A structure-selective endonuclease drives uniparental mitochondrial DNA inheritance

Maternal inheritance of mitochondrial DNA (mtDNA) is a near-universal feature of eukaryotes[1][1], yet the mechanisms that ensure this by preventing paternal mtDNA inheritance have remained unclear. In both Drosophila and humans, mtDNA is actively eliminated from sperm during spermatogenesis, producing mature sperm whose mitochondria lack their genomes[2][2]–[5][3]. Here we identify Hotaru, a previously uncharacterized, testis-specific GIY-YIG endonuclease, as a central player in this process. We find that Hotaru is expressed in elongated spermatids, localizes to the mitochondrial matrix, and is required for paternal mtDNA elimination. In hotaru mutants, sperm retain mtDNA at levels comparable to those present before the elimination process. Genetic and biochemical analyses show that Hotaru selectively recognizes and cleaves cruciform DNA structures within the mtDNA control region. Together, these findings identify a dedicated nuclease that enforces mitochondrial genome elimination in the animal male germline and reveal that an unexpected structural feature of mtDNA serves as the molecular determinant of its destruction. By recognizing DNA structure rather than specific sequence motifs, this mechanism is inherently robust to the high mutation rate of mitochondrial genomes. ### Competing Interest Statement The authors have declared no competing interest. Canadian Institutes of Health Research, https://ror.org/01gavpb45, FRN #542273 (T.R.H), FRN #487044 (H.D.M.W) Canada Research Chairs, #CRC-2021-00346 (H.D.M.W) National Human Genome Research Institute, 5R01HG009190 (J.T.S) Heiwa Nakajima Foundation Scholarship (M.S.) University of Toronto Mito2i (M.S.) NSERC Canada Graduate Scholarship (H.Y.Y.) [1]: #ref-1 [2]: #ref-2 [3]: #ref-5

Age-dependent H3K9 trimethylation by dSetdb1 impairs mitochondrial UPR leading to degeneration of olfactory neurons and loss of olfactory function in Drosophila

Mitochondrial metabolic remodeling drives innate immune activation in Drosophila hemocytes

Innate immune cells rapidly reprogram their metabolism upon activation, yet the metabolic basis of this flexibility in invertebrate systems remains largely unexplored. Here, we investigate the metabolic landscape of Drosophila larval hemocytes, functional analogs of vertebrate myeloid cells, across developmental stages, genotypes, and immune activation states, by combining metabolic flux measurements with single-cell transcriptomics. Under homeostatic conditions, hemocytes rely predominantly on mitochondrial oxidative phosphorylation for ATP production, with minimal glycolytic contribution. Immune activation, particularly lamellocyte differentiation, drives enhanced mitochondrial respiration and metabolic flexibility, accompanied by structural remodeling of the mitochondrial network. Mechanistically, functional lamellocytes require Drp1-mediated mitochondrial fission and utilize glucose and trehalose as primary carbon sources to sustain mitochondrial respiration, which is essential for effective immune responses. Overall, these findings establish that mitochondrial metabolic reprogramming is a conserved feature of innate immune activation in myeloid-like immune cells and reveal an evolutionarily ancient link between mitochondrial dynamics and immune cell activation, with implications for understanding metabolic regulation of innate immunity in invertebrate models and beyond. ### Competing Interest Statement The authors have declared no competing interest.

Developmental regulation of progenitor aging shapes long-term intestinal homeostasis in Drosophila

Aging causes a progressive loss of tissue homeostasis, with stem cell exhaustion as a major hallmark. Age-associated decline in organ function is widely perceived as emanating from progressive accumulation of cellular damage in adult tissues. However, whether aging trajectories are established early on during development remains an open question. Here, we demonstrate that genetic modulation of cellular aging pathways in larval adult midgut progenitors (AMPs), which serve as the precursors of adult intestinal stem cells and differentiated epithelial cells, dictates the long-term trajectory of intestinal aging in Drosophila. Accelerated cellular aging by genetic perturbation employing Toll or Imd pathway overactivation or elevation of reactive oxygen species (ROS) using ND42 (mitochondrial complex I) knockdown in the AMPs results in aberrant progenitor proliferation, skewed lineage allocation, epithelial barrier dysfunction, and genomic instability. These alterations are accompanied by marked destabilization of AMP islet architecture and widespread changes in age-related molecular signatures, as revealed by bulk transcriptomic analysis. In contrast, decelerated cellular aging mediated by Foxo or Atg8a overexpression results in a decrease in enteroendocrine population and the intestinal barrier remained unaffected. Intriguingly, early-life activation of immune and oxidative stress signaling manifested later in the adult gut as elevated enteroendocrine differentiation, highlighting lasting effects on intestinal regenerative capacity and lineage balance. Together, our findings demonstrate that cellular aging is tightly regulated early on in development and its perturbation can cause developmental disruption hampering adult gut homeostasis, establishing AMPs as key developmental determinants that regulate the trajectory of intestinal aging in Drosophila. ### Competing Interest Statement The authors have declared no competing interest. Department of Atomic Energy, https://ror.org/02m388s04, Basic and Translational Research in Cancer grant (no.1/3(7)/2020/TMC/R&D-II/8823 Dt.30.07.2021), Capacity Building and Development of Novel and Cutting-edge Research Activities (no.1/3(4)/2021/TMC/R&D-II/15063 Dt.15.12.2021) Department of Biotechnology, https://ror.org/03tjsyq23, Har Gobind Khorana – Innovative Young Biotechnologist Award (no. BT/13/IYBA/2020/14) to R.J.K., Ramalingaswami Re-entry Fellowship from the Department of Biotechnology, Ministry of Science and Technology, India (BT/RLF/Re-entry/19/2020) to R.J.K. Advanced Centre for Treatment, Research and Education in Cancer, https://ror.org/05b9pgt88, Senior Research Fellowship to A.A.M., Postdoctoral Fellowship to S.M. CEFIPRA-CSRP, Project no. 6704-5 to M.S.

An ARVC-5 Drosophila knock-in model reveals new functions of Tmem43 in lipid homeostasis

Arrhythmogenic right ventricular cardiomyopathy type 5 is caused by the missense mutation S358L in the gene TMEM43 in humans. To date, the molecular mechanisms underlying the disease remain poorly understood. We established a CRISPR/Cas9 knock-in Drosophila model carrying the orthologous Tmem43p.S333L mutation to investigate these mechanisms in vivo. The resulting flies were viable but displayed reduced lifespan, smaller body size, lipid droplet accumulation, and mitochondrial defects. Proteomic and lipidomic profiling revealed a dosage-dependent misregulation of the energy metabolism, concomitant with reduced fatty acid synthesis and ß-oxidation rates, altered peroxisomal pathways, and changes in membrane phospholipid composition. Notably, phosphatidylethanolamine (PE) and phosphatidylinositol (PI) levels were elevated, while triacylglycerols were reduced. Ultrastructural analyses confirmed mitochondrial degradation in the muscle tissue of corresponding mutants. These findings establish Tmem43p.S333L knock-in flies as a robust in vivo model of ARVC-5, and support a role for TMEM43 in linking lipid homeostasis to mitochondrial energy metabolism and integrity. Mutation-derived impairments in these processes result in cardiomyopathy.

Superoxide dismutases maintain niche homeostasis in stem cell populations

Superoxide dismutases, particularly Sod1, differentially tune redox signaling of germline and cyst stem cells, enabling their self-renewal and differentiation, thereby maintaining Drosophila testicular stem cell homeostasis.