The EMBO Journal

In metazoans, mitochondria optimally distribute to sites of need through long-range transport events on microtubules. The prevailing model for this trafficking mechanism is that the tail-anchored calcium-binding GTPase, Miro, recruits cytosolic TRAK and associated molecular motors to the outer mitochondrial membrane. Therefore, Miro is proposed to be an obligate adaptor for TRAK required for bulk mitochondrial transport, a process that is considered particularly important for long-range trafficking in neurons, and thus, for viability. Here, we impaired Miro-TRAK interaction in vivo by introducing a point mutation into the Drosophila TRAK orthologue Milton, that impairs its interaction with Miro, based on recent structural evidence. Flies harbouring this point mutation are viable to adulthood. Moreover, neurons carrying this mutation exhibit little to no observable reduction in axonal mitochondria. Mutant flies, however, display progressive loss of motor function with age and reduced lifespan. We therefore call into question the long-standing view that Miro plays an obligatory role in mitochondrial trafficking and challenge the canonical model for mitochondrial transport.

The mitochondrial Lon protease is essential for proteostasis through ATP-dependent proteolysis and suppression of protein aggregation through an unknown mechanism. Here we show in three independent aggregation systems that human Lon protease (LonP1) directly interacts with fibrillar aggregates to prevent further aggregation: LonP1 binds amyloid fibrils and inhibits their growth, independently of its protease and ATPase activities. This aggregation inhibition depends on hexamer stability, and even the N-domain hexamer of LonP1 lacking all catalytic domains inhibited aggregation, which localizes its fibril-binding interface. We propose that chaperone deficiencies in LonP1 mutants that are associated with genetic disease, are caused by reduced hexamer stability or increased turnover. Our results clarify the observed dual protease and chaperone function of LonP1 by localizing them to different domains and separating the catalytic activities, thereby facilitating targeting the specific functionalities. Further, we identify the structure of the chaperone substrate to be fibrillar aggregates, suggesting that LonP1 may protect against amyloid fibrils in healthy individuals. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=176 SRC="FIGDIR/small/723147v2_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1a8cfforg.highwire.dtl.DTLVardef@11ec6bcorg.highwire.dtl.DTLVardef@1898417org.highwire.dtl.DTLVardef@13f371b_HPS_FORMAT_FIGEXP M_FIG C_FIG SignificanceThe mitochondrial Lon protease has long been proposed to function both as a protease and as a chaperone, though the mechanism of its chaperone activity is debated. Here, we show that human Lon binds to fibrillar protein aggregates and inhibits their elongation, but do not find evidence for chaperoning unfolded chains. Further, our findings challenge the current view that ATPase activity is required for Lon chaperone function. Instead, our results suggest that chaperone deficiency of Lon variants can be explained by variant stability. Our results provide a mechanistic separation of the protease and chaperone-like function LonP1, thereby opening up for targeting one of the functions specifically, and provide new insight into how Lon dysfunction may contribute in multiple ways to age-related and proteostasis-related diseases.

The eukaryotic genome is non-randomly organized within the nucleus, with positioning linked to function. Still, genome-wide radial maps are missing for the majority of experimental model systems. We adapted Genomic loci Positioning by Sequencing (GPSeq) to Saccharomyces cerevisiae, enabling high-resolution mapping along the nuclear center-periphery axis. GPSeq confirms known spatial features and shows that peripheral telomeres and centromeres impose long-range constraints extending up to 200 kb, restricting short chromosome arms from the nuclear interior. Telomere repositioning to the nuclear center, either artificially or during quiescence, reorganizes much of the genome through inward movement of sub telomeric regions and compensatory shifts of mid-arm chromatin outward. In quiescence, reduced centromere peripheral localization further alters genome organization. While transcription has a modest impact on radial positioning in all studied conditions, we uncover that in the absence of centromere or telomere constraints, GC-content functionally organizes chromatin in the nucleus. Graphical abstractThe budding yeast genome is spatially organized in a manner highly dependent on the positioning of centromeres (CENs) and telomeres (TELs). Anchoring of these chromosome landmarks constrains the positioning of adjacent chromatin up to 200 kb within the same radial zone. Beyond this range, genome organization is non-random, with processes like transcription and features such as GC- content associated with specific radial positions in the nucleus. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/722996v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@843adborg.highwire.dtl.DTLVardef@1343eb3org.highwire.dtl.DTLVardef@1009b03org.highwire.dtl.DTLVardef@c10245_HPS_FORMAT_FIGEXP M_FIG C_FIG

In Plasmodium falciparum, DNA replication and asynchronous nuclei formation precede cytokinesis during intraerythrocytic schizogony. Inhibition of fatty acids (FAs) import and impaired membrane biogenesis led to the arrest of mitosis through the inhibition of DNA replication and nuclei formation. On the iRBC surface, parasite ribosomal protein P2 (PfP2) complex mediated FAs import and membrane biogenesis, seemingly prior events before the commitment for DNA replication and nuclei formation. The inhibition of FAs import led to the degradation of histones by the evolutionarily conserved bacterial serine protease ClpP/ClpR in the parasite nucleus. Noncanonical arginine hyperphosphorylation by a novel arginine kinase in the nucleus was subjected for proteostasis and marks histones for degradation by ClpP/ClpR machinery. Inhibition of de novo FAs biosynthesis by an anti-cancer drug, Cerulenin and C75, in HEK293T and HCT116 carcinoma mammalian cells showed histone degradation. Lipid (L) induced histone proteostasis by ClpP/ClpR, seemingly an indispensable L-checkpoint before mitotic commitment.

X-chromosome inactivation, the mechanism for dosage compensation in mammals, is orchestrated by the long non-coding RNA Xist which localises across the X chromosome in cis. The basis for in cis localisation remains poorly understood. To investigate in situ Xist localisation, we established MCPH1-deficient cell models that retain compacted interphase chromosomes, enabling visualisation of individualised chromosome territories. We find that Xist RNA is directed to sites around the periphery of compacted chromosome territories, and moreover that these sites correlate closely with A1-sub-compartments on the X chromosome. We further show that peripheral positioning of A1-sub-compartments occurs on all chromosomes and is a hallmark of early prophase. A key role for compartment interactions in Xist localisation is further supported by analysis of MCPH1-deficient models where Xist is overexpressed (from the X chromosome or an autosomal Xist transgene), and following depletion of HNRNPU, a key factor that anchors Xist RNA to chromosome territories.

Autophagy is a critical cellular process, yet its genomic definition remains inconsistent across digital repositories. This lack of standardisation hinders reproducibility in high-throughput studies and clinical research. Here, we present Au_Sus, a high-confidence human autophagy census established through a frequency-based majority consensus of seven primary databases and literature sources. After rigorous manual curation and nomenclature standardisation, we defined a tiered framework: Maxim_Au (2,581 genes), Au_Sus (the 201-gene core consensus), and Minim_Au (77 universal genes). Functional enrichment and protein-protein interaction analysis confirm that Au_Sus captures a highly integrated and purified autophagic machinery, with significant associations to neurodegeneration and oncology. Furthermore, an analysis of nearly 100 published cancer gene signatures revealed profound functional dilution, with 60% of signature genes absent from our consensus. These findings suggest that many of these models incorporate peripheral stress markers rather than core autophagic effectors. Hence, Au_Sus (freely accessible at ausis.uniovi.es) provides a reliable, ready-to-use benchmark to standardise the study of autophagy in health and disease.

Eukaryotes use several distinct quality control pathways to resolve aberrant ribosomes and mRNAs. For example, the no-go decay mRNA pathway is stimulated after ribosome collisions caused by stalled ribosomes translating damaged or truncated mRNAs. Separate decay pathways for non-functional 40S and 60S subunits containing rRNA mutations affecting decoding and peptidyl transferase activity, respectively, have also been elucidated. To our knowledge, whether eukaryotes have evolved a quality control pathway to sense and process globally stalled ribosomes is unclear; however, such a pathway would be advantageous to eukaryotes during exposure to natural elongation inhibitors such as ricin and diphtheria toxin. Here, we test how prolonged robust inhibition of elongation using a high dose of cycloheximide (CHX) affects ribosome turnover. Despite no decrease in cell viability and that mammalian ribosomes have been classically characterized of having a half-life of 3-5 days, a single 24 hr high dose of CHX resulted in drastically shortened half-lives (<24 hr) of 28S and 18S rRNA in A549 cells. A [~]2-fold reduction in nearly all ribosome species was observed by polysome analysis in HeLa and A549 cells after prolonged CHX treatment. Depletion of ribosomes was also evident when assessing ribosomal proteins from both the 40S and 60S subunits by Western blot. Literature supports that ribosomes can be degraded by autophagy and the ubiquitin (Ub)-proteasome system. Upon testing inhibitors of both pathways, only proteasome inhibitors (i.e., MG132 and bortezomib) rescued both rRNA and ribosomal protein levels. Proteasome inhibitors also rescued ribosome levels in polysome profiling experiments. Remarkably, rRNA levels were not rescued during CHX treatment when co-treated with the Ub activating enzyme E1 inhibitor, TAK243. Polysome analysis also showed that the high prolonged dose of CHX did not cause robust accumulation of collided ribosomes compared to control treatments. Proteasome-dependent turnover of rRNA was also observed with high doses of other elongation inhibitors, namely anisomycin, homoharringtonine, and lactimidomycin. The recognition capabilities of the pathway were further expanded as we observed that 80S ribosomes not trapped on the mRNA were also targeted for degradation by the proteasome. Together, our findings define the framework of a regulatory pathway in mammalian cells that degrades both ribosomal subunits in response to prolonged periods of robust elongation inhibition.

Dormancy is a reversible cellular state characterised by suspended proliferation and increased stress resilience, enabling long-term viability under adverse conditions. Although dormant cells are critical for the life cycle of diverse organisms, from microbes to humans, they are understudied compared to proliferating cells. We present a comparative investigation of dormant cells in two divergent species: spores of fission yeast and diapause embryos of turquoise killifish. A genome-wide screen for genes affecting the lifespan and heat-shock resilience of spores uncovered a trade-off between longevity and heat resistance, and considerable differences in the genetic basis for lifespan between spores and chronologically aging yeast cells. RNA-seq and mass-spectrometry analyses revealed substantial transcriptomic and proteomic changes in spores and diapause embryos, with ribosomal proteins induced as transcripts but repressed as proteins. Transcriptomic regulation of biological processes, but less so of specific genes, is broadly conserved across yeast spores, killifish diapause, and human dormant cancer cells, including the induction of autophagy- and translation-related processes and the repression of cell cycle-related processes. Spores and diapause embryos modulate their transcriptomes and proteomes in response to heat stress and prolonged time. These RNA and protein expression changes are uncoupled and differ from aging-related expression signatures in yeast cells and adult fish. Cells derived from older or stressed spores retain phenotypic differences for several cell divisions, reflected in altered expression signatures, lifespan and stress resilience. Similarly, diapause duration and heat exposure are associated with long-term expression signatures in post-diapause embryos before hatching. This study highlights core biological processes and principles that are remarkably conserved in distinct types of dormant cells.

Sleep is a conserved animal behavior necessary for survival. It is under tight circadian and homeostatic control, and modulated by diet. Here, we identify the amino acid transporter ANIDRA (ANID) as an important sleep regulator in Drosophila. Flies lacking ANID show decreased and poorly consolidated daytime and nighttime sleep. Contrary to wild-type controls, anid mutant flies are unable to adjust their sleep to their diet, behaving as if they were constantly on a complete diet rich in amino acids. ANID is expressed in ensheathing and cortex glia, where it inhibits mTOR activity in a diet-dependent manner. Moreover, pharmacological inhibition of mTOR attenuates the anid mutant sleep phenotypes. Interestingly, DH44-expressing brain neurons, which promote arousal and sense amino acids, are constantly active in ANIDs absence. We therefore propose that ANID mediates detection of dietary amino acids by ensheathing and cortex glia to regulate the activity of arousal-promoting neurons.

Hepatocytes undergo extensive proliferation to facilitate liver repair after injury, yet early adaptive changes prior to proliferation remain unclear. Here, we report that during early acetaminophen (APAP)-induced liver injury, hepatocytes exhibit transient proliferation suppression, most pronounced in mid-zone hepatocytes due to zonal APAP metabolism. Using spatial transcriptomics (ST), immunohistochemistry, and functional studies, we identified a unique mid-zone stress-response program. Central to this adaptation is the Atf4-Chop axis, which actively suppresses proliferation via the cell cycle inhibitor Btg2, prioritizing cytoprotection over cell division. This transient arrest is a critical survival strategy: halting energy-intensive proliferation during peak injury allows mid-zone hepatocytes to redirect resources towards protection, enhancing their survival in early APAP-induced liver injury. Thus, Atf4-Chop-mediated quiescence preserves a hepatocyte reservoir necessary for subsequent regenerative proliferation and effective repair. Our findings reveal a key adaptive trade-off in mid-zone hepatocytes where transient proliferation arrest promotes early survival to enable repair.

Antisense oligonucleotides (ASOs) enter cells efficiently, but the compartment from which productive escape occurs remains uncertain. We used live-cell microscopy, ratiometric pH measurements and 3D focused ion beam scanning electron microscopy (FIB-SEM) in U2OS cells to track a Malat1-targeting ASO from uptake to delivery. The ASO entered by endocytosis and accumulated in late endosomes, endolysosomes and lysosomes, where it induced luminal neutralization without galectin-3 recruitment or limiting-membrane rupture. Under conditions that reduced Malat1-RNA by >90%, quantitative imaging showed that less than 4% of internalized ASOs reached the nucleus. L-leucyl-L-leucine methyl ester (LLOMe)-induced membrane damage released co-internalized dextran but not ASOs, showing that ASOs remain sequestered even in damaged late endocytic compartments. In apilimod-expanded organelles, ASOs concentrated at limiting membranes and intraluminal foci with constrained motion, consistent with association with membrane and luminal structures. Although G3BP1/2 has been proposed to plug damaged endocytic membranes, we detected no recruitment of G3BP1 to endosomes or lysosomes; loss of G3BP1 and G3BP2 increased functional delivery modestly. We therefore propose that productive escape occurs earlier in endocytosis, most likely in early or recycling endosomes, where ASOs would still be unbound within the lumen and where membrane fusion and fission could generate perforations permitting release.

Mosquitoes undergo major metabolic remodeling as they transition from aquatic larvae to terrestrial, blood-feeding adults, yet the biochemical principles supporting these life cycle transitions remain poorly understood1. Early metabolic studies established that mosquitoes are sterol auxotrophs2,3; however, the extent to which this auxotrophy influences developmental progression and stage transitions remains unknown. Here, using sterol-defined culture systems, we uncover a central and stage-specific role for sterol metabolism in mosquitoes. We show that eggs of multiple mosquito species, from Anopheles gambiae (Giles 1902; hereafter A. gambiae) to Aedes species in both wild and laboratory settings, are enriched in cholesterol consistent with maternal provisioning. In the Asian tiger mosquito Aedes albopictus (Skuse, 1894; hereafter Ae. albopictus), egg cholesterol supports early larval development, after which larvae depend on the acquisition of dietary sterols to complete development. Like cholesterol, dietary plant- and fungal-derived sterols support late larval development; however, their utilization is associated with the accumulation of the intermediate sterol desmosterol. Using biochemical assays, we showed that desmosterol is converted to cholesterol through the activity of the Ae. albopictus DHCR-24 enzyme. This metabolic axis of desmosterol-to-cholesterol conversion supports late larval development when mosquitoes rely on plant- and fungal-derived sterols for development. dhcr-24 expression is upregulated during the developmental window of dietary sterol acquisition and is selectively induced by its substrate desmosterol, revealing a diet-encoded regulatory mechanism that coordinates metabolic conversion capacity. Extending the analysis to adulthood, we developed a sterol-defined artificial blood-feeding system that enables precise manipulation of dietary sterols in reproductive females and show that cholesterol availability in the diet of females is an essential metabolic driver of egg laying. These findings reveal a developmentally coordinated sterol-use strategy in the life cycle of Aedes mosquitoes, identifying sterol utilization as a central physiological axis linking development, blood feeding, and reproduction, and revealing a sterol-dependent metabolic vulnerability in mosquitoes. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=178 SRC="FIGDIR/small/725405v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@13c7843org.highwire.dtl.DTLVardef@fb9203org.highwire.dtl.DTLVardef@2f0db8org.highwire.dtl.DTLVardef@12db355_HPS_FORMAT_FIGEXP M_FIG C_FIG

Inflammation increases with aging and contributes to neurodegeneration, yet the principles that determine how immune effectors target host neural tissue remain poorly understood. Antimicrobial peptides (AMPs) are central components of innate immune defenses strongly induced upon infection and upon aging. Studies have shown that AMPs can exhibit cytotoxicity toward host cells, pointing to a role in neurodegeneration. We show that cationic AMPs selectively bind and damage motoneurons that expose phosphatidylserine (PS), an anionic phospholipid normally restricted to the inner leaflet of the plasma membrane. Both infection and aging increase neuronal PS exposure alongside AMP expression. AMP binding occurs in a PS-dependent manner, leading to synaptic bouton fragmentation, accelerated neuronal aging, and locomotor decline. This toxicity is prevented in Drosophila by Turandot proteins, which reduce AMP-PS interactions on motoneurons. Together, our findings define a molecular mechanism underlying neuronal susceptibility to immunopathology and a set of proteins with neuroprotective potential. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=179 HEIGHT=200 SRC="FIGDIR/small/721952v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@ca817aorg.highwire.dtl.DTLVardef@fa8cb0org.highwire.dtl.DTLVardef@12a8bd4org.highwire.dtl.DTLVardef@423907_HPS_FORMAT_FIGEXP M_FIG This study shows that infection or dysbiosis in Drosophila can simultaneously induce antimicrobial peptide expression while promoting phosphatidylserine (PS) exposure on neurons at the neuromuscular junction. Cationic antimicrobial peptides contribute to neurodegeneration by binding to neurons that expose negatively charged phospholipids such as PS. In Drosophila, a family of secreted peptides, the Turandot proteins, can protect the peripheral nervous system by binding to PS-exposed membranes. Together, these findings reveal a role for antimicrobial peptides, a key component of innate immunity, in promoting neurodegeneration as well as a potential protective mechanism by PS masking agent. C_FIG

Citrate is a central metabolite linking tricarboxylic acid (TCA) cycle activity to energy and lipid metabolism and supports the synthesis of inflammatory mediators, including itaconate, in macrophages. While citrate is primarily generated endogenously, extracellular citrate levels are elevated under pathological conditions such as citrate transporter disorder. Cells import extracellular citrate through SLC13 transporters, including the sodium-dependent citrate transporter NaCT (encoded by SLC13A5). However, whether macrophages take up extracellular citrate and how this affects metabolism and function remains unclear. Here, we combined mass spectrometry and tracing approaches to investigate the metabolic fate of citrate in human macrophage cell lines, primary, and iPSC-derived macrophages. We demonstrate that cells take up extracellular citrate, which was enhanced under metabolic stress conditions. Exogenous citrate was not substantially utilized as a carbon source but selectively altered glutamine metabolism and responses to bacterial infection with Salmonella enterica Typhimurium and Legionella pneumophila Corby. Our work identifies extracellular citrate as a context-dependent regulator in macrophages that decouples uptake from metabolic utilization. HighlightsO_LIMacrophages import extracellular citrate via SLC13 transporters C_LIO_LIExtracellular citrate accumulates under hypoxia and inflammatory activation C_LIO_LIExtracellular citrate does not fuel central carbon metabolism in human macrophages C_LIO_LICitrate modulates glutamine immunometabolism and modulates immune responses C_LI eTOC blurbVo{beta}-Willenbockel et al. demonstrate that human macrophages accumulate extracellular citrate without using it as a major carbon source. Instead, citrate modulates glutamine utilization, inflammatory responses, and host-pathogen interactions revealing a context-dependent regulatory role for extracellular metabolites in immune cell function.

The nuclear lamina meshwork is composed of intermediate filaments, referred to as lamins, which function to maintain nuclear integrity and organize the Lamina-Associated chromatin Domains (LADs). Studies have shown lamins support cardiomyocyte nuclear integrity, maturation, and differentiation, as well as epicardial migration. Yet, how these functions are integrated with gene expression programs and cell cycle control during heart development is unknown. We show lamin-A and -B1 are required in cardiomyocytes for perinatal mouse survival. Importantly, lamin-B1 enables cardiomyocyte maturation by maintaining LADs and chromosome territories. By examining changes in cardiomyocyte gene expression and 3D genome organization upon lamin-B1 deletion, we show lamin-B1 maintains chromatin neighborhoods, which can in turn support transcription factors to regulate genes involved in cardiomyocyte structural maturation and gradual cessation of cell division. These findings shed light on the genomic logic by which a nuclear lamina protein can collaborate with transcription factors to promote cell maturation while repressing cell cycle. Highlights1. Lamin-A/B1 dose-dependently set cardiomyocyte nuclear order and postnatal survival. 2. Lamin-B1 loss delays cardiomyocyte maturation and cell cycle exit programs. 3. Lamin-B1 shapes chromatin neighborhoods to gate maturation/cell cycle exit programs.

Cell polarity complexes are essential for embryogenesis, but their regulatory mechanisms during early developmental transitions remain incompletely understood. Here, we individually deleted the Crumbs, Par, and Scrib polarity complexes in mouse embryonic stem cells (mESCs). While loss of any single complex did not affect pluripotency or proliferation, deletion of Par complex disrupted the naive-to-primed transition and impaired subsequent differentiation, particularly lumen formation in neural tube organoids. Mechanistically, Par complex deficiency led to hyperphosphorylation of focal adhesion kinase (FAK) at the primed stage, driving a morphological shift from flat monolayer clusters to dome-shaped colonies. FAK inhibition rescued the aberrant morphology. Upstream, Par complex loss increased AKT phosphorylation, which remodeled extracellular matrix (ECM) and regulated integrin signaling via FURIN-LEFTY, ultimately modulating FAK activity. In addition, conditioned medium from wild-type cells partially rescued differentiation defects in Par knockout cells in a LEFTY-dependent manner. These phenotypes were consistently observed in naive-to-primed transition, neural stem cell differentiation, embryoid body formation, teratoma assays, and neural tube organoid differentiation. Together, these findings establish a Par complex-AKT-FURIN-LEFTY-ECM-integrin-FAK signaling cascade that links apical-basal polarity to early lineage specification and morphogenesis, providing a mechanistic framework for how polarity cues are translated into developmental outcomes. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/722465v1_ufig1.gif" ALT="Figure 1"> View larger version (64K): org.highwire.dtl.DTLVardef@1c62c46org.highwire.dtl.DTLVardef@184d5b5org.highwire.dtl.DTLVardef@1ea9017org.highwire.dtl.DTLVardef@9a0318_HPS_FORMAT_FIGEXP M_FIG C_FIG Significance StatementThis study elucidates the molecular mechanism by which the Par complex regulates the establishment of cell polarity. The authors demonstrate that the Par complex promotes the expression of the protein convertase FURIN via AKT signaling, thereby enhancing the maturation and secretion of LEFTY protein. This process remodels the ECM and modulates integrin signaling, ultimately regulating FAK activity and controlling the establishment of cell polarity. These findings reveal how polarity cues govern early lineage specification and morphogenesis, with implications across multiple developmental contexts.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease of poor prognosis, for which age is the strongest risk factor. Despite significant progress in the discovery of ALS-associated mutations, no model explains how such a diversity of mutations converges in a common pathology. In addition, most ALS cases are sporadic and lack known genetic drivers. We recently reported that arginine-rich peptides arising from the C9ORF72 mutation trigger a widespread accumulation of orphan ribosomal proteins (oRP). Here, we show that oRP accumulation is also observed upon expression of other RNA-related ALS mutations, such as hnRNPA2D290V and TDP-43A315T, as well as upon exposure to the ALS-related neurotoxin {beta}-N-methylamino-L-alanine (BMAA). Furthermore, the transcriptional signature of patients with sporadic ALS resembles that of Diamond-Blackfan anemia (DBA), a known ribosomopathy. Supporting the usefulness of our in vitro data, a transcriptional signature defined from these models provides diagnostic and prognostic value in ALS patients. We propose that the accumulation of oRPs due to dysfunctional ribosome biogenesis is a molecular hallmark of ALS that can contribute to the progressive loss of motor neurons in the disease.

Microbial effectors often act in the apoplast or cytosol, but how they reach host organelles during beneficial plant-fungal interactions remains poorly understood. Here, we identify a class of secreted prion-like effector proteins from the mutualistic root endophyte Serendipita indica, termed PEPSIs. These proteins contain prion-like domains (PrLDs) embedded within intrinsically disordered regions, and three tested PEPSIs require these domains for plastid localization. Focusing on PEPSI6, we show that plastid targeting depends on its PrLD and is associated with engagement of plastid import and proteostasis machinery. In plastids, PEPSI6 associates with 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), promotes DXR accumulation, alters methylerythritol phosphate pathway metabolites, and enhances tolerance to the DXR inhibitor fosmidomycin. PEPSI6 also undergoes apoplastic C-terminal CAP-domain processing, and its colonization-promoting activity persists after PrLD deletion, indicating a plastid-targeting-independent function. Overall, this work identifies PrLDs as noncanonical plastid-targeting elements in PEPSI effectors and reveals DXR as a target of fungal manipulation during root symbiosis. HighlightsO_LIPrion-like domains control plastid targeting of selected PEPSI effectors. C_LIO_LIPEPSI6 associates with DXR and promotes its accumulation in plastids. C_LIO_LIPEPSI6 alters MEP-pathway metabolites and increases tolerance to fosmidomycin. C_LIO_LIApoplastic processing reveals a plastid-independent PEPSI6 activity during root symbiosis. C_LI

Susceptibility to viral infection varies widely but is not fully explained by genetics, immune status, or exposure level. We show that time of day strongly influences infection outcome, with up to 100-fold differences in enteric viral burden depending on infection timing. This temporal gating is abolished in mice lacking a functional circadian clock. We identify the antiviral transcription factor IRF1 as a direct target of the circadian transcription factor BMAL1, resulting in rhythmic expression of a basal antiviral gene program prior to infection. Loss of IRF1 eliminates this program and abrogates time-of-day-dependent differences in viral replication. This circuit operates within intestinal myeloid cells, establishing a preexisting antiviral state. These findings indicate that the circadian clock programs host susceptibility in the intestine, before infection occurs.

Directed cell migration is a vital process that depends on the combined activities of attractive and repulsive cues. As it is essential for normal development, the precise identity of guidance signals and the underlying molecular and cellular mechanisms is being rigorously investigated. In a Drosophila embryo, PGC migration is orchestrated by non-cell autonomous repulsive and attractive cues, controlled by Wunen(s) - Wunen and Wunen2 and, HMGCoA-reductase (Hmgcr), respectively. Hedgehog (Hh), a PGC attractant, is potentiated by Hmgcr. We demonstrate that Wunen(s) employ both nonautonomous and autonomous modes to inhibit Hh signaling. Consistently, in embryos maternally compromised for wunen, mesodermal cells and PGCs accumulate excess Hh, leading to precocious clumping of the PGCs. This behaviour is reminiscent of PGC-specific loss of patched (ptc) - the Hh receptor and an antagonist of Smoothened (Smo), a G protein-coupled receptor (GPCR), involved in Hh signal transduction. Consistently, Wunen(s) inhibit membrane localization of Smo. Conversely, simultaneous overexpression of wunen mitigates PGC scattering induced by ectopic hmgcr expression. Finally, unbiased lipidomics of embryonic extracts after maternal knockdown of wunen confirms disruptions in lipid metabolism. We discuss the mechanistic underpinnings of Wunen(s) involvement in repressing Hh signalling to engineer PGC migration.