EMBO Reports

Mitochondrial dynamics is important for cellular health and includes morphology, fusion, fission, vesicle formation, transport and contact formation with other organelles. Myosin XIX (Myo19) is an actin-based motor which competes with TRAK1/2 adaptors of microtubule-based motors for binding to the outer mitochondrial membrane receptors Mitochondrial Rho GTPases 1/2 (Miro). Currently, it is poorly understood how Myo19 contributes to mitochondrial dynamics. Here, we report on a Myo19-deficient mouse model and the ultrastructure of the mitochondria from cells of Myo19-deficient mice and HEK cells, Miro-deficient HEK cells and TRAK1-deficient HAP1 cells. Myo19-deficient mitochondria in kidney, skeletal and cardiac muscle cells, MEFs and HEK cells have morphological alterations in the inner mitochondrial membrane with reduced numbers of malformed cristae. In addition, mitochondria in Myo19-deficient cells showed fewer ER-mitochondria contact sites (ERMCS). In accordance with the ultrastructural observations, Myo19-deficient MEFs had lower oxygen consumption rates and a reduced abundance of OXPHOS supercomplexes. The simultaneous loss of Miro1 and Miro 2 led to a comparable mitochondria phenotype and reduced ERMCS as observed upon loss of Myo19. However, the loss of TRAK1 caused only a reduction in the number of cristae, but not ERMCS. These results demonstrate that both actin- and microtubule-based motors regulate cristae formation, but only Myo19 and its membrane receptor Miro regulate ERMCS.

BAK and BAX execute intrinsic apoptosis by permeabilising the mitochondrial outer membrane. Their activity is regulated through interactions with pro-survival BCL-2 family proteins and with non-BCL-2 proteins including the mitochondrial porin VDAC2. VDAC2 is important for bringing both BAK and BAX to mitochondria where they execute their apoptotic function. Despite this important function in apoptosis, whilst interactions with pro-survival family members are well characterised and have culminated in the development of drugs that target these interfaces to induce cancer cell apoptosis, the interaction between BAK and VDAC2 remains largely undefined. Deep scanning mutagenesis coupled with cysteine linkage identified key residues in the interaction between BAK and VDAC2. Obstructive labelling of specific residues in the BH3 domain or hydrophobic groove of BAK disrupted this interaction. Conversely, mutating specific residues in a cytosol-exposed region of VDAC2 stabilised the interaction with BAK, and inhibited BAK apoptotic activity. Thus, this VDAC2-BAK interaction site can potentially be targeted to either inhibit BAK-mediated apoptosis in scenarios where excessive apoptosis contributes to disease, or to promote BAK-mediated apoptosis for cancer therapy.

RNA interference, which has a major role in the control of viral infection in insects, is initialized by the sensing of double stranded RNA (dsRNA) by the RNAse III enzyme Dicer-2. Although many in vitro studies have helped understand how Dicer-2 is able to discriminate between different dsRNA substrate termini, much less is known about how this translates to the in vivo recognition of viral dsRNA. Indeed, although Dicer-2 associates with several dsRNA-binding proteins (dsRBPs) that can modify its specificity for a substrate, it remains unknown how Dicer-2 is able to recognize the protected termini of viral dsRNAs. In order to study how the ribonucleoprotein network of Dicer-2 impacts antiviral immunity, we used an IP-MS approach to identify in vivo interactants of different versions of GFP::Dicer-2 in transgenic lines. We provide a global overview of the partners of Dicer-2 in vivo, and reveal how this interactome is modulated by different factors such as the viral infection and/or different point mutations inactivating the helicase or RNase III domains of GFP::Dicer-2. Our analysis uncovers several previously unknown Dicer-2 interactants associated with RNA granules (i.e. Me31B, Rump, eIF4E1 & Syp). Functional characterization of the candidates reveals pro- and antiviral factors in the context of the infection by the picorna-like DCV virus. In particular, the protein Rasputin has been identified as a novel antiviral candidate. The resources provided by this work can be used to gain a better understanding of the molecular complexes assembled around Dicer-2 in the context of antiviral RNAi and beyond.

RIG-I is a cytosolic immune receptor that provides the first line of defense by detecting viral RNA and triggering antiviral responses. Its physiological role in humans remains unclear, as no patients with complete RIG-I deficiency have yet been reported. We identified a critically ill COVID-19 patient with severe RIG-I deficiency caused by heterozygous RIG-I G731R, a novel dominant loss-of-function variant. The G731R mutation in helicase motif VI disrupts the arginine finger, impairing the ATPase activity of RIG-I, but not its RNA-binding ability. However, viral RNA binding fails to expose the signaling domains, thereby impairing the IFN-{beta} response of G731R. Instead, G731R competes with wild-type RIG-I, exerting a dominant negative effect. The loss-of-function is caused by bulky-charged substitutions at G731, as alanine or leucine substitution results in an unexpected gain-of-function phenotype. These findings highlight the importance of uncompromised RIG-I function for human antiviral immunity and the pleiotropic effects of single mutations.

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.

Longevity is determined by diverse signaling pathways including telomere protection and homeostasis master regulators like FOXO3a. We previously showed that the telomeric repeat binding factor 2 (TRF2) expression decreases with age in human skeletal muscle and that, surprisingly, its loss in myofibers does not trigger telomere deprotection. We reveal here that in TERF2-compromised myotubes, FOXO3a is recruited to telomeres where it acts as a protective factor against ATM-dependent DNA damage activation. Moreover, we show that FOXO3a-telomere association increases with age in human skeletal muscle biopsies. In mitotic fibroblasts, the telomere protective properties of FOXO3a are operative if the cells are treated with bleomycin. The telomere function of FOXO3a does not require its Forkhead DNA binding domain but the CR2C. Overall, these findings demonstrate a direct connection between two key longevity pathways, FOXO3a and telomere protection. This unveils an unexpected higher level of integration in the regulation of longevity signaling pathway.

The Mitochondrial contact site and cristae organizing system (MICOS) complex is a multisubunit complex regulating mitochondrial inner membrane (IM) architecture, which is enriched at crista junctions (CJs) and required for cristae membrane dynamics. It modulates various mitochondrial processes including protein and lipid transport and is causally linked to a variety of human diseases. To gain a broad overview of the various pathways modulated by the MICOS complex, we examined its molecular neighbourhood. For this, we employed proximity biotinylation assays using APEX2 fused to four MICOS subunits (MIC10, MIC13, MIC26 and MIC27) in the respective mammalian knockout cells. These four MICOS-APEX2 fusion proteins integrated into the native MICOS complex and properly localised as revealed by electron microscopy combined with DAB staining and STED super-resolution nanoscopy. Here, we identify 119 common and 50 unique proteins, termed MICOS NanoDomain Network (MINDNet) encompassing the versatile proximity proteome of the MIC10/MIC13/MIC26/MIC27 subcomplex playing multifaceted mitochondrial functions. The MINDNet revealed a large number of OXPHOS proteins, protein translocases of the IM and OM, mitochondrial ribosomal proteins and solute carrier family transporters. Using the cues obtained from the proximity interaction studies, we investigated the role of all the MICOS proteins in modulating the function of the OXPHOS complexes. Among all the MICOS proteins, MIC10 and MIC60 consistently regulated the assembly and activity of the OXPHOS complexes. Overall, we propose that the MICOS complex integrates numerous spatial and temporal cues to regulate the dynamic microenvironment, along with IM architecture, which are involved in multiple pathways controlling mitochondrial biogenesis.

Most mitochondrial proteins originate from the cytosol and require active transport into the organelle. Such precursor proteins must be largely unfolded to pass through translocation channels in mitochondrial membranes. Misfolding of transported proteins can result in their arrest and translocation failure. Arrested proteins block further import, disturbing mitochondrial functions and cellular proteostasis. Cellular responses to translocation failure have been defined in yeast. To discover molecular mechanisms that resolve failed import events in human cells, we developed the translocase clogging model using a fusion protein with a rigid domain. The mechanism we uncover differs significantly from these described in fungi, where ATPase-driven extraction of blocked protein is directly coupled with proteasomal processing. We found human cells to rely primarily on mitochondrial factors to clear translocation channel blockage. The mitochondrial membrane depolarization triggered proteolytic cleavage of the stalled protein, which involved mitochondrial protease OMA1. The cleavage allowed releasing the protein fragment that blocked the translocase. The released fragment was further cleared in the cytosol by the valosin containing protein (VCP)/p97 and proteasome.

The centrosome is a cytoplasmic organelle with roles in microtubule organization which has also been proposed to act as a hub for cellular signaling. Some centrosomal components are required for full activation of the DNA Damage Response. However, if the centrosome regulates specific DNA repair pathways is not known. Here, we show that centrosomes presence is required to fully activate recombination, specifically to completely license its initial step, the so-called DNA end resection. Furthermore, we identify a centriolar structure, the subdistal appendages, and a specific factor, CEP170, as the critical centrosomal component involved in the regulation of recombination and resection, albeit it does not control end-joining repair. Cells lacking centrosomes or depleted for CEP170 are, consequently, hyper-sensitive to DNA damaging agents. Moreover, low levels of CEP170 in multiple cancer types correlate with an increase of the mutation burden associated with specific mutational signatures and a better prognosis, suggesting that changes in CEP170 can act as a mutation driver but also could be targeted to improve current oncological treatments.

Upon interaction with immobilized antigens B cells form an immune synapse, where actin remodeling and re-positioning of the microtubule-organizing center (MTOC) together with lysosomes can facilitate antigen extraction. B cells have restricted cytoplasmic space, mainly occupied by a large nucleus, yet the role of nuclear morphology in the formation of the immune synapse has not been addressed. Here we show that, upon activation, B cells re-orientate and adapt the size of their nuclear groove facing the immune synapse, where the MTOC sits and lysosomes accumulate. Silencing nuclear envelope proteins, Nesprin-1 and Sun-1, impairs nuclear reorientation towards the synapse and leads to defects in actin organization at this level. Consequently, B cells are unable to internalize the BCR after antigen activation. Nesprin-1 and Sun-1-silenced B cells also fail to accumulate the tethering factor Exo70 at the center of the synaptic membrane and display defective lysosome positioning, impairing efficient antigen extraction at the immune synapse. Thus, changes in nuclear morphology and positioning emerge as critical regulatory steps to coordinate B cell activation.

Maintenance of the polarity of the epithelial cells facing the lumen of the small intestine is crucial to ensure the vectorial absorption of nutrients as well as the integrity of the apical brush border and the intestinal barrier. Polarized vesicular trafficking plays a key role in this process, and defective transport due to mutations in apical trafficking-related genes has been shown to affect nutrient absorption. Interestingly, it has been demonstrated that downregulation of the polarized sorting clathrin adaptor AP-1B led to both epithelial polarity and proliferation defects in the mouse intestine. This enlightened a new function of polarized trafficking in the gut epithelium and a novel link between trafficking, polarity, and proliferation. Here, using CRISPR-Cas9-mediated mutation of the AP-1B coding gene Ap1m2 in mouse intestinal organoids, we uncovered a novel proliferation pathway controlled by AP-1B. We showed that the polarity defects induced by Ap1m2 mutations led to a defective apical targeting of both Rab11+ apical recycling endosomes and of the polarity determinant Cdc42. Moreover, we showed that these polarity defects were accompanied by an induction of YAP and EGFR/mTOR-dependent proliferation pathways. Finally, we showed that AP-1B additionally controlled a proliferation-independent differentiation pathway towards the secretory lineage. Overall, our results highlighted the pleiotropic roles played by AP-1B in the homeostasis of the gut epithelium.

The cohesin complex performs essential cellular functions including regulation of chromosome cohesion, chromatin organization and DNA repair. Somatic pathogenetic variants in cohesin genes, such as STAG2, have been associated with cancer, but their contribution to brain tumorigenesis is unclear. Here, we report the presence of STAG2 variants in glioblastoma and medulloblastoma patients and determine that loss of STAG2 in human cells leads to DNA damage and apoptosis. Treatment with inhibitors of the Poly ADP-ribose polymerase (PARP), which are used to treat forms of cancer with defects in DNA repair, increased the amount of apoptosis, confirming that synthetic lethality between reduced cohesin and PARP activity could be observed in vitro. Similar results were obtained in vivo by reducing expression of SA1, the Drosophila melanogaster homolog of STAG1/2. Cohesin gene silencing during fly brain development leads to defects in neural stem cells differentiation and tumorigenesis both in the presence of oncogenic activity and per se. Our in vivo and in vitro data suggests that impairment of PARP activity might induce synthetic lethality in cohesin-dependent tumors, highlighting a vulnerability that can be pharmacologically exploited.

Cytochrome c oxidase (CIV) is the last electron acceptor of the mitochondrial respiratory chain. In yeast, it is composed of 12 subunits, three of which are encoded in the mitochondrial genome. CIV assembly is a modular and highly regulated process that requires several specific factors. In this work, we characterized the role of Aim11 in CIV biogenesis. By high-throughput analysis, it was previously detected that Aim11 interacted with some CIV subunits, but the physiological relevance of these interactions was unknown. In the present work, we found that the{Delta} aim11 mutant exhibited reduced respiratory growth and diminished CIV activity. Using mitochondrial complexome profiling, we detected in the{Delta} aim11 mutant accumulation of intermediates of the three CIV-assembly modules, as well as reduction in supercomplexes levels. Aim11 works together with three other uncharacterized proteins: Mtc3, Gep7, and Iai11. The four proteins form a complex that we named AMIGa (Aim11-Mtc3-Iai11-Gep7 association) complex, necessary for the efficient assembly of CIV. Finally, the human protein TMEM242 was identified as Aim11 orthologue.

Oncogene-induced senescence (OIS) is a tumor-suppressive mechanism that arrests cell proliferation in response to oncogene-induced replication stress (RS). OIS also depends on the cGAS-STING pathway, which detects cytosolic DNA and promotes the expression of type I interferons (IFN) and pro-inflammatory cytokines. Whether and how the RS and IFN responses cooperate to promote OIS is currently unknown. Here, we show that the MRE11 nuclease promotes OIS in immortalized human fibroblasts overexpressing the H-RASV12 oncogene both by slowing replication forks and by activating the cGAS-STING pathway in response to RS. Interestingly, overexpression of TREX1, the major nuclease degrading cytosolic DNA, prevented RAS-induced senescence. In contrast, overexpression of a dominant negative mutant of TREX1 was sufficient to induce senescence in human fibroblasts, even in the absence of H-RASV12 induction. Collectively, these data suggest that the RS and IFN responses in OIS are functionally linked through a process involving the nucleases MRE11 and TREX1.

Interleukin (IL)-1{beta} is a pro-inflammatory cytokine implicated in sterile inflammation and tumor development. Investigating the role of MAPKAP kinase 2 (MK2) in IL-1{beta} processing, we found that Il1b mRNA and IL-1{beta} protein levels were elevated in resting MK2-knockout (KO) macrophages and in the serum of MK2/3-double-KO mice. This was linked to activation of the non-canonical NF-{kappa}B pathway in the absence of MK2 or its activator, p38. Rescue by MK2, its kinase-inactive mutant MK2K79R, or p38 suppressed this pathway and reduced Il1b expression. We also observed decreased basal protein levels of tumor suppressor p53 in MK2-or p38-deficient cells. Mechanistically, p53 interacts with mitochondrial caspase-3, promoting cleavage of RelB, thereby inhibiting non-canonical NF-{kappa}B signaling and subsequent Il1b and TP53 expression. These findings explain elevated basal IL-1{beta} levels in MK2-KO macrophages and uncover a new autoregulatory mechanism of TP53 expression. Additionally, they reveal a new mechanism that contributes to the long-discussed link between cancer and inflammation, wherein the tumor suppressor p53 inhibits cytokine production in parallel.

Caspases are cysteine-aspartic proteases that mediate both lethal and non-lethal cellular outcomes, including the promotion of tissue growth. However, the mechanisms underlying the differential regulation of these activities remain unclear. We have previously shown that among the two Drosophila executioner caspases, Dcp-1 and Drice, Dcp-1 promotes tissue growth in a non-lethal manner, independent of canonical apoptotic signaling. Herein, we demonstrated that overexpressed Dcp-1, but not Drice, was activated without canonical apoptosome components. TurboID-based proximity labeling revealed distinct proximal proteomes, among which Sirtuin 1, an Atg8a deacetylase, which promotes autophagy, was specifically required for Dcp-1 activation. Autophagy-related genes, including Bcl-2 family members Debcl and Buffy, are required for Dcp-1 activation. Structure-based prediction using AlphaFold3 further identified Bruce, an autophagy-regulated inhibitor of apoptosis, as a Dcp-1-specific regulator acting outside the apoptosome-mediated pathway. Physiologically, Bruce suppresses wing tissue growth. These findings indicate that non-lethal Dcp-1 activity is governed by the autophagy-Bruce axis, enabling distinct non-lethal functions independent of cell death.

Intracellular trafficking regulates the distribution of transmembrane proteins including the key determinants of epithelial polarity and adhesion. The Adaptor Protein 1 (AP-1) complex is the key regulator of vesicle sorting, which binds many specific cargos. We examined roles of the AP-1 complex in epithelial morphogenesis, using the Drosophila wing as a paradigm. We found that AP-1 knockdown leads to ectopic tissue folding, which is consistent with the observed defects in integrin targeting to the basal cell-extracellular matrix adhesion sites. This occurs concurrently with an integrin-independent induction of cell death, which counteracts elevated proliferation and prevents hyperplasia. We discovered a distinct pool of AP-1, which localizes at the subapical Adherens Junctions. Upon AP-1 knockdown, E-cadherin is hyperinternalized from these junctions and becomes enriched at the Golgi and recycling endosomes. We then provide evidence that E-cadherin hyperinternalization acts upstream of cell death in a potential tumour-suppressive mechanism. Simultaneously, cells compensate for elevated internalization of E-cadherin by increasing its expression to maintain cell-cell adhesion. Author SummaryThe epithelium is one of the four types of tissues found in animals and is essential for the normal development and maintenance of multicellular organisms. In this tissue, the adhesion protein E-cadherin helps keep cells together and facilitates the coordination of their behaviours. E-cadherin is highly dynamic and undergoes constant turnover by intracellular trafficking machinery. Here, we describe the contributions of one of the core components of intracellular trafficking, the AP-1 complex to E-cadherin dynamics. Using epithelial cells from Drosophila melanogaster, we discover that the AP-1 complex limits E-cadherin internalization from the plasma membrane, which is consistent with the localization of a distinct pool of the AP-1 complex at the sites of E-cadherin cell-cell adhesion. We also show that increased E-cadherin internalization triggers programmed cell death, preventing the tissue from hyperplastic overgrowth in a potential tumour-suppressive mechanism. At the same time, cells compensate for the reduction in the membrane presentation of E-cadherin by increasing its expression, therefore protecting tissue integrity.

Mitochondria are increasingly recognized as cellular hubs to orchestrate signaling pathways that regulate metabolism, redox homeostasis, and cell fate decisions. Recent research revealed a role of mitochondria also in innate immune signaling, however, the mechanisms of how mitochondria affect signal transduction are poorly understood. Here we show that the NF-B pathway activated by TNF employs mitochondria as a platform for signal amplification and shuttling of activated NF-B to the nucleus. TNF induces the recruitment of HOIP, the catalytic component of the linear ubiquitin chain assembly complex (LUBAC), and its substrate NEMO to the outer mitochondrial membrane, where M1- and K63-linked ubiquitin chains are generated. NF-B is locally activated and transported to the nucleus by mitochondria, resulting in an increase in mitochondria-nucleus contact sites in a HOIP-dependent manner. Notably, TNF-induced stabilization of the mitochondrial kinase PINK1 contributes to signal amplification by antagonizing the M1-ubiquitin-specific deubiquitinase OTULIN.

Type I interferons (IFNs) are the major host defence against viral infection and are induced following activation of cell surface or intracellular pattern recognition receptors, including retinoic-acid-inducible gene I (RIGI)-like receptors (RLRs). All cellular processes are shaped by the microenvironment and one important factor is the local oxygen tension. The majority of published studies on IFN signalling are conducted under atmospheric (18%) oxygen conditions, that do not reflect the physiological oxygen levels in most organs (1-5% O2). We studied the effect of low oxygen on IFN induction and signalling in induced Pluripotent Stem Cell (iPSC)-derived macrophages as a model for tissue-resident macrophages and assessed the consequence for Zika virus (ZIKV) replication. Hypoxic conditions dampened the expression of interferon-stimulated genes (ISGs) following RLR stimulation or IFN treatment at early time points. RNA-sequencing and bio-informatic analysis uncovered several pathways including changes in transcription factor availability, the presence of HIF binding sites in promoter regions, and CpG content that may contribute to the reduced ISG expression. Importantly, hypoxic conditions increased ZIKV replication at early time points, emphasizing the importance of understanding how low oxygen conditions in the local microenvironment affect pathogen sensing and host defence.

Macroautophagy is a conserved degradation pathway, which mediates cellular homeostasis by the delivery of harmful substances into lysosomes. This is achieved by the sequestration of these substances referred to as cargo within double membrane vesicles, the autophagosomes, which form de novo. Among the many cargoes that are targeted by autophagy are condensates containing p62 and ubiquitinated proteins. p62 recruits the FIP200 protein to initiate autophagosome formation at the condensates. How FIP200 in turn organizes the autophagy machinery is unclear. Here we show that FIP200 is dispensable for the recruitment of the upstream autophagy machinery to the condensates, but it is necessary for phosphatidylinositol 3-phosphate formation and WIPI2 recruitment. We further find that FIP200 is required for the activation of the ULK1 kinase. Surprisingly, ULK1 kinase activity is not strictly required for autophagosome formation at p62 condensates. Super-resolution microscopy of p62 condensates revealed that FIP200 surrounds the condensates where it spatially organizes ATG13 and ATG9A for productive autophagosome formation. Our data provide a mechanistic insight into how FIP200 orchestrates autophagosome initiation at the cargo.