Biology subjects

Locker, N.

Publications and source records attributed to Locker, N..

12 recordsLinked to original sources

bioRxiv2026

Capillary-based Subcellular Sampling Uncovers the Stress Granule Proteome in Single Cells

Many diseases arise from dysfunction within specific organelles or biomolecular condensates, highlighting the value of analysing proteins at subcellular resolution to uncover new biological mechanisms. We report a novel capillary-based subcellular sampling workflow coupled with liquid chromatography-mass spectrometry (LC-MS) for proteomic analysis of defined subcellular regions of individual cells. We applied this methodology to stress granules (SGs), membrane-less biomolecular condensates that form in response to cellular stress (including viral infection), and are implicated in infection, neuropathology and cancer. Comprehensive characterisation of SG protein composition remains limited by technical challenges associated with bulk purification, including loss of spatial context, dynamic behaviour and contamination from cytosolic material. Using our novel method, we identified a high-confidence set of 405 SG-associated proteins, including 46 established SG residents alongside numerous previously unreported candidates. Functional enrichment analysis revealed pathways consistent with known SG biology, while comparison with an independent cytosolic proteome dataset demonstrated minimal overlap, supporting the specificity of the sampling strategy. Selected novel SG protein candidates (AHNAK2, DDX39B, NUDT1 and FKBP2) were validated using immunofluorescence microscopy. These findings establish capillary-based subcellular sampling as a viable approach for proteomic analysis of SGs with preserved spatial context and provide a framework for analysing other subcellular compartments. Table of contentsWe report an LC-MS-based capillary sampling workflow for proteomic analysis of subcellular structures within single cells. This methodology identified 405 high-confidence stress granule-associated proteins, including 46 previously established and numerous novel candidates. The approach demonstrated high specificity and preserved spatial context, expanding the capabilities of subcellular proteomics. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=55 SRC="FIGDIR/small/724230v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1fa0bb0org.highwire.dtl.DTLVardef@1158524org.highwire.dtl.DTLVardef@1d82812org.highwire.dtl.DTLVardef@2ee4d9_HPS_FORMAT_FIGEXP M_FIG C_FIG Figure made in Biorender.com.

cell biology↗

bioRxiv2025

Compositional analysis of yellow fever virus induced stress granules reveals a functional connection to mitochondrial homeostasis

The phase separation of biomolecules into so-called stress granules (SGs) allows the cell to tightly regulate translation activity in response to different stimuli, such as oxidative stress, starvation, or the recognition of non-cellular RNA. Recent reports suggest SGs induced during viral infection, may act as a crossroad between the cellular stress response and the activation of the innate immune response. Here, we aimed to dissect the role of SGs in the context of yellow fever virus (YFV) infection. We found that YFV infection resulted in translational shut-off from 24 hpi on followed by the formation of SGs at 48 hpi, a delay potentially associated with the ability of the YFV capsid to inhibit SG formation, through an interaction with the major SG scaffolding protein G3BP1. To elucidate the role of YFV-induced SGs during infection, we inhibited SG assembly using a small-molecule inhibitor and find that SG formation does not influence viral replication. Uncovering the first proteome of virus-induced SGs, our compositional analysis revealed a specific enrichment of proteins associated with mitochondrial processes in YFV-induced SGs. Indeed, we show that YFV infection results in mitochondrial damage and dysfunction. Together, we propose that YFV-induced SGs may be involved not only in the modulation of cellular homeostasis but also in influencing mitochondrial functions. Author SummaryViruses impose a major burden on the infected host - from structural rearrangements needed to assemble replication complexes, to exploiting cellular energy resources and genetic rewiring associated with antiviral responses. The assembly of membrane-less organelles such as stress granules (SGs) enable cells to rapidly tune cellular processes upon sensing of stresses such as viruses. Moreover, the cells innate immune response is proposed to be regulated by SGs and in turn many viruses disrupt or highjack their components. Yet, the molecular basis for SG functions during infection remain ambiguous. Here we investigated the interplay between yellow fever virus (YFV) infection and SGs. We demonstrate that infection with attenuated or pathogenic viruses result in the formation of SGs. Their compositional analysis reveal that they sequester mitochondrial proteins, correlating with altered mitochondrial functions during infection. This highlights a novel complex interplay between membrane-bound and membrane-less organelles which could present novel opportunities for antiviral therapies.

microbiology↗

bioRxiv2025

The assembly of stress granule-like foci during foot-and-mouth disease virus infection is uncoupled from activation of cellular intrinsic antiviral signalling

Foot-and-mouth disease virus (FMDV) is highly contagious among cloven-hoofed animals and poses a major threat to the livestock industry worldwide. A fundamental gap in knowledge for high consequence viruses such as FMDV is understanding how the virus evolved to evade cellular antiviral responses. FMDV belongs to the Picornaviridae, a family of positive-sense single-stranded RNA viruses. The detection of viral double-stranded viral RNA intermediates during infection can trigger both the assembly of cytoplasmic stress granules (SGs) and the activation of the RIG-I-like receptors (RLR)-mediated innate immune response (IIR). FMDV has been proposed to antagonize these mechanisms, suggesting that both can limit viral replication. In this study, we investigate the dynamic and importance of SG assembly for IIR activation upon dsRNA stimulation or FMDV replication in porcine epithelial kidney cells. First, we show that the formation of SG following a challenge with poly(I:C), a viral dsRNA mimic, does not modulate the activation of IIR. Our data further reveal transient assembly of SG during FMDV infection followed by virus-induced cleavage of G3BP1, a core SG protein. While SG assembly does not impact viral replication or antiviral response activation, we demonstrate that preventing their disassembly negatively impacts FMDV replication. Overall, our data suggests that while SGs assembly is uncoupled from IIR activation, manipulating their disassembly limits replication and might serve as a potential therapeutic target to prevent FMDV infection. ImportanceBiomolecular condensates, including stress granules, are key regulatory compartments that control fundamental cellular processes. By condensing RNAs and proteins, these structures enable cells to rapidly adapt to stress, such as viral infection. Different biomolecular condensates with specific dynamics or compositions have been described during infection, and many viruses are known to disrupt or highjack their components. Moreover, the cell-autonomous innate immune response is proposed to be regulated by biomolecular condensates. However, the molecular mechanisms underpinning these functions remain unclear and controversial. Here we investigated the interplay between stress granules and the innate response upon the stimulation with a viral dsRNA mimic or foot-and-mouth disease virus infection. We demonstrate that infection triggers the formation of stress granules independently from activation of innate signalling pathways. We also show that stress granules persistence attenuates viral replication posing them as part of the cells response that viruses must overcome or subvert to replicate.

microbiology↗

bioRxiv2023

GADD34 mRNA turnover controls the molecular memory of cellular stress experience

The integrated stress response (ISR) is a key cellular signaling pathway activated by environmental alterations that represses protein synthesis to restore homeostasis. To prevent sustained damage, the ISR is counteracted by the upregulation of Growth Arrest and DNA Damage inducible 34 (GADD34), a stress-induced regulatory subunit of protein phosphatase 1 that mediates translation reactivation and stress recovery. Here, we uncover a novel ISR regulatory mechanism that post-transcriptionally controls the stability of PPP1R15A mRNA encoding GADD34. We established that the 3 untranslated region of PPP1R15A mRNA contains an active AU-rich element recognized by proteins of the ZFP36 family, promoting its rapid decay under normal conditions and stabilization for efficient expression of GADD34 in response to stress. We identify the tight temporal control of PPP1R15A mRNA turnover as a key step in establishing the ISR molecular memory, which sets the threshold for cellular responsiveness and mediates adaptation to repeated stress conditions.

cell biology↗

bioRxiv2023

Activation of the mitochondrial unfolded protein response regulates the dynamic formation of stress granules

To rapidly adapt to harmful changes to their environment, cells activate the integrated stress response (ISR). This results in an adaptive transcriptional and translational rewiring, and the formation of biomolecular condensates named stress granules (SGs), to resolve stress. In addition to this first line of defence, the mitochondrial unfolded protein response (UPRmt) activates a specific transcriptional programme to maintain mitochondrial homeostasis. We present evidence that SGs and UPRmt pathways are intertwined and communicate. UPRmt induction results in eIF2 phosphorylation and the initial and transient formation of SGs, which subsequently disassemble. The induction of GADD34 during late UPRmt protects cells from prolonged stress by impairing further assembly of SGs. Furthermore, mitochondrial functions and cellular survival are enhanced during UPRmt activation when SGs are absent, suggesting that UPRmt-induced SGs have an adverse effect on mitochondrial homeostasis. These findings point to a novel crosstalk between SGs and the UPRmt that may contribute to restoring mitochondrial functions under stressful conditions. Summary statementWe describe a novel crosstalk between the mitochondrial unfolded protein response and the integrated stress response involving stress granules that protects cells from further stress.

cell biology↗

bioRxiv2022

The Batten disease protein CLN3 is important for stress granules dynamics and translational activity.

The assembly of membrane-less organelles such as stress granules (SGs) is emerging as central in helping cells rapidly respond and adapt to stress. Following stress sensing, the resulting global translational shutoff leads to the condensation of stalled mRNAs and proteins into SGs. By reorganising cytoplasmic contents, SGs can modulate RNA translation, biochemical reactions and signalling cascades to promote survival until the stress is resolved. While mechanisms for SG disassembly are not widely understood, the resolution of SGs is important for maintaining cell viability and protein homeostasis. Mutations that lead to persistent of aberrant SGs are increasingly associated with neuropathology and a hallmark of several neurodegenerative diseases. Mutations in CLN3 are causative of juvenile neuronal ceroid lipofuscinosis (JNCL), a rare neurodegenerative disease affecting children. CLN3 encodes a transmembrane lysosomal protein implicated in autophagy, endosomal trafficking, metabolism, and response to oxidative stress. Using a HeLa KO model, we now show that CLN3KO is associated with an altered metabolic profile, reduced global translation, and altered stress signalling. We further demonstrate that loss of CLN3 results in perturbations in SG dynamics, resulting in assembly and disassembly defects, and altered expression of the key SG nucleating factor G3BP1. With a growing interest in SG-modulating drugs for the treatment of neurodegenerative diseases, novel insights into the molecular basis of CLN3 Batten disease may reveal avenues for disease-modifying treatments for this debilitating childhood disease.

molecular biology↗

bioRxiv2021

Paracrine granules are cytoplasmic RNP granules distinct from stress granules assembling in response to viral infection.

To rapidly respond and adapt to stresses, such as viral infections, cells have evolved several mechanisms, which include the activation of stress response pathways and the innate immune response. These stress responses result in the rapid inhibition of translation and condensation of stalled mRNAs, together with RNA-binding proteins and signalling components, into cytoplasmic biocondensates called stress granules. Increasing evidence suggests that stress granules contribute to antiviral defense and thus viruses need to evade these response pathways to propagate. In addition, the stress granule pathway is proposed to be dynamic and adaptable to specific stresses. We previously showed that Feline Calicivirus (FCV) impairs SGs assembly by cleaving the scaffolding protein G3BP1. We also observed that uninfected bystander cells assembled G3BP1-granules, suggesting a paracrine response trigged by the infection. We now present evidence that virus-free supernatant generated from infected cells can induce the formation of paracrine granules. They are different from canonical stress granules and exhibit specific kinetics of assembly-disassembly, protein and RNA composition and are linked to antiviral activity. We propose that this paracrine induction reflects a novel cellular defence mechanism to limit viral propagation and promote stress responses in bystander cells. Summary statementWe describe a novel type of paracrine induced RNA granules associated with viruses, highlighting how different stresses results in heterogeneous stress granule-like condensates with specific cellular functions.

cell biology↗

bioRxiv2021

Murine Norovirus infection results in anti-inflammatory response downstream of amino acids depletion in macrophages

Murine norovirus (MNV) infection results in a late translation shut-off, that is proposed to contribute to the attenuated and delayed innate immune response observed both in vitro and in vivo. Recently, we further demonstrated the activation of the eIF2 kinase GCN2 during MNV infection, which has been previously linked to immunomodulation and resistance to inflammatory signalling during metabolic stress. While viral infection is usually associated with activation of dsRNA binding pattern recognition receptor PKR, we hypothesised that the establishment of a metabolic stress in infected cells is a proviral event, exploited by MNV to promote replication through weakening the activation of the innate immune response. In this study, we used multi-omics approaches to characterise cellular responses during MNV replication. We demonstrate the activation of pathways related to the integrated stress response, a known driver of anti-inflammatory phenotypes in macrophages. In particular, MNV infection causes an amino acid imbalance that is associated with GCN2 and ATF2 signalling. Importantly, this reprogramming lacks the features of a typical innate immune response, with the ATF/CHOP target GDF15 contributing to the lack of antiviral responses. We propose that MNV-induced metabolic stress supports the establishment of host tolerance to viral replication and propagation. ImportanceDuring viral infection, host defences are typically characterised by the secretion of pro-inflammatory autocrine and paracrine cytokines, potentiation of the IFN response and induction of the anti-viral response via activation of JAK and Stat signalling. To avoid these and propagate viruses have evolved strategies to evade or counteract host sensing. In this study, we demonstrate that murine norovirus controls the antiviral response by activating a metabolic stress response that activates the amino acid response and impairs inflammatory signalling. This highlights novel tools in the viral countermeasures tool-kit, and demonstrates the importance of the currently poorly understood metabolic reprogramming occurring during viral infections.

microbiology↗

bioRxiv2021

Electrochemical measurements reveal reactive oxygen species in stress granules.

Stress granules (SGs) are membraneless organelles that assemble in the cytoplasm to organize cellular contents and promote rapid adaptation during stress. To understand how SGs contribute to physiological functions, we used electrochemical measurements to detect electroactive species in SGs. With amperometry, we discovered that reactive oxygen species (ROS) are encapsulated inside arseniteinduced SGs, and H2O2 is the main species. The release kinetics of H2O2 from single SGs and the number of H2O2 molecules were quantified. The discovery that SGs contain ROS implicates them as communicators of the cellular stresses rather than a simple end-point. This may explain how SGs regulate cellular metabolism and stress responses. This may also help better understand their cytoprotective functions in pathological conditions associated with SGs such as neurodegenerative diseases, cancers and viral infections.

biochemistry↗

bioRxiv2019

Infectious bronchitis virus regulates cellular stress granule signaling

Viruses must hijack cellular translation machinery to efficiently express viral genes. In many cases, this is impeded by cellular stress responses. These stress responses swiftly relocate and repurpose translation machinery, resulting in global inhibition of translation and the aggregation of stalled 48S mRNPs into cytoplasmic foci called stress granules. This results in translational silencing of all mRNAs excluding those beneficial for the cell to resolve the specific stress. For example, expression of antiviral factors is maintained during viral infection. Here we investigated stress granule regulation by Gammacoronavirus infectious bronchitis virus (IBV), which causes the economically important poultry disease, infectious bronchitis. Interestingly, we found that IBV is able to inhibit multiple cellular stress granule signaling pathways whilst at the same time IBV replication also results in induction of seemingly canonical stress granules in a proportion of infected cells. Moreover, IBV infection uncouples translational repression and stress granule formation and both processes are independent of eIF2 phosphorylation. These results provide novel insights into how IBV modulates cellular translation and antiviral stress signaling.

microbiology↗

bioRxiv2019

Noroviruses subvert the core stress granule component G3BP1 to promote viral VPg-dependent translation.

Knowledge of the host factors required for norovirus replication has been hindered by the challenges associated with culturing human noroviruses. We have combined proteomic analysis of the viral translation and replication complexes with a CRISPR screen, to identify host factors required for norovirus infection. The core stress granule component G3BP1 was identified as a host factor essential for efficient human and murine norovirus infection, demonstrating a conserved function across the Norovirus genus. Furthermore, we show that G3BP1 functions in the novel paradigm of viral VPg-dependent translation initiation, contributing to the assembly of translation complexes on the VPg-linked viral positive sense RNA genome by facilitating 40S recruitment. Our data suggest that G3BP1 functions by providing viral RNA a competitive advantage over capped cellular RNAs, uncovering a novel function for G3BP1 in the life cycle of positive sense RNA viruses and identifying the first host factor with pan-norovirus pro-viral activity.

microbiology↗

bioRxiv2018

Norovirus infection results in assembly of virus-specific G3BP1 granules and evasion of eIF2α signaling.

During viral infection, the accumulation of RNA replication intermediates or viral proteins imposes major stress on the host cell. In response, cellular stress pathways can rapidly impose defence mechanisms by shutting off the protein synthesis machinery, which viruses depend on, and triggering the accumulation of mRNAs into stress granules to limit the use of energy and nutrients. Because this threatens viral gene expression, viruses need to evade these pathways to propagate. Human norovirus is responsible for gastroenteritis outbreaks worldwide. Previously we showed that murine norovirus (MNV) regulates the activity of eukaryotic initiation factors (eIFs). Here we examined how MNV interacts with the eIF2a signaling axis controlling translation and stress granules accumulation. We show that while MNV infection represses host cell translation, it results in the assembly of virus-specific granules rather than stress granules. Further mechanistic analyses revealed that eIF2a signaling is uncoupled from translational stalling. Moreover the interaction of the RNA-binding protein G3BP1 with viral factors together with a redistribution of its cellular interacting partners could explain norovirus evasion of stress granules assembly. These results identify novel strategies by which norovirus ensure efficient replication propagation by manipulating the host stress response.

microbiology↗