Filamentous actin destabilization by H 2 O 2 favors DnmA aggregation, with crucial roles of cysteines 450 and 776 in mitochondrial and peroxisomal division in Aspergillus nidulans

Abstract

ABSTRACT 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.