EMBO Reports

Cell proliferation and fate specification are fundamental processes that ensure the generation of organs with proper size and patterning. Oxidative stress caused by accumulation of reactive oxygen species (ROS) can lead to cell cycle arrest, senescence, cell death and cell fate misspecification, thereby impairing normal development and contributing to many pathological processes. In this study, we identify Drosophila Ciz1 as a critical factor that safeguards epithelial homeostasis and development by preventing oxidative stress. Knockdown of Ciz1 in the Drosophila wing imaginal disc, an epithelial tissue that serves as the larval precursor of the adult wing, results in a small wing phenotype accompanied by thickened and ectopic veins. We further demonstrate that reduced Ciz1 expression leads to accumulation of donut-shaped mitochondria and elevated ROS levels. The increased oxidative stress subsequently suppresses proliferation via activation of JNK and promotes excessive vein formation by upregulating Rhomboid, a positive regulator of EGFR signaling. Interestingly, although Ciz1 is a zinc finger protein that predominantly localizes to the nucleus, neither its zinc finger motifs nor its nuclear localization is required for suppression of oxidative stress. Instead, the prion-like domain in its N-terminal part is essential for this activity. Our work identifies Ciz1 as an important factor in preventing oxidative stress and maintaining epithelial homeostasis.

Intrinsic apoptosis, or programmed cell death, is a vital response to stress and DNA damage in cells, and dysregulation of this pathway is common in cancers. The release of pro-apoptotic factors from mitochondria by the pro-apoptotic protein Bax is preceded by changes in the membrane properties of the outer mitochondrial membrane. We find that the membrane remodeling protein endophilin B1 primes membranes rich in the mitochondria-specific lipid cardiolipin for Bax-mediated membrane permeabilization, via a dual regulatory mechanism. We also show evidence that endophilin B1 translocates to the surface of mitochondria to co-localize with Bax during apoptosis in situ, where it forms biomolecular condensates.

Immune effects of membrane attack complexes (MAC) have been widely attributed to their abilities to cause cell death. Here, we show that the MAC component, C9, forms non-cytolytic aggregates with pro-inflammatory effects. Intracellular aggregates of C9 are detected within inflamed tissues of patients in association with endothelial cell (EC) activation but not increased cell death. We identify NUMBL as a Rab35 effector that directly binds surface-bound C9 to promote C9 internalization and entry into the endolysosomal pathway. Within acidified endolysosomes, C9 forms insoluble aggregates that are targeted for degradative aggrephagy in a process that activates NF-{kappa}B. For C9 aggrephagy to occur, ZFYVE21, a Rab5 effector, complexes with RNF34 to bridge C9 aggregates to LC3B+ aggresome membranes. We detect C9 aggregates in vivo, and we show that a ZFYVE21-RNF34 signaling axis is required for C9 aggrephagy and NF-{kappa}B -dependent EC activation in three separate mouse models. Mice with conditional loss of ZFYVE21 in ECs show reduced aggregraphy, resulting in attenuated systemic inflammation and reduced tissue injury following skin transplantation. Our data show that the C9 component of MACs forms intracellular aggregates with alarmin-like properties.

p53-binding protein 1 (53BP1) is a key mediator of the DNA damage response and genome stability. While its interphase function is well-characterized, its mitotic role remains less understood. Here we show that aberrant activation of 53BP1 through the loss of its negative regulator, TIRR, leads to mitotic abnormalities including altered spindle geometry, kinetochore-microtubule (k-MT) attachment errors and whole chromosome missegregation. We demonstrate that loss of TIRR results in excess interaction between 53BP1 and the key mitotic kinase Polo-like kinase 1 (PLK1), altering PLK1s activation, spatial distribution, and its interaction with known PLK1 substrates at multiple mitotic stages. Moreover, due to PLK1s established role in CENP-A loading, hyperactivation of 53BP1 compromises CENP-A loading, triggers gradual loss of CENP-A from centromeres and generates severe kinetochore assembly defects. These findings uncover a non-canonical mitotic function of 53BP1 as a key regulator of PLK1 activity and chromosome segregation fidelity.

Oxygen is indispensable for survival in aerobic organisms, necessitating mechanisms to sense and respond to fluctuations in oxygen availability. Physiological processes such as early development, proceeds in an oxygen-sensitive environment and this appears conserved across vertebrate evolution. Owing to their avascular nature epithelial tissues routinely experience hypoxia but the epithelial responses to hypoxia and the underlying adaptive molecular regulation remains to be fully understood. We used the bilayered epidermis of Zebrafish embryos to ask how a developing epithelium responds to and copes with hypoxia. We show that under hypoxic conditions, despite the changes in cell morphologies, disruption in E-cadherin polarisation and the presence of intercellular gaps in the outer epidermal layer, the tight junctions are maintained. Our data indicate that ROCK (Rho-associated kinase) mediates the change in cell morphology and the maintenance of barrier function via non-muscle Myosin-II (NM-II). Furthermore, a high level of NM-II activity is essential to suppress Crb3-dependent cell delamination and apoptosis under hypoxia. Genetic perturbations reveal that neither increasing levels of active NMII nor augmenting tight junctions alone improves barrier function defects, indicating both these ROCK-dependent processes are necessary to maintain the barrier function under hypoxia. Our study uncovers the hitherto unappreciated importance of ROCK signaling in the maintenance of epithelial architecture and barrier function in a developing epithelium, ensuring organism survival.

Centrosome amplification is frequently associated with chromosomal instability and tumor progression, but how cells coordinate centriole assembly with the control of centrosome numbers and quality remains poorly understood. TIAM1 is a RAC1 guanine nucleotide exchange factor previously implicated in centrosome-associated signaling and {beta}TrCP-dependent control of PLK4 abundance. Here, we examined how Tiam1 regulates autophagy-lysosome homeostasis in mouse embryonic fibroblasts induced to overexpress PLK4. In contrast to a previous model in which Tiam1 loss promotes productive centriole overduplication, we found, by super-resolution imaging and expansion microscopy, an abnormal distribution of PLK4 on the centrioles centriole-associated structures following TIAM1 depletion, suggesting that TIAM1 may support the organization or maturation of centrioles. TIAM1 depletion also resulted in increased LC3B-positive puncta and enlarged LAMP1-positive compartments, but this was not accompanied by increased LC3B-II accumulation after bafilomycin A1 treatment. These findings suggest that TIAM1 may act at the interface between centriole assembly and endolysosomal/autolysosomal organization, linking TIAM1 to lysosome-associated centrosome quality-control pathways.

Mitochondrial homeostasis is maintained by multiple quality control pathways, including mitophagy, which targets dysfunctional mitochondria for degradation. During receptor-mediated mitophagy, the outer membrane proteins BNIP3 and NIX directly recruit autophagy machinery to the mitochondrial surface, though their precise regulation is still unclear. In recent years, new BNIP3- and NIX-interacting proteins have been identified that influence mitophagic flux. PPTC7 and FBXL4 target BNIP3 and NIX for proteasomal turnover to keep levels of the receptors low, whereas TMEM11 is proposed to spatially control mitophagy by interacting with receptors at active mitophagy sites. However, it is unclear how each of these interactions is controlled and how they interplay with each other. Here, we identify a repressor of mitophagy, ARMC1, which forms a complex with TMEM11, BNIP3, and NIX. During mitophagy activation, ARMC1 dissociates from the complex, freeing the receptors to initiate mitophagy. We find that TMEM11 then acts in an antagonistic relationship with PPTC7, protecting the receptors from proteasomal degradation. Our data are consistent with a two-stage model. At steady state, a population of sentinel receptors is repressed and primed to respond to mitochondrial dysfunction. Once mitophagy is activated, TMEM11 protects BNIP3 and NIX, ensuring a sustained mitophagic response. Our findings provide a framework for understanding how two key regulatory pathways intersect to modulate receptor-mediated mitophagy.

TMEM127 is an adaptor protein that bridges substrates to E3 ubiquitin ligases of the HECT family. Among its interacting partners is the major histocompatibility class I (MHC-I), a critical component of the antigen presentation pathway and the adaptive immune response. MHC-I is ubiquitinated and fated for lysosome-mediated degradation by the WWP2 E3 ligase in a complex that involves TMEM127 and a second adaptor protein, SUSD6. However, the interacting dynamics among complex components remains to be determined, a key knowledge gap towards the development of pharmacological modulators. Here, using in vitro and in vivo models, we report that TMEM127-WWP2 interaction stabilizes the MHC-I degradation complex and reveals an asymmetric role of the two adaptor proteins. Specifically, we find that TMEM127 regulates WWP2 catalytic activity, abundance and localization through its canonical PY motif interaction with the WW domain of WWP2 with contribution of a TMEM127 endocytic motif, providing a mechanism to restrain complex activity. Further, we validate the impact of TMEM127 dosage in the endogenous complex assembly and regulation. Our results nominate TMEM127 as a critical member of the MHC-I degradation complex and highlight the TMEM127-WWP2 interaction as a target for augmenting MHC-I-mediated antigen presentation, a long sought goal in cancer immunology.

Kinesins are microtubule-dependent motors, yet the functions of the kinesin-4 family member KIF27 remain poorly understood. Here, we demonstrate a dynamic and cell-cycle-dependent localization of KIF27, consistent with its functional roles in mitotic progression. Upon mitotic entry, KIF27 relocates to condensed chromosomes. During anaphase, a fraction of KIF27 accumulates at the spindle midzone, and in telophase and late stages of cytokinesis it localizes at the midbody, colocalizing with key cytokinetic regulators at both structures. Recruitment of KIF27 to the midbody depends on KIF23 and CEP55. KIF27 depletion results in profound cell division defects, altered midbody and microtubule morphology, delayed cytokinesis and cytokinesis failure. Beyond cell division, KIF27 depletion directly compromises nuclear morphology, and pan-cancer transcriptomic analyses correlate low KIF27 expression with aneuploidy and poor patient survival in several cancer types. Together, our results identify KIF27 as a novel regulator of mitotic fidelity and genome stability.

The liver is a metabolic hub with a high protein turnover that renders it uniquely susceptible to proteotoxic stress. Perturbation of proteostasis, either by proteasomal inhibitors or in chronic liver diseases, could adversely impact liver physiology. Here, we show that proteasomal inhibition unexpectedly suppresses basal type I interferon (IFN-I) signaling in the murine liver. Proteasomal inhibition by bortezomib selectively downregulates a subset of interferon-stimulated genes (ISGs), among which USP18 and ISG15 emerge as critical determinants of hepatocellular survival. We identify USP18 as a central cytoprotective factor that prevents proteotoxic apoptosis independently of its deubiquitinase activity, but strictly requires its scaffolding function mediated by isoleucine-60 and interaction with STAT2. Mechanistically, proteotoxic stress disrupts IRF9 nuclear translocation, attenuating USP18 transcription, and drives USP18 and other ISGs into insoluble aggregates with kinetics distinct from canonical IFN-I-induced insolubility. Strikingly, IFN-I priming preserves ISG solubility, restores USP18 abundance, and confers resistance to proteotoxic cell death. Together, these findings uncover an unanticipated link between proteostasis and innate immune signaling, and establish the USP18-STAT2 axis to enhance hepatic resilience under proteotoxic stress.

The transition from tumor necrosis factor (TNF)-induced plasma membrane-bound complex I to cytosolic death-inducing complex II switches cells from survival to death. However, the precise regulation of this fatal decision is incompletely understood. Here, we show that mTORC1 promotes this transition by destabilizing later-stage complex I without affecting its initial assembly. Inhibition of mTORC1 unleashes ATG9A and FIP200 activity, thereby promoting the accumulation of CHUK (IKK) in complex I. CHUK scaffolds the kinase-active IKK{beta} to stabilize complex I and prevent complex II formation. Activation of this ATG9A/FIP200-CHUK/IKK{beta} axis protects against TNF-induced fulminant hepatitis while compromises antibacterial defense against Staphylococcus aureus. This mTORC1-governed life-or-death transition provides therapeutic insight into TNF-related pathologies--including cancer, metabolic tissue injury, and microbial infections--where mTORC1 activity is frequently suppressed.

The nuclear envelope (NE) surrounds the genetic material and is continuous with the endoplasmic reticulum (ER). In yeast and other organisms undergoing closed mitosis, nuclear envelope expansion (NME) is strictly required to accommodate spindle elongation and ensure proper chromosome segregation within a single nuclear compartment. Failure to expand the NE during mitosis leads to chromosome missegregation. Here, we show that deletion of the unstructured N-terminal domain of Rgf3, a Rho1-specific guanine nucleotide exchange factor (GEF), causes early mitotic defects that produce the characteristic "cut" phenotype of untimely cell division. The rgf3{Delta}N2 mutant displays spindle buckling, a hallmark of anaphase nuclei unable to properly expand the NE. From yeast to mammals, phosphatidic acid (PA)--a key precursor in phospholipid biosynthesis--is metabolized via two competing pathways, the cytidine diphosphate-diacylglycerol (CDP-DAG) and the Kennedy pathways, both contributing to lipid membrane homeostasis. We provide evidence that impaired Rho1 activation in rgf3{Delta}N2 selectively disrupts phospholipid synthesis through the CDP-choline branch of the Kennedy pathway. Thus, Rho1 promotes mitotic progression by modulating phospholipid biosynthesis to enable efficient NME during anaphase. HighlightsThe N-terminus of Rgf3 is required for proper nuclear envelope expansion (NME) during anaphase. The structurally flexible N-terminal domain of Rgf3 is essential for localized Rho1 activation. Active Rho1 drives mitotic membrane growth by modulating phospholipid synthesis through the Kennedy pathway.

Dendritic cells (DCs) are an important type of antigen presentation cell that regulate immunity by initiating antigen-specific immunity and tolerance through T cell activation. The interaction between DCs and T cells can be mediated through direct cell-cell contact or via the release of extracellular vesicles (EVs) from DCs that harbour antigen presentation machineries. Although small EVs (<200 nm in diameter) such as exosomes released by DCs have been shown to regulate immunity, whether other EV subtypes, in particular those that are released by dying DCs due to homeostatic turnover or following infection, can modulate immune responses is not defined. In this study, we demonstrated that DCs undergoing apoptosis can generate a subclass of large EVs ([~]1,000-5,000 nm in diameter) known as apoptotic bodies (ApoBDs) via distinct morphological steps. Mechanistically, ApoBD formation by apoptotic DCs is regulated by Rho-associated kinase 1 and T-type calcium channels. Functionally, DC-derived ApoBDs were found to mediate direct antigen presentation. These data demonstrate a novel function of ApoBDs and highlight the ability of apoptotic materials derived from dying DCs to continue mediating intercellular communication and regulating immune responses.

Microtubules are polarised polymers that assemble into highly specialised networks in a cell-specific manner. This is controlled in part by microtubule organising centres (MTOCs), which concentrate factors necessary for microtubule nucleation and the organisation of microtubule minus ends. Neurons rely on oppositely polarised microtubule networks, with axons containing mostly plus-end-out microtubules, and dendrites contain many minus-end-out microtubules. How minus-end-out microtubule polarity is established in dendrites remains an important question. Here, we identify a new type of MTOC within the dendrites of Drosophila class I dendritic arborisation neurons, a common model for the neuronal cytoskeleton. We show that membrane swellings distributed intermittently along dendrite shafts, which we term "dendritic varicosities", contain the principal component of the microtubule nucleating complex and repeatedly generate microtubules whose plus ends grow back towards the soma. Varicosities located specifically in distal regions also contain MTOC proteins implicated in minus end anchoring, and this correlates with the accumulation of minus ends specifically in distal varicosities. Depletion of these MTOC proteins leads to major defects in minus end organisation, with microtubule buckles and loops deforming the neuronal membrane. Thus, dendritic varicosities are an important new type of neuronal MTOC that contribute to the generation and organisation of the minus-end-out microtubule network within dendrites.

Microproteins translated from short open reading frames are increasingly understood to play important roles in cell biology and development. Here, we describe a microprotein in Drosophila that is expressed in ovarian follicle cells which surround the developing oocyte. The Dafcin microprotein is predicted to form an amphipathic alpha-helix, a structure known to interact with lipid bilayers. The structure of Dafcin most resembles the influenza HA fusion peptide, which induces negative curvature of endosomal membranes. Dafcin tagged with GFP localizes to the Golgi and is ultimately secreted from the follicle cells. Remarkably, this occurs without the microprotein having a secretory signal sequence. The protein is taken up into the oocyte by endocytosis, localizing to the inner face of storage lysosomes called yolk granules. Mutant analysis shows that Dafcin is required to limit the size of yolk granules. This may occur by inducing negative membrane curvature like HA peptide. In support, liposomes formed in vitro with both Dafcin and HA peptides are smaller in size.

Eukaryotic cells are composed of different organelles that communicate with one another through direct contacts, which are necessary for a host of cellular reactions and for responding to different developmental and environmental changes. Plasma membrane (PM) forms extensive contacts with the endoplasmic reticulum (ER) at specific sites named ER-PM contact sites. These contacts play crucial functions in lipid homeostasis, Ca2+ regulation and signaling in all eukaryotes. However, the mechanisms by which plant ER-PM contact site proteins tether to the PM, as well as the dynamics of these contact sites, remain poorly understood. Here, we investigate the importance of phosphoinositides in the establishment and dynamics of ER-PM contact site proteins in plants. We found that phosphatidylinositol-4-phosphate (PI4P), rather than phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), is required for the association of ER-PM contact site proteins with the PM. Furthermore, we identified a PI4P phosphatase, SUPPRESSOR-OF-ACTIN7 (SAC7), that associates with the ER-PM contact site protein SYNAPTOTAGMIN1 (SYT1) and regulates its dynamic association with the PM. In particular, we found that in growing root hairs, a highly polarized cell type, SAC7 removes SYT1-containing contact sites at the growing tip. Consistently, optogenetic induction of ER-PM tethering reduced root hair elongation within minutes of blue light induction. Altogether, we propose a link between SAC7-mediated regulation of PI4P, dynamic ER-PM contact site establishment and polarized cell growth in plants.

Polo-like kinase 1 (PLK1) is a master regulator of mitosis and is known to dictate DNA repair pathway choice at this stage of the cell cycle. However, its roles in controlling mitotic DNA damage responses remain incompletely characterised. Here, we used acute PLK1 inhibition as a substrate-trapping strategy to stabilise PLK1-target interactions in mitotic cells and identify PLK1-associated DNA repair factors. Proteomic analysis of endogenous HA-tagged PLK1 complexes revealed interactions with multiple genome stability proteins, including SLX4, RAD52, FANCM, and REV1. We demonstrate that PLK1 binds SLX4 and RAD52 via canonical CDK1-dependent phospho-docking motifs centred on SLX4 Ser1453 and RAD52 Thr300. Mutation of these residues abolished PLK1 binding and, at least for RAD52, prevented PLK1-dependent phosphorylation of mitotic targets. Functional studies showed that PLK1 docking to SLX4 is dispensable for interstrand crosslink repair but essential for mitotic DNA synthesis (MiDAS), defining a separation-of-function allele. Likewise, disruption of PLK1 docking to RAD52 impaired MiDAS. Together, these findings identify PLK1 as a key coordinator of mitotic genome maintenance pathways required for MiDAS.

ABSTRACT/SUMMARYTriple negative breast cancer (TNBC) is the most aggressive breast cancer subtype, enriched for cancer stem cells (CSCs), which are the cause of tumor recurrence. CSCs are responsible for tumor initiation and propagation; however, the molecular mechanisms underlying their formation remain largely unclear. We show that carboplatin treated TNBC cells lose their circadian rhythms and promote the enrichment of CSCs. Notably, genetic ablation of the circadian clock by itself, without carboplatin treatment, facilitated the formation of CSCs along with their ability to generate 3D tumorspheres and mouse tumors enriched with CSCs. Mechanistically, we identified an antagonistic interplay between the circadian clock and pluripotency, whereby the core pluripotent factor OCT4 disrupts the expression of circadian timekeeping genes to disable the circadian clock. Furthermore, we uncovered a transcriptional pausing program, controlling the circadian clock, to be perturbed in carboplatin treated TNBC cells. Concordantly, based on gene expression analysis from The Cancer Genome Atlas (TCGA), we found that the uncoupling of transcriptional pausing and the circadian clock correlated with low survivability across diverse cancer types. Moreover, cancer patients with poor prognosis exhibit low expression of the core timekeeping genes Clock, Npas2, Bmal1 and Rorc. Lastly, we restored circadian rhythms in Oct4 deficient TNBC cells and impaired their ability to generate tumorspheres and decreased the number of CSCs in mouse tumors. Overall, our findings demonstrate an unprecedented mechanism for the formation of CSCs that is dependent on the loss of circadian rhythms and thereby has eminent implications for developing new cancer therapies. SIGNIFICANT STATEMENTCircadian rhythms are absent in pluripotent stem cells; however, their presence or absence in cancer stem cells has remained undetermined. Here, we implemented a carboplatin-based paradigm to enrich for cancer stem cells. We found that upon carboplatin treatment, triple negative breast cancer cells lost their circadian rhythms. Strikingly, the formation of cancer stem cells is diminished by partial restoration of circadian cycles achieved by knocking down the core pluripotency gene Oct4. Mechanistically, we observed an alteration of transcriptional pausing in cancer stem cells that may be implicated in the destruction of the circadian clock.

Scaffold integrity is essential for the activity of proteins that function through protein-protein interactions rather than catalytic output. RAF1 exemplifies this duality: although it is a bona fide kinase and a core component of the MAPK cascade, its tumor-promoting role is largely kinase-independent, relying instead on scaffold-mediated suppression of apoptosis. Genetic Raf1 ablation in KRAS-driven lung adenocarcinoma mouse models induces tumor regression without systemic toxicity, making it an attractive candidate for targeted protein degradation. Chemogenetic systems like the dTAG platform are widely used for preclinical target validation. Here, we generated a dTAG-RAF1 mouse model and showed that pharmacological degradation is efficient and systemically well tolerated, but fails to reproduce the tumor regression observed upon genetic Raf1 ablation. Mechanistically, the N-terminal FKBP12F36V tag (dTAG) perturbs the RAF1 interactome, including scaffold associations with apoptotic regulators, thereby blunting the phenotypic consequences of its degradation. These results establish scaffold integrity as a determinant of chemogenetic system fidelity and argue that degradation tools must be validated at the functional level, not only for target elimination, before assessing their therapeutic relevance.

Mouse models have historically been central to studies of TNF-induced cell death and guided pharmaceutical translation into clinic, based on the assumption that TNF signaling is conserved between human and mouse. Here, our work uncovers critical species-specific differences between the two. By systematically dissecting the roles of RIPK1, TRADD, and sensitivity to RIPK1 inhibitors in TNF signaling--including RIPK1 kinase-dependent and-independent apoptosis--we found that both apoptosis modalities diverge between human and mouse cells. In mouse cells, RIPK1 suppresses TRADD-mediated kinase-independent apoptosis, whereas in human cells, RIPK1 and TRADD act redundantly. Moreover, RIPK1 inhibitors block kinase-dependent apoptosis in mouse but not human cells, despite effectively inhibiting RIPK1 S166 phosphorylation. Cross-species complementation revealed that these discrepancies stem not from RIPK1 itself but from cell-context differences. These findings echo the clinical failures of RIPK1 inhibitors despite efficacy in mouse models and underscore the need for humanized models and therapeutics that more faithfully predict clinical outcomes.