Endomembrane damage of intracellular vesicles triggers signals that activate membrane repair in mammalian cells to restore homeostasis. However, the signals that drive diverse membrane repair recruitment at the individual organelle level are unknown. Here by recording Ca2+ leakage history with a newly developed Ca2+ probe in human macrophages, we discovered that Ca²⁺ leakage serves as a conserved signal that triggers ATG8/LC3 lipidation after different types of sterile membrane damage. The damaged compartments consisted of both single membrane and multilayered membrane structures undergoing extensive membrane remodelling. We show the complexity and acidification of these ATG8/LC3-positive compartments depends on the nature of the membrane damage trigger. Functionally, the formation of these multimembrane ATG8/LC3-positive compartments restricted membrane damage independently of canonical autophagy and the recruitment of ESCRT components CHMP2A/CHMP4B. Altogether, we show that endolysosomal Ca²⁺ leakage triggers non-canonical LC3 lipidation on damaged membranes to promote membrane repair in human macrophages.

STING is an evolutionarily conserved key regulator of innate immunity. In the model organism Drosophila melanogaster, STING activates the NF-κB-like transcription factor Relish, initially characterized for its role in the antibacterial IMD pathway. The versatile FADD/Caspase-8 axis is widely used in various immune signaling pathways throughout the animal kingdom, including the IMD pathway. Here, we show that it functions downstream of STING in Drosophila to mediate Relish activation by the Caspase-8 homolog DREDD. We present a detailed structural model illustrating how the adapter protein FADD interacts with two separate STING dimers in the activated oligomerized form of STING, thus providing a molecular explanation for the activation-dependent recruitment of FADD. We further show that FADD interacts with IMD in a structurally distinct but functionally related manner, highlighting how the STING and IMD pathways differentially utilize the adapter protein FADD. Our results illustrate how an ancestral module is incorporated into different innate immune pathways, providing insights into the evolution of host-pathogen interactions.

Actin-rich protrusions densely cover the surface of T cells and are well characterised for their role in migration. Recent studies have uncovered their contribution to antigen surveillance and immune signalling. To further explore how protrusions initiate signalling pathways mediating T-cell activation, we performed live-cell imaging of endogenously tagged proteins in HER2-specific chimeric antigen receptor (CAR) T cells targeting HER2⁺ breast-cancer cells. Quantitative STED microscopy allowed us to monitor protein rearrangement and to correlate it with membrane topology over time. Before activation, key signalling proteins (including Lck, CD45, LAT, and the CAR) were not enriched in protrusions. Upon contact with target cells, rapid protein reorganisation occurred preferentially within protrusions, initiating signalling. HER2-CAR clustering, accompanied by ZAP-70 and LAT recruitment, was enhanced in protrusions. While Lck distribution remained unchanged, exclusion of the phosphatase CD45 was enhanced at protrusion-cell contacts, independently of the CAR signalling domain. Overall, signalling machinery rearranged faster and more effectively at protrusive contacts than at main plasma membrane regions. Together, our data re-frame protrusions as sites of enhanced receptor activation by exclusion and clustering dynamics rather than by pre-enrichment of the signalling machinery.

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.

The in vivo mechanism, cis-acting roadblocks, and biological functions of DNA loop extrusion by eukaryotic SMC complexes remain incompletely defined. Here, we identify condensin-dependent Hi-C contact stripes at the recombination enhancer (RE) and at rDNA in S. cerevisiae. The RE is an autonomous condensin loading site only active in MATa cells from which oriented, unidirectional loop extrusion proceeds with an estimated processivity ~150-250 kb and a density ~0.04-0.18 that varies across the cell cycle. Centromeres, replication forks, and highly transcribed RNA PolII-dependent genes represent roadblocks for condensin. Cohesin is not an obstacle for condensin, while Top2 promotes its loop extrusion activity. A DNA double-strand break (DSB) at MAT blocks loop extrusion, resulting in the establishment of a ~170 kb-long RE-MAT loop. The RE and the DSB are required and sufficient to form this site-specific loop, which promotes RE-proximal homology identification in the early stages of recombinational DNA break-repair. We propose that juxtaposition of the broken MATa site and its target HMLα donor is the relevant structure by which condensin promotes a-to-α mating-type switching.

Bacteriophages have evolved diverse inhibitors targeting key bacterial processes, including virulence and anti-phage defense systems, which could inspire novel antimicrobial strategies and enhance phage therapy approaches. In this study, we characterize Dap2, a protein encoded by a Pseudomonas aeruginosa phage PaoP5, which disrupts host virulence by sequestering the type III secretion system (T3SS) transcriptional activator ExsA, thus suppressing bacterial pathogenicity. Furthermore, Dap2 also directly binds the host Lon protease to prevent degradation of the phage-encoded HNH endonuclease. Deletion of dap2 in PaoP5 strongly impairs phage genome packaging due to insufficient levels of HNH. Finally, Dap2 synergizes with its genomically adjacent partner Dap1, a previously identified HNH-binding protein providing partial Lon resistance, to completely protect HNH against degradation. Together, these findings reveal a dual-function phage protein that simultaneously modulates bacterial virulence and anti-phage immunity, and showcase a synergistic mechanism for complete neutralization of bacterial defense system against which individual components provide only partial protection.

Plasmids are the workhorses of molecular biology: fast, flexible, and often taken for granted. We clone, overexpress, tag, and mutate freely, assuming they will faithfully produce RNA transcripts that match the intended DNA sequence. This assumption is rarely tested and often invalidated. Sequences in plasmid backbones, epitope tags, and codon-optimized regions may inadvertently harbor cryptic promoters or splice sites. The resulting unexpected transcripts and proteins, while often undetected, can distort results and propagate false conclusions through papers, grants, and even clinical trials. In this perspective, we highlight published cases where plasmids have distorted results and misled interpretation. We examine the mechanisms and consequences of plasmid-associated expression artifacts and offer practical strategies to minimize them. Finally, we call for a revision of community standards for experiments using transgenes: deposit complete plasmid sequences and verify the resulting transcripts using RNA-seq.

STIM1 is a transmembrane protein localized in the endoplasmic reticulum (ER), where it acts as a calcium ion sensor, activating store-operated Ca2+ entry upon ER Ca2+ 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 Ca2+ transfer, reduces basal mitochondrial Ca2+ levels, impairs maximal mitochondrial respiration, and reduces ATP production. The STIM1-GRP75 interaction depends on STIM1's Ca2+-sensing ability. ER Ca2+ 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.

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.

Cellular and environmental stress triggers the rapid and global reprogramming of gene transcription by coordinated recruitment of a limited number of key inducible transcription factors to cis-regulatory elements. Here, we performed a comprehensive analysis of different stress models and observed that co-induced genes are generally located in close genomic proximity. By integrating gene expression and transcription factor binding resources across different stress models, we identify an enrichment for clusters in which only one of the clusters’ promoters recruits the key transcription factors, reminiscent of Epromoters—a type of cis-regulatory element that displays both promoter and enhancer function. Epromoter-regulated clusters were frequently found regardless of the stress or inflammatory response. Predicted Epromoters displayed enhancer activity and regulated clusters of stress-response genes independently of their genomic location. These findings imply that Epromoters are central regulatory elements that control gene clusters in response to acute perturbations.