Reiko Kasai

Surface ultrastructure and chemical composition of the cell walls of conidial fungi

Freeze-fractures of the conidial wall surface of the deuteromycetous fungi Penicillium sp., Cladobotryum varium, Cladosporium macrocarpum, and Aspergillus niger and of the sporangiospore surface of the zygomycetous fungus Mycotypha microspora reveal rodlet fascicles. Shadow replicas and deep-etching of these cells demonstrate an outermost amorphous layer adjacent to the rodlet fascicles. In each case, individual rodlets are apparently composed of chains of subunits (approx 50 A diam) while interdigitated fascicles consist of clusters of rodlets arranged in parallel. The conidial wall of A. niger was separated into an outer and inner layer by passing the cells through a cell fractionator. Rodlets were present on the extracellular surface of the isolated and purified outer layer, but absent from the inner layer. The hyphal wall of A. niger was also isolated and purified. A comparison of the chemical composition of the three wall layers demonstrated distinct quantitative differences in total carbohydrate, monosaccharide, total hexosamine, peptide, and lipid content. On the basis of the gross chemical composition of the outer conidial wall layer, as well as data obtained from enzymatic digestion and various chemical treatments of this layer followed by ultrastructural examinations, it is suggested that rodlets of A. niger are mainly proteinaceous.

Correlation between fluidity and fatty acid composition of phospholipid species in Tetrahymena pyriformis during temperature acclimation.

The correlation between the fluidity of phospholipids and their fatty acid composition was studied by spin label technique and gas-liquid chromatography for three major phospholipid species in Tetrahymena pyriformis during temperature acclimation. The fluidity of 2-aminoethylphosphonolipid increased within the first 10 h of the cold-acclimation when the content of gamma-linolenic acid in 2-aminoethylphosphonolipid was highest, and it then decreased up to 24 h. On the other hand, the fluidities of phosphatidylethanolamine and phosphatidylcholine showed a gradual decrease up to 24 h after the temperature shift, although gamma-linolenic acid contents were highest at 10 h after the temperature shift. Thus the fluidity changes of these two phospholipids were interpreted as resulting from the altered content of other fatty acids in addition to gamma-linolenic acid, since the gamma-linolenic acid content was smaller than that of 2-aminoethylphosphonolipid. The results suggest that the content of gamma-linolenic acid in 2-aminoethylphosphonolipid plays a role in regulating the thermal adaptation process.

Studies on thermal adaptation in tetrahymena membrane lipids changes in positional distribution of fatty acids in diacyl-phospholipids and alkyl-acyl-phospholipids during temperature acclimation

The positioning of acyl chains in both 1-O-alkyl-2-acyl- and 1,2-diacyl-phospholipids was analyzed at various time intervals for a thermotolerant strain (NT-1) of Tetrahymena pyriformis cells during cold acclimation. During the 10 h period of adaptation, cells were not able to grow but maintained the ability to divide. The content of palmitate (16 : 0) in phosphatidylcholine and phosphatidylethanolamine was decreased after temperature-shift, with a concurrent increase of palmitoleate (16 : 1 delta 9) and gamma-linolenate (18 : 3 delta 6,9,12). An increase in gamma-linolenate at the 1-position and linoleate at the 2-position was observed in diacyl-phospholipids (phosphatidylethanolamine, phosphatidylcholine and 2-aminoethylphosphonolipid). The 2-position of 1-O-alkyl-2-acyl-phosphatidylcholine and 1-O-alkyl-2-acyl-(2-aminoethyl)phosphonolipid was occupied mainly by gamma-linolenate together with cilienate (18 : 2 delta 6,11) and linoleate (18 : 2 delta 9,12). Cilienate and gamma-linolenate at the 2-position of 1-O-alkyl-2-acyl-phosphatidylcholine were increased after temperature shift, with a small decrease of linoleate. There are little significant changes in alkyl ether lipid content of phosphatidylcholine and 2-aminoethylphosphonolipid after temperature shift. The results indicate that phosphatidylethanolamine, which is most abundant and present only in the diacyl form, would play a crucial role in thermal adaptation of membrane lipids, by replacing palmitate with gamma-linolenate at its 1-position, and also that hexadecyl/gamma-linolenoyl phosphatidylcholine would be an important molecular species in the acclimation.

Regulatory mechanism of palmitoyl-CoA desaturase activity in thermal adaptation. Induction in non-growing Tetrahymena cells deprived of pre-existing desaturase

The regulatory mechanism of palmitoyl-CoA desaturase activity in the downward temperature shift was investigated in the non-growing (starved) Tetrahymena cells. This cell system is advantageous in allowing depletion of the pre-existing palmitoyl-CoA desaturase. An unexpectedly profound increase in its activity was observed following temperature shift from 34 degrees C to 15 degrees C, providing evidence to support the concept that the increased level of the desaturase activity results from induction of the desaturase synthesis, probably by reutilization of degraded cell components, rather than from direct membrane fluidity control as observed in different experiments.

Modifications of membrane lipid composition following the nutritional shift-up of starved cells a comparison with membrane biogenesis in Tetrahymena

Detailed analyses of lipid composition have been made on various membrane fractions isolated at different intervals after 24 h-starved Tetrahymena cells were refed with nutrient-rich medium. During starvation there was a marked alteration in both phospholipid polar headgroup and acyl chain compositions: an increase in 2-aminoethylphospholipid and gamma-linolenic acid (18 : 3) with a concurrent decrease in phosphatidylethanolamine and palmitoleic acid (16 : 1). However, following refeeding, such an altered lipid composition was rather rapidly restored to the initial level of the control cell membranes prior to starvation. This membrane lipid modification was found to occur in good accordance with the recovery of cell size and lipid synthesis. The considerable changes in the principal unsaturated fatty acids, 16 : 1 and 18 : 3, which are formed via the palmitate and stearate desaturation pathways, respectively, were suggested to be accounted for by the levels of desaturases activities. The results of the labeling experiments with radioactive precursors have demonstrated that in the refed cells, there was a more rapid and dynamic transfer or exchange between membranes as compared with that in the exponentially growing control cells. Thus, rapid ameliorative modifications of membrane lipid composition are thought to be required for the urgent growth of membrane systems in the refed cell which should be ready to initiate new division.

Age-dependent modifications in membrane lipids: Lipid composition, fluidity and palmitoyl-CoA desaturase in Tetrahymena membranes

The membrane lipid composition of Tetrahymena pyriformis NT-I was observed to change in a manner markedly dependent on the progress of culture age. The pellicular, mitochondrial and microsomal membranes were isolated from cell harvested at various growth phases (I, early exponential; II, mid-exponential; III, late exponential; IV, early stationary; V, late stationary) and their lipid composition was analyzed by thin-layer and gas-liquid chromatography. Although the phospholipid composition varied somewhat among membrane fractions, the most general age-dependent alteration was a considerable decrease in the content of phosphatidylethanolamine accompanied by a small increase in phosphatidylcholine. The 2-aminoethylphosphonolipid, enriched in the surface membrane pellicle, did not undergo a consistent change. As for fatty acid composition the most notable variation occurred in unsaturated fatty acids; a great increase in oleic and linoleic acids and a compensatory decrease in palmitoleic acid. This resulted in an augmented unsaturation of the overall phospholipid fatty acid profile of the aged membranes. The age-associated drastic decline in the palmitoleic acid content in membrane phospholipids could be accounted for by the markedly lowered activity of palmitoyl-CoA desaturase. The microsomes from the early exponential phase cells possess a 4-fold higher activity of the desaturase as compared to that of the late stationary phase microsomes. The decreased desaturase activity associated with the culture age was also reflected in the corresponding decrease in the conversion rate of [14C]palmitate to [14C]palmitoleate in cells labelled in vivo. The ESR spectra of the spin-labeled phospholipids extracted from the pellicular and microsomal membranes have led to the suggestion that these types of membrane would become more fluid with the age of growth.

Modification of membrane lipids. Phenethyl alcohol-induced alteration of lipid composition in Tetrahymena membranes.

Tetrahymena pyriformis NT-I cells in the early-logarithmic phase were incubated with phenethyl alcohol (2-phenylethanol) and effects on the lipid composition were examined in various membranes. 1. There was a marked modification in phospholipid head, as well as fatty acyl group composition in pellicles, mitochondria and microsomes of the phenethyl alcohol-treated cells. Compared with membranes of the control cells, the membranes from phenethyl alcohol-treated cells were found to contain a higher level of phosphatidylcholine content with the compensating decrease in phosphatidylethanolamine, while 2-aminoethylphosphonolipid showed only a slight decrease in these membranes. The acyl group profile of membrane phospholipids in the presence of phenethyl alcohol was also modified so that a profound elevation of the content of polyunsaturated fatty acids, linoleic and gamma-linolenic acids. The major monounsaturate, palmitoleate decreased. Such lipid alteration is a reversible process, and therefore upon removal of phenethyl alcohol the modified lipid composition returned to normal. 2. By freeze-fracture electron microscopy in combination with temperature quenching, the outer alveolar membrane of the phenethyl alcohol-treated cell was observed to reveal less aggregation of intercalated-membrane particles, as compared with the control membrane. The quantitative analysis of the thermotropic lateral movement of membrane particles provided evidence that the membrane in the phenethyl alcohol-treated cell became more fluid. Such fluidizing effects may result from an increase in the acyl group unsaturation and also in the phosphatidylcholine content. 3. With regard to the mechanism responsible for the marked decrease in palmitoleate in membrane phospholipids, there was found a depressed conversion of the palmitate to palmitoleate in the phenethyl alcohol-treated cells. It was further suggested that the drug may have an inhibitory effect on the synthesis of palmitoyl-CoA desaturase involving the (16 : 0 leads to 16 : 1) conversion. Also, it was demonstrated that the increase in a precursor-product fashion of phosphatidylcholine with the corresponding decrease in phosphatidylethanolamine was not due to transformation of phosphatidylethanolamine to phosphatidylcholine through stepwise methylation.