Biology subjects

Paul, N.

Publications and source records attributed to Paul, N..

13 recordsLinked to original sources

bioRxiv2026

Quantitative fluorescence lifetime imaging of protein microenvironments by genetically encoded compact benzodiazole probes

Quantitative fluorescence lifetime imaging microscopy (FLIM) is limited by a lack of probes that combine single-residue specificity, minimal steric perturbation, visible-light excitation, and strong environment-dependent lifetime contrast. We introduce benzodiazole-based non-canonical amino acids (BD-ncAAs) as compact, genetically encoded sensors for site-specific FLIM. Using an engineered pyrrolysyl-tRNA synthetase pair, we incorporate three different BD-ncAAs into proteins in living cells. Site-specific incorporation of BD-ncAAs into a nanobody at different positions preserved native structure and antigen binding. The lead probe, cyano-substituted CBDS, exhibits visible-light excitation, large Stokes shifts, and pronounced environment-dependent fluorescence lifetimes spanning 1 to 24 ns. Under wash-free, live-cell FLIM conditions, CBDS quantitatively resolves localized structural microenvironments and transmembrane hydration gradients, while also distinguishing distinct states during protein secretion in eukaryotes. BD-ncAAs present genetically encoded, minimally perturbing reporters for tracking target proteins, detecting interactions with binding partners, and quantifying site-specific solvent accessibility in living cells by quantitative FLIM.

biophysics↗

bioRxiv2026

RNF213 M1-ubiquitinates damaged mitochondria to activate pro-inflammatory NF-κB signaling

Through mitochondrial outer membrane permeabilization (MOMP), mitochondria are central to apoptosis. MOMP is pro-inflammatory, impacting diverse processes including anti-tumor immunity and cellular senescence. Despite this, how damaged mitochondria activate inflammation remains unclear. Recent studies have shown that following MOMP, mitochondria are extensively ubiquitinated activating pro-inflammatory NF-{kappa}B signaling. We identify the E3 ligase, RNF213, as essential for pro-inflammatory mitochondrial ubiquitination following MOMP. RNF213 has an established role in pro- inflammatory, cell-autonomous immunity to bacteria and other pathogens. We discover that RNF213 is required for mitochondrial M1-linked (linear) ubiquitination. Interestingly, this was found to be independent of the canonical M1-ubiquitin ligase complex LUBAC. RNF213 can directly catalyze M1-linked ubiquitination. By promoting mitochondrial M1-linked ubiquitination, RNF213 initiates the recruitment of the essential NF{kappa}B adaptor molecule, NEMO, and subsequently activates pro-inflammatory NF-{kappa}B signaling. Collectively, our findings highlight striking similarities between how cells detect damaged mitochondria and intracellular pathogens, leading to inflammation.

cell biology↗

bioRxiv2026

CAGE-TRX expands the scope of time-resolved crystallography through genetically encoded active-site photocaging

We present CAGE-TRX, a broadly applicable time-resolved strategy for release-quench-probe cryocrystallography and release-probe room temperature serial crystallography. These workflows enable light-triggered control of enzyme activity via genetically encoded photocaged amino acids. By decoupling reaction initiation from substrate design, this approach allows synchronized catalysis in crystallo and the capture of transient intermediates. Using {beta}-lactamases as model systems, we demonstrate efficient decaging, restoration of activity, and structural visualization of reaction intermediates.

biochemistry↗

bioRxiv2026

CYCLOPS: an open end-to-end platform for cyclic multiplex imaging and single-cell phenotyping

Multiplex immunofluorescent imaging enables deep spatial profiling of protein expression in tissues but is often limited by reliance on proprietary reagents, dedicated hardware, and closed analysis ecosystems. Here we present CYCLOPS (Cyclic Open Platform for Spatial Proteomics), an end-to-end, open-source workflow for cyclic multiplex imaging and single-cell phenotyping using standard microscopy infrastructure. CYCLOPS integrates an Arduino-based automated fluidics system, an open-chamber stage insert, and antibody-oligonucleotide conjugation based entirely on published chemistries and off-the-shelf components. We demonstrate robust and reproducible antibody conjugation, high-quality multiplexed staining, and stable imaging across >10 cycles with minimal drift (<1 {micro}m) and consistent fluorescence retention with low signal carry-over. The system supports efficient buffer exchange and consistent performance across multiple markers and imaging rounds. Using confocal microscopy, the workflow is compatible with three-dimensional imaging, enabling multiplexed analysis of volumetric tissue structures. To enable quantitative analysis, we establish an open-source image processing and analysis pipeline for single-cell feature extraction and phenotypic classification, avoiding reliance on proprietary software or black-box workflows. This framework integrates image registration, segmentation, and supervised classification to generate biologically interpretable single-cell data. Together, CYCLOPS provides a flexible and accessible platform for cyclic multiplex imaging, lowering barriers to adoption and enabling broader use of spatial proteomics across diverse research settings. This accessible framework democratizes high-plex imaging by enabling any laboratory with a standard confocal microscope to perform iterative multiplexing without reliance on proprietary reagents or hardware.

Cell Biology↗

bioRxiv2024

A Comparison of the Sexual Expression, Biomass, Cannabinoid Content, and Seed Production in XXX and XXY Triploid Cannabis

Cannabis is a multi-billion dollar industry reliant on unpollinated genetically female (XX) plants, as pollination reduces cannabinoid yields and flower quality. The mechanism behind the sexual expression of C. sativa is unclear. This study examines the sexual characteristics of homo- and hetero-gametic triploid cannabis plants (XX(X/Y)) to understand the role of the Y chromosome in cannabis sex determination. Heterogametic triploids were produced by crossing a male diploid C. sativa cv. Durban Poison (2n = 2x = 18 + XY) with a tetraploid female C. sativa cv. Higher Education (2n = 4x = 36 + XXXX) confirmed by flow cytometry and PCR. Measurements include fresh and dry plant weight, dry flower weight, seed counts, seed weights, and cannabinoids quantified using liquid chromatography. Results showed 44.4% of plants were XXY (n = 8), with 75% presenting a monoecious phenotype (n = 6). Of these, 50% produced predominately male flowers (n = 3), while the rest produced predominately female flowers. Plants with predominately female flowers had higher yields and cannabinoids compared to predominantly male plants. XXX genotypes outperformed XXY in several metrics. This study enhances our understanding of C. sativa sex determination and offers insight into breeding and cultivation strategies utilizing triploid cannabis.

plant biology↗

bioRxiv2024

Rendering Proteins Fluorescent Inconspicuously: Genetically Encoded 4-Cyanotryptophan Conserves Their Structure and Enables the Detection of Ligand Binding Sites

Cyano-tryptophans (CN-Trp) are privileged multimodal reporters on protein structure. They are similar in size to the canonical amino acid tryptophan and some of them exhibit bright fluorescence which responds sensitively to changes in the environment. We selected aminoacyl-tRNA synthetases specific for 4-, 5-, 6-, and 7-CN-Trp for high-yield in vivo production of proteins with a single, site-specifically introduced nitrile label. The absorption maximum of 4-CN-Trp is distinct from Trp, allowing the selective excitation of its intense fluorescence. 4-CN-Trp features bright fluorescence in the visible range. Crystal structures of maltose binding protein demonstrate near-complete structural conservation when a native buried Trp residue is replaced by 4-CN-Trp. Besides presenting an inconspicuous tag for live cell microscopy, the high fluorescence of 4-CN-Trp enables measurements of subnanomolar ligand binding affinities in isotropic solution, as demonstrated by the complex between rapamycin and the peptidyl-prolyl isomerase FKBP12 furnished with a 4-CN-Trp residue in the substrate binding pocket. Furthermore, 4-CN-Trp residues positioned at different locations of a protein containing multiple tryptophan residues permits using fluorescence quenching experiments to detect the proximity of individual Trp residues to the binding site of aromatic ligands.

biochemistry↗

bioRxiv2024

mini-Complexome Profiling (mCP), an FDR-controlled workflow for global targeted detection of protein complexes

IntroductionCo-fractionation mass spectrometry couples native-like separations of protein/protein complexes with mass spectrometric proteome analysis for global characterization of protein networks. The technique allows for both de novo detection of complexes and for the detection of subtle changes in their protein composition. The typical requirement for fine-grained fractionation of >80 fractions, however, translates into significant demands on sample quantity and mass spectrometric instrument time, and represents a significant barrier to experimental replication and the use of scarce sample material (ex. Patient biopsies). MethodsWe developed mini-Complexome Profiling (mCP), a streamlined workflow with reduced requirements for fractionation and, thus, biological material and laboratory and instrument time. Soluble and membrane-associated protein complexes are extracted from biological material under mild conditions, and fractionated by Blue Native electrophoresis using commercial equipment. Each fraction is analyzed by data independent acquisition mass-spectrometry, and known protein complexes are detected based on the coelution of known components using a novel R package with a controlled false discovery rate approach. The tool is available to the community on a GitHub repository. ResultsmCP was benchmarked using HEK293 cell lysate and exhibited performance similar to established workflows, but from a significantly reduced number of fractions. We then challenged mCP by performing comparative complexome analysis of cardiomyocytes isolated from different chambers from a single mouse heart, where we identified subtle chamber-specific changes in mitochondrial OxPhos complexes. DiscussionThe reduced sample and instrument time requirements open up new applications of co-fractionation mass spectrometry, specifically for the analysis of sparse samples such as human patient biopsies. The ability to identify subtle changes between similar tissue types (left/right ventricular and atrial cardiomyocytes) serves as a proof of principle for comparative analysis of mild/asymptomatic disease states.

systems biology↗

bioRxiv2024

Effect of pH on the thermostability and redox properties of cytochrome c552 from Wolinella succinogenes

Cytochrome c552 from Wolinella succinogenes is one of the few examples of a low reduction potential class I c-type cytochrome with a mixture of high/low spin state populations observed in its visible spectrum. Analysis of its structural model suggests that the heme is Met/His coordinated and highly solvent-exposed. This supports the hypothesis that it is the solvent accessibility of the propionate groups that controls the reduction potential of small c-type cytochromes. The visible spectra obtained at different pH values reveal the presence of a protonable group with a pKa of 7.3, which also influences the reduction potential of this small cytochrome c552 (Em0 of 97 {+/-} 5 mV, pH 7.0) and can be either an H2O/OH- group distantly coordinating the heme iron, or one of the propionate groups. The thermostability of cytochrome c552 has been studied by circular dichroism and differential scanning calorimetry, indicating a highly stable protein at pH 5-7 (90 {degrees}C to 77 {degrees}C).

biochemistry↗

bioRxiv2023

Primary production dynamics during the decline phase of the North Atlantic annual spring bloom

The second field campaign of the NASA EXport Processes in the Ocean from RemoTe Sensing (EXPORTS) program was conducted in the late spring of 2021 within the vicinity of the Porcupine Abyssal Plain (49.0{degrees}N, 16.5{degrees}W) in the North Atlantic Ocean. Observations from EXPORTS support previous characterizations of this system as highly productive and organic matter rich, with the majority of primary production occurring in large cells ([≥] 5 {micro}m) such as diatoms that are primarily utilizing nitrate. Rates of total euphotic zone depth-integrated net primary production ranged from 36.4 to 146.6 mmol C m- 2 d-1, with an observational period average f-ratio of 0.74, indicating predominantly new production. Substantial variability in the contribution of small (<5 {micro}m) and large cells occurred over the observation period, coinciding with the end of the annual spring phytoplankton bloom. Physical changes associated with storms appear to have impacted the integrated production rates substantially, enhancing rates by [~]10%. These disturbances altered the balance between contributions of the different phytoplankton size fractions, thus highlighting the important role of mixed layer variability in nutrient entrainment into the upper water column and production dynamics. In diatoms, inputs of silicic acid related to deepening of the mixed layer increased silicic acid uptake rates yet concomitant increases in NPP in large cells was not observed. This campaign serves as the high productivity endmember within the EXPORTS program and as such, elucidates how nutrient concentrations and size class play key roles in both low and high productivity systems, but in differing ways.

ecology↗

bioRxiv2021

Increased apoptotic priming of glioblastoma enables therapeutic targeting by BH3-mimetics

IDH wild-type glioblastoma (GBM) is the most prevalent malignant primary brain tumour in adults. GBM typically has a poor prognosis, mainly due to a lack of effective treatment options leading to tumour persistence or recurrence. Tackling this, we investigated the therapeutic potential of targeting anti-apoptotic BCL-2 proteins in GBM. Levels of anti- apoptotic BCL-xL and MCL-1 were consistently increased in GBM compared with non- malignant cells and tissue. Moreover, we found that relative to their differentiated counterparts, patient-derived GBM stem-like cells also displayed higher expression of anti- apoptotic BCL-2 family members. Surprisingly, high anti-apoptotic BCL-xL and MCL-1 expression correlated with heightened susceptibility of GBM to BCL-2 family protein- targeting BH3-mimetics. This is indicative of increased apoptotic priming. Indeed, GBM displayed an obligate requirement for MCL-1 expression in both tumour development and maintenance. Investigating this apoptotic sensitivity, we found that sequential inhibition of BCL-xL and MCL-1 led to robust anti-tumour responses in vivo, in the absence of overt toxicity. These data demonstrate that BCL-xL and MCL-1 pro-survival function is a fundamental prerequisite for GBM survival that can be therapeutically exploited by BH3- mimetics.

cancer biology↗

bioRxiv2020

CYRI-A regulates macropinocytic cup maturation and mediates integrin uptake, limiting invasive migration

The Scar/WAVE complex is the major driver of actin nucleation at the plasma membrane, resulting in lamellipodia and membrane ruffles. While lamellipodia aid migration, membrane ruffles can generate macropinosomes - cup-like structures - important for nutrient uptake and regulation of cell surface receptor levels. How macropinosomes are formed and the role of the actin machinery in their formation and resolution is still not well understood. Mammalian CYRI-B is a recently described negative regulator of the Scar/WAVE complex by RAC1 sequestration, but its other paralogue, CYRI-A has not been characterised. Here we implicate CYRI-A as a key regulator of macropinosome maturation and integrin internalisation from the cell surface. We find that CYRI-A is recruited to nascent macropinosomes in a transient but distinct burst, downstream of PIP3-mediated RAC1 activation and the initial burst of actin assembly driving cup formation, but upstream of internalisation and RAB5 recruitment to the macropinosome. Together, our data place CYRI-A as a local suppressor of actin dynamics, enabling the resolution of the macropinocytic cup. The failure of CYRI-depleted cells to resolve their macropinocytic cups results in reduced integrin a5b1 internalisation, leading to enhanced spreading, invasive behaviour and anchorage-independent 3D growth. We thus describe a new role for CYRI-A as a highly dynamic regulator of RAC1 activity at macropinosomes, modulating homeostasis of integrin surface presentation, with important functional consequences.

cell biology↗

bioRxiv2019

NLRP3- and AIM2-autonomy in a mouse model of MSU crystal-induced acute inflammation in vivo highlights imiquimod-dependent targeting of Il-1 expression as relevant therapy for gout patients.

The role of Monosodium Urate (MSU) crystals in gout pathophysiology is well described, as is the major impact of IL-1b in the inflammatory reaction that constitutes the hallmark of the disease. However, despite the discovery of the NLRP3 inflammasome and its role as a Pattern Recognition Receptor linking the detection of a danger signal (MSU) to IL-1b; secretion in vitro, the precise mechanisms leading to joint inflammation in gout patients are still poorly understood. Here, we provide an extensive clinical, biological and molecular characterization of the acute uratic inflammation mouse model induced by subcutaneous injection of MSU crystals, which accurately mimics human gout. Our work reveals several key features of MSU-dependent inflammation and identifies novel therapeutic opportunities, among which the use of topical application of imiquimod to promote interferon-dependent anti-inflammatory action maybe relevant.

immunology↗

bioRxiv2018

Eps8 is a convergence point integrating EGFR and integrin trafficking and crosstalk

Crosstalk between adhesion and growth factor receptors plays a critical role in tissue morphogenesis and repair, and aberrations contribute substantially to neoplastic disease. However, the mechanisms by which adhesion and growth factor receptor signalling are integrated, spatially and temporally, are unclear.\n\nWe used adhesion complex enrichment coupled with quantitative proteomic analysis to identify rapid changes to adhesion complex composition and signalling following growth factor stimulation. Bioinformatic network and ontological analyses revealed a substantial decrease in the abundance of adhesion regulatory proteins and co-ordinators of endocytosis within 5 minutes of EGF stimulation. Together these data suggested a mechanism of EGF-induced receptor endocytosis and adhesion complex turnover.\n\nCombinatorial interrogation of the networks allowed a global and dynamic view of adhesion and growth factor receptor crosstalk to be assembled. By interrogating network topology we identified Eps8 as a putative node integrating 5{beta}1 integrin and EGFR functions. Importantly, EGF stimulation promoted internalisation of both 5{beta}1 and EGFR. However, perturbation of Eps8 increased constitutive internalisation of 5{beta}1 and EGFR; suggesting that Eps8 constrains 5{beta}1 and EGFR endocytosis in the absence of EGF stimulation. Consistent with this, Eps8 regulated Rab5 activity and was required for maintenance of adhesion complex organisation and for EGF-dependent adhesion complex disassembly. Thus, by co-ordinating 5{beta}1 and EGFR trafficking mechanisms, Eps8 is able to control adhesion receptor and growth factor receptor bioavailability and cellular contractility.\n\nWe propose that during tissue morphogenesis and repair, Eps8 functions to spatially and temporally constrain endocytosis, and engagement, of 5{beta}1 and EGFR in order to precisely co-ordinate adhesion disassembly, cytoskeletal dynamics and cell migration.

cell biology↗