Biology subjects

Spick, M.

Publications and source records attributed to Spick, M..

4 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↗

bioRxiv2024

Rhythmic variation in proteomics: challenges and opportunities for statistical power and biomarker identification

Time-of-day variation in the molecular profile of biofluids and tissues is a well-described phenomenon, but - especially for proteomics - is rarely considered in terms of the challenges this presents to reproducible biomarker identification. In this work we demonstrate these confounding issues using a small-scale proteomics analysis of male participants in a constant routine protocol following an 8-day laboratory study, in which sleep-wake, light-dark and meal timings were controlled. We provide a case study analysis of circadian and ultradian rhythmicity in proteins in the complement and coagulation cascades, as well as apolipoproteins, and demonstrate that rhythmicity increases the risk of Type II errors due to the reduction in statistical power from increased variance. For the proteins analysed herein we show that to maintain statistical power if chronobiological variation is not controlled for, n should be increased (by between 9% and 20%); failure to do so would increase {beta}, the chance of Type II error, from a baseline value of 20% to between 22% and 28%. Conversely, controlling for rhythmic time-of-day variation in study design offers the opportunity to improve statistical power and reduce the chances of Type II errors. Indeed, control of time-of-day sampling is a more cost-effective strategy than increasing sample sizes. We recommend that best practice in proteomics study design should account for temporal variation as part of sampling strategy where possible. Where this is impractical, we recommend that additional variance from chronobiological effects be considered in power calculations, that time of sampling be reported as part of study metadata, and that researchers reference any previously identified rhythmicity in biomarkers and pathways of interest. These measures would mitigate against both false and missed discoveries, and improve reproducibility, especially in studies looking at biomarkers, pathways or conditions with a known chronobiological component.

molecular biology↗

bioRxiv2023

Viral pIC-pocketing: RSV sequestration of eIF4F Initiation Complexes into bi-phasic biomolecular condensates

Orthopneumoviruses characteristically form membrane-less cytoplasmic inclusion bodies (IBs) wherein RNA replication and transcription occur. Herein, we report a strategy whereby the orthopneumoviruses sequester various components of the eiF4F Initiation Complex machinery into viral IBs to facilitate translation of their own mRNAs; pIC-pocketing. Mass spectrometry analysis of sub-cellular fractions from RSV-infected cells identified significant modification of the cellular translation machinery; however; interestingly, ribopuromycylation assays showed no changes to global levels of translation. Electron micrographs of RSV-infected cells revealed bi-phasic organisation of IBs; specifically, spherical "droplets" nested within the larger inclusion. Using correlative light and electron microscopy (CLEM), combined with fluorescence in situ hybridisation (FISH), we showed that the observed bi-phasic morphology represents functional compartmentalisation of the IB and that these domains are synonymous with the previously reported inclusion body associated granules (IBAGs). Detailed analysis demonstrated that IBAGs concentrate nascent viral mRNA, the viral M2-1 protein as well as many components of the eIF4F complex, involved in translation initiation. Interestingly, although ribopuromycylation-based imaging indicates the majority of viral mRNA translation likely occurs in the cytoplasm, there was some evidence for intra-IBAG translation, consistent with the likely presence of ribosomes in a subset of IBAGs imaged by electron microscopy. The mechanistic basis for this pathway was subsequently determined; the viral M2-1 protein interacting with eukaryotic translation initiation factor 4G (eIF4G) to facilitate its transport between the cytoplasm and the separate phases of the viral IB. In summary, our data shows that IBs function to spatially regulate early steps in viral translation within a highly selective biphasic liquid organelle.

microbiology↗

bioRxiv2020

The loss of the pyoverdine secondary receptor in Pseudomonas aeruginosa results in a fitter strain suitable for population invasion

The rapid emergence of antibiotic resistant bacterial pathogens constitutes a critical problem in healthcare and requires the development of novel treatments. Potential strategies include the exploitation of microbial social interactions based on public goods, which are produced at a fitness cost by cooperative microorganisms, but can be exploited by cheaters that do not produce these goods. Cheater invasion has been proposed as a Trojan horse approach to infiltrate pathogen populations with strains deploying built-in weaknesses (e.g. sensitiveness to antibiotics). However, previous attempts have been often unsuccessful because population invasion by cheaters was prevented by various mechanisms including the presence of spatial structure (e.g. growth in biofilms), which limits the diffusion and exploitation of public goods. Here we followed an alternative approach and examined whether the manipulation of public good uptake and not its production could result in potential Trojan horses suitable for population invasion. We focused on the siderophore pyoverdine produced by the human pathogen Pseudomonas aeruginosa MPAO1 and manipulated its uptake by deleting and/or overexpressing the pyoverdine primary (FpvA) and secondary (FpvB) receptors. We found that receptor synthesis feeds back on pyoverdine production and uptake rates, which led to strains with altered pyoverdine-associated costs and benefits. Moreover, we found that the receptor FpvB was advantageous under iron-limited conditions but revealed hidden costs in the presence of an antibiotic stressor (gentamicin). As a consequence, FpvB mutants became the fittest strain under gentamicin exposure, displacing the wildtype in liquid cultures, and in biofilms and during infections of the wax moth larvae Galleria mellonella, which both represent structured environments. Our findings reveal that an evolutionary trade-off associated with the costs and benefits of a versatile pyoverdine uptake strategy can be harnessed for devising a Trojan horse candidate for medical interventions.

microbiology↗