Chromatin must remain competent for responding rapidly to stress signals. Polycomb repressive complex 1 (PRC1) contributes to chromatin compaction through mono-ubiquitylation of histone H2A at Lys119 (H2AK119ub), yet how...Chromatin must remain competent for responding rapidly to stress signals. Polycomb repressive complex 1 (PRC1) contributes to chromatin compaction through mono-ubiquitylation of histone H2A at Lys119 (H2AK119ub), yet how PRC1 complexes are dissociated from chromatin is only incompletely understood. Here, we show that the protease CAPN3 promotes rapid dissociation of PRC1 complexes from chromatin in response to stress. Following liver injury or heat shock, CAPN3 becomes activated and proteolyzes non-core PRC1 subunits to release the complexes from chromatin, leading to a reduction of H2AK119ub levels. These findings demonstrate that CAPN3 facilitates the remodeling of the chromatin landscape, unveiling a protease-mediated epigenetic mechanism for chromatin remodeling, and reveal CAPN3 as a key regulator of stress-induced epigenetic responses.
Inherited mutations in the spindle pole-associated scaffold protein WDR62 cause autosomal recessive primary microcephaly. Previous research has characterised the roles of WDR62 in the regulation of spindle dynamics, cell...Inherited mutations in the spindle pole-associated scaffold protein WDR62 cause autosomal recessive primary microcephaly. Previous research has characterised the roles of WDR62 in the regulation of spindle dynamics, cell division, and brain development. Here, we identify a new function of this protein in regulating purine metabolism. WDR62 interacts directly with BAG2, a co-chaperone of HSP70/90. Under stress conditions, WDR62 and BAG2 re-localise to cytoplasmic granules enriched for enzymes involved in purine synthesis (PFAS) and salvage (HPRT). In WDR62-deficient cells, purine synthesis is impaired, while purine deprivation leads to cytotoxicity and nucleoside accumulation. Furthermore, in these cells elevated BAG2 levels are linked to HPRT destabilisation, which can be reversed by BAG2 knockdown. Notably, microcephaly-associated WDR62 mutations disrupt interaction with BAG2 and fail to restore HPRT levels. In utero depletion of WDR62 or HPRT in the mouse neocortex causes premature delamination and migration of neural precursor cells. Interestingly, HPRT loss enhances self-renewal and proliferation of these precursors, contrasting with the reduced proliferation and precocious differentiation observed upon WDR62 loss. Our study identifies regulatory functions of WDR62 in purine metabolism that may contribute to primary microcephaly.
The discovery of Asgard archaea about a decade ago has greatly reshaped our understanding of archaeal evolution and the origin of eukaryotes. Asgards are currently thought to be the closest prokaryotic relatives of eukar...The discovery of Asgard archaea about a decade ago has greatly reshaped our understanding of archaeal evolution and the origin of eukaryotes. Asgards are currently thought to be the closest prokaryotic relatives of eukaryotes and to represent the archaeal host lineage that participated in the endosymbiotic event leading to the first eukaryotic cell. The presence of numerous eukaryotic signature proteins in Asgard genomes supports this view and provides important insights into the deep evolutionary roots of eukaryotic cellular complexity. However, the close relationship between archaea and eukaryotes had been observed for decades, based on features that are shared in different molecular processes. This review discusses the discovery of Asgard archaea in the broader context of archaeal molecular and cellular biology and highlights how earlier findings foreshadowed their emergence. Primarily targeted at newcomers to the field, the review provides an overview of evolutionary innovations across the Archaea domain and discusses molecular and cellular features of cultivated Asgard strains in light of previous archaeal research.
Nussey DH, d'Adda di Fagagna F, Bardin AJ
… +18 more, Blau HM, Brunet A, Bulavin DV, Guo L, Hara E, Junker JP, Gorbunova V, Mittelbrunn M, Rera M, Reznick J, Seluanov A, Schumacher B, Teeling EC, Valenzano DR, Ye J, Yun MH, Garinis GA, Gilson E
Comparative biology plays a crucial role in uncovering fundamental biological mechanisms and providing evolutionary models for their variation. This approach is particularly valuable for studying aging, given the remarka...Comparative biology plays a crucial role in uncovering fundamental biological mechanisms and providing evolutionary models for their variation. This approach is particularly valuable for studying aging, given the remarkable diversity in aging trajectories across the tree of life. Many evolutionary theories of aging were proposed well before the discovery of the molecular mechanisms involved, and they remain largely theoretical. Moreover, the growing number of model organisms and the expanding array of experimental and theoretical approaches used to study aging have often remained compartmentalized. As a result, integrating these diverse insights into a unified framework has become increasingly important. As a step toward this goal, this field perspective outlines general biological mechanisms that help explain the variability in aging patterns and longevity across the animal kingdom.
Membrane contact sites (MCS) are dynamic regions where the membranes of two organelles come into close apposition. MCSs play many roles in cellular homeostasis by facilitating inter-organelle lipid exchange and organelle...Membrane contact sites (MCS) are dynamic regions where the membranes of two organelles come into close apposition. MCSs play many roles in cellular homeostasis by facilitating inter-organelle lipid exchange and organelle positioning. The late endosome/lysosome (LE/Lys) cholesterol transfer protein STARD3 forms reversible contacts between LE/Lys and the endoplasmic reticulum (ER). This tether protein contains a Phospho-FFAT motif (two phenylalanines in an acidic tract) whose interaction with ER-resident VAPs (vesicle-associated membrane protein-associated proteins) is phosphorylation-dependent. In this study, we identify GSK3α and GSK3β as the kinases responsible for phosphorylating serine 209 within the Phospho-FFAT motif of STARD3. This phosphorylation event is both necessary and sufficient to activate STARD3's tethering activity, thereby promoting ER-LE/Lys contacts. Furthermore, we show that when ER-LE/Lys tethering is prevented, STARD3 triggers LE/Lys homotypic interactions, revealing an additional function for STARD3 on endosome biology. Our findings establish a direct and critical role for GSK3 in regulating MCS via STARD3 phosphorylation, and expand our understanding of the molecular basis of inter-organelle communication.
Two-Pore Domain K+ (K2P) channels are crucial determinants of the resting membrane potential and of cellular electrical excitability in many different cell types. TASK-1 and TASK-3 K2P channel activity is also coupled to...Two-Pore Domain K+ (K2P) channels are crucial determinants of the resting membrane potential and of cellular electrical excitability in many different cell types. TASK-1 and TASK-3 K2P channel activity is also coupled to GPCR signalling pathways via Gαq and their subsequent inhibition is via direct interaction with diacylglycerol (DAG) generated from phosphatidylinositol-4,5-bisphosphate (PIP) hydrolysis. This regulation is defective in two different neurodevelopmental disorders, but the molecular mechanisms underlying this inhibitory process and the reasons for the GPCR-insensitivity of these disease-causing mutations remain unclear. Here we show that GqPCR inhibition inversely correlates with channel open probability, and results from a state-dependent destabilisation of the open state by DAG promoting channel closure. We also identify a DAG interaction-site within a groove between the M2, M3 and M4 domains, and show the crucial role of residues within this site in mediating the inhibitory effect and defining channel sensitivity. These results not only reveal the structural and molecular mechanisms underlying GqPCR regulation of TASK channels, but also explain the pathogenic effect of a common regulatory defect linked to different K2P channelopathies.
During blood feeding, Ixodidae ticks secrete cement proteins, including glycine-rich proteins (GRPs), that facilitate attachment to the vertebrate host. However, the molecular mechanisms underlying exosomal GRP secretion...During blood feeding, Ixodidae ticks secrete cement proteins, including glycine-rich proteins (GRPs), that facilitate attachment to the vertebrate host. However, the molecular mechanisms underlying exosomal GRP secretion at the feeding site and their roles in tick-pathogen interactions remain poorly understood. Here, we analyzed the Ixodes scapularis genome to identify salivary exosomal components involved in modulation of the tick-host skin interface. We identify an arthropod exosomal GRP ( XM_002400035 ) that promotes transmission of Langat virus (LGTV), a tick-borne flavivirus, from ticks to vertebrate hosts. XM_002400035 was consistently upregulated in LGTV-infected I. scapularis ticks, tick-derived cells, and in tick exosomes. RNAi-mediated silencing of this exosomal GRP reduced viral loads, impaired tick blood-feeding efficiency, decreased tick body size and weights, and diminished LGTV acquisition and transmission. Similarly, active immunization of mice with recombinant GRP disrupted tick feeding, reduced tick fitness, and significantly impaired LGTV transmission from infected ticks to naive recipient hosts. Mechanistically, the exosomal GRP modulated host skin chemokine CXCL-12 levels at the feeding site. Together, these findings establish a dual role for a tick exosomal GRP in blood feeding and pathogen transmission and identify this tick exosomal GRP as a potential target for exosome-based transmission-blocking vaccines. More broadly, this work highlights arthropod exosomes as active mediators of flavivirus transmission and suggests new strategies for preventing and controlling tick-borne diseases.
Weiden EM, Serianz Z, Klingl Y
… +27 more, Jörs S, Jaślan D, Keller M, Castro SP, Mkhitaryan M, Jeridi A, Briukhovetska D, Spix B, Scotto Rosato A, Agami A, Schiller HB, Rajan S, Schredelseker J, Fois G, Frick M, Kobold S, Klein M, Geisler F, Garcia-Fortanet J, Murphy LO, Bracher F, Wahl-Schott C, Gudermann T, Dietrich A, Biel M, Yildirim AÖ, Grimm C
In pulmonary fibrosis lung tissue is thickened and scarred, and the lungs become progressively stiffer and smaller, leading to low levels of blood oxygen and shortness of breath. Lung fibrosis is not curable and life exp...In pulmonary fibrosis lung tissue is thickened and scarred, and the lungs become progressively stiffer and smaller, leading to low levels of blood oxygen and shortness of breath. Lung fibrosis is not curable and life expectancy is reduced. Fibrosis is characterized by an increased accumulation of extracellular matrix (ECM) proteins such as collagen and elastin. ECM proteins are degraded predominantly by matrix metalloproteinases (MMPs). Here, we show that the lysosomal cation channel TRPML1, which causes the lysosomal storage disorder mucolipidosis type IV (MLIV) when mutated or lost, regulates the levels of MMPs in the ECM of mouse airways, modulating exocytosis of MMP2, 8, 9, 12, and 19, which mediate collagen/elastin degradation. While TRPML1 loss reduces MMP levels in lung macrophage and fibroblast supernatants, small molecule activation of TRPML1 results in increased levels. MLIV mice display a fibrosis-like lung phenotype similar to the phenotype evoked by bleomycin. We thus identify TRPML1 as a regulator of MMP release in the lung with loss of TRPML1 resulting in lung fibrosis due to excessive extracellular collagen and elastin accumulation.
Moran Y, Coelho SM, Ettema TJG
… +11 more, Feschotte C, Kaltenpoth M, Khila A, Laine AL, Liow LH, Petrov D, Ramakrishnan U, Sarkies P, Srivastava M, Voolstra C, Pulverer B
Molecular ecology and evolution are central to understanding how biological systems function, interact, and diversify. A special issue of this journal reflects the growing synergy of molecular, genomic and cell biology w...Molecular ecology and evolution are central to understanding how biological systems function, interact, and diversify. A special issue of this journal reflects the growing synergy of molecular, genomic and cell biology with ecological and evolutionary reasoning. Accordingly, EMBO Press is recalibrating its editorial practices to better support studies embedded in ecological and evolutionary contexts.
The eukaryotic translation initiation factor 3 (eIF3) is the largest and most complex initiation factor in eukaryotes, functioning as a central hub that integrates signals from cellular stress, metabolism, and developmen...The eukaryotic translation initiation factor 3 (eIF3) is the largest and most complex initiation factor in eukaryotes, functioning as a central hub that integrates signals from cellular stress, metabolism, and developmental pathways to regulate mRNA translation. Recent advances have uncovered subunit-specific roles of eIF3 that extend beyond canonical cap-dependent translation to include specialized mechanisms such as selective mRNA recruitment, noncanonical cap recognition, and translation elongation. This review summarizes the current mechanistic understanding of the contribution of aberrant eIF3 activity to diverse disease processes, including oncogenesis, neurodevelopmental and neurodegenerative disorders, muscle pathology, and infectious disease. We evaluate therapeutic strategies aimed at modulating eIF3 function, including subunit-selective small molecules, RNA-based therapeutics, and CRISPR-based interventions. We discuss the therapeutic promise of both inhibitory approaches-targeting oncogenic or pathogen-hijacked eIF3-and restorative strategies to correct genetic loss-of-function in neurological disease. Finally, we outline key challenges and opportunities for clinical translation, including tissue-specific delivery, subunit selectivity, and the identification of predictive biomarkers. eIF3 emerges as a versatile and druggable node in translational control with broad relevance across human disease.
The outer membrane of Gram-negative bacteria is an asymmetric bilayer in which lipopolysaccharide (LPS), the principal component of the outer leaflet, promotes tight packing and ordering of the membrane components that a...The outer membrane of Gram-negative bacteria is an asymmetric bilayer in which lipopolysaccharide (LPS), the principal component of the outer leaflet, promotes tight packing and ordering of the membrane components that are essential for the barrier and load-bearing functions of this membrane. Lipopolysaccharide mobility is known to be restricted in the outer membrane, but this confinement and the underlying biophysical interactions responsible remain to be fully characterized. Here, we apply a bio-orthogonal strategy for in situ site-specific fluorescent labeling of LPS. Using fluorescence microscopy, we quantify LPS lateral confinement in the outer membrane of Escherichia coli and demonstrate that this confinement is independent of oligosaccharide domain structure. We show that lipopolysaccharide assembles into discrete supramolecular structures, and that restricted lateral mobility arises from a combination of divalent cation-mediated electrostatic interactions in the anionic Lipid A headgroup, and hydrophobic interactions between acyl chains within the lipid milieu. Magnesium cations exert a greater influence than calcium cations on lipopolysaccharide lateral mobility. These traits are conserved across multiple pathogenic bacterial species, irrespective of O-antigen serotype, showing that lipopolysaccharide confinement is a ubiquitous feature of Gram-negative bacteria.
Endosomes are protein sorting stations, where multiple membrane coats form tubulovesicular carriers exporting proteins to the Golgi, the plasma membrane, or endo-lysosomal compartments. Distinct classes of sorting nexins...Endosomes are protein sorting stations, where multiple membrane coats form tubulovesicular carriers exporting proteins to the Golgi, the plasma membrane, or endo-lysosomal compartments. Distinct classes of sorting nexins are assumed to form distinct homogeneous coats that define the endosomal sorting routes and their cargos. Snx3 and the SNX-BAR proteins Vps5-Vps17 belong to different sorting-nexin classes. They can form homogeneous retromer-dependent coats that differ in structure and in their modes of membrane association and cargo recognition. Here, we describe the formation of hybrid coats between purified SNX-BARs, Snx3, and their cargos. Hybrid coats assemble at variable subunit ratios and diameters and show greater membrane-scaffolding activity than homogeneous coats. In vivo, Snx3 and SNX-BARs co-localise and mutually impact the sorting of their respective cargos. Although simultaneous binding of Snx3- and SNX-BARs to Retromer is sterically prohibited, hybrid coats incorporate both SNXs in a common complex, probably linked by retromer oligomerisation. We hence propose that SNX-BARs and Snx3 form retromer-mediated hybrid coats in novel, stoichiometrically adaptable configurations that allow the adjustment of endosomal carriers for transporting varying ratios of cargo.
Brl1 and Brr6 are integral membrane proteins of the yeast nuclear envelope (NE) that transiently associate with nuclear pore complexes (NPCs). The exact roles of Brl1 and Brr6 during NPC assembly are unclear. Here, we de...Brl1 and Brr6 are integral membrane proteins of the yeast nuclear envelope (NE) that transiently associate with nuclear pore complexes (NPCs). The exact roles of Brl1 and Brr6 during NPC assembly are unclear. Here, we demonstrate that Brr6 operates at both early and late stages of NPC assembly. Its early function is supported by amphipathic α-helix mutants, which impact nucleoporin recruitment without nuclear envelope deformation, whereas mutations in conserved cysteine residues result in NE deformation accompanied by defective NE fusion. The N-terminus of Brl1 interacts with the nucleoporin Nic96, promoting Nic96 recruitment to early assembly sites. AlphaFold predictions, the essential role of the conserved PAL motif, and the inhibition of NE fusion upon overexpression of PAL mutants together suggest that the perinuclear domains of Brl1 and Brr6 interact across the perinuclear space. Extending the length of the perinuclear-space regions of Brl1 and Brr6 causes uncontrolled NE fusion, as indicated by nuclear envelope disintegration dependent on the conserved cysteine residues. Together, Brl1 and Brr6 promote NE fusion by bridging the perinuclear space through PAL motif interactions, followed by nuclear envelope fusion and NPC insertion.
Bacterial pathogens must withstand metal-induced stress during infection, yet the mechanisms by which they sense and respond to toxic metal ions remain incompletely understood. Here, we uncover a previously unrecognized...Bacterial pathogens must withstand metal-induced stress during infection, yet the mechanisms by which they sense and respond to toxic metal ions remain incompletely understood. Here, we uncover a previously unrecognized mechanism in Mycobacterium tuberculosis, the causative agent of tuberculosis, which assembles dynamic, membrane-associated platforms organized by PacL proteins to mediate resistance to multiple metals. The small membrane-associated proteins PacL1, PacL2, and PacL3 coordinate the clustering of P-type ATPase pumps, namely CtpC, CtpG, and CtpV, into functional complexes that we term effluxosomes. Using single-particle tracking, we reveal distinct dynamic populations, with highly mobile PacL proteins integrating into more slowly mobile effluxosomes. PacL proteins stabilize CtpC and CtpG within these assemblies, promoting cross-resistance to zinc and cadmium, with PacL1 acting as a multi-substrate metallochaperone that binds zinc, cadmium, and copper via a conserved C-terminal motif. Single-molecule-based super-resolution microscopy shows that conserved residues within the PacL transmembrane domain are essential for effluxosome assembly. Strikingly, proximity labeling reveals a broad PacL1 interaction network, suggesting that effluxosomes contribute to a wider stress adaptation program. These findings establish effluxosomes as dynamic membrane machineries that orchestrate coordinated multi-metal resistance in M. tuberculosis, opening new avenues for antimicrobial targeting.
Ubiquitin-binding shuttle proteins are important components of stress-induced biomolecular condensates in cells. Yeast Dsk2 scaffolds proteasome-containing condensates via multivalent interactions with proteasomes and po...Ubiquitin-binding shuttle proteins are important components of stress-induced biomolecular condensates in cells. Yeast Dsk2 scaffolds proteasome-containing condensates via multivalent interactions with proteasomes and polyubiquitinated substrates under stress conditions. Here, we identify the chaperone-binding STI1 domain as the main driver of Dsk2 self-association and phase separation. Using nuclear magnetic resonance (NMR) spectroscopy and computational simulations, we find that the STI1 domain interacts with three transient amphipathic helices within the intrinsically disordered regions of Dsk2. Removal of either the STI1 domain or these helices significantly reduces Dsk2's propensity to form condensates. In vivo, perturbing STI1-helix interactions, specifically removal of the transient helices, reduces the formation of azide stress-induced Dsk2/proteasome condensates, in line with our in vitro results. Modeling of Dsk2 STI1-helix interactions reveals a binding mode reminiscent of chaperone STI1/DP2 domains interacting with client helices. Our findings support a model whereby STI1-helix interactions important for Dsk2 condensate formation can be replaced by STI1-client interactions for downstream chaperone or other protein quality control outcomes.
STIM1 is a transmembrane protein localized in the endoplasmic reticulum (ER), where it acts as a calcium ion sensor, activating store-operated Ca entry upon ER Ca depletion. Via cellular calcium influx, STIM1 is thought...STIM1 is a transmembrane protein localized in the endoplasmic reticulum (ER), where it acts as a calcium ion sensor, activating store-operated Ca entry upon ER Ca depletion. Via cellular calcium influx, STIM1 is thought to indirectly affect mitochondrial calcium content. Here we show that STIM1 also interacts with mitochondrial proteins such as PTPIP51 and GRP75, suggesting its presence in mitochondria-associated ER membranes (MAMs), which are specialized ER regions that facilitate ER-mitochondria communication. Lowering STIM1 expression disrupts ER-to-mitochondria Ca transfer, reduces basal mitochondrial Ca levels, impairs maximal mitochondrial respiration, and reduces ATP production. The STIM1-GRP75 interaction depends on STIM1's Ca-sensing ability. ER Ca depletion or the constitutive-open R429C mutation both reduce STIM1 binding to GRP75, suggesting that conformational changes in STIM1 play a role in this interaction. Deletion analysis revealed that the STIM1 (551-611) segment is crucial for GRP75 binding, as the peptide STIM1(551-611) binds GRP75, while STIM1(Δ551-611) shows reduced binding. These findings reveal a previously unrecognized role of STIM1 in direct inter-organelle communication.
Chromatin organization, through the assembly of DNA with histones and the folding of nucleosome chains, regulates DNA accessibility for transcription, DNA replication and repair. Although models derived from in vitro stu...Chromatin organization, through the assembly of DNA with histones and the folding of nucleosome chains, regulates DNA accessibility for transcription, DNA replication and repair. Although models derived from in vitro studies have proposed distinct nucleosome chain geometries, the organization of chromatin within the crowded cell nucleus remains elusive. Using cryo-electron tomography of thin vitreous sections, we directly observed the path of nucleosomal and linker DNA in situ from a flash-frozen organism - Drosophila embryos. We quantified linker length and curvature, characterizing an irregular zig-zag chromatin-folding motif, with a low degree of linker bending. Nucleosome conformations could be identified on individual particles in favorable orientations without structure averaging. Additionally, we observed particles that accommodate a number of DNA gyres ranging from less than one to up to three, which resemble previously proposed non-octameric nucleosomal particles with variable DNA wrapping.
Fu HH, Wang MC, Wang ZQ
… +14 more, Sang YH, Li ZK, Li FF, Liu JR, Qin QL, Zhu XY, Wang N, Wan JJ, Teng ZJ, Zhang WP, Gates AJ, Li CY, Todd JD, Zhang YZ
Dimethylsulfoniopropionate (DMSP) catabolism by marine Roseobacters is important for global biogeochemical cycling and the climate. Many Roseobacters contain competing DMSP demethylation and cleavage pathways, but only c...Dimethylsulfoniopropionate (DMSP) catabolism by marine Roseobacters is important for global biogeochemical cycling and the climate. Many Roseobacters contain competing DMSP demethylation and cleavage pathways, but only cleavage produces the climate-cooling gas dimethylsulfide. Here, we identify the "switch" regulator in Roseobacters, DmdR, which transcriptionally represses demethylation (dmdA, encoding DMSP demethylase), cleavage (acuI, encoding acryloyl-CoA reductase) and oxidative stress protection (dmdEF, dinB) genes under low intracellular DMSP levels. Increased DMSP levels lead to DMSP cleavage and accumulation of cytotoxic cleavage product acryloyl-CoA. Acryloyl-CoA binding to DmdR derepresses dmdA-acuI transcription to stimulate acryloyl-CoA catabolism and DMSP demethylation. Upregulation of the newly identified peroxidase DmdF, and possibly also of DmdE and DinB, counteracts oxidative stress associated with DMSP demethylation. Thus, DmdR, along with DmdR-independent regulators of DMSP cleavage, likely maintains cellular DMSP levels to allow its antistress functions, but accelerates demethylation and catabolism of toxic intermediates at higher DMSP levels. Of note, DmdR appears to control acryloyl-CoA catabolism/detoxification even in abundant marine bacteria lacking dmdA, suggesting additional mechanisms. DmdR and DmdEF are widespread in Earth's oceans and important for biogeochemical cycling and climate-active gas production.
Gene drives are engineered alleles that bias their own inheritance in offspring, enabling the spread of specific traits throughout a population. Targeting female fertility genes in a gene drive can be an efficient strate...Gene drives are engineered alleles that bias their own inheritance in offspring, enabling the spread of specific traits throughout a population. Targeting female fertility genes in a gene drive can be an efficient strategy for population suppression. In this study, we investigated nine female fertility genes in Drosophila melanogaster using CRISPR-based homing gene drives. Employing a multiplexed gRNA approach to prevent the formation of functional resistance alleles, we aimed to maintain high drive-conversion efficiency with low fitness costs in female drive-carriers. Drive efficiency was assessed in individual crosses and had varied performance across different target genes. Notably, drives targeting the octopamine β2 receptor (oct) and stall (stl) genes exhibited the highest drive-conversion rates and were further tested in cages. A drive targeting stl successfully suppressed a cage population with a high release frequency, though suppression failed in another replicate cage with a lower initial release frequency. Fitness costs in female drive carriers were observed in test cages, impacting the overall efficiency of population suppression. Further tests on the fertility of these lines using individual crosses indicated that some fitness costs were due to maternal deposition of Cas9 combined with new gRNA expression, which would only occur in progeny of drive males when testing split drives with separate Cas9 (when mimicking cages with complete drives) but not for complete drive systems. This could enable success in complete drives with higher maternal Cas9 deposition, even if cage experiments in split drives fail. Overall, our findings identify oct and stl as promising fertility targets and demonstrate both the potential and the constraints of fertility-based suppression drives, providing empirical evidence to guide the design and assessment of more efficient population control strategies.