Copper Homeostasis in Aspergillus nidulans Involves Coordinated Transporter Function, Expression and Cellular Dynamics.

Copper ion homeostasis involves a finely tuned and complex multi-level response system. This study expands on various aspects of the system in the model filamentous fungus Aspergillus nidulans. An RNA-seq screen in standard growth and copper toxicity conditions revealed expression changes in key copper response elements, providing an insight into their coordinated functions. The same study allowed for the deeper characterization of the two high-affinity copper transporters: AnCtrA and AnCtrC. In mild copper deficiency conditions, the null mutant of AnctrC resulted in secondary level copper limitation effects, while deletion of AnctrA resulted in primary level copper limitation effects under extreme copper scarcity conditions. Each transporter followed a characteristic expression and cellular localization pattern. Although both proteins partially localized at the plasma membrane, AnCtrC was visible at membranes that resembled the ER, whilst a substantial pool of AnCtrA accumulated in vesicular structures resembling endosomes. Altogether, our results support the view that AnCtrC plays a major role in covering the nutritional copper requirements and AnCtrA acts as a specific transporter for extreme copper deficiency scenarios.

Tolerance to alkaline ambient pH in Aspergillus nidulans depends on the activity of ENA proteins.

Tolerance of microorganisms to abiotic stress is enabled by regulatory mechanisms that coordinate the expression and activity of resistance genes. Alkalinity and high salt concentrations are major environmental physicochemical stresses. Here, we analyzed the roles of sodium-extrusion family (ENA) transporters EnaA, EnaB and EnaC in the response to these stress conditions in the filamentous fungus Aspergillus nidulans. While EnaC has a minor role, EnaB is a key element for tolerance to Na+ and Li+ toxicity. Adaptation to alkaline pH requires the concerted action of EnaB with EnaA. Accordingly, expression of enaA and enaB was induced by Na+, Li+ and pH 8. These expression patterns are altered in a sltAΔ background and completely inhibited in a mutant expressing non-functional PacC protein (palH72). However, a constitutively active PacC form was not sufficient to restore maximum enaA expression. In agreement with their predicted role as membrane ATPases, EnaA localized to the plasma membrane while EnaB accumulated at structures resembling the endoplasmic reticulum. Overall, results suggest different PacC- and SltA-dependent roles for EnaB in pH and salt homeostasis, acting in coordination with EnaA at pH 8 but independently under salt stress.

New insights into copper homeostasis in filamentous fungi.

Copper is a metal ion that is required as a micronutrient for growth and proliferation. However, copper accumulation generates toxicity by multiple mechanisms, potentially leading to cell death. Due to its toxic nature at high concentrations, different chemical variants of copper have been extensively used as antifungal agents in agriculture and medicine. Most studies on copper homeostasis have been carried out in bacteria, yeast, and mammalian organisms. However, knowledge on filamentous fungi is less well documented. This review summarizes the knowledge gathered in the last few years about copper homeostasis in the filamentous fungi Aspergillus fumigatus and Aspergillus nidulans: The mechanism of action of copper, the uptake and detoxification systems, their regulation at the transcriptional level, and the role of copper homeostasis in fungal pathogenicity are presented.

Copper Resistance in Aspergillus nidulans Relies on the PI-Type ATPase CrpA, Regulated by the Transcription Factor AceA.

Copper homeostasis has been extensively studied in mammals, bacteria, and yeast, but it has not been well-documented in filamentous fungi. In this report, we investigated the basis of copper tolerance in the model fungus Aspergillus nidulans. Three genes involved in copper homeostasis have been characterized. First, crpA the A. nidulans ortholog of Candida albicans CaCRP1 gene encoding a PI-type ATPase was identified. The phenotype of crpA deletion led to a severe sensitivity to Cu+2 toxicity and a characteristic morphological growth defect in the presence of high copper concentration. CrpA displayed some promiscuity regarding metal species response. The expression pattern of crpA showed an initial strong elevation of mRNA and a low continuous gene expression in response to long term toxic copper levels. Coinciding with maximum protein expression level, CrpA was localized close to the cellular surface, however protein distribution across diverse organelles suggests a complex regulated trafficking process. Secondly, aceA gene, encoding a transcription factor was identified and deleted, resulting in an even more extreme copper sensitivity than the ΔcrpA mutant. Protein expression assays corroborated that AceA was necessary for metal inducible expression of CrpA, but not CrdA, a putative metallothionein the function of which has yet to be elucidated.

Tip-to-nucleus migration dynamics of the asexual development regulator FlbB in vegetative cells.

In Aspergillus nidulans, asexual differentiation requires the presence of the transcription factor FlbB at the cell tip and apical nuclei. Understanding the relationship between these two pools is crucial for elucidating the biochemical processes mediating conidia production. Tip-to-nucleus communication was demonstrated by photo-convertible FlbB::Dendra2 visualization. Tip localization of FlbB depends on Cys382 in the C-terminus and the bZIP DNA-binding domain in the N-terminus. FlbE, a critical FlbB interactor, binds the bZIP domain. Furthermore, the absence of FlbE results in loss of tip localization but not nuclear accumulation. flbE deletion also abrogates transcriptional activity indicating that FlbB gains transcriptional competence from interactions with FlbE at the tip. Finally, a bipartite nuclear localization signal is required for nuclear localization of FlbB. Those motifs of FlbB may play various roles in the sequence of events necessary for the distribution and activation of this transcriptionally active developmental factor. The tip accumulation, FlbE-dependent activation, transport and nuclear import sketch out a process of relaying an environmentally triggered signal from the tip to the nuclei. As the first known instance of transcription factor-mediated tip-to-nucleus communication in filamentous fungi, this provides a general framework for analyses focused on elucidating the set of molecular mechanisms coupling apical signals to transcriptional events.

Integrative structure-function mapping of the nucleoporin Nup133 suggests a conserved mechanism for membrane anchoring of the nuclear pore complex.

The nuclear pore complex (NPC) is the sole passageway for the transport of macromolecules across the nuclear envelope. Nup133, a major component in the essential Y-shaped Nup84 complex, is a large scaffold protein of the NPC's outer ring structure. Here, we describe an integrative modeling approach that produces atomic models for multiple states of Saccharomyces cerevisiae (Sc) Nup133, based on the crystal structures of the sequence segments and their homologs, including the related Vanderwaltozyma polyspora (Vp) Nup133 residues 55 to 502 (VpNup133(55-502)) determined in this study, small angle X-ray scattering profiles for 18 constructs of ScNup133 and one construct of VpNup133, and 23 negative-stain electron microscopy class averages of ScNup133(2-1157). Using our integrative approach, we then computed a multi-state structural model of the full-length ScNup133 and validated it with mutational studies and 45 chemical cross-links determined via mass spectrometry. Finally, the model of ScNup133 allowed us to annotate a potential ArfGAP1 lipid packing sensor (ALPS) motif in Sc and VpNup133 and discuss its potential significance in the context of the whole NPC; we suggest that ALPS motifs are scattered throughout the NPC's scaffold in all eukaryotes and play a major role in the assembly and membrane anchoring of the NPC in the nuclear envelope. Our results are consistent with a common evolutionary origin of Nup133 with membrane coating complexes (the protocoatomer hypothesis); the presence of the ALPS motifs in coatomer-like nucleoporins suggests an ancestral mechanism for membrane recognition present in early membrane coating complexes.

Cytoplasmic dynamics of the general nuclear import machinery in apically growing syncytial cells.

Karyopherins are transporters involved in the bidirectional, selective and active transport of macromolecules through nuclear pores. Importin-ß1 is the paradigm of karyopherins and, together with its cargo-adapter importin-α, mediates the general nuclear import pathway. Here we show the existence of different cellular pools of both importin-α and -ß1 homologues, KapA and KapB, in the coenocytic ascomycete Aspergillus nidulans. Fluorescence analysis of haploid and diploid strains expressing KapB::GFP and/or KapA::mRFP showed patches of both karyopherins concurrently translocating long distances in apically-growing cells. Anterograde and retrograde movements allowed those patches to reach cell tips and distal regions with an average speed in the range of µm/s. This bidirectional traffic required microtubules as well as kinesin and dynein motors, since it is blocked by benomyl and also by the inactivation of the dynein/dynactin complex through nudA1 or nudK317 mutations. Deletion of Kinesin-3 motor UncA, required for the transport through detyrosinated microtubules, strongly inhibited KapA and KapB movement along hyphae. Overall, this is the first report describing the bidirectional dynamics of the main nuclear import system in coenocytic fungi. A functional link is proposed between two key cellular machines of the filamentous fungal cell: nuclear transport and the tip-growth apparatus.

The Aspergillus nidulans peripheral ER: disorganization by ER stress and persistence during mitosis.

The genetically amenable fungus Aspergillus nidulans is well suited for cell biology studies involving the secretory pathway and its relationship with hyphal tip growth by apical extension. We exploited live-cell epifluorescence microscopy of the ER labeled with the translocon component Sec63, endogenously tagged with GFP, to study the organization of 'secretory' ER domains. The Sec63 A. nidulans ER network includes brightly fluorescent peripheral strands and more faintly labeled nuclear envelopes. In hyphae, the most abundant peripheral ER structures correspond to plasma membrane-associated strands that are polarized, but do not invade the hyphal tip dome, at least in part because the subapical collar of endocytic actin patches constrict the cortical strands in this region. Thus the subapical endocytic ring might provide an attachment for ER strands, thereby ensuring that the growing tip remains 'loaded' with secretory ER. Acute disruption of secretory ER function by reductive stress-mediated induction of the unfolded protein response results in the reversible aggregation of ER strands, cessation of exocytosis and swelling of the hyphal tips. The secretory ER is insensitive to brefeldin A treatment and does not undergo changes during mitosis, in agreement with the reports that apical extension continues at normal rates during this period.

Nuclear transporters in a multinucleated organism: functional and localization analyses in Aspergillus nidulans.

Nuclear transporters mediate bidirectional macromolecule traffic through the nuclear pore complex (NPC), thus participating in vital processes of eukaryotic cells. A systematic functional analysis in Aspergillus nidulans permitted the identification of 4 essential nuclear transport pathways of a hypothetical number of 14. The absence of phenotypes for most deletants indicates redundant roles for these nuclear receptors. Subcellular distribution studies of these carriers show three main distributions: nuclear, nucleocytoplasmic, and in association with the nuclear envelope. These locations are not specific to predicted roles as exportins or importins but indicate that bidirectional transport may occur coordinately in all nuclei of a syncytium. Coinciding with mitotic NPC rearrangements, transporters dynamically modified their localizations, suggesting supplementary roles to nucleocytoplasmic transport specifically during mitosis. Loss of transportin-SR and Mex/TAP from the nuclear envelope indicates absence of RNA transport during the partially open mitosis of Aspergillus, whereas nucleolar accumulation of Kap121 and Kap123 homologues suggests a role in nucleolar disassembly. This work provides new insight into the roles of nuclear transporters and opens an avenue for future studies of the molecular mechanisms of transport among nuclei within a common cytoplasm, using A. nidulans as a model organism.

Analysis of a novel calcium auxotrophy in Aspergillus nidulans.

In Aspergillus nidulans a combination of null mutations in halA, encoding a protein kinase, and sltA, encoding a zinc-finger transcription factor having no yeast homologues, results in an elevated calcium requirement ('calcium auxotrophy') without impairing net calcium uptake. sltA(-) (+/-halA(-)) mutations result in hypertrophy of the vacuolar system. In halA(-)sltA(-) (and sltA(-)) strains, transcript levels for pmcA and pmcB, encoding vacuolar Ca(2+)-ATPase homologues, are highly elevated, suggesting a regulatory relationship between vacuolar membrane area and certain vacuolar membrane ATPase levels. Deletion of both pmcA and pmcB strongly suppresses the 'calcium auxotrophy'. Therefore the 'calcium auxotrophy' possibly results from excessive vacuolar calcium sequestration, causing cytosolic calcium deprivation. Null mutations in nhaA, homologous to Saccharomyces cerevisiae NHA1, encoding a plasma membrane Na(+)/H(+) antiporter effluxing Na(+) and K(+), and a non-null mutation in trkB, homologous to S. cerevisiae TRK1, encoding a plasma membrane high affinity K(+) transporter, also suppress the calcium auxotrophy.

KapI, a non-essential member of the Pse1p/Imp5 karyopherin family, controls colonial and asexual development in Aspergillus nidulans.

Asexual development in the filamentous fungus Aspergillus nidulans is governed by the timely expression and cellular localization of multiple transcription factors. Hence, factors mediating import and export across the nuclear pore complexes (karyopherins) are expected to play a key role in coordinating the developmental programme. Here we characterize KapI, a putative homologue of the Saccharomyces cerevisiae Kap121/Pse1p karyopherin. KapI is a non-essential importin-beta-like protein located in the nucleus during vegetative growth and conidiophore development. The DeltakapI phenotype is aconidial with many aerial hyphae. This phenotype can be suppressed under abiotic stress. In this regard, it resembles that of the null allele of the bZIP transcription factor FlbB. However a DeltaflbB; DeltakapI double mutant exhibited an additive phenotype with totally impaired conidiation, unresponsive to abiotic stress. In contrast to DeltaflbB, the null kapI mutant is not a fluffy-low-bristle expression mutant. Taken together the findings indicate that KapI is required during asexual development, mediating the nuclear transport of factors acting in a different pathway(s) from those involving the upstream developmental activators.