H. van den Ende

The cell wall architecture of Candida albicans wild-type cells and cell wall-defective mutants

In Candida albicans wild‐type cells, the β1,6‐glucanase‐extractable glycosylphosphatidylinositol (GPI)‐dependent cell wall proteins (CWPs) account for about 88% of all covalently linked CWPs. Approximately 90% of these GPI‐CWPs, including Als1p and Als3p, are attached via β1,6‐glucan to β1,3‐glucan. The remaining GPI‐CWPs are linked through β1,6‐glucan to chitin. The β1,6‐glucanase‐resistant protein fraction is small and consists of Pir‐related CWPs, which are attached to β1,3‐glucan through an alkali‐labile linkage. Immunogold labelling and Western analysis, using an antiserum directed against Saccharomyces cerevisiae Pir2p/Hsp150, point to the localization of at least two differentially expressed Pir2 homologues in the cell wall of C. albicans. In mnn9Δ and pmt1Δ mutant strains, which are defective in N‐ and O‐glycosylation of proteins respectively, we observed enhanced chitin levels together with an increased coupling of GPI‐CWPs through β1,6‐glucan to chitin. In these cells, the level of Pir‐CWPs was slightly upregulated. A slightly increased incorporation of Pir proteins was also observed in a β1,6‐glucan‐deficient hemizygous kre6Δ mutant. Taken together, these observations show that C. albicans follows the same basic rules as S. cerevisiae in constructing a cell wall and indicate that a cell wall salvage mechanism is activated when Candida cells are confronted with cell wall weakening.

Low external pH induces HOG1‐dependent changes in the organization of the Saccharomyces cerevisiae cell wall

Low environmental pH strongly affected the organization of the Saccharomyces cerevisiae cell wall, resulting in rapidly induced resistance to β1,3‐glucanase. At a molecular level, we found that a considerable amount of Cwp1p became anchored through a novel type of linkage for glycosylphosphatidylinositol (GPI)‐dependent cell wall proteins, namely an alkali‐labile linkage to β1,3‐glucan. This novel type of modification for Cwp1p did not require the presence of a GPI‐derived structure connecting the protein with β1,6‐glucan. In addition, we found high levels of Cwp1p, which was double‐anchored through both the novel alkali‐sensitive bond to β1,3‐glucan and the alkali‐resistant GPI‐derived linkage to β1,6‐glucan. Further cell wall analyses demonstrated that Pir2p/Hsp150 and possibly other Pir cell wall proteins, which were already known to be linked to the β1,3‐glucan framework by an alkali‐sensitive linkage, were also more efficiently retained in the cell wall at pH 3.5 than at pH 5.5. Consequently, the alkali‐sensitive type of linkage of cell wall proteins to β1,3‐glucan was induced by low pH. The low pH‐induced alterations in yeast cell wall architecture were demonstrated to be dependent on a functional HOG1 gene, but not on the Slt2p‐mediated MAP kinase pathway. Consistent with this observation, DNA microarray studies revealed transcriptional induction of many known high‐osmolarity glycerol (HOG) pathway‐dependent genes, including four cell wall‐related genes, namely CWP1, HOR7, SPI1 and YGP1.

The contribution of the O-glycosylated protein Pir2p/Hsp150 to the construction of the yeast cell wall in wild-type cells and β1,6-glucan-deficient mutants

The cell wall of yeast contains a major structural unit, consisting of a cell wall protein (CWP) attached via a glycosylphosphatidylinositol (GPI)‐derived structure to β1,6‐glucan, which is linked in turn to β1,3‐glucan. When isolated cell walls were digested with β1,6‐glucanase, 16% of all CWPs remained insoluble, suggesting an alternative linkage between CWPs and structural cell wall components that does not involve β1,6‐glucan. The β1,6‐glucanase‐resistant protein fraction contained the recently identified GPI‐lacking, O‐glycosylated Pir‐CWPs, including Pir2p/Hsp150. Evidence is presented that Pir2p/Hsp150 is attached to β1,3‐glucan through an alkali‐sensitive linkage, without β1,6‐glucan as an interconnecting moiety. In β1,6‐glucan‐deficient mutants, the β1,6‐glucanase‐resistant protein fraction increased from 16% to over 80%. This was accompanied by increased incorporation of Pir2p/Hsp150. It is argued that this is part of a more general compensatory mechanism in response to cell wall weakening caused by low levels of β1,6‐glucan.

Chemical structure of algaenans from the fresh water algae Tetraedron minimum, Scenedesmus communis and Pediastrum boryanum

Abstract The cell walls of the fresh water green microalgae Tetraedron minimum, Scenedesmus communis and Pediastrum boryanum are composed of highly resistant, non-hydrolyzable aliphatic biopolymers as revealed by 13C-NMR, FTIR and thermal and chemical degradations. The biopolymers are composed of long-chain even-carbon-numbered ω9-unsaturated ω-hydroxy fatty acid monomers varying in chain length from 30 to 34 carbon atoms. These monomers are intermolecularly ester linked to form linear chains in which the unsaturations act as the starting position of ether cross-linking. S. communis biosynthesises a more densely cross-linked algaenan than T. minimum and P. boryanum. The monomers of T. minimum have, on average, larger chain lengths than those of P. boryanum and S. communis. The polyether nature of these algaenans makes them highly resistant against degradation, so that they are selectively preserved in the sedimentary record. Therefore, these algaenans probably are important precursors for Type I kerogens.

Hyperosmotic stress induces rapid synthesis of phosphatidyl-D-inositol 3,5-bisphosphate in plant cells

Abstract. Cells from several different plant species synthesised a polyphosphoinositide (PPI)-like lipid when osmo-stressed. Synthesis was maximal after about 10 min and was stimulated by a variety of osmolytes. Using NaCl, the strongest response centred around 200 mM. The lipid was shown to be the novel PPI isomer phosphatidyl-inositol 3,5-bisphosphate [PtdIns-(3,5)P2] by analytical thin-layer chromatography and conversion to PtdIns(3,4,5)P3 using recombinant phosphoinositide 4-OH kinase. The results indicate that PtdIns-(3,5)P2 plays a role in a general osmo-signalling pathway in plants. Its potential role is discussed.

Detailed analysis of the turnover of polyphosphoinositides and phosphatidic acid upon activation of phospholipases C and D in Chlamydomonas cells treated with non-permeabilizing concentrations of mastoparan

Abstract. Treating Chlamydomonas moewusii cells with non-permeabilizing concentrations of mastoparan (1–5 μM) increased inositol 1,4,5-trisphosphate (InsP3) levels up to 20-fold in a dose-dependent manner and rapidly induced deflagellation and mating-structure activation, two well-defined Ca2+-responses. When metabolism of the phospholipid precursors was monitored in 32Pi-labelled cells, as much as 70% of the radioactivity in phosphatidylinositol bisphosphate (PtdInsP2) was lost within 20 s. Thereafter, the 32P-label in PtdInsP2 increased to twice the control level within 10 min. A similar pattern of 32P-labelling was also exhibited by PtdInsP. An HPLC-headgroup analysis revealed that only PtdIns4P and PtdIns(4,5)P2 were involved and not the D3-phosphorylated isomers. Correlated with the increased polyphosphoinositide (PPI) turnover, there was a massive (5- to 10-fold) increase in 32P-labelled phosphatidic acid (PtdOH) and, slightly later, an increase in its metabolic product, diacylglycerol pyrophosphate (DGPP), reflecting the phosphorylation of the resulting diacylglycerol (DAG) and PtdOH, respectively. Mastoparan-treatment of 32P-labelled cells in the presence of 0.2% n-butanol increased the formation of radioactive phosphatidylbutanol (PtdBut), a specific reporter of phospholipase D (PLD) activity. This means that mastoparan activates both phospholipase C (PLC) and PLD, and thus both pathways could contribute to the increase in PtdOH. To distinguish between them, a differential labelling strategy was applied based on the fact that 32Pi-label is slowly incorporated into structural phospholipids but rapidly incorporated into ATP. Since PLD hydrolyses a structural lipid, radioactivity only appears slowly in PtdOHPLD (and PtdBut). In contrast, PtdOHPLC is synthesised by phosphorylation of DAG, and therefore should rapidly incorporate radioactivity. In practice, PtdOH formed on addition of mastoparan was rapidly labelled, reflecting the specific radioactivity of the [32P]ATP pool. Based on the production of [32P]PtdBut, we estimate that about 5–17% of the PtdOH was generated through the PLD pathway, while the majority originated from PLC activity. Together, this is the first demonstration (i) that PLC activation is correlated with increases in Ca2+, InsP3, PtdOH and DGPP, at the cost of PtdInsP and PtdInsP2, all in one and the same cell, (ii) of the characteristics of stimulated and unstimulated PPI turnover, (iii) that stimulated turnover affects the D-4 PPI and not the 3-isomers, (iv) that PLC and PLD are activated at the same time, (v) of a simple labelling method to discriminate between the two in terms of PtdOH production.

Cyclic AMP is involved in sexual reproduction of Chlamydomonas eugametos

When plus and minus mating type gametes of Chlamydomonas eugametos were mixed, a rapid transient increase in the amount of cAMP was observed with a maximum at 20 s after the start of the sexual agglutination reaction. The transient increase only occurred within the cells and was also exhibited when cell suspensions of single mating type were presented with isolated flagella of the other mating type. Cyclic AMP‐dependent protein kinase and cyclic AMP‐phosphodiesterase activities were found in cell homogenates. Since the rise in cAMP concentration preceded all known morphological and physiological changes in the cells that prepare them for fusion, it might be a primary response, induced by sexual agglutination.

Evidence for a functional membrane barrier in the transition zone between the flagellum and cell body of Chlamydomonas eugametos gametes

Evidence is presented which supports the concept of a functional membrane barrier in the transition zone at the base of each flagellum of Chlamydomonas eugametos gametes. This makes it unlikely that agglutination factors present on the surface of the cell body can diffuse or be transported to the flagellar membrane. The evidence is as follows: 1) The glycoprotein composition of the flagellar membrane is very different to that of the cell-body plasma membrane. 2) The flagella of gametes treated with cycloheximide, tunicamycin or α, α′-dipyridyl become non-agglutinable but the source of agglutination factors on the cell body is not affected. 3) Even under natural conditions when the flagella are non-agglutinable, for example in vis-à-vis pairs or in appropriate cell strains that are non-agglutinable in the dark, the cell bodies maintain the normal complement of active agglutinins. 4) When flagella of living cells are labeled with antibodies bound to fluorescein, the label does not diffuse onto the cell-body surface. 5) When gametes fuse to form vis-à-vis pairs, the original mating-type-specific antigenicity of each cell body is slowly lost (probably due to the antigens diffusing over both cell bodies), while the specific antigenicity of the flagellar surface is maintained. Even when the flagella of vis-à-vis pairs are regenerated from cell bodies with mixed antigenicity, the antigenicity of the flagella remains matingtype-specific. 6) Evidence is presented for the existence of a pool of agglutination factors within the cell bodies but not on the outer surface of the cells.

The Effect of Sex and Deleterious Mutations on Fitness in Chlamydomonas

In this paper we present an experimental test of the deterministic mutation hypothesis on the evolution of sex. We studied the direct effect (i. e. before selection) of sex on offspring fitness of two strains of the unicellular alga Chlamydomonas moewusii, that had been kept in the laboratory for over 60 years. The logistic parameters r and K of each genotype, estimated in batch culture, were used as a measure of fitness. Strains were treated with uv to cause additional deleterious mutations. By comparing mean log fitness of parents and offspring in relation to the fitness difference of the parents, we tested whether and how deleterious mutations interact. No significant recombinational load was found in the offspring of the untreated strains. However, a significant negative effect of sex on log r and log K was found after crossing uv treated strains. We argue that this negative effect of sex on fitness suggests synergistic interaction, at least between the UV-induced and the naturally accumulated deleterious mutations. The latter result therefore supports the deterministic mutation hypothesis.

Chlamydomonas eugametos (chlorophyta) stores phosphate in polyphosphate bodies together with calcium

When Chlamydomonas eugametos Moewus cells are starved of phosphate, they accumulate 32Pi much faster than before starvation. Phosphate accumulation is stimulated by calcium. Less than 5% of the 32Pi taken up by the cell is present in soluble molecules, suggesting that most is in a metabolically inactive, storage form. Nuclear magnetic resonance and X‐ray microanalysis data are presented to show that it is stored as polyphosphate in electron‐dense bodies hi the cytoplasm. The same bodies accumulate divalent cations, in particular calcium. The P/Ca ratio in the bodies was maintained between 5.4 (1‐week‐old cells) and 3.3 (5‐week‐old cells) during cultivation, suggesting that the calcium and phosphorus relations of the bodies are coupled. The possibility that these electron‐dense bodies represent calcium stores that can be released to activate calcium signaling is discussed.