p25 of the dynactin complex plays a dual role in cargo binding and dynactin regulation.

Cytoplasmic dynein binds its cargoes via the dynactin complex and cargo adapters, and the dynactin pointed-end protein p25 is required for dynein-dynactin binding to the early endosomal dynein adapter HookA (Hook in the fungus Aspergillus nidulans). However, it is unclear whether the HookA-dynein-dynactin interaction requires p27, another pointed-end protein forming heterodimers with p25 within vertebrate dynactin. Here, live-cell imaging and biochemical pulldown experiments revealed that although p27 is a component of the dynactin complex in A. nidulans, it is dispensable for dynein-dynactin to interact with ΔC-HookA (cytosolic HookA lacking its early endosome-binding C terminus) and is not critical for dynein-mediated early endosome transport. Using mutagenesis, imaging, and biochemical approaches, we found that several p25 regions are required for the ΔC-HookA-dynein-dynactin interaction, with the N terminus and loop1 being the most critical regions. Interestingly, p25 was also important for the microtubule (MT) plus-end accumulation of dynactin. This p25 function in dynactin localization also involved p25's N terminus and the loop1 critical for the ΔC-HookA-dynein-dynactin interaction. Given that dynactin's MT plus-end localization does not require HookA and that the kinesin-1-dependent plus-end accumulation of dynactin is unnecessary for the ΔC-HookA-dynein-dynactin interaction, our results indicate that p25 plays a dual role in cargo binding and dynactin regulation. As cargo adapters are implicated in dynein activation via binding to dynactin's pointed end to switch the conformation of p150, a major dynactin component, our results suggest p25 as a critical pointed-end protein involved in this process.

COPI localizes to the early Golgi in Aspergillus nidulans.

Coatomer-I (COPI) is a heteromeric protein coat that facilitates the budding of membranous carriers mediating Golgi-to-ER and intra-Golgi transport. While the structural features of COPI have been thoroughly investigated, its physiological role is insufficiently understood. Here we exploit the amenability of A. nidulans for studying intracellular traffic, taking up previous studies by Breakspear et al. (2007) with the α-COP/CopA subunit of COPI. Endogenously tagged α-COP/CopA largely localizes to SedVSed5 syntaxin-containing early Golgi cisterna, and acute inactivation of ER-to-Golgi traffic delocalizes COPI to a haze, consistent with the cisternal maturation model. In contrast, the Golgi localization of COPI is independent of the TGN regulators HypBSec7 and HypATrs120, implying that COPI budding predominates at the SedVSed5 early Golgi, with lesser contribution of the TGN. This finding agrees with the proposed role of COPI-mediated intra-Golgi retrograde traffic in driving cisternal maturation, which predicts that the capacity of the TGN to generate COPI carriers is low. The COPI early Golgi compartments intimately associates with Sec13-containing ER exit sites. Characterization of the heat-sensitive copA1ts (sodVIC1) mutation showed that it results in a single residue substitution in the ε-COP-binding Carboxyl-Terminal-Domain of α-COP that likely destabilizes its folding. However, we show that Golgi disorganization by copA1ts necessitates >150 min-long incubation at 42 °C. This weak subcellular phenotype makes it unsuitable for inactivating COPI traffic acutely for microscopy studies, and explains the aneuploidy-stabilizing role of the mutation at subrestrictive temperatures.

Ultrastructural and SINS analysis of the cell wall integrity response of Aspergillus nidulans to the absence of galactofuranose.

With lethal opportunistic fungal infections on the rise, it is imperative to explore new methods to examine virulence mechanisms. The fungal cell wall is crucial for both the virulence and viability of Aspergillus nidulans. One wall component, Galf, has been shown to contribute to important fungal processes, integrity of the cell wall and pathogenesis. Here, we explore gene deletion strains lacking the penultimate enzyme in Galf biosynthesis (ugmAΔ) and the protein that transports Galf for incorporation into the cell wall (ugtAΔ). In applying gene deletion technology to the problem of cell wall integrity, we have employed multiple micro- and nano-scale imaging tools, including confocal fluorescence microscopy, electron microscopy, X-Ray fluorescence and atomic force microscopy. Atomic force microscopy allows quantification of ultrastructural cell wall architecture while near-field infrared spectroscopy provides spatially resolved chemical signatures, both at the nanoscale. Here, for the first time, we demonstrate correlative data collection with these two emerging modalities for the multiplexed in situ study of the nanoscale architecture and chemical composition of fungal cell walls.

Deletion of Aspergillus nidulans GDP-mannose transporters affects hyphal morphometry, cell wall architecture, spore surface character, cell adhesion, and biofilm formation.

Systemic human fungal infections are increasingly common. Aspergillus species cause most of the airborne fungal infections. Life-threatening invasive aspergillosis was formerly found only in immune-suppressed patients, but recently some strains of A. fumigatus have become primary pathogens. Many fungal cell wall components are absent from mammalian systems, so they are potential drug targets. Cell-wall-targeting drugs such as echinocandins are used clinically, although echinocandin-resistant strains were discovered shortly after their introduction. Currently there are no fully effective anti-fungal drugs. Fungal cell wall glycoconjugates modulate human immune responses, as well as fungal cell adhesion, biofilm formation, and drug resistance. Guanosine diphosphate (GDP) mannose transporters (GMTs) transfer GDP-mannose from the cytosol to the Golgi lumen prior to mannosylation. Aspergillus nidulans GMTs are encoded by gmtA and gmtB. Here we elucidate the roles of A. nidulans GMTs. Strains engineered to lack either or both GMTs were assessed for hyphal and colonial morphology, cell wall ultrastructure, antifungal susceptibility, spore hydrophobicity, adherence and biofilm formation. The gmt-deleted strains had smaller colonies with reduced sporulation and with thicker hyphal walls. The gmtA deficient spores had reduced hydrophobicity and were less adherent and less able to form biofilms in vitro. Thus, gmtA not only participates in maintaining the cell wall integrity but also plays an important role in biofilm establishment and adherence of A. nidulans. These findings suggested that GMTs have roles in A. nidulans growth and cell-cell interaction and could be a potential target for new antifungals that target virulence determinants.

Linkage of oxidative stress and mitochondrial dysfunctions to spontaneous culture degeneration in Aspergillus nidulans.

Filamentous fungi including mushrooms frequently and spontaneously degenerate during subsequent culture maintenance on artificial media, which shows the loss or reduction abilities of asexual sporulation, sexuality, fruiting, and production of secondary metabolites, thus leading to economic losses during mass production. To better understand the underlying mechanisms of fungal degeneration, the model fungus Aspergillus nidulans was employed in this study for comprehensive analyses. First, linkage of oxidative stress to culture degeneration was evident in A. nidulans. Taken together with the verifications of cell biology and biochemical data, a comparative mitochondrial proteome analysis revealed that, unlike the healthy wild type, a spontaneous fluffy sector culture of A. nidulans demonstrated the characteristics of mitochondrial dysfunctions. Relative to the wild type, the features of cytochrome c release, calcium overload and up-regulation of apoptosis inducing factors evident in sector mitochondria suggested a linkage of fungal degeneration to cell apoptosis. However, the sector culture could still be maintained for generations without the signs of growth arrest. Up-regulation of the heat shock protein chaperones, anti-apoptotic factors and DNA repair proteins in the sector could account for the compromise in cell death. The results of this study not only shed new lights on the mechanisms of spontaneous degeneration of fungal cultures but will also provide alternative biomarkers to monitor fungal culture degeneration.

The cell-end marker TeaA and the microtubule polymerase AlpA contribute to microtubule guidance at the hyphal tip cortex of Aspergillus nidulans to provide polarity maintenance.

In the absence of landmark proteins, hyphae of Aspergillus nidulans lose their direction of growth and show a zigzag growth pattern. Here, we show that the cell-end marker protein TeaA is important for localizing the growth machinery at hyphal tips. The central position of TeaA at the tip correlated with the convergence of the microtubule (MT) ends to a single point. Conversely, in the absence of TeaA, the MTs often failed to converge to a single point at the cortex. Further analysis suggested a functional connection between TeaA and AlpA (an ortholog of the MT polymerase Dis1/CKAP5/XMAP215) for proper regulation of MT growth at hyphal tips. AlpA localized at MT plus-ends, and bimolecular fluorescence complementation assays suggested that it interacted with TeaA after MT plus-ends reached the tip cortex. In vitro MT polymerization assays showed that AlpA promoted MT growth up to sevenfold. Addition of the C-terminal region of TeaA increased the catastrophe frequency of the MTs. Thus, the control of the AlpA activity through TeaA might be a novel principle for MT growth regulation after reaching the cortex. In addition, we present evidence that the curvature of hyphal tips also could be involved in the control of MT growth at hyphal tips.

Investigation of the antimicrobial effect of Neosartorya fischeri antifungal protein (NFAP) after heterologous expression in Aspergillus nidulans.

Neosartorya fischeri antifungal protein (NFAP) is a ß-defensin-like peptide produced by the N. fischeri NRRL 181 isolate. In this study, we investigated the manifestation of the antimicrobial effect of NFAP via heterologous expression of the nfap gene in an NFAP-sensitive fungus, Aspergillus nidulans. Heterologous expression of the nfap gene was carried out in A. nidulans CS2902 using a pAMA1-based autonomous replicative vector construct. The effect of the produced NFAP on the germination of A. nidulans conidia was investigated by scanning electron microscopy (SEM), and by DAPI and Calcofluor white (CFW) staining. 2',7'-Dichlorodihydrofluorescein diacetate staining and an Annexin V-FITC Apoptosis Detection kit were used to reveal the accumulation of reactive oxygen species (ROS) and the possible apoptotic, necrotic effect. The impact of mono- and divalent cations on the antimicrobial activity of NFAP was also examined. Transformants expressing the nfap gene showed reduced hyphal growth compared with the untransformed strain. This effect was absent in the presence of mono- and divalent cations (50 and 100 mM KCl, Mg(2)SO(4), Na(2)SO(4)). Delayed and abnormal germination was observed in the case of transformants. Conidia developed short branching germination tubes with swollen tips. The great majority of germinating conidia were destroyed after 8 h of cultivation, although a few survived and developed into abnormal hyphae. Damage in the organization of the cell wall, the destruction of chitin filaments and the accumulation of nuclei at the broken hyphal tips were detected by SEM, DAPI and CFW staining. The accumulation of ROS and more frequent apoptotic, necrotic events were also observed in the case of the NFAP-producing A. nidulans strain.

The septin AspB in Aspergillus nidulans forms bars and filaments and plays roles in growth emergence and conidiation.

In yeast, septins form rings at the mother-bud neck and function as diffusion barriers. In animals, septins form filaments that can colocalize with other cytoskeletal elements. In the filamentous fungus Aspergillus nidulans there are five septin genes, aspA (an ortholog of Saccharomyces cerevisiae CDC11), aspB (an ortholog of S. cerevisiae CDC3), aspC (an ortholog of S. cerevisiae CDC12), aspD (an ortholog of S. cerevisiae CDC10), and aspE (found only in filamentous fungi). The aspB gene was previously reported to be the most highly expressed Aspergillus nidulans septin and to be essential. Using improved gene targeting techniques, we found that deletion of aspB is not lethal but results in delayed septation, increased emergence of germ tubes and branches, and greatly reduced conidiation. We also found that AspB-green fluorescent protein (GFP) localizes as rings and collars at septa, branches, and emerging layers of the conidiophore and as bars and filaments in conidia and hyphae. Bars are found in dormant and isotropically expanding conidia and in subapical nongrowing regions of hyphae and display fast movements. Filaments form as the germ tube emerges, localize to hyphal and branch tips, and display slower movements. All visible AspB-GFP structures are retained in ΔaspD and lost in ΔaspA and ΔaspC strains. Interestingly, in the ΔaspE mutant, AspB-GFP rings, bars, and filaments are visible in early growth, but AspB-GFP rods and filaments disappear after septum formation. AspE orthologs are only found in filamentous fungi, suggesting that this class of septins might be required for stability of septin bars and filaments in highly polar cells.

Quantifying the importance of galactofuranose in Aspergillus nidulans hyphal wall surface organization by atomic force microscopy.

The fungal wall mediates cell-environment interactions. Galactofuranose (Galf), the five-member ring form of galactose, has a relatively low abundance in Aspergillus walls yet is important for fungal growth and fitness. Aspergillus nidulans strains deleted for Galf biosynthesis enzymes UgeA (UDP-glucose-4-epimerase) and UgmA (UDP-galactopyranose mutase) lacked immunolocalizable Galf, had growth and sporulation defects, and had abnormal wall architecture. We used atomic force microscopy and force spectroscopy to image and quantify cell wall viscoelasticity and surface adhesion of ugeAΔ and ugmAΔ strains. We compared the results for ugeAΔ and ugmAΔ strains with the results for a wild-type strain (AAE1) and the ugeB deletion strain, which has wild-type growth and sporulation. Our results suggest that UgeA and UgmA are important for cell wall surface subunit organization and wall viscoelasticity. The ugeAΔ and ugmAΔ strains had significantly larger surface subunits and lower cell wall viscoelastic moduli than those of AAE1 or ugeBΔ hyphae. Double deletion strains (ugeAΔ ugeBΔ and ugeAΔ ugmAΔ) had more-disorganized surface subunits than single deletion strains. Changes in wall surface structure correlated with changes in its viscoelastic modulus for both fixed and living hyphae. Wild-type walls had the largest viscoelastic modulus, while the walls of the double deletion strains had the smallest. The ugmAΔ strain and particularly the ugeAΔ ugmAΔ double deletion strain were more adhesive to hydrophilic surfaces than the wild type, consistent with changes in wall viscoelasticity and surface organization. We propose that Galf is necessary for full maturation of A. nidulans walls during hyphal extension.

Intimate bacterial-fungal interaction triggers biosynthesis of archetypal polyketides in Aspergillus nidulans.

Fungi produce numerous low molecular weight molecules endowed with a multitude of biological activities. However, mining the full-genome sequences of fungi indicates that their potential to produce secondary metabolites is greatly underestimated. Because most of the biosynthesis gene clusters are silent under laboratory conditions, one of the major challenges is to understand the physiological conditions under which these genes are activated. Thus, we cocultivated the important model fungus Aspergillus nidulans with a collection of 58 soil-dwelling actinomycetes. By microarray analyses of both Aspergillus secondary metabolism and full-genome arrays and Northern blot and quantitative RT-PCR analyses, we demonstrate at the molecular level that a distinct fungal-bacterial interaction leads to the specific activation of fungal secondary metabolism genes. Most surprisingly, dialysis experiments and electron microscopy indicated that an intimate physical interaction of the bacterial and fungal mycelia is required to elicit the specific response. Gene knockout experiments provided evidence that one induced gene cluster codes for the long-sought after polyketide synthase (PKS) required for the biosynthesis of the archetypal polyketide orsellinic acid, the typical lichen metabolite lecanoric acid, and the cathepsin K inhibitors F-9775A and F-9775B. A phylogenetic analysis demonstrates that orthologs of this PKS are widespread in nature in all major fungal groups, including mycobionts of lichens. These results provide evidence of specific interaction among microorganisms belonging to different domains and support the hypothesis that not only diffusible signals but intimate physical interactions contribute to the communication among microorganisms and induction of otherwise silent biosynthesis genes.

Structure and activity of Aspergillus nidulans copper amine oxidase.

Aspergillus nidulans amine oxidase (ANAO) has the unusual ability among the family of copper and trihydroxyphenylalanine quinone-containing amine oxidases of being able to oxidize the amine side chains of lysine residues in large peptides and proteins. We show here that in common with the related enzyme from the yeast Pichia pastoris, ANAO can promote the cross-linking of tropoelastin and oxidize the lysine residues in α-casein proteins and tropoelastin. The crystal structure of ANAO, the first for a fungal enzyme in this family, has been determined to a resolution of 2.4 Å. The enzyme is a dimer with the archetypal fold of a copper-containing amine oxidase. The active site is the most open of any of those of the structurally characterized enzymes in the family and provides a ready explanation for its lysine oxidase-like activity.

In vivo analysis of the functions of gamma-tubulin-complex proteins.

To enhance our understanding of the function(s) of gamma-tubulin-complex proteins (GCPs), we identified and analyzed the functions of the Aspergillus nidulans homologs of GCP2-GCP6 (here designated GCPB-GCBF). The gamma-tubulin small complex (gamma-TuSC) components, gamma-tubulin, GCPB and GCPC, are essential for viability and mitotic spindle formation, whereas GCPD-GCPF are not essential for viability, spindle formation or sexual reproduction. GCPD-GCPF function in reducing the frequency of chromosome mis-segregation and in the assembly of large gamma-tubulin complexes. Deletion of any of the gamma-TuSC components eliminates the localization of all GCPs to the spindle pole body (SPB), whereas deletion of GCPD-GCPF does not affect localization of gamma-TuSC components. Thus, GCPD-GCPF do not tether the gamma-TuSC to the SPB, but, rather, the gamma-TuSC tethers them to the SPB. GCPD-GCPF exhibit a hierarchy of localization to the SPB. Deletion of GCPF eliminates GCPD-GCPE localization to the SPB, and deletion of GCPD eliminates GCPE (but not GCPF) localization. All GCPs localize normally in a GCPE deletion. We propose a model for the structure of the gamma-tubulin complex and its attachment to polar microtubule organizing centers.

Microtubule organizing centers and gamma-tubulin.

The polar assembly of cellular microtubules is organized by microtubule organizing centers (MTOCs). Eukaryotic cells across different species, and different cell types within single species, have morphologically diverse MTOCs, which have the common function of organizing microtubule arrays by initiating microtubule assembly and anchoring microtubules by their slow-growing 'minus' ends, thus ensuring that the rapidly growing 'plus' ends extend distally. The past few years have witnessed a variety of approaches aimed at defining the molecular components of the MTOC that are responsible for regulating microtubule assembly by defining molecules common to all MTOCs.

Eisosome organization in the filamentous ascomycete Aspergillus nidulans.

Eisosomes are subcortical organelles implicated in endocytosis and have hitherto been described only in Saccharomyces cerevisiae. They comprise two homologue proteins, Pil1 and Lsp1, which colocalize with the transmembrane protein Sur7. These proteins are universally conserved in the ascomycetes. We identify in Aspergillus nidulans (and in all members of the subphylum Pezizomycotina) two homologues of Pil1/Lsp1, PilA and PilB, originating from a duplication independent from that extant in the subphylum Saccharomycotina. In the aspergilli there are several Sur7-like proteins in each species, including one strict Sur7 orthologue (SurG in A. nidulans). In A. nidulans conidiospores, but not in hyphae, the three proteins colocalize at the cell cortex and form tightly packed punctate structures that appear different from the clearly distinct eisosome patches observed in S. cerevisiae. These structures are assembled late during the maturation of conidia. In mycelia, punctate structures are present, but they are composed only of PilA, while PilB is diffused in the cytoplasm and SurG is located in vacuoles and endosomes. Deletion of each of the genes does not lead to any obvious growth phenotype, except for moderate resistance to itraconazole. We could not find any obvious association between mycelial (PilA) eisosome-like structures and endocytosis. PilA and SurG are necessary for conidial eisosome organization in ways that differ from those for their S. cerevisiae homologues. These data illustrate that conservation of eisosomal proteins within the ascomycetes is accompanied by a striking functional divergence.

Adaptation of the Tokuyasu method for the ultrastructural study and immunogold labelling of filamentous fungi.

The study of filamentous fungi is fundamental not only to extend their biotechnological applications, but also to develop new drugs to fight pathological species. Morphological analyses are particularly relevant when investigating their development and differentiation. The need to maintain the orientation of hypahe and the presence of a cell wall, which hampers the sample infiltration with cryoprotectants and other reagents necessary to preserve the cell ultrastructure, creates difficulties with the use of electron microscopy (EM). Here, we present an immunoelectron microscopy (IEM) procedure that combines the Tokuyasu protocol adapted to yeast and the flat-embedding technique. While the first method leads to a fine resolution of the ultrastructure of Aspergillus nidulans because of both the cell wall permeabilization and the negative membrane coloration, the second permits us to preserve the spatial distribution of the hypahe of this fungus. The presented data demonstrate the advantages of this combination and the unprecedented potential of this relatively simple and rapid protocol in resolving the morphology of filamentous fungi and performing localization studies.

Aspergillus nidulans biofilm formation modifies cellular architecture and enables light-activated autophagy.

After growing on surfaces, including those of medical and industrial importance, fungal biofilms self-generate internal microenvironments. We previously reported that gaseous microenvironments around founder Aspergillus nidulans cells change during biofilm formation causing microtubules to disassemble under control of the hypoxic transcription factor SrbA. Here we investigate if biofilm formation might also promote changes to structures involved in exocytosis and endocytosis. During biofilm formation, the endoplasmic reticulum (ER) remained intact but ER exit sites and the Golgi apparatus were modified as were endocytic actin patches. The biofilm-driven changes required the SrbA hypoxic transcription factor and could be triggered by nitric oxide, further implicating gaseous regulation of biofilm cellular architecture. By tracking green fluorescent protein (GFP)-Atg8 dynamics, biofilm founder cells were also observed to undergo autophagy. Most notably, biofilm cells that had undergone autophagy were triggered into further autophagy by spinning disk confocal light. Our findings indicate that fungal biofilm formation modifies the secretory and endocytic apparatus and show that biofilm cells can also undergo autophagy that is reactivated by light. The findings provide new insights into the changes occurring in fungal biofilm cell biology that potentially impact their unique characteristics, including antifungal drug resistance.

Class III chitin synthase ChsB of Aspergillus nidulans localizes at the sites of polarized cell wall synthesis and is required for conidial development.

Class III chitin synthases play important roles in tip growth and conidiation in many filamentous fungi. However, little is known about their functions in those processes. To address these issues, we characterized the deletion mutant of a class III chitin synthase-encoding gene of Aspergillus nidulans, chsB, and investigated ChsB localization in the hyphae and conidiophores. Multilayered cell walls and intrahyphal hyphae were observed in the hyphae of the chsB deletion mutant, and wavy septa were also occasionally observed. ChsB tagged with FLAG or enhanced green fluorescent protein (EGFP) localized mainly at the tips of germ tubes, hyphal tips, and forming septa during hyphal growth. EGFP-ChsB predominantly localized at polarized growth sites and between vesicles and metulae, between metulae and phialides, and between phalides and conidia in asexual development. These results strongly suggest that ChsB functions in the formation of normal cell walls of hyphae, as well as in conidiophore and conidia development in A. nidulans.

The cell end marker protein TeaC is involved in growth directionality and septation in Aspergillus nidulans.

Polarized growth in filamentous fungi depends on the correct spatial organization of the microtubule (MT) and actin cytoskeleton. In Schizosaccharomyces pombe it was shown that the MT cytoskeleton is required for the delivery of so-called cell end marker proteins, e.g., Tea1 and Tea4, to the cell poles. Subsequently, these markers recruit several proteins required for polarized growth, e.g., a formin, which catalyzes actin cable formation. The latest results suggest that this machinery is conserved from fission yeast to Aspergillus nidulans. Here, we have characterized TeaC, a putative homologue of Tea4. Sequence identity between TeaC and Tea4 is only 12.5%, but they both share an SH3 domain in the N-terminal region. Deletion of teaC affected polarized growth and hyphal directionality. Whereas wild-type hyphae grow straight, hyphae of the mutant grow in a zig-zag way, similar to the hyphae of teaA deletion (tea1) strains. Some small, anucleate compartments were observed. Overexpression of teaC repressed septation and caused abnormal swelling of germinating conidia. In agreement with the two roles in polarized growth and in septation, TeaC localized to hyphal tips and to septa. TeaC interacted with the cell end marker protein TeaA at hyphal tips and with the formin SepA at hyphal tips and at septa.

Identification of novel cell wall destabilizing antifungal compounds using a conditional Aspergillus nidulans protein kinase C mutant.

OBJECTIVES: Despite the need for novel drugs to combat fungal infections, antifungal drug discovery is currently limited by both the availability of suitable drug targets and assays to screen corresponding targets. The aim of this study was to screen a library of small chemical compounds to identify cell wall inhibitors using a conditional protein kinase C (PKC)-expressing strain of Aspergillus nidulans. This mutant is specifically susceptible to cell wall damaging compounds under PKC-repressive growth conditions. METHODS: The inhibitory effect of a library of small chemical compounds was examined in vitro using the conditional A. nidulans PKC strain and a panel of pathogenic fungal isolates. Microscopy was used to assess alterations to fungal ultrastructure following treatment. RESULTS: Three 'hit' compounds affecting cell wall integrity were identified from a screen of 5000 small chemical compounds. The most potent compound, CW-11, was further characterized and shown to specifically affect cell wall integrity. In clinical isolates of Aspergillus fumigatus, CW-11 induces morphological changes characteristic of damage to the cell wall, including wall thickening and rupturing. Analysis of the susceptibility of A. fumigatus and A. nidulans cell wall and signalling pathway mutants to CW-11 suggests that its mode of action differs from that of the antifungals caspofungin and voriconazole. CONCLUSIONS: This work demonstrates the feasibility of using a conditional Aspergillus mutant to conduct a small-molecule library screen to identify novel 'hit' compounds affecting cell wall integrity.

Nuclear migration in a nud mutant of Aspergillus nidulans is inhibited in the presence of a quantitatively normal population of cytoplasmic microtubules.

Nuclear migration was studied in germinating conidia of a temperature-sensitive mutant of the fungus Aspergillus nidulans. At the restrictive temperature motility was demonstrably impaired because significantly fewer nuclei migrated into the germ tube relative to a population of similarly sized germlings grown at the permissive temperature. Further comparison of these populations showed that the mutant was leaky in that an increasing number of nuclei migrated as the total nuclear content increased in each germling. The restrictive temperature also induced elevated mitotic asynchrony and increased numbers of nuclei per germling. Serial section-based reconstruction of the microtubules in a freeze-substituted germling showed that they were not attached to the nucleus-associated organelles, were approximately parallel to the long axis of the germ tube, and seemed to be randomly distributed between the central and peripheral cytoplasm. Five germlings from each temperature were selected for quantitative analysis of cytoplasmic microtubules. All 10 germlings had typical nuclear migration phenotypes. No significant temperature-related difference in microtubule density was found. We conclude that inhibition of nuclear migration in the mutant is the effect of some defect other than the failure of cytoplasmic microtubules to assemble to their normal population density. We also suggest that nuclear motility is not dependent on mitosis-related microtubules.