Hyphae Of Aspergillus Nidulans Research Articles

Cell cycle progression and cell polarity require sphingolipid biosynthesis in Aspergillus nidulans.

Sphingolipids are major components of the plasma membrane of eukaryotic cells and were once thought of merely as structural components of the membrane. We have investigated effects of inhibiting sphingolipid biosynthesis, both in germinating spores and growing hyphae of Aspergillus nidulans. In germinating spores, genetic or pharmacological inactivation of inositol phosphorylceramide (IPC) synthase arrests the cell cycle in G(1) and also prevents polarized growth during spore germination. However, inactivation of IPC synthase not only eliminates sphingolipid biosynthesis but also leads to a marked accumulation of ceramide, its upstream intermediate. We therefore inactivated serine palmitoyltransferase, the first enzyme in the sphingolipid biosynthesis pathway, to determine effects of inhibiting sphingolipid biosynthesis without an accumulation of ceramide. This inactivation also prevented polarized growth but did not affect nuclear division of germinating spores. To see if sphingolipid biosynthesis is required to maintain polarized growth, and not just to establish polarity, we inhibited sphingolipid biosynthesis in cells in which polarity was already established. This inhibition rapidly abolished normal cell polarity and promoted cell tip branching, which normally never occurs. Cell tip branching was closely associated with dramatic changes in the normally highly polarized actin cytoskeleton and found to be dependent on actin function. The results indicate that sphingolipids are essential for the establishment and maintenance of cell polarity via control of the actin cytoskeleton and that accumulation of ceramide is likely responsible for arresting the cell cycle in G(1).

Protein synthesis in mycelial protoplasts of Aspergillus nidulans

The capacity for protein synthesis in protoplasts isolated from hyphae of Aspergillus nidulans during staged lytic digestions was investigated by measurement of uptake and incorporation of [ 14 C]leucine into protoplast samples. All protoplast fractions were active in [ 14 C]leucine uptake but differences in uptake rates and levels of incorporation between different fractions were attributed to protoplast release patterns in the two stabilizer systems used. The effect of suspension of mycelium in buffer/stabilizer solutions of the loss of [ 14 C]leucine from pre-labelled material was investigated to assess the effect of plasmolysis and prolonged suspension in the protoplast isolation medium. Losses of [ 14 C]leucine were a maximum of 22–23% on plasmolysis and prolonged suspension had little further effect; mycelium remained synthetically active under these conditions.

Measurements of respiratory activity in isolated protoplasts of Aspergillus nidulans

The respiratory capacities of protoplasts isolated from hyphae of Aspergillus nidulans during staged lytic digestions were investigated. Oxygen exchange and cytochrome oxidase activity were measured using enzyme assay, manometric and oxygen electrode techniques. The suitability of these methods for measurements involving protoplast samples was assessed. Metabolic activity of protoplasts was related to that within intact mycelium. Data obtained were consistent with the view that lytic digestion represents sequential fractionation of fungal mycelium.

Interaction of thiabendazole with fungal tubulin

Thiabendazole, 2-(4′-thiazolyl)benzimidazole, at 80 μM completely inhibits mitosis in hyphae of Aspergillus nidulans, growing in liquid culture. DNA and RNA synthesis and mycelial growth are only partially inhibited at this concentration. Binding studies with cell-free mycelial extracts from Penicillium expansum showed that thiabendazole competitively inhibits [ 14C]carbendazim binding to tubulin, which suggests that the antimitotic activity of thiabendazole is based on interference with microtubule assembly. Tubulin from a thiabendazole-resistant and carbendazim-highly sensitive mutant of P. expansum has a lower affinity to thiabendazole and a higher affinity to carbendazim than tubulin from a wild-type strain. This indicates that in this mutant the structure of the binding site is affected. The data presented suggest that several sites of both the tubulin and ligand molecule are involved in the binding of benzimidazole compounds to fungal tubulin.

Distribution of autolysins in hyphae of Aspergillus nidulans: evidence for a lipid-mediated attachment to hyphal walls

Preparations of broken Aspergillus nidulans hyphae contained both free and wall-bound autolysins. The bound enzymes were not solubilized by 8 M LiCl or neutral or anionic detergents; they were readily detached from walls by a cationic detergent or by autodigestion. Once detached, the enzymes did not reassociate with wall to give salt-resistant complexes. Six enzymes hydrolyzing wall polymers were bound to the envelope, and the same activities were also detected among soluble proteins in the cytoplasmic fraction. It is suggested that cytoplasmic vesicles, containing autolysins, are inserted into or trapped by newly formed wall in the growing hypha; these constitute the wall-bound autolysin fraction. Starvation for a carbon source derepressed the synthesis of five out of the six autolysins, and the amounts of both soluble and wall-bound activities increased by one to two orders of magnitude.

Autolytic enzymes in hyphae of Aspergillus nidulans: their action on old and newly formed walls

Walls, purified from hyphae of the ascomycete Aspergillus nidulans, autolyzed on incubation and liberated glucose, mannose, galactose, N-acetylglucosamine, and soluble oligosaccharides. Digestion proceeded at linear rates until approximately 3% of the wall polymers had been hydrolyzed and then slowed markedly. The change in the rate of autolysis was not due to loss of enzyme activity but was caused by the disappearance of a fraction of the wall which was highly susceptible to digestion. Radioactive labeling showed that this fraction was the newly formed wall. The new wall was highly susceptible to enzyme action both when it was deposited at the apex in growing hyphae or when deposited laterally in hyphae treated with cycloheximide. The relations between wall modification and apical growth are discussed.

The membraneous type of lomasome (membranosome) in the hyphae of Aspergillus nidulans.

General ultrastructure of vegetative hyphae ofAspergillus nidulans has been studied. Walls, nuclei, mitochondria, endoplasmic reticulum (but no Golgi apparatus), have been described and discussed. A continuity between the outer mitochondrial membrane and other cytomembranes as well as continuity between the endoplasmic reticulum and plasmalemma was sometimes observed. Interest has been concentrated on the several varieties of the membraneous-type of lomasome figures. These appear to constitute a sequentially ordered developmental series of a single type of complex organelle called membranosome. The dense core of these organelles appears to be their initiation center around which the surrounding membrane becomes multilayered in the mature membranosome. Possible functional relationship of this membraneous type of lomasome with both interrelated plasmalemma and wall productions was considered.

Influence of cytochalasin B on hyphal morphogenesis of Aspergillus nidulans.

Cytochalasin B causes the hyphae ofAspergillus nidulans either to branch more frequently or to form “beaded” filaments. This is interpreted as failure to sustain the concentration of wall growth materials at a single point near the cell surface; remoter sites are unaffected.

Regulation and timing of deoxyribonucleic acid synthesis in hyphae of Aspergillus nidulans.

Pulse labeling of deoxyribonucleic acid (DNA) and radioautography have been used to study the effect of growth rate on nuclear replication in Aspergillus nidulans. When conidia were germinated in media supporting a fast growth rate, the radioactive pulse labeled either all of the nuclei in a cell or none of them. At slower growth rates, hyphae contained both labeled and unlabeled nuclei. Altering the growth rate thus changed nuclear replication from simultaneous to sequential. The time taken to duplicate the DNA in a nucleus, estimated from the ratio of labeled to total nuclei, remained constant at the different doubling times. The distribution of label showed that nuclei in the same hypha spent unequal times in both the postmitotic gap (G1) and the premitotic gap (G2) periods when grown at slow rates. These unequal G1 and G2 periods are considered to cause asynchrony. Once DNA synthesis was out of phase through growth on a poor medium, transferring the hypha to a rich medium did not resynchronize the nuclei. To interpret the data, two initiator mechanisms, one starting DNA synthesis and the other mitosis, are postulated to control nuclear replication in A. nidulans.