Biology subjects

Fernandes, J.

Publications and source records attributed to Fernandes, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Microtubule Lattice Spacing Governs MAP-Motor Regulation

Microtubules (MTs) serve as intracellular tracks that enable molecular motors to transport cargos to specific cellular destinations. It has been proposed that the signals directing motor-driven transport are encoded on MTs through different isotypes, lattice conformations, and post- translational modifications (PTMs) of tubulin, or MT-associated proteins (MAPs) that decorate the MT surface. However, molecular predictions of these models have not been rigorously tested in vitro. Using isotypically pure recombinant tubulin and biochemical reconstitution, we examined how tubulin PTMs and MT lattice spacing influence MAP binding and kinesin-1 motility. We found that kinesin-1 is largely insensitive to tubulin PTMs but is strongly regulated by MT lattice spacing. Likewise, the MAPs tau, MAP7, MAP4, DCX, and MAP9 exhibit little sensitivity to tubulin PTMs, whereas the MT-binding affinities of tau, DCX, and MAP7 depend on lattice spacing. In the presence of activating (MAP7) and inhibitory (tau) MAPs, lattice spacing determines MAP occupancy and thereby controls kinesin-1 motility. These findings support a two-layer transport code in which MT lattice spacing directs MAP binding, and MAPs determine which motors can move along individual MT tracks.

biochemistry↗

bioRxiv2026

Toxin-triggered activation of regulated exocytosis enhances bacterial egress from the intestinal layer

Bacterial exit from host cells is essential for dissemination yet remains poorly understood. Here, we define a non-lytic egress pathway exploited by the enteric pathogen Yersinia pseudotuberculosis that co-opts the host exocytosis machinery in intestinal epithelial cells. The bacteria secrete a CNF-family toxin that activates the Cdc42-PLC {gamma}1-IP3-IP3R signaling cascade, triggering SNARE-dependent fusion of the Yersinia-containing vacuoles with the cell membrane via VAMP7, Stx4, and SNAP23. This controlled exocytotic release preserves epithelial barrier integrity and occurs infrequently, representing a key rate-limiting step in systemic spread. These findings establish the host exocytotic machinery as an active determinant of bacterial egress, uncover a conserved vesicle trafficking pathway hijacked by intracellular pathogens for dissemination, and assign a new role for bacterial toxins in regulating host cell exit. Authors SummaryMany bacterial pathogens penetrate and cross protective host cell barriers to spread within the body. While the mechanisms by which enteric bacteria enter host cells are well characterized, far less is known about how they exit host cells after invasion. In this study, we investigated how the enteric pathogen Yersinia pseudotuberculosis exits from human gut epithelial cells after crossing them. We found that they egress at the basolateral side of the intestinal cells through a controlled, non-destructive process that preserves the integrity of the epithelial barrier. To achieve this, the bacteria co-opt host signaling pathways involved in host cell vesicle release (exocytosis) to promote controlled egress. These exit events are rare, suggesting that bacterial escape from cells is a major bottleneck during infection. Importantly, we show that this process is enhanced by a secreted toxin of the Cytotoxic Necrotizing Factor (CNF) family (CNFY), which promotes the fusion of the bacteria-containing vacuole with the basolateral cell membrane. This finding uncovers a previously unrecognized role of bacterial toxins in facilitating bacterial cell egress.

microbiology↗

bioRxiv2026

IMAGENE: Single-cell association of live cell imaging and gene expression profiles of non-adherent cells through photoactivatable adhesives

Live cell imaging is uniquely placed to study cell behavior as it preserves spatial context and enables non-destructive observations over time. Integrating live cell imaging and molecular phenotypes with single-cell resolution is key to uncovering the relationship between the behavioral and morphological signatures of cells, and their molecular states. Non-adherent cells - as are most immune cells - however, present unique challenges in linking live cell imaging and fixed cell assays with single-cell resolution due to the difficulty of identifying individual cells across experimental modalities. To overcome this issue, we developed IMAGENE, an experimental and computational pipeline that leverages previously reported photoactivatable biocompatible adhesive material (PA-BAM) coatings for on-the-fly cell immobilization. We demonstrate the IMAGENE experimental and computational pipeline by generating a dataset of label-free time-lapse videos of primary human naive CD8+ T cells following 24 hours of polyclonal stimulation. Individual cells, including highly motile cells, can be matched to expression profiles of genes of interest obtained through KrakenFISH, a modified version of the previously reported autoFISH setup for automated, single-molecule fluorescence in situ hybridization (smFISH) experiments that supports sample parallelization. We use this data to train explainable machine learning models that predict expression levels of individual genes, with variable performance, from hand-crafted dynamic and spatial features obtained from live cell imaging.

bioengineering↗

bioRxiv2025

Chromosome-scale Solanum pennellii and Solanum cheesmaniae genome assemblies reveal structural variants, repeat content and recombination barriers of the tomato clade

Crop wild relatives are important resources for improving cultivated crops, yet the precise introgression of wild genetic material into cultivated crops is often difficult and cannot be fully defined without high-quality genome assemblies. Here we used PacBio HiFi, ONT ultra-long and Hi-C sequencing approaches to generate chromosome-scale de novo genome assemblies of two wild species related to the domesticated tomato (Solanum lycopersicum) -- the broadly stress-resistant Solanum pennellii (accession LA0716) and the salt-resistant Solanum cheesmaniae (accession LA1039). K-mer and BUSCO analysis of both assemblies demonstrated above 99% completeness and the improved S. pennellii genome adds 146 Mbp to the 12 chromosomes compared with the original reference. We aligned the new assemblies with seven gold-standard assemblies from the Lycopersicon clade, using Solanum tuberosum as an outgroup, and identified shared and species-specific structural variants. The repeat content of all nine assemblies was characterized, providing evidence for independent explosions of Tekay (gypsy superfamily) retrotransposons in S. pennellii and S. peruvianum. Whole genome sequencing of 709 recombinant plants derived from male and female backcrosses of three different hybrids (S.pennellii, S. cheesemaniae and Solanum lycopersicum cv. Micro-Tom crossed with Solanum lycopersicum cv. Moneyberg-TMV) revealed higher crossover rate in female meiosis. Recombination landscape analysis identified conserved female-enhanced recombination regions, and coldspots that were completely devoid of meiotic crossovers including megabase-scale inversions and insertion-deletion polymorphisms between S. lycopersicum and S. pennellii. In summary, we harnessed our high-quality S. pennellii and S. cheesmaniae genome assemblies to reveal how repeat content diverged in nature and during breeding, and uncovered how reproductive gender interacts with structural variants to dictate the recombination landscape in tomato hybrids.

plant biology↗

bioRxiv2025

Predicting and Controlling Collective Fate in Multicellular Systems

Collective behavior is a defining property of multicellular systems, where coordinated outcomes emerge from local cell-cell interactions. Yet the quantitative rules linking single-cell decision-making to tissue-scale organization remain poorly resolved. Here, we develop a quantitative framework that defines an order parameter predicting when initially disordered colonies undergo a transition to ordered fate alignment and when minimal, localized inputs can redirect their collective state. This analysis reveals a distinct control regime in which multicellular assemblies become susceptible to a single engineered "guide" cell. We validate these predictions by introducing guide cells that integrate into unperturbed colonies and redirect fate patterns within the theoretically defined control windows. Together, these results connect single-cell decision rules to emergent tissue-level organization and establish a generalizable biological control strategy in which a minority engineered subset can reliably redirect the developmental trajectory of a much larger multicellular population. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=65 SRC="FIGDIR/small/693804v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@37baeforg.highwire.dtl.DTLVardef@d1d86aorg.highwire.dtl.DTLVardef@d7375eorg.highwire.dtl.DTLVardef@1741e9a_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOSummary figure:C_FLOATNO Emergence, Scaling, and Control of Multicellular Collective Order. (A) Cell-number-dependent collective order. Sparse colonies lack coordination and yield disordered fate distributions; dense colonies exhibit coordinated, ordered outcomes. (B) Collective order rises with colony compactness ({propto} cell density) and collapses across colony radii R onto a single curve. (C) Once coordination emerges, a single "guide cell" can steer fate-analogous to a sheepdog guiding a flock. The herding efficacy exhibits a biphasic window versus cell number. C_FIG

systems biology↗

bioRxiv2025

Structure and mechanism of microtubule stabilization and motor regulation by MAP9

Microtubule-associated proteins (MAPs) regulate the organization of microtubules and control intracellular transport, but their individual contributions to microtubule dynamics and motor regulation remain poorly understood. Here, we identify MAP9 as a critical factor that stabilizes microtubules and facilitates neuronal morphogenesis. MAP9 knockdown abolishes the outgrowth of neurites, a phenotype not observed through the loss of other neuronal MAPs. Cryo-electron microscopy revealed that, unlike other MAPs that bind along protofilaments, MAP9 binds around the microtubule as a long alpha helix using five consecutive repeats. This unique binding mode enables MAP9 to staple adjacent protofilaments, thereby preventing microtubule depolymerization. We also showed that MAP9 selectively permits kinesin-3 and dynein motility while hindering kinesin-1 through interactions with a divergent loop-8 of the kinesin motor domain. Our results establish MAP9 as a key MAP required for neuronal growth and uncover how it differentially regulates intracellular transport driven by kinesin motors.

biophysics↗

bioRxiv2025

CDM1 is required for meiotic progression and genome integrity during male meiosis in Arabidopsis

Microsporogenesis involves the integration of meiosis with the developmental program leading to formation of microspores from meiocytes. The CALLOSE DEFICIENT MICROSPORE 1 (CDM1) gene of Arabidopsis has been previously characterized as being required for callose metabolism and its loss results in defects in microspore wall development, degeneration of meiotic products, and male sterility. We report here that CDM1 is required for normal meiosis I progression, meiotic chromosome organization, and maintenance of DNA integrity. CDM1 is part of the ATM dependent DNA damage response in dividing vegetative tissues. The cdm1 mutant shows defects in meiotic chromosome condensation, arrest at metaphase I, susceptibility to chromosome breakage in rDNA regions, defects in positioning of the products of meiosis, and increased expression of recombination related genes. However, fidelity of chromosome segregation is not affected. Furthermore, the meiotic phenotype of cdm1 is independent of SPO11 induced meiotic double strand breaks. CDM1 protein displays granular localization in meiosis throughout the cytoplasm that resembles that of mRNP granules. The results show that CDM1 is required for maintenance of proper chromosome organization and DNA integrity in the course of male meiosis. The chromosomal defects in cdm1 may originate from regions of defective or incomplete replication. HighlightCDM1 expression is DNA damage inducible in Arabidopsis mitotic cells. CDM1 is required for genome integrity in male meiosis independent of SPO11-induced DNA breaks, suggesting a role in regulation of premeiotic replication repair.

plant biology↗

bioRxiv2025

Purification protocol of hypertrophied hepatic stellate cells for their transcriptomic characterization from CDAHFD mice liver

Hepatic stellate cells (HSC) are known for their major role in hepatic fibrosis. Recently, it has been shown that different subpopulations of HSC co-exist during fibrogenesis and play different roles in the establishment of fibrosis. We previously highlighted, in murine model and human biopsies, a specific subpopulation of hypertrophied HSC (hypHSC) which exhibit exacerbated retinoid droplets and were closely associated to collagen fibers. The present study describes the purification protocol of hypHSC developed from a murine model of metabolic liver fibrosis. Liver dissociation followed by density gradient and fluorescence assisted cell sorting allowed us to obtain higlhly pure hypHSC preparations. Then, a transcriptomic analysis (bulk RNAseq) of hypHSCs versus quiescent HSCs purified from healthy mouse liver, was performed. This showed that hypHSCs molecular signature differs from HSC subtypes already described in the literature, with a "hybrid" profile of both inflammation and matrix remodeling. Our study highlights that a phenotype-to-molecular approach can provide complementary elements to single-cell molecular approaches.

cell biology↗

bioRxiv2025

Impaired stem cell migration and divisions in Duchenne Muscular Dystrophy revealed by live imaging

Dysregulation of stem cell properties is a hallmark of many pathologies, but the dynamic behaviour of stem cells in their microenvironment during disease progression remains poorly understood. Using the mdx mouse model of Duchenne Muscular Dystrophy, we developed innovative live-imaging of muscle stem cells (MuSCs) in vivo, and ex vivo on isolated myofibres. We show that mdx MuSCs have impaired migration and precocious differentiation through unbalanced symmetric divisions, driven by p38 and PI3K signalling pathways, in contrast to the p38-only dependence of healthy MuSCs. Cross-grafting shows that MuSC fate decisions are governed by intrinsic cues, whereas their migration behaviour is determined by the extracellular niche. This study provides the first dynamic analysis of dystrophic MuSC properties in vivo, reconciling conflicting reports on their function. Our findings establish DMD as a MuSC disease with intrinsic defects and niche dysfunction, offering strategies to restore stem cell functions for improved muscle regeneration.

cell biology↗

bioRxiv2024

miR-184 controls Dilp8 to regulate the timing of a critical developmental transition in Drosophila melanogaster

Organismal development depends on the precise coordination of growth and developmental timing, which is regulated by a complex interplay of intrinsic and extrinsic factors. However, the mechanisms underlying this regulation are not fully understood. Post-transcriptional regulation by microRNAs (miRNAs) plays a pivotal role in ensuring the proper timing of gene expression necessary for growth and development. In this study, we conducted a genetic screen to identify microRNAs that regulate developmental timing in Drosophila. Our screen identified miR-184, previously implicated in germline maturation and embryonic development, as a regulator of pupariation timing by acting in the larval imaginal discs. Using genetic and molecular approaches, we identified Drosophila insulin-like peptide 8 (Dilp8), a paracrine factor critical for regulating developmental stability, as a target of miR-184. During normal larval development miR-184 facilitates timely pupariation by regulating dilp8 levels. Furthermore, we demonstrate that miR-184 plays a critical role in tissue damage responses by inducing dilp8 expression, which delays pupariation to enable damage repair mechanisms. These findings reveal a novel post-transcriptional regulatory mechanism that links miR-184 to the control of developmental timing under normal growth conditions and in response to tissue damage.

developmental biology↗

bioRxiv2024

Immune regulators EDS1 and PAD4 constrain ADR1-dependent disease resistance in rice

Withdrawal StatementThe authors have withdrawn this manuscript owing to finding inconsistencies in genotyping of kitaake rice eds1 pad4 double mutant lines and to establishing that combined loss of EDS1 and PAD4 does not underlie the stunted autoimmune-like phenotype reported in this preprint. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding authors: Jane Parker, Thomas Kroj and Haitao Cui (co-corresponding)

plant biology↗

bioRxiv2023

Torques within and outside the human spindle balance twist at anaphase

At each cell division, nanometer-scale motors and microtubules give rise to the micron-scale spindle. Many mitotic motors step helically around microtubules in vitro, and most are predicted to twist the spindle in a left-handed direction. However, the human spindle exhibits only slight global twist, raising the question of how these molecular torques are balanced. Here, using lattice light sheet microscopy, we find that anaphase spindles in the epithelial cell line MCF10A have a high baseline twist, and we identify factors that both increase and decrease this twist. The midzone motors KIF4A and MKLP1 are redundantly required for left-handed twist at anaphase, and we show that KIF4A generates left-handed torque in vitro. The actin cytoskeleton also contributes to left-handed twist, but dynein and its cortical recruitment factor LGN counteract it. Together, our work demonstrates that force generators regulate twist in opposite directions from both within and outside the spindle, preventing strong spindle twist during chromosome segregation.

cell biology↗

bioRxiv2023

ERK1/2 Inhibition Alleviates Noise-Induced Hearing Loss While Tempering Down the Immune Response

Noise-induced hearing loss (NIHL) is a major cause of hearing impairment, yet no FDA-approved drugs exist to prevent it. Targeting the mitogen activated protein kinase (MAPK) cellular pathway has emerged as a promising approach to attenuate NIHL. Tizaterkib is an orally bioavailable, highly specific ERK1/2 inhibitor, currently in Phase-1 anticancer clinical trials. Here, we tested tizaterkibs efficacy against permanent NIHL in mice at doses equivalent to what humans are currently prescribed in clinical trials. The drug given orally 24 hours after noise exposure, protected an average of 20-25 dB SPL in three frequencies, in female and male mice, had a therapeutic window >50, and did not confer additional protection to KSR1 genetic knockout mice, showing the drug works through the MAPK pathway. Tizaterkib shielded from noise-induced cochlear synaptopathy, and a 3-day, twice daily, treatment with the drug was the optimal determined regimen. Importantly, tizaterkib was shown to decrease the number of CD45 and CD68 positive immune cells in the cochlea following noise exposure, which could be part of the protective mechanism of MAPK inhibition.

neuroscience↗

bioRxiv2021

Nanotrap Particles Improve Nanopore Sequencing of SARS-CoV-2 and Other Respiratory Viruses

Presented here is a magnetic hydrogel particle enabled workflow for capturing and concentrating SARS-CoV-2 from diagnostic remnant swab samples that significantly improves sequencing results using the Oxford Nanopore Technologies MinION sequencing platform. Our approach utilizes a novel affinity-based magnetic hydrogel particle, circumventing low input sample volumes and allowing for both rapid manual and automated high throughput workflows that are compatible with nanopore sequencing. This approach enhances standard RNA extraction protocols, providing up to 40x improvements in viral mapped reads, and improves sequencing coverage by 20-80% from lower titer diagnostic remnant samples. Furthermore, we demonstrate that this approach works for contrived influenza virus and respiratory syncytial virus samples, suggesting that it can be used to identify and improve sequencing results of multiple viruses in VTM samples. These methods can be performed manually or on a KingFisher Apex system.

microbiology↗

bioRxiv2021

INTEROCEPTIVE INFORMATION OF PHYSICAL VIGOR: OREXIN NEURONS GAUGE CIRCULATING IGF-I FOR MOTIVATIONAL MOTOR OUTPUT

The brain relies on interoceptive feedback signals to regulate bodily functions. Mice with low serum IGF-1 levels (LID mice) exhibit reduced spontaneous running, a behavior that normalizes after sustained systemic IGF-1 treatment. This observation led us to hypothesize that circulating IGF-1--a key regulator of skeletal muscle and bone mass that crosses the blood-brain barrier during physical activity--may convey body vigor information to the brain. Since hypothalamic orexin neurons, that are involved in regulating physical activity, express IGF-1 receptors (IGF-1R) and are modulated by this growth factor, we hypothesized that these neurons might gauge circulating IGF-1 levels to modulate physical activity. Indeed, inactivation of IGF-1R in mouse orexin neurons (Firoc mice) was associated to less time spent in free running. These mice maintain physical fitness but display altered mood and are less sensitive to the rewarding actions of exercise. Further, in response to exercise, Firoc mice showed limited c-fos activation of hypothalamic orexin neurons and monoaminergic neurons of the ventro-tegmental area (VTA) in the brainstem. This area is involved in the rewarding component of exercise that seems to be modulated by IGF-1, as mice receiving systemic IGF-1 showed increased c-fos expression in VTA neurons, while mice with reduced IGF-1R expression in VTA neurons showed no improved mood after exercise. Collectively, these results suggest that circulating IGF-1 is gauged by orexin neurons to modulate physical activity, and that VTA neurons convey the rewarding properties of exercise through direct actions of IGF-1 on them. Hence, serum IGF-1 may constitute an interoceptive signal acting onto orexin/VTA neurons to modulate physical activity according to physical vigor (muscle and bone mass).

neuroscience↗

bioRxiv2021

A simple, inexpensive and multi scale 3D fluorescent test sample for optical sectioning microscopies

Fluorescence standards allow for quality control and for the comparison of data sets across instruments and laboratories in applications of quantitative fluorescence. For example, users of microscopy core facilities expect a homogenous and time-invariant illumination and a uniform detection sensitivity, which are prerequisites for quantitative imaging analysis, particle tracking or fluorometric pH or Ca2+-concentration measurements. Similarly, confirming the three-dimensional (3-D) resolution of optical sectioning micro-scopes prior to volumetric reconstructions calls for a regular calibration with a standardised point source. Typically, the test samples required for such calibration measurements are different ones, and they depend much on the very microscope technique used. Also, the ever-increasing choice among these techniques increases the demand for comparison and metrology across instruments. Here, we advocate and demonstrate the multiple uses of a surprisingly versatile and simple 3-D test sample that can complement existing and much more expensive calibration samples: simple commercial tissue paper labelled with a fluorescent highlighter pen. We provide relevant sample characteristics and show examples ranging from the sub-{micro}m to cm scale, acquired on epifluorescence, confocal, image scanning, two-photon (2P) and light-sheet microscopes. Graphical abstractPyranine-labeled tissue paper, imaged upon 405-nm epifluorescence excitation through a 455LP LP dichroic and 465LP emission filter. Objective x20/NA0.25. Overlaid are the normalised absorbance (dashed) and emission spectra (through line), respectively. In the present work we show that this "primitive" and inexpensive three-dimensional (3-D) test sample is a surprisingly versatile and powerful tool for quality assessment, comparison across microscopes as well as routine metrology for optical sectioning techniques, both for research labs and imaging core facilities. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=172 SRC="FIGDIR/small/441588v1_ufig1.gif" ALT="Figure 1"> View larger version (84K): org.highwire.dtl.DTLVardef@13a11dforg.highwire.dtl.DTLVardef@16b818corg.highwire.dtl.DTLVardef@1061c64org.highwire.dtl.DTLVardef@32015a_HPS_FORMAT_FIGEXP M_FIG C_FIG Research highlights- highlighter-pen marked tissue paper is a surprisingly powerful and versatile test sample for 3-D fluorescence microscopies - standard tissue paper presents features ranging from 400 nm to centimetres - our sample can simultaneously be used for testing intensity, field homogeneity, resolution, optical sectioning and image contrast - it is easy to prepare, versatile, photostable and inexpensive

biophysics↗

bioRxiv2021

INSULIN-LIKE GROWTH FACTOR I SENSITIZATION REJUVENATES SLEEP PATTERNS IN OLD MICE

Sleep disturbances are common during aging. Compared to young animals, old mice show altered sleep structure, with changes in both slow and fast electrocorticographic (ECoG) activity and fewer transitions between sleep and wake stages. Insulin-like growth factor I (IGF-I), which is involved in adaptive changes during aging, was previously shown to increase ECoG activity in young mice and monkeys. Furthermore, IGF-I shapes sleep architecture by modulating the activity of mouse orexin neurons in the lateral hypothalamus (LH). We now report that both ECoG stimulation and activation of orexin neurons by systemic IGF-I is abrogated in old mice. Moreover, stimulation of orthodromically activated LH neurons by either systemic or local IGF-I in young mice is absent in old mice. As orexin neurons of old mice show markedly increased IGF-I receptor (IGF-IR) levels, suggesting loss of sensitivity to IGF-I, we treated old mice with AIK3a305, a novel IGF-IR sensitizer, and observed restored responses to IGF-I and rejuvenation of sleep patterns. Thus, disturbed sleep structure in aging mice may be related to impaired IGF-I signaling onto orexin neurons, reflecting a broader loss of IGF-I activity in the aged mouse brain.

neuroscience↗

bioRxiv2021

Hybrid multisite silicon neural probe with integrated flexible connector for interchangeable packaging

Multisite neural probes are a fundamental tool to study brain function. Hybrid silicon/polymer neural probes, in particular, allow the integration of complex probe geometries, such as multi-shank designs, with flexible biocompatible cabling. Despite these advantages and benefiting from the highly reproducible fabrication methods on both silicon and polymer substrates, they have not been widely available. This paper presents the development, fabrication, characterization, and in vivo electrophysiological assessment of a hybrid multisite multi-shank silicon probe with a monolithically integrated polyimide flexible interconnect cable. The fabrication process was optimized on wafer level and several neural probes with 64 gold electrode sites equally distributed along 8 shanks with an integrated 8 m-thick highly flexible polyimide interconnect cable were produce. To avoid the time-consuming bonding of the probe to definitive packaging, the flexible cable was designed to terminate in a connector pad that can mate with commercial zero-insertion force (ZIF) connectors for electronics interfacing. This allows great experimental flexibility since interchangeable packaging can be used according to experimental demands. High-density distributed in vivo electrophysiological recordings were obtained from the hybrid neural probes with low intrinsic noise and high signal-to-noise ratio (SNR).

bioengineering↗