Biology subjects

Campelo, F.

Publications and source records attributed to Campelo, F..

At least 19 recordsLinked to original sources

bioRxiv2026

A PKD-caveolin axis drives secretory carrier biogenesis at the TGN

Constitutive secretion from the trans-Golgi network (TGN) to the cell surface proceeds via carriers thought to form without a canonical cytoplasmic coat, yet how these carriers are generated remains poorly understood. Here, we identify a distinct population of TGN-to-cell surface carriers transporting influenza hemagglutinin (HA) and uncover a coat-like mechanism underlying their formation. HA carrier biogenesis requires non-vesicular lipid transfer at endoplasmic reticulum (ER)-Golgi membrane contact sites (MCSs) and protein kinase D (PKD) activity. We show that caveolin promotes membrane budding by assembling into cholesterol- and PKD-associated oligomers that act as a membrane-embedded, coat-like scaffold at lipid nanodomain-enriched TGN subdomains. These findings establish caveolin as a structural and regulatory component of TGN export and support a model in which a PKD-caveolin axis couples ER-Golgi lipid transfer to cargo sorting, membrane remodeling and fission during secretory carrier biogenesis.

cell biology↗

bioRxiv2025

Mechanical forces stimulate Golgi export

Cells face diverse mechanical stimuli that vary with cell type, state, and pathological conditions. Mechanobiology investigates how cells sense and respond to these forces. While most work has focused on the cell surface and nucleus as primary mechanosensors, how intracellular organelles adapt to extracellular mechanical forces remains largely unknown. Here, we show that extracellular mechanical signals influence the secretory function of the Golgi apparatus. By subjecting adherent cells to mechanical challenges -cell spreading on different ligands, altered substrate stiffness, or equibiaxial strain-, we reveal that extracellular forces modulate Golgi-to-cell surface carrier biogenesis, thereby regulating exocytosis. Together with changes in Golgi membrane tension, we identify molecular determinants of the mechanotransduction pathway, including microtubule acetylation, diacylglycerol production, and protein kinase D activity. In turn, inhibition of Golgi export suppresses this mechanoresponse and causes impaired cell spreading. These findings uncover a bidirectional mechanotransduction axis in which extracellular mechanics tune Golgi secretory output, providing a framework for investigating organelle-based mechanoadaptation in physiology and disease.

cell biology↗

bioRxiv2025

EpitopeTransfer: a Phylogeny-aware transfer learning framework for taxon-specific linear B-cell epitope prediction

Generalist linear B-cell epitope predictors are typically trained on large, heterogeneous datasets, which can lead to biased representations and degraded performance for under-represented or emerging pathogens. We present a transfer learning framework leveraging the ESM family of protein language models (PLMs) as sequence feature embedders, adapting them to specific evolutionary contexts through phylogeny-informed fine-tuning. By coupling pretrained PLM representations with hierarchical, taxon-aware adaptation, our approach enables efficient knowledge transfer from sets of related pathogens to data-scarce targets while preserving lineage-specific signals. This strategy consistently improves predictive performance over state-of-the-art epitope prediction methods across a diverse set of targets. We further demonstrate that these gains arise from the targeted adaptation and downstream models guided by evolutionary relatedness, indicating the value of this structured transfer learning for epitope prediction and highlighting its potential usability for further predictive applications with evolutionarily-structured data.

bioinformatics↗

bioRxiv2024

Tensing Flipper: Photosensitized manipulation of membrane tension, lipid phase separation and raft protein sorting in biological membranes

The lateral organization of proteins and lipids in the plasma membrane is fundamental to regulating a wide range of cellular processes. Compartmentalized ordered membrane domains enriched with specific lipids, often termed lipid rafts, have been shown to modulate the physicochemical and mechanical properties of membranes and to drive protein sorting. Novel methods and tools enabling the visualization, characterization and/or manipulation of membrane compartmentalization are crucial to link the properties of the membrane with cell functions. Flipper, a commercially-available fluorescent membrane tension probe, has become a reference tool for quantitative membrane tension studies in living cells. Here, we report on a so far unidentified property of Flipper, namely, its ability to photosensitize singlet oxygen (1O2) under blue light when embedded into lipid membranes. This in turn results in the production of lipid hydroperoxides that increase membrane tension and trigger phase separation. In biological membranes, the photo-induced segregated domains retain the sorting ability of intact phase-separated membranes, directing raft and non-raft proteins into ordered and disordered regions, respectively, in contrast to radical-based photo-oxidation reactions that disrupt raft protein partitioning. The dual tension reporting and photosensitizing abilities of Flipper enable simultaneous visualization and manipulation of the mechanical properties and lateral organization of membranes, providing a powerful tool to optically control lipid raft formation and to explore the interplay between membrane biophysics and cell function.

biophysics↗

bioRxiv2023

Differential spatial regulation and activation of integrin nanoclusters inside focal adhesions

5{beta}1 and v{beta}3 integrins are core components of focal adhesions (FAs) involved in cell attachment, migration, and mechanobiology-dependent processes. Recent works indicate that both integrins organize in nanoclusters inside FAs, with sub-populations of active and inactive {beta}1 nanoclusters. However, whether both integrins work in concert or their activities are spatially regulated is not fully understood. Using dual-color super-resolution microscopy (STORM, STED and DNA-PAINT) we show that integrins 5{beta}1 and v{beta}3 and their main adaptor proteins exhibit similar functional nanoscale segregation across all adhesion proteins examined, independent of FA maturation state. Notably, both integrins never mix at the nanoscale indicating that their functions might be spatially regulated. We find a nearly 1:1 relationship between active integrin and adaptor nanoclusters suggesting that coordinated integrin activation occurs via the concurrent engagement of adaptor nanoclusters. Interestingly, 5{beta}1 nanoclusters preferentially localize at the FA periphery near adaptor nanoclusters, establishing regions of multi-nanocluster enrichment, whereas v{beta}3 nanoclusters uniformly distribute throughout FAs. Overall, our results show that adhesion proteins are arranged as modular nanoscale units that distinctively organize inside FAs to spatially regulate integrin activation and function.

cell biology↗

bioRxiv2023

Plant plasmodesmata bridges form through ER-driven incomplete cytokinesis

Diverging from conventional cell division models, plant cells undergo incomplete division to generate plasmodesmata communication bridges between daughter cells. While fundamental for plant multicellularity, the molecular events leading to bridge stabilization, as opposed to severing, remain unknown. Using electron tomography, we mapped the transition from cell plate fenestrae to plasmodesmata. We show that the ER connects daughter cells across fenestrae, and as the cell plate matures, fenestrae contract, causing the PM to mold around constricted ER tubes. The ERs presence prevents fenestrae fusion, forming plasmodesmata, while its absence results in closure. The ER-PM tethers MCTP3, 4, and 6 further stabilize nascent plasmodesmata during fenestrae contraction. Genetic deletion in Arabidopsis reduces plasmodesmata formation. Our findings reveal how plants undergo incomplete division to promote intercellular communication. One-Sentence SummaryThe ER is important for stabilizing nascent plasmodesmata, a process integral to incomplete cytokinesis in plants.

plant biology↗

bioRxiv2023

Nanoscale imaging reveals the mechanisms of ER-to-Golgi transport via a dynamic tubular-vesicular network

The endoplasmic reticulum (ER) and the Golgi apparatus are the first sorting stations along the secretory pathway of mammalian cells and have a crucial role in protein quality control and cellular homeostasis. While machinery components mediating ER-to-Golgi transport have been mapped, it is unclear how exchange between the two closely juxtaposed organelles is coordinated in living cells. Here, using gene editing to tag machinery components, live-cell confocal and stimulated emission depletion (STED) super-resolution microscopy, we show that ER-to-Golgi transport occurs via a dynamic network of tubules positive for the small GTPase ARF4. swCOPI machinery is tightly associated to this network and moves with tubular-vesicular structures. Strikingly, the ARF4 network appears to be continuous with the ER and ARF4 tubules remodel around static ER exit sites (ERES) defined by COPII machinery. We were further able to dissect the steps of ER-to-Golgi transport with functional trafficking assays. A wave of cargo released from the ER percolates through peripheral and Golgi-tethered ARF4 structures before filling the cis-Golgi. Perturbation via acute degradation of ARF4 shows an active regulatory role for the GTPase and COPI in anterograde transport. Our data supports a model in which anterograde ER-to-Golgi transport occurs via an ARF4 tubular-vesicular network directly connecting the ER and Golgi-associated pre-cisternae.

cell biology↗

bioRxiv2023

Partitioning to ordered membrane domains regulates the kinetics of secretory traffic

The organelles of eukaryotic cells maintain distinct protein and lipid compositions required for their specific functions. The mechanisms by which many of these components are sorted to their specific locations remain unknown. While some motifs mediating subcellular protein localization have been identified, many membrane proteins and most membrane lipids lack known sorting determinants. A putative mechanism for sorting of membrane components is based on membrane domains known as lipid rafts, which are laterally segregated nanoscopic assemblies of specific lipids and proteins. To assess the role of such domains in the secretory pathway, we applied a robust tool for synchronized secretory protein traffic (RUSH, Retention Using Selective Hooks) to protein constructs with defined affinity for raft phases. These constructs consist solely of single-pass transmembrane domains (TMDs) and, lacking other sorting determinants, constitute probes for membrane domain-mediated trafficking. We find that while raft affinity can be sufficient for steady-state PM localization, it is not sufficient for rapid exit from the endoplasmic reticulum (ER), which is instead mediated by a short cytosolic peptide motif. In contrast, we find that Golgi exit kinetics are highly dependent on raft affinity, with raft preferring probes exiting Golgi [~]2.5-fold faster than probes with minimal raft affinity. We rationalize these observations with a kinetic model of secretory trafficking, wherein Golgi export can be facilitated by protein association with raft domains. These observations support a role for raft-like membrane domains in the secretory pathway and establish an experimental paradigm for dissecting its underlying machinery.

cell biology↗

bioRxiv2022

Phylogeny-aware linear B-cell epitope predictor detects candidate targets for specific immune responses to Monkeypox virus

Monkeypox is a disease caused by the Monkeypox virus (MPXV), a double-stranded DNA virus from genus Orthopoxvirus under family Poxviridae, that has recently emerged as a global health threat after decades of local outbreaks in Central and Western Africa. Effective epidemiological control against this disease requires the development of cheaper, faster diagnostic tools to monitor its spread, including antigen and serological testing. There is, however, little available information about MPXV epitopes, particularly those that would be effective in discriminating between MPXV infections and those by other virus from the same family. We used the available data from the Immune Epitope Database (IEDB) to generate and validate a predictive model optimised for detecting linear B-cell epitopes (LBCEs) from Orthopoxvirus, based on a phylogeny-aware data selection strategy. By coupling this predictive approach with conservation and similarity analyses, we identified nine specific peptides from MPXV that are likely to represent distinctive LBCEs for the diagnostic of Monkeypox infections, including the independent detection of a known epitope experimentally characterised as a potential specific diagnostic target for MPXV. The results obtained indicate ability of the proposed pipeline to uncover promising targets for the development of cheaper, more specific diagnostic tests for this emerging viral disease. A full reproducibility package (including code, data, and outputs) is available at https://doi.org/10.5281/zenodo.7838331.

bioinformatics↗

bioRxiv2022

Functional characterization of the Met50Val substitution in SLC30A9 as a novel case of adaptive introgression in humans

SLC30A9 encodes a ubiquitously zinc transporter (ZnT9) and has been consistently suggested as a candidate for positive selection in humans. However, no direct adaptive molecular phenotype has been demonstrated. Our results provide evidence for directional selection operating in two major complementary haplotypes in Africa and East Asia. These haplotypes are associated with differential gene expression but also differ in the Met50Val substitution (rs1047626) in ZnT9, which we show is found in homozygosis in the Denisovan genome and displays accompanying signatures suggestive of archaic introgression. Although we found no significant differences in systemic zinc content between individuals with different rs1047626 genotypes, we demonstrate that the expression of the derived isoform (ZnT9 50Val) in HEK293 cells shows a gain of function when compared with the ancestral (ZnT9 50Met) variant. Notably, the ZnT9 50Val variant was found associated with differences in zinc handling by the mitochondria and endoplasmic reticulum, with an impact on mitochondrial metabolism. Given the essential role of the mitochondria in skeletal muscle and since the derived allele at rs1047626 is known to be associated with greater susceptibility to several neuropsychiatric traits, we propose that adaptation to cold may have driven this selection event, while also impacting predisposition to neuropsychiatric disorders in modern humans. Author SummaryContrasting continental signatures of positive natural selection have been previously found in the human SLC30A9 gene encoding the protein ZnT9, which transports zinc across cell membranes. Here we investigate the genetic variants that have been targeted by natural selection in the surrounding region of this gene and which molecular and whole-body changes may have brought about. We found that two major SLC30A9 variant combinations (haplotypes) that are extremely frequent in Africa and East Asia, respectively, are expressed differentially. These two haplotypes also differ at one site that creates an amino acid difference at ZnT9; the version most often found outside Africa avoiding zinc overload in the endoplasmic reticulum and mitochondria and directly influencing mitochondrial activity. Moreover, we found that this substitution, which is known to be associated with greater susceptibility to several neuropsychiatric disorders, is present in the Denisova and displays accompanying patterns of variation that could be suggestive of adaptive introgression. Since mitochondria play an important role in skeletal muscle energy metabolism, we speculate that adaptation to cold may have driven this selection event outside Africa, while also impacting predisposition to neuropsychiatric disorders in modern humans.

evolutionary biology↗

bioRxiv2022

Actin-regulated Siglec-1 nanoclustering influences HIV-1 capture and virus-containing compartment formation in dendritic cells

The immunoglobulin-like lectin receptor CD169 (Siglec-1) mediates the capture of HIV-1 by activated dendritic cells (DC) through binding to sialylated ligands. These interactions result in a more efficient virus capture as compared to resting DCs, although the underlying mechanisms are poorly understood. Using a combination of super-resolution microscopy, single particle tracking and biochemical perturbations we studied the nanoscale organization of Siglec-1 on activated DCs and its impact on viral capture and its trafficking to a single viral-containing compartment. We found that activation of DCs leads to Siglec-1 basal nanoclustering at specific plasma membrane regions where receptor diffusion is constrained by Rho-ROCK activation and formin-dependent actin polymerization. Using liposomes with varying ganglioside concentrations, we further demonstrate that Siglec-1 nanoclustering enhances the receptor avidity to limiting concentrations of gangliosides carrying sialic ligands. Binding to either HIV-1 particles or ganglioside-bearing liposomes lead to enhanced Siglec-1 nanoclustering and global actin rearrangements characterized by a drop in RhoA activity, facilitating the final accumulation of viral particles in a single sac-like compartment. Overall, our work provides new insights on the role of the actin machinery of activated DCs in regulating the formation of basal Siglec-1 nanoclustering, being decisive for the capture and actin-dependent trafficking of HIV-1 into the virus-containing compartment.

cell biology↗

bioRxiv2022

Sorting of secretory proteins at the trans-Golgi network by TGN46

Secretory proteins are sorted at the trans-Golgi network (TGN) for export into specific transport carriers. However, the molecular players involved in this fundamental process remain largely elusive. Here, we identified the human transmembrane protein TGN46 as a receptor for the export of secretory cargo protein PAUF in CARTS - a class of protein kinase D-dependent TGN-to-plasma membrane carriers. We show that TGN46 is necessary for cargo sorting and loading into nascent carriers at the TGN. By combining quantitative fluorescence microscopy and mutagenesis approaches, we further discovered that the lumenal domain of TGN46 encodes for its cargo sorting function. In summary, our results define a cellular function of TGN46 in sorting secretory proteins for export from the TGN.

cell biology↗

bioRxiv2022

Particle flow modulates growth dynamics and nanoscale-arrested growth of transcription factor condensates in living cells

Liquid-liquid phase separation (LLPS) is emerging as key physical principle for biological organization inside living cells, forming condensates that play important roles in the regulation of multiple functions. Inside living nuclei, transcription factor (TF) condensates regulate transcriptional initiation and amplify transcriptional output of expressed genes. Yet, the biophysical parameters controlling TF condensation are still poorly understood. Here we applied a battery of single molecule imaging tools, theory and simulations to investigate the physical properties of TF condensates of the Progesterone Receptor (PR) in vivo. Analysis of individual PR trajectories at different ligand concentrations showed marked signatures of a ligand-tunable and regulated LLPS process. Using a machine learning architecture, we uncovered that diffusion within condensates follows fractional Brownian motion, reflecting viscoelastic interactions between PR and chromatin within condensates. High density single molecule localization maps further revealed that condensate growth dynamics is dominated by Brownian motion coalescence (BMC) at shorter times, but deviate at longer timescales reaching a growth plateau with nanoscale condensate sizes. To understand our observations we developed an extension of the BMC model by including stochastic unbinding of particles within condensates. The model reproduced the BMC behavior together with finite condensate sizes a steady-state, fully recapitulating our experimental data. Our results are thus consistent with droplet growth dynamics being regulated by the escaping probability of TFs molecules from condensates. The interplay between condensation assembly and molecular escaping maintains an optimum physical condensate size. Such phenomena must have implications for the biophysical regulation of other TF condensates and could also operate in multiple biological scenarios.

biophysics↗

bioRxiv2021

Estimated limits of organism-specific training for epitope prediction

BackgroundThe identification of linear B-cell epitopes remains an important task in the development of vaccines, therapeutic antibodies and several diagnostic tests. Machine learning predictors are trained to flag potential epitope candidates for experimental validation and currently, most predictors are trained as generalist models using large, heterogeneous data sets. Recently, organism-specific training has been shown to improve prediction performance for data-rich organisms. Unfortunately, for most organisms, large volumes of validated epitope data are not yet available. This article investigates the limits of organism-specific training for epitope prediction. It explores the validity of organism-specific training for data-poor organisms by examining how the size of the training data set affects prediction performance. It also compares the performance of organism-specific training under simulated data-poor conditions to that of models trained using traditional large heterogeneous and hybrid data sets. ResultsThis work shows how models trained on small organism-specific data sets can outperform similar models trained on (potentially much larger) heterogeneous and mixed data sets. The results reported indicate that as few as 20 labelled peptides from a given pathogen can be sufficient to generate models that outperform widely-used predictors from the literature, which are trained on heterogeneous data. Models trained using more than about 100 to 150 organism-specific peptides perform consistently better than most generalist models across a wide variety of performance measures, and in some cases can even approach the performance of organism-specific models trained on considerably larger data sets. ConclusionsOrganism-specific training improves linear B-cell epitope prediction performance even in situations when only small training sets are available, which opens new possibilities for the development of bespoke, high-performance predictive models when studying data-poor organisms such as emerging or neglected pathogens.

bioinformatics↗

bioRxiv2021

Multi-objective prioritisation of candidate epitopes for diagnostic test development

BackgroundThe development of peptide-based diagnostic tests requires the identification of epitopes that are at the same time highly immunogenic and, ideally, unique to the pathogen of interest, to minimise the chances of cross-reactivity. Existing computational pipelines for the prediction of linear B-cell epitopes tend to focus exclusively on the first objective, leaving considerations of cross-reactivity to later stages of test development. ResultsWe present a multi-objective approach to the prioritisation of candidate epitopes for experimental validation, in the context of diagnostic test development. The dual objectives of uniqueness (measured as dissimilarity from known epitope sequences from other pathogens) and predicted immunogenicity (measured as the probability score returned by the prediction model) are considered simultaneously. Validation was performed using data from three distinct pathogens (namely the nematode Onchocerca volvulus, the Epstein-Barr Virus and the Hepatitis C Virus), with predictions derived using an organism-specific prediction approach. The multi-objective rankings returned sets of non-dominated solutions as potential targets for the development of diagnostic tests with lower probability of false positives due to cross-reactivity. ConclusionsThe application of the proposed approach to three test pathogens led to the identification of 20 new potential epitopes, with both high probability and a high degree of exclusivity to the target organisms. The results indicate the potential of the proposed approach to provide enhanced filtering and ranking of potential candidates, highlighting potential cross-reactivities and including this information into the test development process right from the target identification and prioritisation step.

bioinformatics↗

bioRxiv2021

CALANGO: an annotation-based, phylogeny-aware comparative genomics framework for exploring and interpreting complex genotypes and phenotypes

The increasing availability of genomic, annotation, evolutionary and phenotypic data for species contrasts with the lack of studies that adequately integrate these heterogeneous data sources to produce biologically meaningful knowledge. Here, we present CALANGO, a phylogeny-aware comparative genomics tool that uncovers functional molecular convergences and homologous regions associated with quantitative genotypes and phenotypes across species, enabling the fast discovery of novel statistically sound, biologically relevant phenotype-genotype associations. We demonstrate the usefulness of CALANGO in two case studies. The first one unveils potential causal links between prophage density and the pathogenicity phenotype in Escherichia coli, and confidently demonstrates how CALANGO supports the investigation of basic causal relationships by enabling a level of counterfactual investigation of observed associations in the data. As a second case study, we used our tool to search for homologous regions associated with a complex phenotypic trait in a major group of eukaryotes: the evolution of maximum height in angiosperms. We confidently identify a previously unknown association between maximum plant height and the expansion of the self-incompatibility system, a molecular mechanism that prevents inbreeding and increases genetic diversity. Taller species also have lower rates of molecular evolution due to their longer generation times, a critical concern for their long-term viability. The new mechanism we report could counterbalance this fact, and have far-reaching consequences for fields as diverse as conservation biology and agriculture. CALANGO is provided as a fully operational R package that can be freely installed from CRAN.

bioinformatics↗

bioRxiv2020

Membrane receptor MerTK is a newly identified transcriptional regulator that associates to chromatin as nanoclusters during human DC differentiation

MerTK is a transmembrane receptor tyrosine kinase (RTK) mainly expressed in dendritic cells (DCs) and macrophages where it plays an important role in immunotolerance, but also in activating oncogenic signalling pathways. Albeit MerTK is exploited as clinical target in cancer and auto-immune disorders, the mechanisms that regulate its diverse functions are poorly understood. Here, we identified a remarkably high pool of the full receptor in the nucleus of human DCs. Nuclear translocation was ligand-dependent. Importantly, MerTK nuclear levels correlated to DC differentiation and were spatiotemporally regulated by the transmembrane receptor LRP-1. Using dual-colour super-resolution nanoscopy we discovered that nuclear MerTK forms nanoclusters, whose strength strongly depends on chromatin accessibility during DC differentiation. We finally revealed high transcription capacity of MerTK. Overall, our work indicates that nuclear MerTK acts as a transcription factor regulating DC differentiation, thus implicating for the first time a physiological function for RTK nuclear translocation in immunity.

immunology↗

bioRxiv2020

TANGO1 membrane helices create a lipid diffusion barrier at curved membranes

We have previously shown TANGO1 organises membranes at the interface of the endoplasmic reticulum (ER) and ERGIC/Golgi (Raote et al., 2018). TANGO1 corrals retrograde membranes at ER exit sites to create an export conduit. Here the retrograde membrane is, in itself, an anterograde carrier. This mode of forward transport necessitates a mechanism to prevent membrane mixing between ER and the retrograde membrane. TANGO1 has an unusual membrane helix organisation, composed of one membrane-spanning helix (TM) and another that penetrates the inner leaflet (IM). We have reconstituted these membrane helices in model membranes and shown that TM and IM together reduce the flow of lipids at a region of defined shape. We have also shown that the helices align TANGO1 around an ER exit site. We suggest this is a mechanism to prevent membrane mixing during TANGO1-mediated transfer of bulky secretory cargos from the ER to the ERGIC/Golgi via a tunnel.

cell biology↗