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A global existential and interlocking environmental, climate and cooperation/equity polycrisis is being faced, which increasingly impacts and is impacted by water and land systems. As a result, transformations in response are gaining increasing traction. Advances in approaches to visualising and communicating how innovations and changes in landscape features enable shifts, transformations and transitions are more crucial than ever before. Visions help focus the actions, collaboration and alignment of multiple actors in working towards a common purpose, whilst also entering people’s consciousness at the deep level of values, transforming beliefs and consequently, thinking and action. They give direction to effort and pull on transformational innovation. The evidence-based ‘Water Visions Visualisation Platform’ presents an innovative and accessible way to illustrate, communicate and support future water visioning and strategising at the landscape scale based on composite paradigms, scenarios, horizons and concepts. Plausible visions of the future are envisioned, illustrated, narratively described and qualitatively assessed, as well as connected with real-world examples and resources through the interactive platform. This paper outlines the co-creation methodology, the architecture and the initial co-development of the platform, as well as a preliminary evaluation of its efficacy through literature-based criteria and the sharing of the platform with stakeholders.

Elissa Burton,1,2 Vinicius Cavalheri,1 Richard Adams,3 Colleen Oakley Browne,4 Petra Bovery-Spencer,4 Audra M Fenton,3 Bruce W Campbell,5 Keith D Hill1,6 1School of Physiotherapy and Exercise Science, Curtin University, Perth, WA, Australia; 2Research Department, Silver Chain, Perth, WA, Australia; 3Community Services, West Gippsland Healthcare Group, Warragul, VIC, Australia; 4Falls Prevention for People Living with Dementia Project, Central West Gippsland Primary Care Partnership, Moe, VIC, Australia; 5Allied Health, Latrobe Regional Hospital, Traralgon, VIC, Australia; 6Preventive and Public Health Division, National Ageing Research Institute, Melbourne, VIC, Australia Objective: The objective of this systematic review and meta-analysis is to evaluate the effectiveness of exercise programs to reduce falls in older people with dementia who are living in the community.Method: Peer-reviewed articles (randomized controlled trials [RCTs] and quasi-experimental trials) published in English between January 2000 and February 2014, retrieved from six electronic databases – Medline (ProQuest), CINAHL, PubMed, PsycInfo, EMBASE and Scopus – according to predefined inclusion criteria were included. Where possible, results were pooled and meta-analysis was conducted.Results: Four articles (three RCT and one single-group pre- and post-test pilot study) were included. The study quality of the three RCTs was high; however, measurement outcomes, interventions, and follow-up time periods differed across studies. On completion of the intervention period, the mean number of falls was lower in the exercise group compared to the control group (mean difference [MD] [95% confidence interval {CI}] =-1.06 [-1.67 to -0.46] falls). Importantly, the exercise intervention reduced the risk of being a faller by 32% (risk ratio [95% CI] =0.68 [0.55–0.85]). Only two other outcomes were reported in two or more of the studies (step test and physiological profile assessment). No between-group differences were observed in the results of the step test (number of steps) (MD [95% CI] =0.51 [-1.77 to 2.78]) or the physiological profile assessment (MD [95% CI] =-0.10 [-0.62 to 0.42]).Conclusion: Findings from this review suggest that an exercise program may potentially assist in preventing falls of older people with dementia living in the community. However, further research is needed with studies using larger sample sizes, standardized measurement outcomes, and longer follow-up periods, to inform evidence-based recommendations. Keywords: cognitive impairment, older people, physical activity, fallers, community dwelling

Abstract The genomic DNA sequence of a novel enteric uncultured microphage, ΦCA82 from a turkey gastrointestinal system was determined utilizing metagenomics techniques. The entire circular, single-stranded nucleotide sequence of the genome was 5,514 nucleotides. The ΦCA82 genome is quite different from other microviruses as indicated by comparisons of nucleotide similarity, predicted protein similarity, and functional classifications. Only three genes showed significant similarity to microviral proteins as determined by local alignments using BLAST analysis. ORF1 encoded a predicted phage F capsid protein that was phylogenetically most similar to the Microviridae ΦMH2K member's major coat protein. The ΦCA82 genome also encoded a predicted minor capsid protein (ORF2) and putative replication initiation protein (ORF3) most similar to the microviral bacteriophage SpV4. The distant evolutionary relationship of ΦCA82 suggests that the divergence of this novel turkey microvirus from other microviruses may reflect unique evolutionary pressures encountered within the turkey gastrointestinal system.

Abstract Background Because biotechnological uses of bacteriophage gene products as alternatives to conventional antibiotics will require a thorough understanding of their genomic context, we sequenced and analyzed the genomes of four closely related phages isolated from Clostridium perfringens, an important agricultural and human pathogen. Results Phage whole-genome tetra-nucleotide signatures and proteomic tree topologies correlated closely with host phylogeny. Comparisons of our phage genomes to 26 others revealed three shared COGs; of particular interest within this core genome was an endolysin (PF01520, an N-acetylmuramoyl-L-alanine amidase) and a holin (PF04531). Comparative analyses of the evolutionary history and genomic context of these common phage proteins revealed two important results: 1) strongly significant host-specific sequence variation within the endolysin, and 2) a protein domain architecture apparently unique to our phage genomes in which the endolysin is located upstream of its associated holin. Endolysin sequences from our phages were one of two very distinct genotypes distinguished by variability within the putative enzymatically-active domain. The shared or core genome was comprised of genes with multiple sequence types belonging to five pfam families, and genes belonging to 12 pfam families, including the holin genes, which were nearly identical. Conclusions Significant genomic diversity exists even among closely-related bacteriophages. Holins and endolysins represent conserved functions across divergent phage genomes and, as we demonstrate here, endolysins can have significant variability and host-specificity even among closely-related genomes. Endolysins in our phage genomes may be subject to different selective pressures than the rest of the genome. These findings may have important implications for potential biotechnological applications of phage gene products.

Abstract Background The origin and evolution of the homologous GTP-binding cytoskeletal proteins FtsZ typical of Bacteria and tubulin characteristic of eukaryotes is a major question in molecular evolutionary biology. Both FtsZ and tubulin are central to key cell biology processes – bacterial septation and cell division in the case of FtsZ and in the case of tubulins the function of microtubules necessary for mitosis and other key cytoskeleton-dependent processes in eukaryotes. The origin of tubulin in particular is of significance to models for eukaryote origins. Most members of domain Bacteria possess FtsZ, but bacteria in genus Prosthecobacter of the phylum Verrucomicrobia form a key exception, possessing tubulin homologs BtubA and BtubB. It is therefore of interest to know whether other members of phylum Verrucomicrobia possess FtsZ or tubulin as their FtsZ-tubulin gene family representative. Results Verrucomicrobium spinosum, a member of Phylum Verrucomicrobia of domain Bacteria, has been found to possess a gene for a protein homologous to the cytoskeletal protein FtsZ. The deduced amino acid sequence has sequence signatures and predicted secondary structure characteristic for FtsZ rather than tubulin, but phylogenetic trees and sequence analysis indicate that it is divergent from all other known FtsZ sequences in members of domain Bacteria. The FtsZ gene of V. spinosum is located within a dcw gene cluster exhibiting gene order conservation known to contribute to the divisome in other Bacteria and comparable to these clusters in other Bacteria, suggesting a similar functional role. Conclusion Verrucomicrobium spinosum has been found to possess a gene for a protein homologous to the cytoskeletal protein FtsZ. The results suggest the functional as well as structural homology of the V. spinosum FtsZ to the FtsZs of other Bacteria implying its involvement in cell septum formation during division. Thus, both bacteria-like FtsZ and eukaryote-like tubulin cytoskeletal homologs occur in different species of the phylum Verrucomicrobia of domain Bacteria, a result with potential major implications for understanding evolution of tubulin-like cytoskeletal proteins and the origin of eukaryote tubulins.

Quorum sensing (QS) is a cell-cell signaling system that enables bacteria to coordinate population density-dependent changes in behavior. This chemical communication pathway is mediated by diffusible N -acyl L-homoserine lactone signals and cytoplasmic signal-responsive LuxR-type receptors in Gram-negative bacteria. As many common pathogenic bacteria use QS to regulate virulence, there is significant interest in disrupting QS as a potential therapeutic strategy. Prior studies have implicated the natural products salicylic acid, cinnamaldehyde, and other related benzaldehyde derivatives as inhibitors of QS in the opportunistic pathogen Pseudomonas aeruginosa, yet we lack an understanding of the mechanisms by which these compounds function. Herein, we evaluate the activity of a set of benzaldehyde derivatives using heterologous reporters of the P. aeruginosa LasR and RhlR QS signal receptors. We find that most tested benzaldehyde derivatives can antagonize LasR or RhlR reporter activation at micromolar concentrations, although certain molecules also cause mild growth defects and nonspecific reporter antagonism. Notably, several compounds showed promising RhlR or LasR-specific inhibitory activities over a range of concentrations below that causing toxicity. ortho -Vanillin, a previously untested compound, was the most promising within this set. Competition experiments against the native ligands for LasR and RhlR revealed that ortho -vanillin can interact competitively with RhlR but not with LasR. Overall, these studies expand our understanding of benzaldehyde activities in the LasR and RhlR receptors and reveal potentially promising effects of ortho -vanillin as a small molecule QS modulator against RhlR., Importance: Quorum sensing (QS) regulates many aspects of bacterial pathogenesis and has attracted much interest as a target for anti-virulence therapies over the past 30 years, for example, antagonists of the LasR and RhlR QS receptors in Pseudomonas aeruginosa . Potent and selective QS inhibitors remain relatively scarce. However, natural products have provided a bounty of chemical scaffolds with anti-QS activities, but their molecular mechanisms are poorly characterized. The current study serves to fill this void by examining the activity of an important and wide-spread class of natural product QS modulators, benzaldehydes, and related derivatives, in LasR and RhlR. We demonstrate that ortho -vanillin can act as a competitive inhibitor of RhlR, a receptor that has emerged and may supplant LasR in certain settings as a target for P. aeruginosa QS control. The results and insights provided herein will advance the design of chemical tools to study QS with improved activities and selectivities., Competing Interests: The authors declare no conflict of interest.

Wild-type Aspergillus nidulans asexual spores (conidia) are green due to a pigment that protects the spores against ultraviolet light. The pigment is produced by a biosynthetic pathway, the genes of which are dispersed in the genome. The backbone molecule of the pigment is a polyketide synthesized by a polyketide synthase encoded by the wA gene. If wA is not functional, the conidia are white. The polyketide is modified by a laccase encoded by the yA gene and inactivation of yA in an otherwise wild-type background results in yellow spores. Additional spore color mutations have been isolated and mapped to a locus genetically, but the genes that correspond to these loci have not been determined. Spore color markers have been useful historically, and they remain valuable in the molecular genetics era. One can determine if a transforming fragment has been successfully integrated at the wA or yA locus by simply looking at the color of transformant conidia. The genes of the potentially useful color loci chaA (chartreuse conidia) and fwA (fawn conidia) have not been identified previously. We chose a set of candidate genes for each locus by comparing the assembled genome with the genetic map. By systematically deleting these candidate genes, we identified a cytochrome P450 gene (AN10028) corresponding to chaA . Deletions of this gene result in chartreuse conidia and chartreuse mutations can be complemented in trans by a functional copy of this gene. With fwA , we found that the existing fawn mutation, fwA1 , is a deletion of 2241 base pairs that inactivates three genes. By deleting each of these genes, we determined that fwA is AN1088, an EthD domain protein. Deletion of AN1088 results in fawn conidia as expected. Neither deletion of chaA nor fwA restricts growth and both should be valuable target loci for transformations. Combinations of deletions have allowed us to investigate the epistasis relationships of wA , yA , chaA and fwA .

Functions of protein SUMOylation remain incompletely understood in different cell types. Via forward genetics, here we identified ubaBQ247*, a loss-of-function mutation in a SUMO activation enzyme UbaB in the filamentous fungus Aspergillus nidulans. The ubaBQ247*, ΔubaB, and ΔsumO mutants all produce abnormal chromatin bridges, indicating the importance of SUMOylation in the completion of chromosome segregation. The bridges are enclosed by nuclear membrane containing peripheral nuclear pore complex proteins that normally get dispersed during mitosis, and the bridges are also surrounded by cytoplasmic microtubules typical of interphase cells. Time-lapse sequences further indicate that most bridges persist through interphase prior to the next mitosis, and anaphase chromosome segregation can produce new bridges that persist into the next interphase. When the first mitosis happens at a higher temperature of 42°C, SUMOylation deficiency produces not only chromatin bridges but also many abnormally shaped single nuclei that fail to divide. UbaB-GFP localizes to interphase nuclei just like the previously studied SumO-GFP, but the nuclear signals disappear during mitosis when the nuclear pores are partially open, and the signals reappear after mitosis. The nuclear localization is consistent with many SUMO targets being nuclear proteins. Finally, although the budding yeast SUMOylation machinery interacts with LIS1, a protein critical for dynein activation, loss of SUMOylation does not cause any obvious defect in dynein-mediated transport of nuclei and early endosomes, indicating that SUMOylation is unnecessary for dynein activation in A. nidulans., Competing Interests: Conflicts of interest The authors declare no conflict of interest., (Published by Oxford University Press on behalf of The Genetics Society of America 2023.)

Production of secondary metabolites is controlled by a complicated regulatory network in eukaryotic cells. Several layers of regulators are involved in this process, ranging from pathway-specific regulation, to epigenetic control, to global regulation. Here, we discover that interaction of an RNA-binding protein CsdA with a regulator RsdA coordinates fungal secondary metabolism. Employing a genetic deletion approach and transcriptome analysis as well as metabolomics analysis, we reveal that CsdA and RsdA synergistically regulate fungal secondary metabolism comprehensively. Mechanistically, comprehensive genetic and biochemical studies prove that RsdA and CsdA co-localize in the nucleus and physically interact to achieve their functions. In particular, we demonstrate that CsdA mediates rsdA expression by binding specific motif "GUCGGUAU" of its pre-mRNA at a post-transcriptional level. We thus uncover a mechanism in which RNA-binding protein physically interacts with, and controls the expression level of, the RsdA to coordinate fungal secondary metabolism., (© 2023. The Author(s).)

Aspergillus fumigatus is a serious human pathogen causing life-threatening Aspergillosis in immunocompromised patients. Secondary metabolites (SMs) play an important role in pathogenesis, but the products of many SM biosynthetic gene clusters (BGCs) remain unknown. In this study, we have developed a heterologous expression platform in Aspergillus nidulans , using a newly created genetic dereplication strain, to express a previously unknown BGC from A. fumigatus and determine its products. The BGC produces sartorypyrones, and we have named it the spy BGC. Analysis of targeted gene deletions by HRESIMS, NMR, and microcrystal electron diffraction (MicroED) enabled us to identify 12 products from the spy BGC. Seven of the compounds have not been isolated previously. We also individually expressed the polyketide synthase (PKS) gene spyA and demonstrated that it produces the polyketide triacetic acid lactone (TAL), a potentially important biorenewable platform chemical. Our data have allowed us to propose a biosynthetic pathway for sartorypyrones and related natural products. This work highlights the potential of using the A. nidulans heterologous expression platform to uncover cryptic BGCs from A. fumigatus and other species, despite the complexity of their secondary metabolomes., Competing Interests: The authors declare no conflicts of interest., (This journal is © The Royal Society of Chemistry.)

Secondary metabolites (SMs) are biologically active small molecules, many of which are medically valuable. Fungal genomes contain vast numbers of SM biosynthetic gene clusters (BGCs) with unknown products, suggesting that huge numbers of valuable SMs remain to be discovered. It is challenging, however, to identify SM BGCs, among the millions present in fungi, that produce useful compounds. One solution is resistance gene-guided genome mining, which takes advantage of the fact that some BGCs contain a gene encoding a resistant version of the protein targeted by the compound produced by the BGC. The bioinformatic signature of such BGCs is that they contain an allele of an essential gene with no SM biosynthetic function, and there is a second allele elsewhere in the genome. We have developed a computer-assisted approach to resistance gene-guided genome mining that allows users to query large databases for BGCs that putatively make compounds that have targets of therapeutic interest. Working with the MycoCosm genome database, we have applied this approach to look for SM BGCs that target the proteasome β6 subunit, the target of the proteasome inhibitor fellutamide B, or HMG-CoA reductase, the target of cholesterol reducing therapeutics such as lovastatin. Our approach proved effective, finding known fellutamide and lovastatin BGCs as well as fellutamide- and lovastatin-related BGCs with variations in the SM genes that suggest they may produce structural variants of fellutamides and lovastatin. Gratifyingly, we also found BGCs that are not closely related to lovastatin BGCs but putatively produce novel HMG-CoA reductase inhibitors., One-Sentence Summary: A new computer-assisted approach to resistance gene-directed genome mining is reported along with its use to identify fungal biosynthetic gene clusters that putatively produce proteasome and HMG-CoA reductase inhibitors., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)

For many years, two central, unanswered questions in cytoskeleton research were how microtubule assembly is nucleated and microtubule polarity established. The discoveries of γ-tubulin and γ-tubulin ring complexes were key advances that allowed these questions to be substantially answered. The discovery of γ-tubulin was the product of a genetic screen in Aspergillus nidulans for genes important for microtubule function. γ-Tubulin is a member of the tubulin superfamily of proteins, closely related to α- and β-tubulin but distinct from both. It is ubiquitous in eukaryotes, and in many organisms there are small families of γ-tubulin genes. γ-Tubulin and associated proteins form ring-like complexes that localize to microtubule-organizing centers (MTOCs) and play an important role in the nucleation of microtubule assembly from MTOCs and the establishment of microtubule polarity.

RNA-binding protein Musashi-1 (MSI1) is a key regulator of several stem cell populations. MSI1 is involved in tumor proliferation and maintenance, and it regulates target mRNAs at the translational level. The known mRNA targets of MSI1 include Numb , APC , and P21 WAF-1 , key regulators of Notch/Wnt signaling and cell cycle progression, respectively. In this study, we aim to identify small molecule inhibitors of MSI1-mRNA interactions, which could block the growth of cancer cells with high levels of MSI1. Using a fluorescence polarization (FP) assay, we screened small molecules from several chemical libraries for those that disrupt the binding of MSI1 to its consensus RNA. One cluster of hit compounds is the derivatives of secondary metabolites from Aspergillus nidulans . One of the top hits, Aza-9, from this cluster was further validated by surface plasmon resonance and nuclear magnetic resonance spectroscopy, which demonstrated that Aza-9 binds directly to MSI1, and the binding is at the RNA binding pocket. We also show that Aza-9 binds to Musashi-2 (MSI2) as well. To test whether Aza-9 has anti-cancer potential, we used liposomes to facilitate Aza-9 cellular uptake. Aza-9-liposome inhibits proliferation, induces apoptosis and autophagy, and down-regulates Notch and Wnt signaling in colon cancer cell lines. In conclusion, we identified a series of potential lead compounds for inhibiting MSI1/2 function, while establishing a framework for identifying small molecule inhibitors of RNA binding proteins using FP-based screening methodology.

In order for eukaryotes to efficiently detect and respond to environmental stimuli, a myriad of protein signaling pathways are utilized. An example of highly conserved signaling pathways in eukaryotes are the mitogen-activated protein kinase (MAPK) pathways. In fungi, MAPK pathways have been shown to regulate a diverse array of biological processes, such as asexual and sexual development, stress responses and the production of secondary metabolites (SMs). In the model fungus Aspergillus nidulans , a MAPK pathway known as the pheromone module is utilized to regulate both development and SM production. This signaling cascade consists of the three kinases SteC, MkkB, and MpkB, as well as the SteD adaptor protein and the HamE scaffold. In this study, homologs of each of these proteins have been identified in the opportunistic human pathogen A. fumigatus. By performing epitope tagging and mass spectrometry experiments, we have shown that these proteins form a pentameric complex, similar to what is observed in A. nidulans. This complex has been shown to assemble in the cytoplasm and MpkB enters the nucleus, where it would presumably interact with various transcription factors. Pheromone module mutant strains exhibit drastic reductions in asexual sporulation, vegetative growth rate and production of SMs, such as gliotoxin. Mutants also display increased sensitivity to cell wall and oxidative stress agents. Overall, these data provide evidence of the existence of a conserved MAP kinase signaling pathway in Aspergillus species and suggest that this pathway is critical for the regulation of fungal development and secondary metabolism., (Copyright © 2020 Frawley, Stroe, Oakley, Heinekamp, Straßburger, Fleming, Brakhage and Bayram.)

Aspergillus nidulans is an opportunistic fungal pathogen in patients with immunodeficiency, and virulence of A. nidulans isolates has mainly been studied in the context of chronic granulomatous disease (CGD), with characterization of clinical isolates obtained from non-CGD patients remaining elusive. This study therefore carried out a detailed biological characterization of two A. nidulans clinical isolates (CIs), obtained from a patient with breast carcinoma and pneumonia and from a patient with cystic fibrosis that underwent lung transplantation, and compared them to the reference, nonclinical FGSC A4 strain. Both CIs presented increased growth in comparison to that of the reference strain in the presence of physiologically relevant carbon sources. Metabolomic analyses showed that the three strains are metabolically very different from each other in these carbon sources. Furthermore, the CIs were highly susceptible to cell wall-perturbing agents but not to other physiologically relevant stresses. Genome analyses identified several frameshift variants in genes encoding cell wall integrity (CWI) signaling components. Significant differences in CWI signaling were confirmed by Western blotting among the three strains. In vivo virulence studies using several different models revealed that strain MO80069 had significantly higher virulence in hosts with impaired neutrophil function than the other strains. In summary, this study presents detailed biological characterization of two A. nidulans sensu stricto clinical isolates. Just as in Aspergillus fumigatus , strain heterogeneity exists in A. nidulans clinical strains that can define virulence traits. Further studies are required to fully characterize A. nidulans strain-specific virulence traits and pathogenicity. IMPORTANCE Immunocompromised patients are susceptible to infections with opportunistic filamentous fungi from the genus Aspergillus Although A. fumigatus is the main etiological agent of Aspergillus species-related infections, other species, such as A. nidulans , are prevalent in a condition-specific manner. A. nidulans is a predominant infective agent in patients suffering from chronic granulomatous disease (CGD). A. nidulans isolates have mainly been studied in the context of CGD although infection with A. nidulans also occurs in non-CGD patients. This study carried out a detailed biological characterization of two non-CGD A. nidulans clinical isolates and compared the results to those with a reference strain. Phenotypic, metabolomic, and genomic analyses highlight fundamental differences in carbon source utilization, stress responses, and maintenance of cell wall integrity among the strains. One clinical strain had increased virulence in models with impaired neutrophil function. Just as in A. fumigatus , strain heterogeneity exists in A. nidulans clinical strains that can define virulence traits., (Copyright © 2020 Bastos et al.)

Eukaryotic striatin forms striatin-interacting phosphatase and kinase (STRIPAK) complexes that control many cellular processes including development, cellular transport, signal transduction, stem cell differentiation and cardiac functions. However, detailed knowledge of complex assembly and its roles in stress responses are currently poorly understood. Here, we discovered six striatin (StrA) interacting proteins (Sips), which form a heptameric complex in the filamentous fungus Aspergillus nidulans. The complex consists of the striatin scaffold StrA, the Mob3-type kinase coactivator SipA, the SIKE-like protein SipB, the STRIP1/2 homolog SipC, the SLMAP-related protein SipD and the catalytic and regulatory phosphatase 2A subunits SipE (PpgA), and SipF, respectively. Single and double deletions of the complex components result in loss of multicellular light-dependent fungal development, secondary metabolite production (e.g. mycotoxin Sterigmatocystin) and reduced stress responses. sipA (Mob3) deletion is epistatic to strA deletion by supressing all the defects caused by the lack of striatin. The STRIPAK complex, which is established during vegetative growth and maintained during the early hours of light and dark development, is mainly formed on the nuclear envelope in the presence of the scaffold StrA. The loss of the scaffold revealed three STRIPAK subcomplexes: (I) SipA only interacts with StrA, (II) SipB-SipD is found as a heterodimer, (III) SipC, SipE and SipF exist as a heterotrimeric complex. The STRIPAK complex is required for proper expression of the heterotrimeric VeA-VelB-LaeA complex which coordinates fungal development and secondary metabolism. Furthermore, the STRIPAK complex modulates two important MAPK pathways by promoting phosphorylation of MpkB and restricting nuclear shuttling of MpkC in the absence of stress conditions. SipB in A. nidulans is similar to human suppressor of IKK-ε(SIKE) protein which supresses antiviral responses in mammals, while velvet family proteins show strong similarity to mammalian proinflammatory NF-KB proteins. The presence of these proteins in A. nidulans further strengthens the hypothesis that mammals and fungi use similar proteins for their immune response and secondary metabolite production, respectively., Competing Interests: The authors have declared that no competing interests exist.

We have identified the cyclin domain-containing proteins encoded by the genomes of 17 species of Aspergillus as well as 15 members of other genera of filamentous ascomycetes. Phylogenetic analyses reveal that the cyclins fall into three groups, as in other eukaryotic phyla, and, more significantly, that they are remarkably conserved in these fungi. All 32 species examined, for example, have three group I cyclins, cyclins that are particularly important because they regulate the cell cycle, and these are highly conserved. Within the group I cyclins there are three distinct clades, and each fungus has a single member of each clade. These findings are in marked contrast to the yeasts Saccharomyces cerevisiae, Schizosaccharomyces pombe , and Candida albicans , which have more numerous group I cyclins. These results indicate that findings on cyclin function made with a model Aspergillus species, such as A. nidulans , are likely to apply to other Aspergilli and be informative for a broad range of filamentous ascomycetes. In this regard, we note that the functions of only one Aspergillus group I cyclin have been analysed (NimE Cyclin B of A. nidulans ). We have consequently carried out an analysis of the members of the other two clades using A. nidulans as our model. We have found that one of these cyclins, PucA, is essential, but deletion of PucA in a strain carrying a deletion of CdhA, an activator of the anaphase promoting complex/cyclosome (APC/C), is not lethal. These data, coupled with data from heterokaryon rescue experiments, indicate that PucA is an essential G 1 /S cyclin that is required for the inactivation of the APC/C-CdhA, which, in turn, allows the initiation of the S phase of the cell cycle. Our data also reveal that PucA has additional, non-essential, roles in the cell cycle in interphase. The A. nidulans member of the third clade (AN2137) has not previously been named or analyzed. We designate this gene clbA . ClbA localizes to kinetochores from mid G 2 until just prior to chromosomal condensation. Deletion of clbA does not affect viability. However, by using a regulatable promoter system new to Aspergillus , we have found that expression of a version of ClbA in which the destruction box sequences have been removed is lethal and causes a mitotic arrest and a high frequency of non-disjunction. Thus, although ClbA is not essential, its timely destruction is essential for viability, chromosomal disjunction, and successful completion of mitosis.

Spore pigmentation is very common in the fungal kingdom. The best studied pigment in fungi is melanin which coats the surface of single cell spores. What and how pigments function in a fungal species with multiple cell conidia is poorly understood. Here, we identified and deleted a polyketide synthase (PKS) gene PfmaE and showed that it is essential for multicellular conidial pigmentation and development in a plant endophytic fungus, Pestalotiopsis fici. To further characterize the melanin pathway, we utilized an advanced Aspergillus nidulans heterologous system for the expression of the PKS PfmaE and the Pfma gene cluster. By structural elucidation of the pathway metabolite scytalone in A. nidulans, we provided chemical evidence that the Pfma cluster synthesizes DHN melanin. Combining genetic deletion and combinatorial gene expression of Pfma cluster genes, we determined that the putative reductase PfmaG and the PKS are sufficient for the synthesis of scytalone. Feeding scytalone back to the P. fici ΔPfmaE mutant restored pigmentation and multicellular adherence of the conidia. These results cement a growing understanding that pigments are essential not simply for protection of spores from biotic and abiotic stresses but also for spore structural development., (© 2017 John Wiley & Sons Ltd.)

The RNA-binding protein Hu antigen R (HuR) binds to AU-rich elements (ARE) in the 3'-untranslated region (UTR) of target mRNAs. The HuR-ARE interactions stabilize many oncogenic mRNAs that play important roles in tumorigenesis. Thus, small molecules that interfere with the HuR-ARE interaction could potentially inhibit cancer cell growth and progression. Using a fluorescence polarization (FP) competition assay, we identified the compound azaphilone-9 (AZA-9) derived from the fungal natural product asperbenzaldehyde, binds to HuR and inhibits HuR-ARE interaction (IC50 ~1.2 μM). Results from surface plasmon resonance (SPR) verified the direct binding of AZA-9 to HuR. NMR methods mapped the RNA-binding interface of HuR and identified the involvement of critical RNA-binding residues in binding of AZA-9. Computational docking was then used to propose a likely binding site for AZA-9 in the RNA-binding cleft of HuR. Our results show that AZA-9 blocks key RNA-binding residues of HuR and disrupts HuR-RNA interactions in vitro. This knowledge is needed in developing more potent AZA-9 derivatives that could lead to new cancer therapy.

Fungal secondary metabolites (SMs) are extremely important in medicine and agriculture, but regulation of their biosynthesis is incompletely understood. We have developed a genetic screen in Aspergillus nidulans for negative regulators of fungal SM gene clusters and we have used this screen to isolate mutations that upregulate transcription of the non-ribosomal peptide synthetase gene required for nidulanin A biosynthesis. Several of these mutations are allelic and we have identified the mutant gene by genome sequencing. The gene, which we designate mcrA, is conserved but uncharacterized, and it encodes a putative transcription factor. Metabolite profiles of mcrA deletant, mcrA overexpressing, and parental strains reveal that mcrA regulates at least ten SM gene clusters. Deletion of mcrA stimulates SM production even in strains carrying a deletion of the SM regulator laeA, and deletion of mcrA homologs in Aspergillus terreus and Penicillum canescens alters the secondary metabolite profile of these organisms. Deleting mcrA in a genetic dereplication strain has allowed us to discover two novel compounds as well as an antibiotic not known to be produced by A. nidulans. Deletion of mcrA upregulates transcription of hundreds of genes including many that are involved in secondary metabolism, while downregulating a smaller number of genes., Competing Interests: The authors declare no conflict of interests., (© 2016 John Wiley & Sons Ltd.)

In secondary metabolite biosynthesis, core synthetic genes such as polyketide synthase genes usually encode proteins that generate various backbone precursors. These precursors are modified by other tailoring enzymes to yield a large variety of different secondary metabolites. The number of core synthesis genes in a given species correlates, therefore, with the number of types of secondary metabolites the organism can produce. In our study, heterologous expression of all the A. terreus NRPS-like genes showed that two NRPS-like proteins, encoded by atmelA and apvA , release the same natural product, aspulvinone E. In hyphae this compound is converted to aspulvinones whereas in conidia it is converted to melanin. The genes are expressed in different tissues and this spatial control is probably regulated by their own specific promoters. Comparative genomics indicates that atmelA and apvA might share a same ancestral gene and the gene apvA is located in a highly conserved region in Aspergillus species that contains genes coding for life-essential proteins. Our data reveal the first case in secondary metabolite biosynthesis in which the tissue specific production of a single compound directs it into two separate pathways, producing distinct compounds with different functions. Our data also reveal that a single trans -prenyltransferase, AbpB, prenylates two substrates, aspulvinones and butyrolactones, revealing that genes outside of contiguous secondary metabolism gene clusters can modify more than one compound thereby expanding metabolite diversity. Our study raises the possibility of incorporation of spatial, cell-type specificity in expression of secondary metabolites of biological interest and provides new insight into designing and reconstituting their biosynthetic pathways.

Tremendous progress has been made in understanding the functions of γ-tubulin and, in particular, its role in microtubule nucleation since the publication of its discovery in 1989. The structure of γ-tubulin has been determined, and the components of γ-tubulin complexes have been identified. Significant progress in understanding the structure of the γ-tubulin ring complex and its components has led to a persuasive model for how these complexes nucleate microtubule assembly. At the same time, data have accumulated that γ-tubulin has important but less well understood functions that are not simply a consequence of its function in microtubule nucleation. These include roles in the regulation of plus-end microtubule dynamics, gene regulation, and mitotic and cell cycle regulation. Finally, evidence is emerging that γ-tubulin mutations or alterations of γ-tubulin expression play an important role in certain types of cancer and in other diseases., (© 2015 Oakley et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

The spindle assembly checkpoint (SAC) plays a critical role in preventing mitotic errors by inhibiting anaphase until all kinetochores are correctly attached to spindle microtubules. In spite of the economic and medical importance of filamentous fungi, relatively little is known about the behavior of SAC proteins in these organisms. In our efforts to understand the role of γ-tubulin in cell cycle regulation, we have created functional fluorescent protein fusions of four SAC proteins in Aspergillus nidulans, the homologs of Mad2, Mps1, Bub1/BubR1 and Bub3. Time-lapse imaging reveals that SAC proteins are in distinct compartments of the cell until early mitosis when they co-localize at the spindle pole body. SAC activity is, thus, spatially regulated in A. nidulans. Likewise, Cdc20, an activator of the anaphase-promoting complex/cyclosome, is excluded from interphase nuclei, but enters nuclei at mitotic onset and accumulates to a higher level in mitotic nuclei than in the surrounding nucleoplasm before leaving in anaphase/telophase. The activity of this critical cell cycle regulatory complex is likely regulated by the location of Cdc20. Finally, the γ-tubulin mutation mipAD159 causes a nuclear-specific failure of nuclear localization of Mps1 and Bub1/R1 but not of Cdc20, Bub3 or Mad2., (© 2014 John Wiley & Sons Ltd.)

Fungi are prolific producers of secondary metabolites (SMs) that show a variety of biological activities. Recent advances in genome sequencing have shown that fungal genomes harbor far more SM gene clusters than are expressed under conventional laboratory conditions. Activation of these "silent" gene clusters is a major challenge, and many approaches have been taken to attempt to activate them and, thus, unlock the vast treasure chest of fungal SMs. This review will cover recent advances in genome mining of SMs in Aspergillus nidulans. We will also discuss current updates in gene annotation of A. nidulans and recent developments in A. nidulans as a molecular genetic system, both of which are essential for rapid and efficient experimental verification of SM gene clusters on a genome-wide scale. Finally, we will describe advances in the use of A. nidulans as a heterologous expression system to aid in the analysis of SM gene clusters from other fungal species that do not have an established molecular genetic system.

Regulation of secondary metabolite (SM) gene clusters in Aspergillus nidulans has been shown to occur through cluster-specific transcription factors or through global regulators of chromatin structure such as histone methyltransferases, histone deacetylases, or the putative methyltransferase LaeA. A multicopy suppressor screen for genes capable of returning SM production to the SM deficient ΔlaeA mutant resulted in identification of the essential histone acetyltransferase EsaA, able to complement an esa1 deletion in Saccharomyces cereviseae. Here we report that EsaA plays a novel role in SM cluster activation through histone 4 lysine 12 (H4K12) acetylation in four examined SM gene clusters (sterigmatocystin, penicillin, terrequinone and orsellinic acid), in contrast to no increase in H4K12 acetylation of the housekeeping tubA promoter. This augmented SM cluster acetylation requires LaeA for full effect and correlates with both increased transcript levels and metabolite production relative to wild type. H4K12 levels may thus represent a unique indicator of relative production potential, notably of SMs., (© 2012 Blackwell Publishing Ltd.)

A γ-tubulin mutation in Aspergillus nidulans, mipA-D159, causes failure of inactivation of the anaphase-promoting complex/cyclosome (APC/C) in interphase, resulting in failure of cyclin B (CB) accumulation and removal of nuclei from the cell cycle. We have investigated the role of CdhA, the A. nidulans homologue of the APC/C activator protein Cdh1, in γ-tubulin-dependent inactivation of the APC/C. CdhA was not essential, but it targeted CB for destruction in G(1), and APC/C(CdhA) had to be inactivated for the G(1)-S transition. mipA-D159 altered the localization pattern of CdhA, and deletion of the gene encoding CdhA allowed CB to accumulate in all nuclei in strains carrying mipA-D159. These data indicate that mipA-D159 causes a failure of inactivation of APC/C(CdhA) at G(1)-S, perhaps by altering its localization to the spindle pole body, and, thus, that γ-tubulin plays an important role in inactivating APC/C(CdhA) at this point in the cell cycle.

Because of the industrial and medical importance of members of the fungal genus Aspergillus, there is considerable interest in the functions of cytoskeletal components in growth and secretion in these organisms. We have analyzed the genome of Aspergillus nidulans and found that there are two previously unstudied myosin genes, a myosin II homolog, myoB (product = MyoB) and a myosin V homolog, myoE (product = MyoE). Deletions of either cause significant growth defects. MyoB localizes in strings that coalesce into contractile rings at forming septa. It is critical for septation and normal deposition of chitin but not for hyphal extension. MyoE localizes to the Spitzenkörper and to moving puncta in the cytoplasm. Time-lapse imaging of SynA, a v-SNARE, reveals that in myoE deletion strains vesicles no longer localize to the Spitzenkörper. Tip morphology is slightly abnormal and branching occurs more frequently than in controls. Tip extension is slower than in controls, but because hyphal diameter is greater, growth (increase in volume/time) is only slightly reduced. Concentration of vesicles into the Spitzenkörper before incorporation into the plasma membrane is, thus, not required for hyphal growth but facilitates faster tip extension and a more normal hyphal shape.

Secondary metabolite (SM) production by fungi is hypothesized to provide some fitness attribute for the producing organisms. However, most SM clusters are "silent" when fungi are grown in traditional laboratory settings, and it is difficult to ascertain any function or activity of these SM cluster products. Recently, the creation of a chromatin remodeling mutant in Aspergillus nidulans induced activation of several cryptic SM gene clusters. Systematic testing of nine purified metabolites from this mutant identified an emodin derivate with efficacy against both human fungal pathogens (inhibiting both spore germination and hyphal growth) and several bacteria. The ability of catalase to diminish this antimicrobial activity implicates reactive oxygen species generation, specifically, the generation of hydrogen peroxide, as the mechanism of emodin hydroxyl activity.

Gene-silencing mechanisms are being shown to be associated with an increasing number of fungal developmental processes. Telomere position effect (TPE) is a eukaryotic phenomenon resulting in gene repression in areas immediately adjacent to telomere caps. Here, TPE is shown to regulate expression of transgenes on the left arm of chromosome III and the right arm of chromosome VI in Aspergillus nidulans. Phenotypes found to be associated with transgene repression included reduction in radial growth and the absence of sexual spores; however, these pleiotropic phenotypes were remedied when cultures were grown on media with appropriate supplementation. Simple radial growth and ascosporogenesis assays provided insights into the mechanism of TPE, including a means to determine its extent. These experiments revealed that the KU70 homologue (NkuA) and the heterochromatin-associated proteins HepA, ClrD and HdaA were partially required for transgene silencing. This study indicates that TPE extends at least 30 kb on chromosome III, suggesting that this phenomenon may be important for gene regulation in subtelomeric regions of A. nidulans.

A cold-sensitive gamma-tubulin allele of Aspergillus nidulans, mipAD159, causes defects in mitotic and cell cycle regulation at restrictive temperatures that are apparently independent of microtubule nucleation defects. Time-lapse microscopy of fluorescently tagged mitotic regulatory proteins reveals that cyclin B, cyclin-dependent kinase 1, and the Ancdc14 phosphatase fail to accumulate in a subset of nuclei at restrictive temperatures. These nuclei are permanently removed from the cell cycle, whereas other nuclei, in the same multinucleate cell, cycle normally, accumulating and degrading these proteins. After each mitosis, additional daughter nuclei fail to accumulate these proteins, resulting in an increase in noncycling nuclei over time and consequent inhibition of growth. Extensive analyses reveal that these noncycling nuclei result from a nuclear autonomous, microtubule-independent failure of inactivation of the anaphase-promoting complex/cyclosome. Thus, gamma-tubulin functions to regulate this key mitotic and cell cycle regulatory complex.

Deletion of cclA, a component of the COMPASS complex of Aspergillus nidulans, results in the production of monodictyphenone and emodin derivatives. Through a set of targeted deletions in a cclA deletion strain, we have identified the genes required for monodictyphenone and emodin analog biosynthesis. Identification of an intermediate, endocrocin, from an mdpHDelta strain suggests that mdpH might encode a decarboxylase. Furthermore, by replacing the promoter of mdpA (a putative aflJ homolog) and mdpE (a putative aflR homolog) with the inducible alcA promoter, we have confirmed that MdpA functions as a coactivator. We propose a biosynthetic pathway for monodictyphenone and emodin derivatives based on bioinformatic analysis and characterization of biosynthetic intermediates.

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.

In the filamentous fungus Aspergillus nidulans, cytokinesis/septation is triggered by the septation initiation network (SIN), which first appears at the spindle pole body (SPB) during mitosis. The coiled-coil protein SNAD is associated with the SPB and is required for timely septation and conidiation. We have determined that SNAD acted as a scaffold protein that is required for the localization of the SIN proteins of SIDB and MOBA to the SPB. Another scaffold protein SEPK, whose localization at the SPB was dependent on SNAD, was also required for SIDB and MOBA localization to the SPB. In the absence of either SEPK or SNAD, SIDB/MOBA successfully localized to the septation site, indicating that their earlier localization at SPB was not essential for their later appearance at the division site. Unlike their functional counterparts in fission yeast, SEPK and SNAD were not required for vegetative growth but only for timely septation. Furthermore, down-regulation of negative regulators of the SIN suppressed the septation and conidiation phenotypes due to the loss of SNAD. Therefore, we conclude that SPB localization of SIN components is not essential for septation per se, but critical for septation to take place in a timely manner in A. nidulans.

The sequencing of Aspergillus genomes has revealed that the products of a large number of secondary metabolism pathways have not yet been identified. This is probably because many secondary metabolite gene clusters are not expressed under normal laboratory culture conditions. It is, therefore, important to discover conditions or regulatory factors that can induce the expression of these genes. We report that the deletion of sumO, the gene that encodes the small ubiquitin-like protein SUMO in A. nidulans, caused a dramatic increase in the production of the secondary metabolite asperthecin and a decrease in the synthesis of austinol/dehydroaustinol and sterigmatocystin. The overproduction of asperthecin in the sumO deletion mutant has allowed us, through a series of targeted deletions, to identify the genes required for asperthecin synthesis. The asperthecin biosynthesis genes are clustered and include genes encoding an iterative type I polyketide synthase, a hydrolase, and a monooxygenase. The identification of these genes allows us to propose a biosynthetic pathway for asperthecin.

The recently sequenced genomes of several Aspergillus species have revealed that these organisms have the potential to produce a surprisingly large range of natural products, many of which are currently unknown. We have found that A. nidulans produces emericellamide A, an antibiotic compound of mixed origins with polyketide and amino acid building blocks. Additionally, we describe the discovery of four previously unidentified, related compounds that we designate emericellamide C-F. Using recently developed gene targeting techniques, we have identified the genes involved in emericellamide biosynthesis. The emericellamide gene cluster contains one polyketide synthase and one nonribosomal peptide synthetase. From the sequences of the genes, we are able to deduce a biosynthetic pathway for the emericellamides. The identification of this biosynthetic pathway opens the door to engineering novel analogs of this structurally complex metabolite.

Hyphal tip growth in fungi is important because of the economic and medical importance of fungi, and because it may be a useful model for polarized growth in other organisms. We have investigated the central questions of the roles of cytoskeletal elements and of the precise sites of exocytosis and endocytosis at the growing hyphal tip by using the model fungus Aspergillus nidulans. Time-lapse imaging of fluorescent fusion proteins reveals a remarkably dynamic, but highly structured, tip growth apparatus. Live imaging of SYNA, a synaptobrevin homologue, and SECC, an exocyst component, reveals that vesicles accumulate in the Spitzenkörper (apical body) and fuse with the plasma membrane at the extreme apex of the hypha. SYNA is recycled from the plasma membrane by endocytosis at a collar of endocytic patches, 1-2 mum behind the apex of the hypha, that moves forward as the tip grows. Exocytosis and endocytosis are thus spatially coupled. Inhibitor studies, in combination with observations of fluorescent fusion proteins, reveal that actin functions in exocytosis and endocytosis at the tip and in holding the tip growth apparatus together. Microtubules are important for delivering vesicles to the tip area and for holding the tip growth apparatus in position.

Aspergillus nidulans is an important experimental organism, and it is a model organism for the genus Aspergillus that includes serious pathogens as well as commercially important organisms. Gene targeting by homologous recombination during transformation is possible in A. nidulans, but the frequency of correct gene targeting is variable and often low. We have identified the A. nidulans homolog (nkuA) of the human KU70 gene that is essential for nonhomologous end joining of DNA in double-strand break repair. Deletion of nkuA (nkuA delta) greatly reduces the frequency of nonhomologous integration of transforming DNA fragments, leading to dramatically improved gene targeting. We have also developed heterologous markers that are selectable in A. nidulans but do not direct integration at any site in the A. nidulans genome. In combination, nkuA delta and the heterologous selectable markers make up a very efficient gene-targeting system. In experiments involving scores of genes, 90% or more of the transformants carried a single insertion of the transforming DNA at the correct site. The system works with linear and circular transforming molecules and it works for tagging genes with fluorescent moieties, replacing genes, and replacing promoters. This system is efficient enough to make genomewide gene-targeting projects feasible.

The aspergilli comprise a diverse group of filamentous fungi spanning over 200 million years of evolution. Here we report the genome sequence of the model organism Aspergillus nidulans, and a comparative study with Aspergillus fumigatus, a serious human pathogen, and Aspergillus oryzae, used in the production of sake, miso and soy sauce. Our analysis of genome structure provided a quantitative evaluation of forces driving long-term eukaryotic genome evolution. It also led to an experimentally validated model of mating-type locus evolution, suggesting the potential for sexual reproduction in A. fumigatus and A. oryzae. Our analysis of sequence conservation revealed over 5,000 non-coding regions actively conserved across all three species. Within these regions, we identified potential functional elements including a previously uncharacterized TPP riboswitch and motifs suggesting regulation in filamentous fungi by Puf family genes. We further obtained comparative and experimental evidence indicating widespread translational regulation by upstream open reading frames. These results enhance our understanding of these widely studied fungi as well as provide new insight into eukaryotic genome evolution and gene regulation.

In Aspergillus nidulans, cytoplasmic dynein and NUDF/LIS1 are found at the spindle poles during mitosis, but they seem to be targeted to this location via different mechanisms. The spindle pole localization of cytoplasmic dynein requires the function of the anaphase-promoting complex (APC), whereas that of NUDF does not. Moreover, although NUDF's localization to the spindle poles does not require a fully functional dynein motor, the function of NUDF is important for cytoplasmic dynein's targeting to the spindle poles. Interestingly, a gamma-tubulin mutation, mipAR63, nearly eliminates the localization of cytoplasmic dynein to the spindle poles, but it has no apparent effect on NUDF's spindle pole localization. Live cell analysis of the mipAR63 mutant revealed a defect in chromosome separation accompanied by unscheduled spindle elongation before the completion of anaphase A, suggesting that gamma-tubulin may recruit regulatory proteins to the spindle poles for mitotic progression. In A. nidulans, dynein is not apparently required for mitotic progression. In the presence of a low amount of benomyl, a microtubule-depolymerizing agent, however, a dynein mutant diploid strain exhibits a more pronounced chromosome loss phenotype than the control, indicating that cytoplasmic dynein plays a role in chromosome segregation.

The filamentous fungus Aspergillus nidulans grows by polarized extension of hyphal tips. The actin cytoskeleton is essential for polarized growth, but the role of microtubules has been controversial. To define the role of microtubules in tip growth, we used time-lapse microscopy to measure tip growth rates in germlings of A. nidulans and in multinucleate hyphal tip cells, and we used a green fluorescent protein-alpha-tubulin fusion to observe the effects of the antimicrotubule agent benomyl. Hyphal tip cells grew approximately 5 times faster than binucleate germlings. In germlings, cytoplasmic microtubules disassembled completely in mitosis. In hyphal tip cells, however, microtubules disassembled through most of the cytoplasm in mitosis but persisted in a region near the hyphal tip. The growth rate of hyphal tip cells did not change significantly in mitosis. Benomyl caused rapid disassembly of microtubules in tip cells and a 10x reduction in growth rate. When benomyl was washed out, microtubules assembled quickly and rapid tip growth resumed. These results demonstrate that although microtubules are not strictly required for polarized growth, they are rate-limiting for the growth of hyphal tip cells. These data also reveal that A. nidulans exhibits a remarkable spatial regulation of microtubule disassembly within hyphal tip cells.

We have developed a dual-color imaging system based on cyan fluorescent protein-labeled histone H2A and green fluorescent protein-labeled alpha tubulin to visualize DNA and spindles simultaneously in the same living cell of Aspergillus nidulans. This system allows new details of mitosis and nuclear movement to be revealed.

Recent data from multiple organisms indicate that gamma-tubulin has essential, but incompletely defined, functions in addition to nucleating microtubule assembly. To investigate these functions, we examined the phenotype of mipAD159, a cold-sensitive allele of the gamma-tubulin gene of Aspergillus nidulans. Immunofluorescence microscopy of synchronized material revealed that at a restrictive temperature mipAD159 does not inhibit mitotic spindle formation. Anaphase A was inhibited in many nuclei, however, and after a slight delay in mitosis (approximately 6% of the cell cycle period), most nuclei reentered interphase without dividing. In vivo observations of chromosomes at a restrictive temperature revealed that mipAD159 caused a failure of the coordination of late mitotic events (anaphase A, anaphase B, and chromosomal disjunction) and nuclei reentered interphase quickly even though mitosis was not completed successfully. Time-lapse microscopy also revealed that transient mitotic spindle abnormalities, in particular bent spindles, were more prevalent in mipAD159 strains than in controls. In experiments in which microtubules were depolymerized with benomyl, mipAD159 nuclei exited mitosis significantly more quickly (as judged by chromosomal condensation) than nuclei in a control strain. These data reveal that gamma-tubulin has an essential role in the coordination of late mitotic events, and a microtubule-independent function in mitotic checkpoint control.

gamma-Tubulin localizes to microtubule-organizing centers in animal and fungal cells where it is important for microtubule nucleation. Plant cells do not have morphologically defined microtubule organizing centers, however, and gamma-tubulin is distributed in small, discrete structures along microtubules. The great difference in distribution has prompted speculation that plant gamma-tubulins function differently from animal and fungal gamma-tubulins. We tested this possibility by expressing Arabidopsis gamma-tubulin in the fission yeast Schizosaccharomyces pombe. At high temperatures, the plant gamma-tubulin was able to bind to microtubule-organizing centers, nucleate microtubule assembly, and support the growth and replication of S. pombe cells lacking endogenous gamma-tubulin. However, the distribution of microtubules was abnormal as was cell morphology, and at low temperatures, cells were arrested in mitosis. These results reveal that Arabidopsis gamma-tubulin can carry out essential functions in S. pombe and is, thus, functionally conserved. The morphological abnormalities reveal that it cannot carry out some nonessential functions, however, and they underscore the importance of gamma-tubulin in morphogenesis of fission yeast cells and in maintaining normal interphase microtubule arrays.

The tinA gene of Aspergillus nidulans encodes a protein that interacts with the NIMA mitotic protein kinase in a cell cycle-specific manner. Highly similar proteins are encoded in Neurospora crassa and Aspergillus fumigatus. TINA and NIMA preferentially interact in interphase and larger forms of TINA are generated during mitosis. Localization studies indicate that TINA is specifically localized to the spindle pole bodies only during mitosis in a microtubule-dependent manner. Deletion of tinA alone is not lethal but displays synthetic lethality in combination with the anaphase-promoting complex/cyclosome mutation bimE7. At the bimE7 metaphase arrest point, lack of TINA enhanced the nucleation of bundles of cytoplasmic microtubules from the spindle pole bodies. These microtubules interacted to form spindles joined in series via astral microtubules as revealed by live cell imaging. Because TINA is modified and localizes to the spindle pole bodies at mitosis, and lack of TINA causes enhanced production of cytoplasmic microtubules at metaphase arrest, we suggest TINA is involved in negative regulation of the astral microtubule organizing capacity of the spindle pole bodies during metaphase.

In many important organisms, including many algae and most fungi, the nuclear envelope does not disassemble during mitosis. This fact raises the possibility that mitotic onset and/or exit might be regulated, in part, by movement of important mitotic proteins into and out of the nucleoplasm. We have used two methods to determine whether tubulin levels in the nucleoplasm are regulated in the fungus Aspergillus nidulans. First, we have used benomyl to disassemble microtubules and create a pool of free tubulin that can be readily observed by immunofluorescence. We find that tubulin is substantially excluded from interphase nuclei, but is present in mitotic nuclei. Second, we have observed a green fluorescent protein/alpha-tubulin fusion in living cells by time-lapse spinning-disk confocal microscopy. We find that tubulin is excluded from interphase nuclei, enters the nucleus seconds before the mitotic spindle begins to form, and is removed from the nucleoplasm during the M-to-G1 transition. Our data indicate that regulation of intranuclear tubulin levels plays an important, perhaps essential, role in the control of mitotic spindle formation in A. nidulans. They suggest that regulation of protein movement into the nucleoplasm may be important for regulating mitotic onset in organisms with intranuclear mitosis.

Growth of most eukaryotic cells requires directed transport along microtubules (MTs) that are nucleated at nuclear-associated microtubule organizing centers (MTOCs), such as the centrosome and the fungal spindle pole body (SPB). Herein, we show that the pathogenic fungus Ustilago maydis uses different MT nucleation sites to rearrange MTs during the cell cycle. In vivo observation of green fluorescent protein-MTs and MT plus-ends, tagged by a fluorescent EB1 homologue, provided evidence for antipolar MT orientation and dispersed cytoplasmic MT nucleating centers in unbudded cells. On budding gamma-tubulin containing MTOCs formed at the bud neck, and MTs reorganized with >85% of all minus-ends being focused toward the growth region. Experimentally induced lateral budding resulted in MTs that curved out of the bud, again supporting the notion that polar growth requires polar MT nucleation. Depletion or overexpression of Tub2, the gamma-tubulin from U. maydis, affected MT number in interphase cells. The SPB was inactive in G2 phase but continuously recruited gamma-tubulin until it started to nucleate mitotic MTs. Taken together, our data suggest that MT reorganization in U. maydis depends on cell cycle-specific nucleation at dispersed cytoplasmic sites, at a polar MTOC and the SPB.

Previous research has found that a gamma-tubulin mutation in Schizosaccharomyces pombe is synthetically lethal with a deletion of the C-terminal motor domain kinesin-like protein gene pkl1, but the lethality of the double mutant prevents a phenotypic analysis of the synthetic interaction. We have investigated interactions between klpA1, a deletion of an Aspergillus nidulans homolog of pkl1, and mutations in the mipA, gamma-tubulin gene. We find that klpA1 dramatically increases the cold sensitivity and slightly reduces the growth rate at all temperatures, of three mipA alleles. In synchronized cells we find that klpA1 causes a substantial but transient inhibition of the establishment of spindle bipolarity. At a restrictive temperature, mipAD123 causes a slight, transient inhibition of spindle bipolarity and a more significant inhibition of anaphase A. In the mipAD123/klpA1 strain, formation of bipolar spindles is more strongly inhibited than in the klpA1 single mutant and many spindles apparently never become bipolar. These results indicate, surprisingly, that gamma-tubulin and the klpA kinesin have overlapping roles in the establishment of spindle bipolarity. We propose a model to account for these data.