Light in the Fungal World: From Photoreception to Gene Transcription and Beyond.

Fungi see light of different colors by using photoreceptors such as the White Collar proteins and cryptochromes for blue light, opsins for green light, and phytochromes for red light. Light regulates fungal development, promotes the accumulation of protective pigments and proteins, and regulates tropic growth. The White Collar complex (WCC) is a photoreceptor and a transcription factor that is responsible for regulating transcription after exposure to blue light. In Neurospora crassa, light promotes the interaction of WCCs and their binding to the promoters to activate transcription. In Aspergillus nidulans, the WCC and the phytochrome interact to coordinate gene transcription and other responses, but the contribution of these photoreceptors to fungal photobiology varies across fungal species. Ultimately, the effect of light on fungal biology is the result of the coordinated transcriptional regulation and activation of signal transduction pathways.

GPCR-mediated glucose sensing system regulates light-dependent fungal development and mycotoxin production.

Microorganisms sense environmental fluctuations in nutrients and light, coordinating their growth and development accordingly. Despite their critical roles in fungi, only a few G-protein coupled receptors (GPCRs) have been characterized. The Aspergillus nidulans genome encodes 86 putative GPCRs. Here, we characterise a carbon starvation-induced GPCR-mediated glucose sensing mechanism in A. nidulans. This includes two class V (gprH and gprI) and one class VII (gprM) GPCRs, which in response to glucose promote cAMP signalling, germination and hyphal growth, while negatively regulating sexual development in a light-dependent manner. We demonstrate that GprH regulates sexual development via influencing VeA activity, a key light-dependent regulator of fungal morphogenesis and secondary metabolism. We show that GprH and GprM are light-independent negative regulators of sterigmatocystin biosynthesis. Additionally, we reveal the epistatic interactions between the three GPCRs in regulating sexual development and sterigmatocystin production. In conclusion, GprH, GprM and GprI constitute a novel carbon starvation-induced glucose sensing mechanism that functions upstream of cAMP-PKA signalling to regulate fungal development and mycotoxin production.

Endocytic recycling via the TGN underlies the polarized hyphal mode of life.

Intracellular traffic in Aspergillus nidulans hyphae must cope with the challenges that the high rates of apical extension (1µm/min) and the long intracellular distances (>100 µm) impose. Understanding the ways in which the hyphal tip cell coordinates traffic to meet these challenges is of basic importance, but is also of considerable applied interest, as fungal invasiveness of animals and plants depends critically upon maintaining these high rates of growth. Rapid apical extension requires localization of cell-wall-modifying enzymes to hyphal tips. By combining genetic blocks in different trafficking steps with multidimensional epifluorescence microscopy and quantitative image analyses we demonstrate that polarization of the essential chitin-synthase ChsB occurs by indirect endocytic recycling, involving delivery/exocytosis to apices followed by internalization by the sub-apical endocytic collar of actin patches and subsequent trafficking to TGN cisternae, where it accumulates for ~1 min before being re-delivered to the apex by a RAB11/TRAPPII-dependent pathway. Accordingly, ChsB is stranded at the TGN by Sec7 inactivation but re-polarizes to the apical dome if the block is bypassed by a mutation in geaAgea1 that restores growth in the absence of Sec7. That polarization is independent of RAB5, that ChsB predominates at apex-proximal cisternae, and that upon dynein impairment ChsB is stalled at the tips in an aggregated endosome indicate that endocytosed ChsB traffics to the TGN via sorting endosomes functionally located upstream of the RAB5 domain and that this step requires dynein-mediated basipetal transport. It also requires RAB6 and its effector GARP (Vps51/Vps52/Vps53/Vps54), whose composition we determined by MS/MS following affinity chromatography purification. Ablation of any GARP component diverts ChsB to vacuoles and impairs growth and morphology markedly, emphasizing the important physiological role played by this pathway that, we propose, is central to the hyphal mode of growth.

Velvet domain protein VosA represses the zinc cluster transcription factor SclB regulatory network for Aspergillus nidulans asexual development, oxidative stress response and secondary metabolism.

The NF-κB-like velvet domain protein VosA (viability of spores) binds to more than 1,500 promoter sequences in the filamentous fungus Aspergillus nidulans. VosA inhibits premature induction of the developmental activator gene brlA, which promotes asexual spore formation in response to environmental cues as light. VosA represses a novel genetic network controlled by the sclB gene. SclB function is antagonistic to VosA, because it induces the expression of early activator genes of asexual differentiation as flbC and flbD as well as brlA. The SclB controlled network promotes asexual development and spore viability, but is independent of the fungal light control. SclB interactions with the RcoA transcriptional repressor subunit suggest additional inhibitory functions on transcription. SclB links asexual spore formation to the synthesis of secondary metabolites including emericellamides, austinol as well as dehydroaustinol and activates the oxidative stress response of the fungus. The fungal VosA-SclB regulatory system of transcription includes a VosA control of the sclB promoter, common and opposite VosA and SclB control functions of fungal development and several additional regulatory genes. The relationship between VosA and SclB illustrates the presence of a convoluted surveillance apparatus of transcriptional control, which is required for accurate fungal development and the linkage to the appropriate secondary metabolism.

Alternative transcription start sites of the enolase-encoding gene enoA are stringently used in glycolytic/gluconeogenic conditions in Aspergillus oryzae.

Gene expression using alternative transcription start sites (TSSs) is an important transcriptional regulatory mechanism for environmental responses in eukaryotes. Here, we identify two alternative TSSs in the enolase-encoding gene (enoA) in Aspergillus oryzae, an industrially important filamentous fungus. TSS use in enoA is strictly dependent on the difference in glycolytic and gluconeogenic carbon sources. Transcription from the upstream TSS (uTSS) or downstream TSS (dTSS) predominantly occurs under gluconeogenic or glycolytic conditions, respectively. In addition to enoA, most glycolytic genes involved in reversible reactions possess alternative TSSs. The fbaA gene, which encodes fructose-bisphosphate aldolase, also shows stringent alternative TSS selection, similar to enoA. Alignment of promoter sequences of enolase-encoding genes in Aspergillus predicted two conserved regions that contain a putative cis-element required for enoA transcription from each TSS. However, uTSS-mediated transcription of the acuN gene, an enoA ortholog in Aspergillus nidulans, is not strictly dependent on carbon source, unlike enoA. Furthermore, enoA transcript levels in glycolytic conditions are higher than in gluconeogenic conditions. Conversely, acuN is more highly transcribed in gluconeogenic conditions. This suggests that the stringent usage of alternative TSSs and higher transcription in glycolytic conditions in enoA may reflect that the A. oryzae evolutionary genetic background was domesticated by exclusive growth in starch-rich environments. These findings provide novel insights into the complexity and diversity of transcriptional regulation of glycolytic/gluconeogenic genes among Aspergilli.

Aspergillus nidulans in the post-genomic era: a top-model filamentous fungus for the study of signaling and homeostasis mechanisms.

The accessibility to next-generation sequencing (NGS) techniques has enabled the sequencing of hundreds of genomes of species representing all kingdoms. In the case of fungi, genomes of more than a thousand of species are publicly available. This is far from covering the number of 2.2-3.8 million fungal species estimated to populate the world but has significantly improved the resolution of the fungal tree of life. Furthermore, it has boosted systematic evolutionary analyses, the development of faster and more accurate diagnostic analyses of pathogenic strains or the improvement of several biotechnological processes. Nevertheless, the diversification of the nature of fungal species used as model has also weakened research in other species that were traditionally used as reference in the pre-genomic era. In this context, and after more than 65 years since the first works published by Pontecorvo, Aspergillus nidulans remains as one of the most referential model filamentous fungus in research fields such as hyphal morphogenesis, intracellular transport, developmental programs, secondary metabolism, or stress response. This mini-review summarizes how A. nidulans has contributed to the progress in these fields during the last years, and discusses how it could contribute in the future, assisted by NGS and new-generation molecular, microscopy, or cellular tools.

Why outcross? The abandon-ship hypothesis in a facultative outcrossing/selfing fungal species.

Most species able to reproduce both sexually and asexually (facultative sexual species) invest more in sexual reproduction in stressful environment conditions. According to the abandon-ship hypothesis, plasticity for investment in sexual reproduction may have been selected in these species, allowing unfit genotypes to generate progeny carrying new advantageous allelic combinations. We tested this hypothesis in Aspergillus nidulans, a fungus able to reproduce asexually, or sexually, by outcrossing and/or haploid selfing (i.e. fusion of genetically identical haploid nuclei, causing immediate genome-wide homozygosity). We crossed various strains of A. nidulans in a non-stressful environment and a stressful environment containing a non-lethal dose of fungicide. Without stress, crosses preferentially generated haploselfed fruiting bodies, whereas stressful conditions significantly increased the outcrossing rate. Our results strongly support the abandon-ship hypothesis and suggest that, for parents with low fitness, the costs of investing in sexual reproduction may be compensated by the production of fitter progeny carrying beneficial allele combinations. Similarly, the progeny generated by outcrossing was less fit than that produced by haploid selfing in non-stressful environments, but fitter in stressful conditions, suggesting that outcrossing may have short-term advantages in stressful environments in A. nidulans.

Sterigmatocystin production is restricted to hyphae located in the proximity of hülle cells.

Aspergillus nidulans produces sterigmatocystin, a secondary metabolite mycotoxin, for the protection of its reproductive structures. Previous studies on grazing behavior of fungivore arthropods, regulation of sexual development, and secondary metabolite biosynthesis have revealed the association of sterigmatocystin biosynthesis with sexual reproduction, but the spatial distribution of sterigmatocystin producing hyphae within the colony has never been investigated. In this work, we aimed to locate the site of sterigmatocystin production within the colony by employing a yCFP reporter system. We demonstrated that the stcO promoter is active only in vegetative hyphae that surround groups of hülle cells and the activity decreases and eventually ceases as the distance between the hypha and the hülle cells increases. This phenomenon indicates that the vegetative mycelium might consist of morphologically uniform, but functionally different hyphae.

The Aspergillus nidulans syntaxin PepA(Pep12) is regulated by two Sec1/Munc-18 proteins to mediate fusion events at early endosomes, late endosomes and vacuoles.

Syntaxins are target-SNAREs that crucially contribute to determine membrane compartment identity. Three syntaxins, Tlg2p, Pep12p and Vam3p, organize the yeast endovacuolar system. Remarkably, filamentous fungi lack the equivalent of the yeast vacuolar syntaxin Vam3p, making unclear how these organisms regulate vacuole fusion. We show that the nearly essential Aspergillus nidulans syntaxin PepA(Pep12) , present in all endocytic compartments between early endosomes and vacuoles, shares features of Vam3p and Pep12p, and is capable of forming compositional equivalents of all known yeast endovacuolar SNARE bundles including that formed by yeast Vam3p for vacuolar fusion. Our data further indicate that regulation by two Sec1/Munc-18 proteins, Vps45 in early endosomes and Vps33 in early and late endosomes/vacuoles contributes to the wide domain of PepA(Pep12) action. The syntaxin TlgB(Tlg2) localizing to the TGN appears to mediate retrograde traffic connecting post-Golgi (sorting) endosomes with the TGN. TlgB(Tlg2) is dispensable for growth but becomes essential if the early Golgi syntaxin SedV(Sed5) is compromised, showing that the Golgi can function with a single syntaxin, SedV(Sed5) . Remarkably, its pattern of associations with endosomal SNAREs is consistent with SedV(Sed5) playing roles in retrograde pathway(s) connecting endocytic compartments downstream of the post-Golgi endosome with the Golgi, besides more conventional intra-Golgi roles.

Seeing the world differently: variability in the photosensory mechanisms of two model fungi.

Light plays an important role for most organisms on this planet, serving either as a source of energy or information for the adaptation of biological processes to specific times of day. The fungal kingdom is estimated to contain well over a million species, possibly 10-fold more, and it is estimated that a majority of the fungi respond to light, eliciting changes in several physiological characteristics including pathogenesis, development and secondary metabolism. Two model organisms for photobiological studies have taken centre-stage over the last few decades--Neurospora crassa and Aspergillus nidulans. In this review, we will first discuss our understanding of the light response in N. crassa, about which the most is known, and will then juxtapose N. crassa with A. nidulans, which, as will be described below, provides an excellent template for understanding photosensory cross-talk. Finally, we will end with a commentary on the variability of the light response among other relevant fungi, and how our molecular understanding in the aforementioned model organisms still provides a strong base for dissecting light responses in such species.

Calcineurin and Calcium Channel CchA Coordinate the Salt Stress Response by Regulating Cytoplasmic Ca2+ Homeostasis in Aspergillus nidulans.

The eukaryotic calcium/calmodulin-dependent protein phosphatase calcineurin is crucial for the environmental adaption of fungi. However, the mechanism of coordinate regulation of the response to salt stress by calcineurin and the high-affinity calcium channel CchA in fungi is not well understood. Here we show that the deletion of cchA suppresses the hyphal growth defects caused by the loss of calcineurin under salt stress in Aspergillus nidulans Additionally, the hypersensitivity of the ΔcnaA strain to extracellular calcium and cell-wall-damaging agents can be suppressed by cchA deletion. Using the calcium-sensitive photoprotein aequorin to monitor the cytoplasmic Ca(2+) concentration ([Ca(2+)]c) in living cells, we found that calcineurin negatively regulates CchA on calcium uptake in response to external calcium in normally cultured cells. However, in salt-stress-pretreated cells, loss of either cnaA or cchA significantly decreased the [Ca(2+)]c, but a deficiency in both cnaA and cchA switches the [Ca(2+)]c to the reference strain level, indicating that calcineurin and CchA synergistically coordinate calcium influx under salt stress. Moreover, real-time PCR results showed that the dysfunction of cchA in the ΔcnaA strain dramatically restored the expression of enaA (a major determinant for sodium detoxification), which was abolished in the ΔcnaA strain under salt stress. These results suggest that double deficiencies of cnaA and cchA could bypass the requirement of calcineurin to induce enaA expression under salt stress. Finally, YvcA, a member of the transient receptor potential channel (TRPC) protein family of vacuolar Ca(2+) channels, was proven to compensate for calcineurin-CchA in fungal salt stress adaption.IMPORTANCE The feedback inhibition relationship between calcineurin and the calcium channel Cch1/Mid1 has been well recognized from yeast. Interestingly, our previous study (S. Wang et al., PLoS One 7:e46564, 2012, http://dx.doi.org/10.1371/journal.pone.0046564) showed that the deletion of cchA could suppress the hyphal growth defects caused by the loss of calcineurin under salt stress in Aspergillus nidulans In this study, our findings suggest that fungi are able to develop a unique mechanism for adapting to environmental salt stress. Compared to cells cultured normally, the NaCl-pretreated cells had a remarkable increase in transient [Ca(2+)]c Furthermore, we show that calcineurin and CchA are required to modulate cellular calcium levels and synergistically coordinate calcium influx under salt stress. Finally, YvcA, a member of of the TRPC family of vacuolar Ca(2+) channels, was proven to compensate for calcineurin-CchA in fungal salt stress adaption. The findings in this study provide insights into the complex regulatory links between calcineurin and CchA to maintain cytoplasmic Ca(2+) homeostasis in response to different environments.

The velvet family of fungal regulators contains a DNA-binding domain structurally similar to NF-κB.

Morphological development of fungi and their combined production of secondary metabolites are both acting in defence and protection. These processes are mainly coordinated by velvet regulators, which contain a yet functionally and structurally uncharacterized velvet domain. Here we demonstrate that the velvet domain of VosA is a novel DNA-binding motif that specifically recognizes an 11-nucleotide consensus sequence consisting of two motifs in the promoters of key developmental regulatory genes. The crystal structure analysis of the VosA velvet domain revealed an unforeseen structural similarity with the Rel homology domain (RHD) of the mammalian transcription factor NF-κB. Based on this structural similarity several conserved amino acid residues present in all velvet domains have been identified and shown to be essential for the DNA binding ability of VosA. The velvet domain is also involved in dimer formation as seen in the solved crystal structures of the VosA homodimer and the VosA-VelB heterodimer. These findings suggest that defence mechanisms of both fungi and animals might be governed by structurally related DNA-binding transcription factors.

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.

Modelling, substrate docking and mutational analysis identify residues essential for function and specificity of the major fungal purine transporter AzgA.

The AzgA purine/H(+) symporter of Aspergillus nidulans is the founding member of a functionally and phylogenetically distinct transporter family present in fungi, bacteria and plants. Here a valid AzgA topological model is built based on the crystal structure of the Escherichia coli uracil transporter UraA, a member of the nucleobase-ascorbate transporter (NAT/NCS2) family. The model consists of 14 transmembrane, mostly α-helical, segments (TMSs) and cytoplasmic N- and C-tails. A distinct compact core of 8 TMSs, made of two intertwined inverted repeats (TMSs 1-4 and 8-11), is topologically distinct from a flexible domain (TMSs 5-7 and 12-14). A putative substrate binding cavity is visible between the core and the gate domains. Substrate docking, molecular dynamics and mutational analysis identified several residues critical for purine binding and/or transport in TMS3, TMS8 and TMS10. Among these, Asn131 (TMS3), Asp339 (TMS8) and Glu394 (TMS10) are proposed to directly interact with substrates, while Asp342 (TMS8) might be involved in subsequent substrate translocation, through H(+) binding and symport. Thus, AzgA and other NAT transporters use topologically similar TMSs and amino acid residues for substrate binding and transport, which in turn implies that AzgA-like proteins constitute a distant subgroup of the ubiquitous NAT family.

A dually located multi-HMG-box protein of Aspergillus nidulans has a crucial role in conidial and ascospore germination.

Seven HMG-box proteins of Aspergillus nidulans have been identified in the genomic databases. Three of these have the characteristics of non-specific DNA-binding proteins. One of these, AN1267 (HmbB), comprises one canonical HMG-box in its C-terminus and upstream of the canonical box two structurally related boxes, to be called Shadow-HMG-boxes. This protein defines, together with the Podospora anserina mtHMG1, a clade of proteins present in the Pezizomycotina, with orthologues in some of the Taphrinomycotina. HmbB localizes primarily to the mitochondria but occasionally in nuclei. The deletion of the cognate gene results in a number of pleiotropic effects, including those on hyphal morphology, sensitivity to oxidative stress, absence of sterigmatocystin production and changes in the profile of conidial metabolites. The most striking phenotype of deletion strains is a dramatic decrease in conidial and ascospore viability. We show that this is most likely due to the protein being essential to maintain mitochondrial DNA in spores.

Regulated inactivation of the spindle assembly checkpoint without functional mitotic spindles.

The spindle assembly checkpoint (SAC) arrests mitosis until bipolar attachment of spindle microtubules to all chromosomes is accomplished. However, when spindle formation is prevented and the SAC cannot be satisfied, mammalian cells can eventually overcome the mitotic arrest while the checkpoint is still activated. We find that Aspergillus nidulans cells, which are unable to satisfy the SAC, inactivate the checkpoint after a defined period of mitotic arrest. Such SAC inactivation allows normal nuclear reassembly and mitotic exit without DNA segregation. We demonstrate that the mechanisms, which govern such SAC inactivation, require protein synthesis and can occur independently of inactivation of the major mitotic regulator Cdk1/Cyclin B or mitotic exit. Moreover, in the continued absence of spindle function cells transit multiple cell cycles in which the SAC is reactivated each mitosis before again being inactivated. Such cyclic activation and inactivation of the SAC suggests that it is subject to cell-cycle regulation that is independent of bipolar spindle function.

Further characterization of the role of the mitochondrial high-mobility group box protein in the intracellular redox environment of Aspergillus nidulans.

HmbB, a predominantly mitochondrial high-mobility group box (HMGB) protein, of Aspergillus nidulans affects diverse biological activities, such as sterigmatocystin production, the maintenance of mitochondrial DNA copy number, germination of asexual and sexual spores, and protection against oxidative stress agents. We hypothesized that the latter correlates with an unbalanced intracellular redox state, in which case, a not yet fully characterized physiological function could be attributed to this mitochondrial HMGB protein. Here, we studied the intracellular redox environment and oxidative stress tolerance in hmbB+ and hmbBΔ strains under normal and oxidative stress conditions by measuring glutathione redox couple, intracellular reactive oxygen species (ROS) content and ROS-protecting enzyme activities. Our results revealed that the intracellular redox environment is different in hmbBΔ conidia and mycelia from that of hmbB+, and shed light on the seemingly contradictory difference in the tolerance of hmbBΔ mycelia to diamide and menadione oxidative stressors.

A mutation in the converter subdomain of Aspergillus nidulans MyoB blocks constriction of the actomyosin ring in cytokinesis.

We have identified a mutant allele of the Aspergillus nidulans homologue of myosin II (myoB; AN4706), which prevents normal septum formation. This is the first reported myosin II mutation in a filamentous fungus. Strains expressing the myoB(G843D) allele produce mainly aberrant septa at 30 °C and are completely aseptate at temperatures above 37 °C. Conidium formation is greatly reduced at 30 °C and progressively impaired with increasing temperature. Sequencing of the myoB(G843D) allele identified a point mutation predicted to result in a glycine-to-aspartate amino acid substitution at residue 843 in the myosin II converter domain. This residue is conserved in all fungal, plant, and animal myosin sequences that we have examined. The mutation does not prevent localization of the myoB(G843D) gene product to contractile rings, but it does block ring constriction. MyoB(G843D) rings at sites of abortive septation disassemble after an extended period and dissipate into the cytoplasm. During contractile ring formation, both wild type and mutant MyoB::GFP colocalize with actin--an association that begins at the pre-ring "string" stage. Down-regulation of wild-type myoB expression under control of the alcA promoter blocks septation but does not prevent actin from aggregating at putative septation sites--the actin rings, however, do not fully coalesce. Both septation and targeting of MyoB are blocked by disruption of filamentous actin using latrunculin B. We propose a model in which myosin assembly at septation sites depends upon the presence of F-actin, but assembly of the actin component of contractile rings depends upon normal levels of myosin only for the final stages of ring compaction.

Protein phosphatase 2A (PP2A) regulatory subunits ParA and PabA orchestrate septation and conidiation and are essential for PP2A activity in Aspergillus nidulans.

Protein phosphatase 2A (PP2A) is a major intracellular protein phosphatase that regulates multiple aspects of cell growth and metabolism. Different activities of PP2A and subcellular localization are determined by its regulatory subunits. Here we identified and characterized the functions of two protein phosphatase regulatory subunit homologs, ParA and PabA, in Aspergillus nidulans. Our results demonstrate that ParA localizes to the septum site and that deletion of parA causes hyperseptation, while overexpression of parA abolishes septum formation; this suggests that ParA may function as a negative regulator of septation. In comparison, PabA displays a clear colocalization pattern with 4',6-diamidino-2-phenylindole (DAPI)-stained nuclei, and deletion of pabA induces a remarkable delayed-septation phenotype. Both parA and pabA are required for hyphal growth, conidiation, and self-fertilization, likely to maintain normal levels of PP2A activity. Most interestingly, parA deletion is capable of suppressing septation defects in pabA mutants, suggesting that ParA counteracts PabA during the septation process. In contrast, double mutants of parA and pabA led to synthetic defects in colony growth, indicating that ParA functions synthetically with PabA during hyphal growth. Moreover, unlike the case for PP2A-Par1 and PP2A-Pab1 in yeast (which are negative regulators that inactivate the septation initiation network [SIN]), loss of ParA or PabA fails to suppress defects of temperature-sensitive mutants of the SEPH kinase of the SIN. Thus, our findings support the previously unrealized evidence that the B-family subunits of PP2A have comprehensive functions as partners of heterotrimeric enzyme complexes of PP2A, both spatially and temporally, in A. nidulans.

The NIMA kinase is required to execute stage-specific mitotic functions after initiation of mitosis.

The G2-M transition in Aspergillus nidulans requires the NIMA kinase, the founding member of the Nek kinase family. Inactivation of NIMA results in a late G2 arrest, while overexpression of NIMA is sufficient to promote mitotic events independently of cell cycle phase. Endogenously tagged NIMA-GFP has dynamic mitotic localizations appearing first at the spindle pole body and then at nuclear pore complexes before transitioning to within nuclei and the mitotic spindle and back at the spindle pole bodies at mitotic exit, suggesting that it functions sequentially at these locations. Since NIMA is indispensable for mitotic entry, it has been difficult to determine the requirement of NIMA for subaspects of mitosis. We show here that when NIMA is partially inactivated, although mitosis can be initiated, a proportion of cells fail to successfully generate two daughter nuclei. We further define the mitotic defects to show that normal NIMA function is required for the formation of a bipolar spindle, nuclear pore complex disassembly, completion of chromatin segregation, and the normal structural rearrangements of the nuclear envelope required to generate two nuclei from one. In the remaining population of cells that enter mitosis with inadequate NIMA, two daughter nuclei are generated in a manner dependent on the spindle assembly checkpoint, indicating highly penetrant defects in mitotic progression without sufficient NIMA activity. This study shows that NIMA is required not only for mitotic entry but also sequentially for successful completion of stage-specific mitotic events.