Microtubule dynamics in mitosis in Aspergillus nidulans

Mitosis in Aspergillus nidulans is very rapid, requiring less than 5 min at 37 °C in germlings (Bergen and Morris, 1983). In this time the cytoplasmic microtubules (MTs) must disassemble, the mitotic spindle assemble, function and disassemble, and cytoplasmic MTs reassemble. It follows that cytoplasmic MTs must be extremely dynamic in this period and we were interested, in particular, in examining the processes of MT disassembly in prophase and reassembly in anaphase and telophase. We observed a diploid strain that expressed GFP-α-tubulin. We used a spinning disk confocal microscope that allowed rapid image capture, which proved necessary because microtubule dynamics were extremely rapid. We found, for the first time, that microtubule severing occurs in prophase in a filamentous fungus and that catastrophe rather than nucleation limits astral microtubule growth.     

The genetic analysis of mitosis in Aspergillus nidulans

We describe here recent work on the molecular genetics of mitosis in the filamentous fungus Aspergillus nidulans. Aspergillus is one of three simple eukaryotes with powerful genetic systems that have been used to analyze mitosis. The modern molecular biological techniques available with this organism have made it possible to use mutations to identify genes and proteins that play an important role in mitosis. Three Aspergillus genes that affect mitosis are described. One gene, nimA, is specifically expressed late in the cell cycle and codes for a putative protein kinase that induces mitosis, even in cells blocked in S-phase. The second gene, bimG, codes for a putative phosphatase that interacts functionally with the nimA kinase. The third gene, bimE, codes for a protein that suppresses mitosis during interphase, apparently by keeping nimA turned off. None of these genes appear to be similar to any of the genes affecting mitosis that have been characterized in other eukaryotes, but rather appear to be elements of a system that prevents mitosis from occurring during interphase.     

Growth rate of Aspergillus nidulans hyphae is independent of a prominent array of microtubules

Roles for the microtubule (MT) cytoskeleton in fungal growth include mitosis and nuclear migration but otherwise are less clearly understood. Confocal microscopy was used to quantify MT abundance and growth rate in hyphae of a haploid Aspergillus nidulans strain containing green fluorescent protein (GFP)-α-tubulin. There was no correlation between growth rate and MT abundance for 112 growing hyphae in an untreated population. However, 109 nongrowing hyphae from the same group had lower average MT abundance. Results for untreated cells were compared with cells treated for 30–120 min with the MT drugs benomyl and taxol, the actin drug latrunculin B, and with solvents used for the drug treatments. Compared with their respective controls, MT abundance was significantly increased by dimethyl sulfoxide (DMSO), significantly reduced by benomyl, and moderately increased by latrunculin, but was unaffected by ethanol. In the same cells, growth rates were significantly increased by ethanol and taxol, significantly reduced by latrunculin, and unaffected by DMSO. Average hyphal growth rate in the first 60 min following 1 μg/ml benomyl treatment was statistically similar to untreated cells, despite the absence of visible MTs after 2 min of treatment. However, growth rate was significantly reduced by 2.5 μg/ml benomyl over the same time period, implying additional effects at the higher concentration. For individual hyphae in each treatment, growth rates varied over short time periods; treatment with 0.1% ethanol substantially increased this variability. Growth rates of taxol-treated hyphae decreased following fluorescence observation, suggesting a possible application to cancer chemotherapy. Overall, there was no correlation between cytoplasmic MT abundance and A. nidulans growth rate within 2 h of cytoskeletal drug or solvent treatment.     

Regulation of apical dominance in Aspergillus nidulans hyphae by reactive oxygen species

In fungal hyphae, apical dominance refers to the suppression of secondary polarity axes in the general vicinity of a growing hyphal tip. The mechanisms underlying apical dominance remain largely undefined, although calcium signaling may play a role. Here, we describe the localized accumulation of reactive oxygen species (ROS) in the apical region of Aspergillus nidulans hyphae. Our analysis of atmA (ATM) and prpA (PARP) mutants reveals a correlation between localized production of ROS and enforcement of apical dominance. We also provide evidence that NADPH oxidase (Nox) or related flavoproteins are responsible for the generation of ROS at hyphal tips and characterize the roles of the potential Nox regulators NoxR, Rac1, and Cdc42 in this process. Notably, our genetic analyses suggest that Rac1 activates Nox, whereas NoxR and Cdc42 may function together in a parallel pathway that regulates Nox localization. Moreover, the latter pathway may also include Bem1, which we propose represents a p40phox analog in fungi. Collectively, our results support a model whereby localized Nox activity generates a pool of ROS that defines a dominant polarity axis at hyphal tips.     

Apical growth and mitosis are independent processes in Aspergillus nidulans

Summary. It is well established that cytoplasmic microtubules are depolymerized during nuclear division and reassembled as mitotic microtubules. Mounting evidence showing that cytoplasmic microtubules were also involved in apical growth of fungal hyphae posed the question of whether apical growth became disrupted during nuclear division. We conducted simultaneous observations of mitosis (fluorescence microscopy) and apical growth (phase-contrast microscopy) in single hyphae of Aspergillus nidulans to determine if the key parameters of apical growth (elongation rate and Spitzenkörper behavior) were affected during mitosis. To visualize nuclei during mitosis, we used a strain of A. nidulans, SRS27, in which nuclei are labeled with the green-fluorescent protein. To reveal the Spitzenkörper and measure growth with utmost precision, we used computer-enhanced videomicroscopy. Our analysis showed that there is no disruption of apical growth during mitosis. There was no decrease in the rate of hyphal elongation or any alteration in Spitzenkörper presence before, during, or after mitosis. Our findings suggest that apical growth and mitosis do not compete for internal cellular resources. Presumably, the population of cytoplasmic microtubules involved in apical growth operates independently of that involved in mitosis.Present address: Department of Plant Sciences, University of Oxford, Oxford, United Kingdom.     

Partial nuclear pore complex disassembly during closed mitosis in Aspergillus nidulans

BACKGROUND: Many organisms undergo closed mitosis and locate tubulin and mitotic kinases to nuclei only during mitosis. How this is regulated is unknown. Interestingly, the NIMA kinase of Aspergillus nidulans interacts with two nuclear pore complex (NPC) proteins and NIMA is required for mitotic localization of the Cdk1 kinase to nuclei. Therefore, we wished to define the mechanism by which the NPC is regulated during A. nidulans' closed mitosis. RESULTS: The structural makeup of the NPC is dramatically changed during A. nidulans' mitosis. At least five NPC proteins disperse throughout the cell during mitosis while at least three structural components remain at the NPC. These modifications correlate with marked changes in the function of the NPC. Notably, during mitosis, An-RanGAP is not excluded from nuclei, and five other nuclear or cytoplasmic proteins investigated fail to locate as they do during interphase. Mitotic modification of the NPC requires NIMA and Cdk1 kinase activation. NIMA appears to be particularly important. Most strikingly, ectopic induction of NIMA promotes mitotic-like changes in NPC structure and function during S phase. Furthermore, NIMA locates to the NPC during entry into mitosis, and a dominant-negative version of NIMA that causes G2 delay dwells at the NPC. CONCLUSIONS: We conclude that partial NPC disassembly under control of NIMA and Cdk1 in A. nidulans may represent a new mechanism for regulating closed mitoses. We hypothesize that proteins locate by their relative binding affinities within the cell during A. nidulans' closed mitosis, analogous to what occurs during open mitosis.     

The role of actin, fimbrin and endocytosis in growth of hyphae in Aspergillus nidulans

Filamentous fungi are ideal systems to study the process of polarized growth, as their life cycle is dominated by hyphal growth exclusively at the cell apex. The actin cytoskeleton plays an important role in this growth. Until now, there have been no tools to visualize actin or the actin-binding protein fimbrin in live cells of a filamentous fungus. We investigated the roles of actin (ActA) and fimbrin (FimA) in hyphal growth in Aspergillus nidulans . We examined the localization of ActA::GFP and FimA::GFP in live cells, and each displayed a similar localization pattern. In actively growing hyphae, cortical ActA::GFP and FimA::GFP patches were highly mobile throughout the hypha and were concentrated near hyphal apices. A patch-depleted zone occupied the apical 0.5 μm of growing hypha. Both FimA::GFP and Act::GFP also localize transiently to septa. Movement and later localization of both was compromised after cytochalasin treatment. Disruption of fimA resulted in delayed polarity establishment during conidium germination, abnormal hyphal growth and endocytosis defects in apolar cells. Endocytosis was severely impaired in apolar fimA disruption cells. Our data support a novel apical recycling model which indicates a critical role for actin patch-mediated endocytosis to maintain polarized growth at the apex.     

A putative high affinity hexose transporter, hxtA, of Aspergillus nidulans is induced in vegetative hyphae upon starvation and in ascogenous hyphae during cleistothecium formation

Fungi employ different carbohydrate uptake systems to adapt to certain environmental conditions and to different carbon source concentrations. The hydrolysis of polymeric carbohydrates and the subsequent uptake of monomeric forms may also play a role in development. Aspergillus nidulans accumulates cell wall components during vegetative growth and degrades them during sexual development. We have identified the hxtA (high affinity hexose transporter) gene in a differential library, which was enriched for sexual-specific genes. The hxtA gene is disrupted by 6 introns and predicted to encode a 531 amino acid protein with high similarity to major facilitator superfamily members including the high affinity hexose transporter Gtt1 from Trichoderma harzianum. A. nidulans HxtA contains the 12 predicted transmembrane domains characteristic for this family. Deletion of hxtA did not impair growth of A. nidulans on a variety of carbon sources nor did it inhibit sexual development suggesting redundant sugar uptake systems. We found at least 17 putative hexose transporters in the genome of A. nidulans. Despite the high similarity of HxtA to fungal high affinity glucose transporters, the hxtA gene did not restore growth on glucose of a Saccharomyces cerevisiae mutant, in which all hexose transporters were deleted. Northern blot analysis revealed that the A. nidulans hxtA gene was repressed under high glucose conditions and expressed in vegetative hyphae upon carbon starvation and during sexual development. We found hxtA(p)::sgfp expression in developing cleistothecia specifically in ascogenous hyphae and propose that HxtA is a high affinity glucose transporter involved in sugar metabolism during sexual development.     

Extracellular siderophores of rapidly growing Aspergillus nidulans and Penicillium chrysogenum

The highly active extracellular siderophores previously detected in young cultures of Aspergillus nidulans and Penicillium chrysogenum have been identified as the cyclic ester fusigen (fusarinine C), and its open-chain form, fusigen B (fusarinine B).     

The polo-like kinase PLKA is required for initiation and progression through mitosis in the filamentous fungus Aspergillus nidulans

Polo-like kinases (PLK) function during multiple stages of mitotic progression and in cytokinesis. We identified and cloned a PLK homologue in Aspergillus nidulans, plkA, which is the first PLK reported in a filamentous fungus and the largest member of the PLK family to date. As plkA was essential, the effects of overexpression and localization of protein in living cells were explored to determine PLKA function. Overexpression of PLKA permitted hyphal formation, but blocked nuclear division in interphase. In NIMA or NIMT temperature-sensitive backgrounds, overexpression of PLKA impaired normal entry into mitosis upon release from restrictive temperature, supporting a role for PLKA during G2/M. In the few mitotic cells present, spindles were monopolar or disorganized, and chromatin condensation and segregation were impaired, suggesting additional roles for PLKA in spindle formation and in chromosome dynamics. Consistent with this, green fluorescent protein (GFP)-tagged PLKA could localize to the spb during interphase, and to the spb and nucleus throughout mitosis. Intriguingly, PLKA remained on the spb during telophase and into G1, in contrast to other PLK. In addition, spb localization was independent of NIMA function, unlike that demonstrated in Schizosaccharomyces pombe where PLK localization to the spb required the NIMA homologue Fin1. PLKA was not detected at cortical, septation-associated sites, and overexpression did not drive septum formation, also in contrast to that observed with other PLK. Therefore, PLKA is important for multiple events during mitosis, similar to PLK in higher organisms, but exhibits differences in size, localization and influence on septation/cytokinesis, suggesting additional novel regulatory features.     

Checkpoint defects leading to premature mitosis also cause endoreplication of DNA in Aspergillus nidulans

The G2 DNA damage and slowing of S-phase checkpoints over mitosis function through tyrosine phosphorylation of NIMX(cdc2) in Aspergillus nidulans. We demonstrate that breaking these checkpoints leads to a defective premature mitosis followed by dramatic rereplication of genomic DNA. Two additional checkpoint functions, uvsB and uvsD, also cause the rereplication phenotype after their mutation allows premature mitosis in the presence of low concentrations of hydroxyurea. uvsB is shown to encode a rad3/ATR homologue, whereas uvsD displays homology to rad26, which has only previously been identified in Schizosaccharomyces pombe. uvsB(rad3) and uvsD(rad26) have G2 checkpoint functions over mitosis and another function essential for surviving DNA damage. The rereplication phenotype is accompanied by lack of NIME(cyclinB), but ectopic expression of active nondegradable NIME(cyclinB) does not arrest DNA rereplication. DNA rereplication can also be induced in cells that enter mitosis prematurely because of lack of tyrosine phosphorylation of NIMX(cdc2) and impaired anaphase-promoting complex function. The data demonstrate that lack of checkpoint control over mitosis can secondarily cause defects in the checkpoint system that prevents DNA rereplication in the absence of mitosis. This defines a new mechanism by which endoreplication of DNA can be triggered and maintained in eukaryotic cells.     

Characterization of an Aspergillus nidulans mutant with abnormal distribution of nuclei in hyphae, metulae, phialides and conidia

The V10 deteriorated variant of Aspergillus nidulans has hyphae, metulae, phialides and conidia with abnormal nuclear distributions. The alterations observed were: increase in the number of nuclei in hyphae, metulae and phialides, presence of anucleate, uninucleate and multinucleate conidia, abnormal vegetative growth and defective conidiation. When 0.5 M NaCl was added to the medium, an increase in the number of conidia was observed but their morphology and number of nuclei were not modified. The gene responsible for these alterations was named anuA1. The anuA1 gene is located on linkage group VII and is possibly involved in nuclear migration to hyphae, metulae, phialides and conidia.     

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.     

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.