Jesús Aguirre

The Saccharomyces cerevisiae adaptor Opy2 couples cell-surface cues to MAPK cascades, but its roles outside yeasts remain largely unexplored. We characterize OpyA, the Aspergillus nidulans Opy2 ortholog, and identify it as a major regulator of polarized growth and hyphal tip homeostasis. OpyA retains Opy2 domain organization, including the eight extracellular cysteines required for Sho1-branch signaling in yeast. ΔopyA mutants display reduced, irregular growth, hyperbranching, calcofluor white/Congo red sensitivity, and lysis of ~50% of hyphal tips, indicating compromised cell wall integrity and tip stability. Conidiation is strongly reduced, and sexual development arrests at sterile cleistothecial primordia. Signaling is selectively affected: ΔopyA mutants tolerate H₂O₂ and phosphorylate SakA normally in response to it, whereas SakA phosphorylation after osmotic shock is diminished. Additive conidiation defects in double mutants place OpyA parallel to the SakA and SrrA axes. Strikingly, the ΔopyA mutant phenotype is suppressed by KCl or NaCl, but not by sorbitol, and by CaCl₂, but not by MgCl₂, indicating ion-specific rather than purely osmotic rescue. Calcium restores colony growth and branching pattern, improving hyphal tip integrity, conidiation, and the formation of fertile cleistothecia, suggesting that OpyA loss creates a calcium-responsive defect. OpyA::GFP localizes to the plasma membrane with apical enrichment and to cytoplasmic puncta that include CMAC-positive acidic compartments, while non-lysed ΔopyA hyphae display a tubular tip vacuolar network, consistent with altered vesicle trafficking. We propose that OpyA integrates cell-surface surveillance with calcium-dependent signaling to sustain hyphal tip growth, cell wall integrity and developmental competence. Its conservation across fungi makes OpyA a potential antifungal target.

The dynamin-like protein DnmA and its receptor FisA are essential for mitochondrial division in the fungus Aspergillus nidulans . Our research has demonstrated that H ₂ O ₂ triggers significant changes in mitochondrial morphology, including constriction, division, and remodeling of the outer membrane. Here, we used genetics and confocal microscopy approaches to examine the impact of H ₂ O ₂ on the cellular distribution of DnmA. Additionally, we evaluated the roles of conserved cysteines in mitochondrial and peroxisomal division. We show that H ₂ O ₂ induces a DnmA aggregation consistent with higher-order oligomerization and its recruitment to mitochondria. This response is partially attributed to a rapid depolymerization and reorganization of actin caused by H ₂ O ₂ , implying that actin dynamics is also regulated by redox mechanisms. C295S, C380S, and C462S substitutions have minimal or no impact on DnmA function. In contrast, the C450S and C776S substitutions severely impair mitochondrial and peroxisomal division, despite not notably affecting the general distribution of DnmA and having opposite effects on DnmA oligomerization in the absence of FisA. Molecular dynamics simulations show that C450S and C776S substitutions as well as C450 oxidation have important effects on DnmA bundle signaling element-stalk domain angle, solvent-accessible surface area, and salt bridge interactions. The high probability of C450 oxidation and its associated changes in DnmA structure indicate that C450 oxidation by H ₂ O ₂ would impact DnmA multimeric structure. We propose that H ₂ O ₂ regulates mitochondrial division by orchestrating the generation of mitochondrial constrictions and the oligomerization of DnmA. In this model, DnmA C450 oxidation would constitute a necessary priming event for transition to the productive self-assembly required for mitochondrial scission. IMPORTANCE: Mitochondria constitute major sources of H ₂ O ₂ and other reactive oxygen species in eukaryotic cells. The division of these organelles is crucial for multiple processes in cell biology and relies on highly regulated mechano-GTPases that are oligomerization dependent and belong to the dynamin-related protein family, like A. nidulans DnmA. Our previous work demonstrated that H ₂ O ₂ induces mitochondrial constriction, division, and remodeling of the outer membrane. Here, we show that H ₂ O ₂ also induces a DnmA aggregation consistent with higher-order oligomerization and its recruitment to mitochondria. The study of this response uncovered that H ₂ O ₂ induces the depolymerization and reorganization of actin as well as the critical role that cysteines 450 and 776 play in DnmA function. Our results provide new insights into the mechanisms of reactive oxygen species cell signaling and how they can regulate the dynamics of the actin cytoskeleton and the division of mitochondria and peroxisomes.

The dynamin-like protein DnmA and its receptor FisA are essential for H₂O₂-induced mitochondrial division in Aspergillus nidulans. Here, we show that in the absence of DnmA or FisA, mitochondria show few spontaneous transient constrictions, the frequency of which is extensively increased by H₂O₂ or the carbonyl cyanide m-chlorophenyl hydrazone (CCCP). While H₂O₂-induced constrictions are transient, CCCP induces a drastic and irreversible alteration of mitochondrial filaments. H₂O₂ induces a gradual mitochondrial depolarization, while CCCP-induced depolarization is abrupt. The calcium chelator BAPTA-AM prevents the formation of mitochondrial constrictions induced by either H₂O₂ or CCCP. H₂O₂ also induces major rearrangements of the mitochondrial outer membrane, which remain after constrictions dissipate, as well as changes in endoplasmic reticulum (ER) and nuclear morphology. Similar mitochondrial constriction, ER and nuclear morphology changes are detected during the early stages of asexual development. ER and ER-Mitochondria encounter structure (ERMES) complex—composed of proteins Mdm10, Mmm1, Mdm43 and Mdm12—are important for mitochondrial division in Saccharomyces cerevisiae. As the Mdm10 ortholog MdmB was found to be essential in A. nidulans, we evaluated its functions in ΔmdmB terminal mutants and ΔmdmB heterokaryons. ΔmdmB conidia produce a short germ tube that fails to grow further, in which inherited mitochondria become gigantic and round shaped, lacking clear contacts with the ER. In slow-growing ΔmdmB heterokaryotic mycelia, multiple hyphae contain very long mitochondria with high ROS levels, as occur in ΔdnmA and ΔfisA mutants. In this hyphae, H₂O₂ fails to induce mitochondrial constrictions but not outer mitochondrial membrane reshaping, indicating that these are two separate effects of H₂O₂. Our results indicate that H₂O₂ induces a generalized mitochondrial constriction response, prior to actual division, involving gradual depolarization; they also indicate that Ca²⁺ and the ERMES complex are critical for both mitochondrial constriction and division. This supports a view of mitochondrial dynamics as the result of a cascade of signaling events that can be initiated in vivo by H₂O₂.

Reactive oxygen species (ROS) regulate several aspects of cell physiology in filamentous fungi including the antioxidant response and development. However, little is known about the signaling pathways involved in these processes. Here, we report Aspergillus nidulans global phosphoproteome during mycelial growth and show that under these conditions, H₂O₂ induces major changes in protein phosphorylation. Among the 1964 phosphoproteins we identified, H₂O₂ induced the phosphorylation of 131 proteins at one or more sites as well as the dephosphorylation of a larger set of proteins. A detailed analysis of these phosphoproteins shows that H₂O₂ affected the phosphorylation of critical regulatory nodes of phosphoinositide, MAPK, and TOR signaling as well as the phosphorylation of multiple proteins involved in the regulation of gene expression, primary and secondary metabolism, and development. Our results provide a novel and extensive protein phosphorylation landscape in A. nidulans, indicating that H₂O₂ induces a shift in general metabolism from anabolic to catabolic, and the activation of multiple stress survival pathways. Our results expand the significance of H₂O₂ in eukaryotic cell signaling.

Aspergillus sydowii is a moderate halophile fungus extensively studied for its biotechnological potential and halophile responses, which has also been reported as a coral reef pathogen. In a recent publication, the transcriptomic analysis of this fungus, when growing on wheat straw, showed that genes related to cell wall modification and cation transporters were upregulated under hypersaline conditions but not under 0.5 M NaCl, the optimal salinity for growth in this strain. This led us to study osmolyte accumulation as a mechanism to withstand moderate salinity. In this work, we show that A. sydowii accumulates trehalose, arabitol, mannitol, and glycerol with different temporal dynamics, which depend on whether the fungus is exposed to hypo- or hyperosmotic stress. The transcripts coding for enzymes responsible for polyalcohol synthesis were regulated in a stress-dependent manner. Interestingly, A. sydowii contains three homologs (Hog1, Hog2 and MpkC) of the Hog1 MAPK, the master regulator of hyperosmotic stress response in S. cerevisiae and other fungi. We show a differential regulation of these MAPKs under different salinity conditions, including sustained basal Hog1/Hog2 phosphorylation levels in the absence of NaCl or in the presence of 2.0 M NaCl, in contrast to what is observed in S. cerevisiae. These findings indicate that halophilic fungi such as A. sydowii utilize different osmoadaptation mechanisms to hypersaline conditions.

The dynamin-like protein Drp1 and its receptor Fis-1 are required for mitochondria and peroxisome fission in animal and yeast cells. Here, we show that in the fungus Aspergillus nidulans the lack of Drp1 and Fis-1 homologs DnmA and FisA has strong developmental defects, leading to a notable decrease in hyphal growth and asexual and sexual sporulation, with some of these defects being aggravated or partially remediated by different carbon sources. Although both DnmA and FisA, are essential for mitochondrial fission, participate in peroxisomal division and are fully required for H₂O₂-induced mitochondrial division, they also appear to play differential functions. Despite their lack of mitochondrial division, ΔdnmA and ΔfisA mutants segregate mitochondria to conidiogenic cells and produce viable conidia that inherit a single mitochondrion. During sexual differentiation, ΔdnmA and ΔfisA mutants develop fruiting bodies (cleistothecia) that differentiate excessive ascogenous tissue and a reduced number of viable ascospores. ΔdnmA and ΔfisA mutants show decreased respiration and notably high levels of mitochondrial reactive oxygen species (ROS), which likely correspond to superoxide. Regardless of this, ΔdnmA mutants can respond to an external H₂O₂ challenge by re-localizing the MAP kinase-activated protein kinase (MAPKAP) SrkA from the cytoplasm to the nuclei. Our results show that ROS levels regulate mitochondrial dynamics while a lack of mitochondrial fission results in lower respiration, increased mitochondrial ROS and developmental defects, indicating that ROS, mitochondrial division and development are critically interrelated processes.