Organization and Dynamics of the Aspergillus nidulans Golgi during Apical Extension and Mitosis

Aspergillus nidulans hyphae grow exclusively by apical extension. Golgi equivalents (GEs) labeled with mRFP-tagged PH^OSBP domain form a markedly polarized, dynamic network of ring-shaped and fenestrated cisternae that remains intact during “closed” mitosis. mRFP-PH^OSBP GEs advance associated with the growing apex where secretion predominates but do not undergo long-distance movement toward the tip that could account for their polarization. mRFP-PH^OSBP GEs overlap with the trans-Golgi resident Sec7 but do not colocalize with also polarized accretions of the early Golgi marker GrhA^Grh1-GFP, indicating that early and late Golgi membranes segregate spatially. AnSec23-GFP ER exit sites (ERES) are numerous, relatively static foci localizing across the entire cell. However, their density is greatest near the tip, correlating with predominance of early and trans-Golgi elements in this region. Whereas GrhA-GFP structures and ERES reach the apical dome, mRFP-PH^OSBP GEs are excluded from this region, which contains the endosome dynein loading zone. After latrunculin-mediated F-actin disruption, mRFP-PH^OSBP GEs fragment and, like AnSec23-GFP ERES, depolarize. Brefeldin A transiently collapses late and early GEs into distinct aggregates containing Sec7/mRFP-PH^OSBP and GrhA-GFP, respectively, temporarily arresting apical extension. Rapid growth reinitiates after washout, correlating with reacquisition of the normal Golgi organization that, we conclude, is required for apical extension.     

An InCytes from MBC Selection: Nucleolar Separation from Chromosomes during Aspergillus nidulans Mitosis Can Occur Without Spindle Forces

How the nucleolus is segregated during mitosis is poorly understood and occurs by very different mechanisms during closed and open mitosis. Here we report a new mechanism of nucleolar segregation involving removal of the nucleolar-organizing regions (NORs) from nucleoli during Aspergillus nidulans mitosis. This involves a double nuclear envelope (NE) restriction which generates three NE-associated structures, two daughter nuclei (containing the NORs), and the nucleolus. Therefore, a remnant nucleolar structure can exist in the cytoplasm without NORs. In G1, this parental cytoplasmic nucleolus undergoes sequential disassembly releasing nucleolar proteins to the cytoplasm as nucleoli concomitantly reform in daughter nuclei. By depolymerizing microtubules and mutating spindle assembly checkpoint function, we demonstrate that a cycle of nucleolar “segregation” can occur without a spindle in a process termed spindle-independent mitosis (SIM). During SIM physical separation of the NOR from the nucleolus occurs, and NE modifications promote expulsion of the nucleolus to the cytoplasm. Subsequently, the cytoplasmic nucleolus is disassembled and rebuilt at a new site around the nuclear NOR. The data demonstrate the existence of a mitotic machinery for nucleolar segregation that is normally integrated with mitotic spindle formation but that can function without it.     

Interference of Griseofulvin with the Segregation of Chromosomes at Mitosis in Diploid Aspergillus nidulans

Low concentrations of the antibiotic griseofulvin were found to cause increased frequencies of somatic segregation due to chromosome nondisjunction in a diploid strain of Aspergillus nidulans.     

Xylanase production by Aspergillus nidulans

Abstract The effect of phagocyte activation by TNF-α on the ability to trigger a chemiluminescence (CL) response, associated with the release of oxidizing species was evaluated in healthy human mononuclear cells in the presence of Mycobacterium leprae . Recombinant TNF-α (r-TNF-α) increased the CL response of unstimulated M. bovis BCG- and PMA-stimulated cells but did not reverse the M. leprae defective activation of the human phagocyte oxidative burst. M. leprae was less well phagocytosed than M. bovis BCG but phagocytosis of mycobacteria was not altered by addition of r-TNF-α. The failure of activation of oxygen-free radical production might have some relevance to the pathogenesis of leprosy.     

Cellulase formation by Aspergillus nidulans

The optimum pH, temperature and concentration of the substrate, carboxymethyl-cellulose (CMC), for the production of cellulases by Aspergillus nidulans were found to be 3.05, 37°C and 1%, respectively. When grown on CMC under optimum conditions, it produced the three components of the cellulase complex, exo-β-1,4-glucanase, endo-β-1,4-glucanase and β-1,4-glucosidase, both in cell free as well as cell-associated states. The enzyme yields in shake cultures were lower than those obtained during stationary cultivation. Among the defined substrates, lactose emerged as the best inducer for exo-glucanase and endo-glucanase, while β-glucosidase was best induced by pectin. Endo-glucanase production increased significantly when A. nidulans was grown on insoluble delignified lognocellulosic substrates, with the maximum being on paddy straw.It appears that the synthesis of individual components of the cellulase system of A. nidulans may not be regulated in a strictly coordinated manner.     

Proteome map of Aspergillus nidulans during osmoadaptation

The model filamentous fungus Aspergillus nidulans, when grown in a moderate level of osmolyte (+0.6M KCl), was previously found to have a significantly reduced cell wall elasticity (Biotech Prog, 21:292, 2005). In this study, comparative proteomic analysis via two-dimensional gel electrophoresis (2de) and matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) mass spectrometry was used to assess molecular level events associated with this phenomenon. Thirty of 90 differentially expressed proteins were identified. Sequence homology and conserved domains were used to assign probable function to twenty-one proteins currently annotated as "hypothetical." In osmoadapted cells, there was an increased expression of glyceraldehyde-3-phosphate dehydrogenase and aldehyde dehydrogenase, as well as a decreased expression of enolase, suggesting an increased glycerol biosynthesis and decreased use of the TCA cycle. There also was an increased expression of heat shock proteins and Shp1-like protein degradation protein, implicating increased protein turnover. Five novel osmoadaptation proteins of unknown functions were also identified.     

Transformation by integration in Aspergillus nidulans

DNA-mediated genetic transformation of Aspergillus nidulans has been achieved by incubating protoplasts from a strain of A. nidulans carrying a deletion in the acetamidase structural gene with DNA of derivatives of plasmid pBR322 containing the cloned structural gene for acetamidase [Hynes et al., Mol. Cell. Biol. 3 (1983) 1430-1439; p3SR2] in the presence of polyethylene glycol and CaCl2. The highest frequency obtained was 25 transformants per microgram of DNA. No enhancement of the transformation frequency was observed when DNAs of plasmids carrying either a fragment of the A. nidulans ribosomal repeat (p3SR2rr) or a fragment containing a possible A. nidulans mitochondrial origin of replication (p3SR2mo) in addition to the acetamidase gene were used. Both pBR322 and acetamidase gene sequences become integrated into the genome of A. nidulans in transformant strains. Integration events into the residual sequences adjacent to the deletion in the acetamidase gene, and probably (for p3SR2rr and p3SR2mo) into the ribosomal repeat unit are described.     

The amdS gene of Aspergillus nidulans: control by multiple regulatory signals

The amdS gene of A. nidulans has proved extremely favourable for the isolation of mutations affecting gene regulation. Trans-acting regulatory genes involved in amdS induction by small molecular weight effectors have been identified – amdR (ω-amino acids) facB (acetate) and amdA (acetate). Another gene, the areA gene, has properties expected of a major activator gene involved in nitrogen metabolite repression of amdS. All of these regulatory genes are also involved in the control of various other functions encoded by structural genes unlinked to amdS. Mutations in the 5′-region adjacent to amdS have been isolated and allow the identification of independent cis-acting sequences which are the target sites for the regulatory genes. The involvement of these sequences in regulatory product binding has been deduced from titration studies using transformants containing multiple copies of the 5′ sequences. A combination of genetics and molecular analysis is allowing a detailed characterization of this system.     

Biosynthetic Pathways of Glycerol Accumulation under Salt Stress in Aspergillus nidulans

Redkar, R. J., Locy. R. D., and Singh, N. K. 1995. Biosynthetic pathways of glycerol accumulation under salt stress in Aspergillus nidulans. Experimental Mycology 19, 241-246. A culture of Aspergillus nidulans (FGSC 359) was gradually adapted for growth in media containing up to 2 M NaCl or was exposed to a salt shock with 2 M NaCl. The intracellular glycerol level increased by about 7.9-fold in salt-adapted and 2.4-fold in salt-shocked cultures when compared to the unadapted culture. The biosynthetic pathway involved in the accumulation of glycerol was investigated under long-term salt adaptation and short-term salt shock. Glycerol 3-phosphate dehydrogenase (EC 1.1.1.8) was induced 1.4-fold in salt-shocked but not in salt-adapted cultures. An alternate enzymatic pathway involving glycerol dehydrogenase (NADP⁺-dependent) utilizing dihydroxyacetone (DHA) and/or DL-glyceraldehyde (DL-GAD) was induced by NaCl. DHA-dependent glycerol dehydrogenase activity was induced about 6.3-fold in salt adapted and 1.35-fold in salt-shocked cultures, while DL-GAD-dependent activity was induced about 6.1-fold in salt-adapted and 1.2-fold in salt shocked cultures. However, the level of glycerol dehydrogenase activity with DL-GAD as substrate was 7% of the DHA-dependent activity. We conclude that a salt-inducible NADP⁺-dependent glycerol dehydrogenase activity electrophoretically indistinguishable from previously described glycerol dehydrogenase I results in glycerol accumulation in salt-stressed A. nidulans.     

Starch digestion and adsorption by beta-amylase of Emericella nidulans (Aspergillus nidulans).

A mutant strain of Emericella nidulans MNU 82 was isolated by multistep mutation. The beta-amylase produced by the mutant was able to digest raw starch. It was readily and strongly adsorbed onto raw starch at pH 5.0. The enzyme to starch ratio was 1950 U/g starch. The enzyme showed no correlation between the capacity of raw starch digestion and adsorption of the enzyme.