fragility for older cells is higher than that of younger cells. However, cellular aging does not produce a gradual increase in osmotic fragility. Instead, it seems to produce changes which can both increase and decrease the fragility, resulting in a broader distribution of fragilities with some of the older cells actually less fragile than the younger ones.     

Effects of temperature, oxygen and carbon dioxide on osmotic fragility of carp, Cyprinus carpio L., erythrocytes

The in vitro effects of gases and temperature on the osmotic fragility of carp erythrocytes were studied. At the three different temperatures analyzed (5, 11 and 20°C) there was no noticeable modification in erythrocyte membrane osmotic resistance. Osmotic fragility of red blood cells was altered by CO₂ and air treatment, as compared to the standard procedure. This suggests the need to take into account a possible moderate hypoxia that develops in the routine procedure of nucleated erythrocyte osmotic fragility tests.     

Danazol therapy renders red cells resistant to osmotic lysis

Danazol, an attenuated androgen, is useful in endometriosis, idiopathic thrombocytopenic purpura (ITP), and autoimmune hemolytic anemia (AIHA). However, its mechanism of action is unknown. We investigated the possibility that danazol affects cell membranes directly. Red cell osmotic fragility was studied in patients receiving danazol. A significant decrease in osmotic fragility was observed. Accompanying the change, peripheral blood smears showed many target cells and electron microscopy revealed extra folds in erythrocyte membranes. Twenty-two patients were studied prospectively before and after danazol. Osmotic fragility decreased significantly (P less than 0.001) in 1 month of therapy and progressed with further treatment. A rebound increase (P less than 0.01) was observed in 1 month after discontinuation of danazol among 16 patients. Incubation experiments showed that danazol-induced changes are not reversed with normal sera. Patient sera did not induce the changes in normal red cells. Danazol in vitro protected red cells from osmotic lysis at low concentrations but enhanced lysis at high concentrations. We suggest that danazol alters red cell membranes directly to increase their surface area, inducing target cell formation and increasing their resistance to osmotic lysis.     

Locus-specific Changes in Cell Wall Composition Characteristic of Osmotic Mutants of Neurospora crassa

The osmotic phenotype of Neurospora crassa is characterized by inhibition of growth at high osmolalities of growth medium. Mutations at six osmotic loci of linkage group I were examined to assess the biochemical and physiological effects of these mutants. Isolated cell walls from 23 osmotic strains were compared with the wild type with regard to quantitative levels of the following components: percentage of total dry weight, total glucose, alkali-soluble glucose, nonglucose carbohydrates, amino acids, glucosamine, galactosamine, and a compound tentatively identified as quinovosamine. The last component has not previously been observed in N. crassa cell walls. Although the cell wall dry weight content of osmotic mutants was not altered, walls isolated from all of the osmotic strains had less alkali-insoluble glucose than those from the wild type. In addition, all of the loci except cut exhibited other consistent differences from the wild type. The os-1, os-3, and os-5 mutants had low levels of alkali-soluble glucose. The os-3 and os-5 mutants had high levels of nonglucose carbohydrates, and flm-2 had a low level of nonglucose carbohydrates. The os-4 mutants had low levels of galactosamine and amino acids and high levels alkali-soluble glucose. An os-1 mutant, B135, produced less of the whole alkali-soluble fraction of the cell wall.     

The structure of cell wall alpha-glucan from fission yeast

Morphology and structural integrity of fungal cells depend on cell wall polysaccharides. The chemical structure and biosynthesis of two types of these polysaccharides, chitin and (1-->3)-beta-glucan, have been studied extensively, whereas little is known about alpha-glucan. Here we describe the chemical structure of alpha-glucan isolated from wild-type and mutant cell walls of the fission yeast Schizosaccharomyces pombe. Wild-type alpha-glucan was found to consist of a single population of linear glucose polymers, approximately 260 residues in length. These glucose polymers were composed of two interconnected linear chains, each consisting of approximately 120 (1-->3)-linked alpha-d-glucose residues and some (1-->4)-linked alpha-D-glucose residues at the reducing end. By contrast, alpha-glucan of an alpha-glucan synthase mutant with an aberrant cell morphology and reduced alpha-glucan levels consisted of a single chain only. We propose that alpha-glucan biosynthesis involves an ordered series of events, whereby two alpha-glucan chains are coupled to create mature cell wall alpha-glucan. This mature form of cell wall alpha-glucan is essential for fission-yeast morphogenesis.     

Regulation of Cell Wall Synthesis by the Clathrin Light Chain Is Essential for Viability in Schizosaccharomyces pombe

The regulation of cell wall synthesis by the clathrin light chain has been addressed. Schizosaccharomyces pombe clc1Δ mutant was inviable in the absence of osmotic stabilization; when grown in sorbitol-supplemented medium clc1Δ cells grew slowly, formed aggregates, and had strong defects in morphology. Additionally, clc1Δ cells exhibited an altered cell wall composition. A mutant that allowed modulating the amount of Clc1p was created to analyze in more detail the dependence of cell wall synthesis on clathrin. A 40% reduction in the amount of Clc1p did not affect acid phosphatase secretion and bulk lipid internalization. Under these conditions, β(1,3)glucan synthase activity and cell wall synthesis were reduced. Also, the delivery of glucan synthases to the cell surface, and the secretion of the Eng1p glucanase were defective. These results suggest that the defects in the cell wall observed in the conditional mutant were due to a defective secretion of enzymes involved in the synthesis/remodelling of this structure, rather than to their endocytosis. Our results show that a reduction in the amount of clathrin that has minor effects on general vesicle trafficking has a strong impact on cell wall synthesis, and suggest that this is the reason for the lethality of clc1Δ cells in the absence of osmotic stabilization.     

Hyperpolarized growth of Saccharomyces cerevisiae cak1P212S and cla4 mutants weakens cell walls and renders cells dependent on chitin synthase 3.

In a screen for cell wall defects in Saccharomyces cerevisiae, we isolated a strain carrying a mutation in the Cdc28-activating kinase CAK1. The cak1P212S mutant cells exhibit multiple, elongated and branched buds, beta(1,3)glucan-poor regions of the cell periphery and lysed upon osmotic shock after treatment with the chitin synthase III inhibitor Nikkomycin Z. Ultrastructural examination of cak1P212S mutants revealed a thin, uneven cell wall and marked abnormalities in septum formation. In all of the above aspects, the cak1P212S mutants are similar to previously described cla4 mutants, suggesting that the cell wall defects are common to mutants with hyperpolarized growth. In cak1P212S mutants, chitin accumulates all over the surface of the cells and glucan synthase activity is located preferentially to the tips of elongated buds. We conclude that the cell wall weakness in cak1P212S mutants is caused by hyperpolarized secretion of glucan synthase and lack of reinforcement of the lateral cell walls. Showing that the defect depends at least in part on Cdc28, the cak1P212S hyperpolarized growth phenotype can be suppressed by a Cak1-independent Cdc28-allele. The results underline the importance of a minor cell wall component, the chitin of lateral walls, for the integrity of the cell in a stress situation.     

Characterization of a fission yeast mutant which displays defects in cell wall integrity and cytokinesis.

The fission yeast cps6-153 mutant was originally isolated based on its hypersensitivity to the spindle poison isopropyl N-3-chlorophenyl carbamate (CIPC). The mutant also shows defects in both cell wall integrity and cytokinesis, resulting in the accumulation of unseparated cells with weakened cell walls. The arrested cells display a disoriented alignment of cytoplasmic microtubules. When the mutant cells are cultivated at high temperature (35 degrees C), both cell walls and septa become very thick. Electron microscopy revealed the disorganized structure of the thickened cell walls and septa, in which fibrillar components were not completely masked with an amorphous matrix. rad25+ was cloned from a genomic library by complementation of the mutant phenotypes, suggesting the involvement of Rad25p, one of two 14-3-3 proteins in S. pombe, in the pathway of cell wall integrity and cytokinesis.     

Osmotic properties of bovine erythrocytes aged in vivo

The osmotic properties of bovine erythrocytes aged in vivo were studied by the modified microhematocrit method. The osmotic fragility of older red cells decreases due to their larger relative osmotically non-active volume. Relative critical cell volume of bovine erythrocytes does not alter significantly with cell age. The age dependent change in the osmotic fragility of human red blood cells, the reverse of that found for bovine erythrocytes, is due to a different alteration of the critical cell volume during intravascular erythrocyte aging.     

Temperature-Sensitive Divisionless Mutant of Bacillus subtilis Defective in the Initiation of Septation

A temperature-sensitive divisionless mutant of Bacillus subtilis 168, tms-12, is shown to be defective in an early step in septum formation at the restrictive temperature. The nature of this defect has been studied by comparing the growth and composition of mutant and wild-type (tms-12(+)) cells at the restrictive (48 C) and permissive (34 C) temperatures. At 48 C, tms-12 cells grow as nonseptate, multinucleate filaments. Filamentation does not appear to be a result of alterations in properties of the cell wall, since the ratio of mucopeptide to teichoic acid, the autolytic activity, and the ability of the walls to protect cells against osmotic shock are comparable in tms-12 filaments and tms-12(+) bacilli grown at 48 C. Synthesis of deoxyribonucleic acid and the segregation of nucleoids also proceed normally during filamentation. The synthesis of membrane, however, is delayed during filamentation of tms-12. No gross alterations were observed in the protein or lipid composition of membranes isolated from mutant filaments. Septum formation resumes when filaments are returned to 34 C and appears to be associated with an increased synthesis of membrane. The occurrence of septa was monitored both by microscopic observation of cross walls and by assays of the number of viable protoplasts released from bacillary filaments upon removal of the cell wall. Septation recovery can be blocked by inhibitors of ribonucleic acid and protein synthesis added during, but not after, the first 7 min of recovery at 34 C. By contrast, inhibition of deoxyribonucleic synthesis does not block recovery.     

Physiological (osmotic fragility) and morphological effects on red blood cells: action of phytic acid and stannous fluoride

Phytic acid occurs in foods derived from plants. We have investigated the possibility that phytic acid and stannous fluoride are capable of altering the physiological properties (osmotic fragility) and morphological properties of red blood cells (RBC). Osmotic fragility was unchanged by the presence of phytic acid and stannous fluoride in the studied concentrations, but RBC morphology was modified in the presence of the studied substances. In conclusion, the alterations to RBC morphology were not sufficient to promote modifications in osmotic fragility. Our results suggest that the chelating properties of phytic acid could be responsible for the observed effects.     

Influence of lipid components of Mycoplasma laidlawii membranes on osmotic fragility of cells

Razin, S. (University of Connecticut, Storrs), M. E. Tourtellotte, R. N. McElhaney, and J. D. Pollack. Influence of lipid components of Mycoplasma laidlawii membranes on osmotic fragility of cells. J. Bacteriol. 91:609-616. 1966.-Lipid composition of Mycoplasma laidlawii membranes could be significantly changed by variations in the growth medium. The effect of these changes on the osmotic fragility of the cells was studied. Cholesterol, incorporated into the membrane from the growth medium, had no significant effect on osmotic fragility. Carotenoids, synthesized by the cells from acetate, were likewise without effect. Unsaturated long-chain fatty acids increased markedly the resistance of M. laidlawii to osmotic lysis and promoted growth. The fatty acids of the growth medium were incorporated mainly into membrane phospholipids. The ratio between saturated and unsaturated fatty acids in membrane lipids depended on that of the growth medium.     

Guard cell wall: immunocytochemical detection of polysaccharide components

The composition of guard cell walls in sugar beet leaves (Beta vulgaris L.) was studied by using histochemical staining and immunocytochemical detection of cell wall antigens. The findings were compared with those in the walls of epidermal and mesophyll cells. Probing of leaf sections with monoclonal antibodies against pectins, terminal fucosyl residues linked alpha-(1-->2) to galactose, beta-(1-->3)-glucans and arabinogalactan-proteins revealed several specific features of guard cells. Pectic epitopes recognized by JIM7 were homogeneously distributed in the wall, whereas pectins recognized by JIM5 were not found in the walls themselves, but were abundant in the cuticular layer. Large amounts of molecules bearing terminal fucose were located predominantly in ventral and lateral guard cell walls. Much smaller amounts were detected in dorsal walls of these cells, as well as in the walls of pavement and mesophyll cells. Conspicuous accumulation of these compounds was observed in the vicinity of the guard cell plasmalemma, whereas labelling was scarce in the areas of the wall adjacent to the cell surface. The presence of callose clearly marked the ventral wall between the recently formed, very young guard cells. Callose also appeared in some mature walls, where it was seen as punctate deposits that probably reflected a specific physiological state of the guard cells. Large amounts of arabinogalactan-proteins were deposited within the cuticle, and smaller amounts of these proteoglycans were also detected in other tissues of the leaf. The histochemical and immunocytochemical structure of the guard cell wall is discussed in the light of its multiple functions, most of which involve changes in cell size and shape.     

Changes in cell wall biomechanical properties in the xyloglucan-deficient xxt1/xxt2 mutant of Arabidopsis

The main load-bearing network in the primary cell wall of most land plants is commonly depicted as a scaffold of cellulose microfibrils tethered by xyloglucans. However, a xyloglucan-deficient mutant (xylosyltransferase1/xylosyltransferase2 [xxt1/xxt2]) was recently developed that was smaller than the wild type but otherwise nearly normal in its development, casting doubt on xyloglucan's role in wall structure. To assess xyloglucan function in the Arabidopsis (Arabidopsis thaliana) wall, we compared the behavior of petiole cell walls from xxt1/xxt2 and wild-type plants using creep, stress relaxation, and stress/strain assays, in combination with reagents that cut or solubilize specific components of the wall matrix. Stress/strain assays showed xxt1/xxt2 walls to be more extensible than wild-type walls (supporting a reinforcing role for xyloglucan) but less extensible in creep and stress relaxation processes mediated by α-expansin. Fusicoccin-induced "acid growth" was likewise reduced in xxt1/xxt2 petioles. The results show that xyloglucan is important for wall loosening by α-expansin, and the smaller size of the xxt1/xxt2 mutant may stem from the reduced effectiveness of α-expansins in the absence of xyloglucan. Loosening agents that act on xylans and pectins elicited greater extension in creep assays of xxt1/xxt2 cell walls compared with wild-type walls, consistent with a larger mechanical role for these matrix polymers in the absence of xyloglucan. Our results illustrate the need for multiple biomechanical assays to evaluate wall properties and indicate that the common depiction of a cellulose-xyloglucan network as the major load-bearing structure is in need of revision.     

Biochemical and immunohistochemical analysis of pectic polysaccharides in the cell walls of Arabidopsis mutant QUASIMODO 1 suspension-cultured cells: implications for cell adhesion

Mutation in the Arabidopsis thaliana QUASIMODO 1 gene (QUA1), which encodes a putative glycosyltransferase, reduces cell wall pectin content and cell adhesion. Suspension-cultured calli were generated from roots of wild-type (wt) and qua1-1 A. thaliana plants. The altered cell adhesion phenotype of the qua1-1 plant was also found with its suspension-cultured calli. Cell walls of both wt and qua1-1 calli were analysed by chemical, enzymatic and immunohistochemical techniques in order to assess the role of pectic polysaccharides in the mutant phenotype. Compared with the wt, qua1-1 calli cell walls contained more arabinose (23.6 versus 21.6 mol%), rhamnose (3.1 versus 2.7 mol%), and fucose (1.4 versus 1.2 mol%) and less uronic acid (24.2 versus 27.6 mol%), and they were less methyl-esterified (DM: 22.9% versus 30.3%). When sequential pectin extraction of calli cell walls was performed, qua1-1 water-soluble and chelator-soluble extracts contained more arabinose and less uronic acid than wt. Water-soluble pectins were less methyl-esterified in qua1-1 than in wt. Chelator-soluble pectins were more acetyl-esterified in qua1-1. Differences in the cell wall chemistry of wt and mutant calli were supported by a reduction in JIM7 labelling (methyl-esterified homogalacturonan) of the whole wall in small cells and particularly by a reduced labelling with 2F4 (calcium-associated homogalacturonan) in the middle lamella at tricellular junctions of large qua1-1 cells. Differences in the oligosaccharide profile obtained after endopolygalacturonase degradation of alkali extracts from qua1-1 and wt calli indicated variations in the structure of covalently bonded homogalacturonan. About 29% more extracellular polymers rich in pectins were recovered from the calli culture medium of qua1-1 compared with wt. These results show that perturbation of QUASIMODO 1-1 gene expression in calli resulted in alterations of homogalacturonan content and cell wall location. The consequences of these structural variations are discussed with regard to plant cell adhesion.     

Interaction between wall deposition and cell elongation in dark-grown hypocotyl cells in Arabidopsis

A central problem in plant biology is how cell expansion is coordinated with wall synthesis. We have studied growth and wall deposition in epidermal cells of dark-grown Arabidopsis hypocotyls. Cells elongated in a biphasic pattern, slowly first and rapidly thereafter. The growth acceleration was initiated at the hypocotyl base and propagated acropetally. Using transmission and scanning electron microscopy, we analyzed walls in slowly and rapidly growing cells in 4-d-old dark-grown seedlings. We observed thick walls in slowly growing cells and thin walls in rapidly growing cells, which indicates that the rate of cell wall synthesis was not coupled to the cell elongation rate. The thick walls showed a polylamellated architecture, whereas polysaccharides in thin walls were axially oriented. Interestingly, innermost cellulose microfibrils were transversely oriented in both slowly and rapidly growing cells. This suggested that transversely deposited microfibrils reoriented in deeper layers of the expanding wall. No growth acceleration, only slow growth, was observed in the cellulose synthase mutant cesA6(prc1-1) or in seedlings, which had been treated with the cellulose synthesis inhibitor isoxaben. In these seedlings, innermost microfibrils were transversely oriented and not randomized as has been reported for other cellulose-deficient mutants or following treatment with dichlorobenzonitrile. Interestingly, isoxaben treatment after the initiation of the growth acceleration in the hypocotyl did not affect subsequent cell elongation. Together, these results show that rapid cell elongation, which involves extensive remodeling of the cell wall polymer network, depends on normal cellulose deposition during the slow growth phase.