Biology subjects

Bernardino de la Serna, J.

Publications and source records attributed to Bernardino de la Serna, J..

15 recordsLinked to original sources

bioRxiv2026

Protein Crowding Buffers Membrane Fluidity

Like cholesterol, protein crowding has been shown to alter the temperature dependence of lateral diffusion in biomembranes, eventually eliminating the distinct liquid-gel phase transition. Here, we resolve the molecular origin of this protein effect with coarse-grained and all-atom simulations of protein-crowded phosphatidylcholine bilayers, which give access to lipid structure and dynamics on both sides of the transition temperature. Unlike cholesterol, which is known to induce a continuous liquid ordered phase by having opposite effects on the two phases, we find that membrane proteins decrease lipid order at all temperatures studied. The disordering is stronger at low temperatures, so that once the proteins cover roughly half of the membrane area the low- and high-temperature phases become alike and the transition is lost. Our results suggest that in addition to local cholesterol concentration, local protein concentration may also help dictate membrane fluidity and especially its response to temperature change.

biophysics↗

bioRxiv2026

A Cherry-Flavoured E-Cigarette Adduct, BPGA, Reprograms Alveolar Epithelial Cell Fate Through Epithelial-to-Mesenchymal Transition and Evasion of Apoptosis

E-cigarettes have attracted significant attention as a safer substitute for conventional tobacco smoking. However, they have introduced new inhalable toxicants, including benzaldehyde-propylene glycol acetal (BPGA)--a chemical adduct produced by cherry-flavoured e-cigarettes. The health risks associated with such flavour-derived acetals remain insufficiently elucidated at the cellular level. This study investigated the role of BPGA in the progression of epithelial-to-mesenchymal transition (EMT)-like changes in alveolar epithelial cells (A549 cells). A549 cells exposed to various concentrations of BPGA were analysed for cell viability, morphology, mitochondrial function, lysosomal health, and cytoskeletal integrity using viability assays and fluorescence imaging. Intracellular reactive oxygen species (ROS) production was quantified using the 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) assay. Antioxidant enzyme expression, inflammatory responses, and EMT-associated phenotypic alterations were evaluated using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunofluorescence (IF) assays. Exposure of alveolar epithelial cells to BPGA caused a concentration-dependent decrease in cell viability. BPGA exposure resulted in mitochondrial membrane depolarisation, lysosomal damage, cytoskeletal changes, and stress fibre formation, which altered cell morphology. It significantly increased intracellular ROS production. As a result, antioxidant enzyme levels were upregulated as a protective response. However, during severe oxidative stress, this response was overwhelmed. Excess ROS disrupted cellular homeostasis and initiated apoptosis, though not completely. ROS also acted as a signalling molecule, promoting the upregulation of inflammatory mediators. These changes were associated with altered EMT marker expression, suggesting that BPGA might drive EMT-like remodelling. In conclusion, BPGA, a chemical adduct from e-cigarette vapour, induces alveolar injury by promoting oxidative stress, inflammation, and EMT-related changes, which may explain a mechanism by which e-cigarette exposure could lead to lung injury and pulmonary fibrosis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=169 SRC="FIGDIR/small/724520v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@2e9797org.highwire.dtl.DTLVardef@e2543eorg.highwire.dtl.DTLVardef@108bb1aorg.highwire.dtl.DTLVardef@33e710_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO C_FIG

pharmacology and toxicology↗

bioRxiv2025

Airway smooth muscle--on-a-chip: a microfluidic approach to study alveolar smooth muscle remodelling

Respiratory illnesses, like chronic obstructive pulmonary disease (COPD) and asthma, pose significant global health challenges due to their chronic nature and limited treatment options. Airway smooth muscle (ASM) plays a vital role in respiratory diseases, particularly in airway remodelling and obstruction. ASM, which encircles the bronchial tree and extends to the trachea, plays a vital yet not fully understood role in lung physiology. However, its dysfunction is strongly associated with asthma and COPD progression, leading to excessive contraction, increased inflammatory mediator release, and ASM hypertrophy. However, identifying its precise function is challenging due to limitations in existing research models for assessing ASM contraction. In vivo models offer a comprehensive physiological perspective but possess ethical concerns and they do not allow for the direct measurement of ASM contraction. Meanwhile, ex vivo and in vitro models provide a more direct assessment; however, they lack crucial physiological factors. Understanding how ASM cells interact with their surroundings is essential for gaining deeper insights into respiratory disorders. To address this gap, we aimed to mimic the human airway smooth muscle-on-a-chip model, incorporating ASM cells in a 3D microenvironment. This microfluidic platform provides a physiologically relevant environment, allowing for studying complex mechanisms that drive airway remodelling and dysfunction in respiratory diseases. The ASM-on-a-chip is designed for long-term 3D cell culture of ASM cells that reorient itself to form a smooth muscle fibre. The design provides side channels for manipulating the constituent of the hydrogel to study the effect of compounds on AMS remodelling.

bioengineering↗

bioRxiv2025

A rapidly prototyped, simple yet versatile dynamic breathing Exposure-on-a-Chip for investigating nanoparticle deposition in the alveoli

We developed and characterized a three-layer microfluidic Exposure-on-a-Chip (EOC) model to replicate the alveolar microenvironment and simulate breathing motions, offering a physiologically relevant platform for studying inhaled nanomedicines. The EOC chip, fabricated from biocompatible polydimethylsiloxane (PDMS), features a fluidic chamber for cell culture, a pneumatic chamber for pressure application, and a thin PDMS membrane (150 {micro}m) separating the chambers to support cell growth and enable mechanical stretching. Xurography and 3D printing were validated as efficient and reproducible fabrication methods. Mechanical characterization, using fluorescent bead tracing, confirmed that the PDMS membrane accurately mimics alveolar breathing motions under physiological conditions (1-8% strain). Biological validation showed that alveolar epithelial and endothelial cells cultured on the EOC formed functional monolayers, maintaining barrier integrity under cyclic and static stretching, separately. To study nanoparticle behavior, we examined the deposition of nanoparticles under dynamic stretching versus static conditions. Significantly fewer nanoparticles accumulated in cells under continuous dynamic exposure with stretching compared to static culture, highlighting the critical role of mechanical forces in nanoparticle-cell interactions. The EOC platform provides a robust and scalable tool for evaluating nanomedicine efficacy in dynamic environments, representing a significant advancement in alveolus-on-chip technology.

bioengineering↗

bioRxiv2024

In vivo functional profiling and structural characterisation of the human Glp1r A316T variant

Glucagon-like peptide-1 receptor agonists (GLP-1RAs) are effective therapies for type 2 diabetes (T2D) and obesity, yet patient responses are variable. Variation in the human Glp1r gene might be directly linked to therapeutic responses. A naturally occurring missense variant, A316T, protects against T2D and cardiovascular disease. Here, we have generated and characterised a human Glp1r A316T mouse model. Human Glp1rA316T/A316T mice displayed lower fasting blood glucose versus wildtype littermates, even under metabolic stress, and exhibited alterations in islet cytoarchitecture and /{beta}-cell identity under a high-fat, high-sucrose diet. This was however associated with blunted responses to GLP-1RAs in vivo. Further investigations in rodent and human {beta}-cell models demonstrated that human Glp1r A316T exhibits characteristics of constitutive activation but dampened GLP-1RA responses. Results are further supported by cryo-EM analyses and molecular dynamics simulations of GLP-1R A316T structure, collectively demonstrating that the A316T variant governs basal GLP-1R activity and pharmacological responses to GLP-1R-targeting therapies. TeaserThe Glp1r A316T missense variant displays improved glucose tolerance but impaired pharmacological incretin responses in vivo.

cell biology↗

bioRxiv2024

Molecular mapping and functional validation of GLP-1R cholesterol binding sites in pancreatic beta cells

G protein-coupled receptors (GPCRs) are integral membrane proteins which closely interact with their plasma membrane lipid microenvironment. Cholesterol is a lipid enriched at the plasma membrane with pivotal roles in the control of membrane fluidity and maintenance of membrane microarchitecture, directly impacting on GPCR stability, dynamics and function. Cholesterol extraction from pancreatic beta cells has previously been shown to disrupt the internalisation, clustering and cAMP responses of the glucagon-like peptide-1 receptor (GLP-1R), a class B1 GPCR with key roles in the control of blood glucose levels via the potentiation of insulin secretion in beta cells and weight reduction via the modulation of brain appetite control centres. Here, we unveil the detrimental effect of a high cholesterol diet on GLP-1R-dependent glucoregulation in vivo, and the improvement in GLP-1R function that a reduction in cholesterol synthesis using simvastatin exerts in pancreatic islets. We next identify and map sites of cholesterol high occupancy and residence time on active versus inactive GLP-1Rs using coarse-grained molecular dynamics (cgMD) simulations, followed by a screen of key residues selected from these sites and detailed analyses of the effects of mutating one of these residues, Val229, to alanine on GLP-1R interactions with cholesterol, plasma membrane behaviours, clustering, trafficking and signalling in pancreatic beta cells and primary islets, unveiling an improved insulin secretion profile for the V229A mutant receptor. This study 1) highlights the role of cholesterol in regulating GLP-1R responses in vivo; 2) provides a detailed map of GLP-1R - cholesterol binding sites in model membranes; 3) validates their functional relevance in beta cells; and 4) highlights their potential as locations for the rational design of novel allosteric modulators with the capacity to fine-tune GLP-1R responses.

cell biology↗

bioRxiv2023

Exposure to aldehyde cherry e-liquid flavouring and its vape by-product disrupts pulmonary surfactant biophysical function

Over the last decade there has been a surge in vaping device usage, especially among adolescents, raising concerns for potentially related lung damage. Notoriously, there have been many e-cigarette or vaping-related lung injury (EVALI) cases resulting in hospitalisations and deaths. Although the vaping component vitamin E acetate has been linked to a large proportion of EVALI cases resulting in its widespread banning, one fifth of the cases remain unexplained. Furthermore, the overall long-term impact of vaping on respiratory health is poorly understood. A likely driver behind EVALI is pulmonary surfactant disruption, as it is the first point of contact for any inhaled toxicant in the alveoli, and abnormalities of its function are linked to some symptoms presented in EVALI cases. Aberrant biophysical function of the surfactant results in alveolar surface tension increase, causing alveolar collapse. Vaping chemicals with the potential to disrupt surfactant function must be hydrophobic molecules able to interact with surfactant components at the alveolar air-liquid interface. Recent findings have recorded the synthesis of highly hydrophobic acetal by-products of the base vaping chemical propylene glycol and common flavouring aldehydes, including the cherry flavouring benzaldehyde, not identified in preliminary e-liquid safety tests. This study provides evidence that benzaldehyde and its by-product, benzaldehyde propylene glycol acetal, have the potential to significantly disrupt surfactant biophysical function via interactions with surfactant proteins SP-B and/or SP-C, which may provide stable interactions within the surfactant film by forming associations with the sublayer of surfactant three-dimensional structure present at high lateral compression, i.e., expiration breathing. Data also suggest considerable vaping chemical loss to the experimental subphase, indicating potential further implications to the alveolar epithelial layer beneath.

pharmacology and toxicology↗

bioRxiv2022

Surfactant Proteins SP-B and SP-C in Pulmonary Surfactant Monolayers: Physical Properties Controlled by Specific Protein-Lipid Interactions

The lining of the alveoli is covered by pulmonary surfactant, a complex mixture of surface-active lipids and proteins that enables efficient gas exchange between inhaled air and the circulation. Despite decades of advancements in the study of the pulmonary surfactant, the molecular scale behavior of the surfactant and the inherent role of the number of different lipids and proteins in surfactant behavior are not fully understood. The most important proteins in this complex system are the surfactant proteins SP-B and SP-C. Given this, in this work we performed non-equilibrium all-atom molecular dynamics simulations to study the interplay of SP-B and SP-C with multi-component lipid monolayers mimicking the pulmonary surfactant in composition. The simulations were complemented by z-scan fluorescence correlation spectroscopy and atomic force microscopy measurements. Our state-of-the-art simulation model reproduces experimental pressure-area isotherms and lateral diffusion coefficients. In agreement with previous research, the inclusion of either SP-B and SP-C increases surface pressure, and our simulations provide a molecular scale explanation for this effect: The proteins display preferential lipid interactions with phosphatidylglycerol, they reside predominantly in the lipid acyl chain region, and they partition into the liquid expanded phase or even induce it in an otherwise packed monolayer. The latter effect is also visible in our atomic force microscopy images. The research done contributes to a better understanding of the roles of specific lipids and proteins in surfactant function, thus helping to develop better synthetic products for surfactant replacement therapy used in the treatment of many fatal lung-related injuries and diseases.

biophysics↗

bioRxiv2022

An examination of the divergent spatiotemporal signaling of GLP-1R versus GIPR in pancreatic beta cells

The incretin receptors, glucagon-like peptide-1 receptor (GLP-1R) and glucose-dependent insulinotropic polypeptide receptor (GIPR), are class B GPCRs and prime therapeutic targets for the treatment of type 2 diabetes (T2D) and obesity. They are expressed in pancreatic beta cells where they potentiate insulin release in response to food intake. Despite GIP being the main incretin in healthy individuals, GLP-1R has been favoured versus GIPR as a therapeutic target due to GIPR responses being blunted in T2D patients and the conflicting effects of GIPR agonists and antagonists in improving glucose tolerance and preventing weight gain. There is, however, a recently renewed interest in GIPR biology following the realisation that GIPR responses can be restored after an initial period of blood glucose normalization and the recent development of dual GLP-1R-GIPR agonists with superior capacity for the control of blood glucose levels and weight. The importance of GLP-1R trafficking and subcellular signaling in the control of receptor outputs is well established, but little is known about the pattern of spatiotemporal signaling from the GIPR in beta cells. Here we have directly compared the main trafficking and signaling characteristics of both receptors in pancreatic beta cells, finding striking differences in their propensities for internalization, recycling, and degradation, as well as plasma membrane versus endosomal activity, with potential implications for receptor-specific control of beta cell function.

cell biology↗

bioRxiv2021

Multi-dimensional and spatiotemporal correlative imaging at the plasma membrane of live cells to determine the continuum nano-to-micro scale lipid adaptation and collective motion

The primary cilium is a specialized plasma membrane protrusion with important receptors for signalling pathways. In polarized epithelial cells, the primary cilium assembles after the midbody remnant (MBR) encounters the centrosome at the apical surface. The membrane surrounding the MBR, namely remnant associated membrane patch (RAMP) once situated next to the centrosome, releases some of its lipid components to form a centrosome-associated membrane patch (CAMP) from which the ciliary membrane stems. The RAMP undergoes a spatiotemporal membrane refinement during the formation of the CAMP, which becomes highly enriched in condensed membranes with low lateral mobility. To better understand this process, we have developed a correlative imaging approach that yields quantitative information about the lipid lateral packing, its mobility and collective assembly at the plasma membrane at different spatial scales over time. Our work paves the way towards a quantitative understanding of lipid collective assembly at the plasma membrane spatiotemporally as a functional determinant in cell biology and its direct correlation with the membrane physicochemical state. These findings allowed us to gain a deeper insight into the mechanisms behind the biogenesis of the ciliary membrane of polarized epithelial cells.

biophysics↗

bioRxiv2020

Addressing differentiation in live human keratinocytes by assessment of membrane packing order

Differentiation of keratinocytes is critical for epidermal stratification and formation of a protective stratum corneum. It involves a series of complex processes leading through gradual changes in characteristics and functions of keratinocytes up to their programmed cell death via cornification. The stratum corneum is an impermeable barrier, comprised of dead cell remnants (corneocytes) embedded within lipid matrix. Corneocyte membranes are comprised of specialized lipids linked to late differentiation proteins, contributing to the formation of a highly stiff and mechanically strengthen layer. To date, the assessment of the progression of keratinocyte differentiation is only possible by determination of specific differentiation markers, e.g. by using proteomics-based approaches. Unfortunately, this requires fixation or cell lysis, and currently there is no robust methodology available to study differentiation in living cells, neither at a single cell, nor in high throughput. Here, we explore a new live-cell based approaches for screening differentiation advancement in keratinocytes, in a "calcium switch" model. We employ a polarity-sensitive dye, Laurdan, and Laurdan general polarization function (GP) as a reporter of the degree of membrane lateral packing order or condensation, as an adequate marker of differentiation. We show that the assay is straightforward and can be conducted either on a single cell level using confocal spectral imaging or on the ensemble level using a fluorescence plate reader. Such systematic quantification may become useful for understanding mechanisms of keratinocyte differentiation, such as the role of membrane inhomogeneities in stiffness, and for future therapeutic development.

developmental biology↗

bioRxiv2019

The ciliary membrane of polarized epithelial cells stems from a midbody remnant-associated membrane patch with condensed nanodomains

The primary cilium is a specialized plasma membrane protrusion that harbors receptors involved in important signaling pathways. Despite its central role in regulating cellular behavior, the biogenesis of the primary cilium is not fully understood. In fact, the source of the ciliary membrane remains a mystery in cell types that assemble their primary cilium entirely at the cell surface, such as polarized renal epithelial cells. After cytokinesis, the remnant of the midbody of these cells moves to the center of the apical surface, where it licenses the centrosome for ciliogenesis through an unidentified mechanism. Here, to investigate the origin of the ciliary membrane and the role of the midbody remnant, we analyzed membrane compaction and lipid dynamics at the microscale and nanoscale in living renal epithelial MDCK cells. We found that a specialized patch made of condensed membranes with restricted lipid lateral mobility surrounds the midbody remnant. This patch accompanies the remnant on its journey towards the centrosome and, once the two structures have met, the remnant delivers part of membranes of the patch to build the ciliary membrane. In this way, we have determined the origin of the ciliary membrane and the contribution of the midbody remnant to primary cilium formation in cells whose primary cilium is assembled at the plasma membrane.

cell biology↗

bioRxiv2018

Nanoscale spatio-temporal diffusion modes measured by simultaneous confocal and STED imaging

The diffusion dynamics in the cellular plasma membrane provides crucial insights into the molecular interactions, organization and bioactivity. Fluorescence correlation spectroscopy combined with super-resolution stimulated emission depletion nanoscopy (STED-FCS) measures such dynamics with high spatial and temporal resolution and reveals nanoscale diffusion characteristics by measuring the molecular diffusion in conventional confocal mode and super-resolved STED mode sequentially. However, to directly link the spatial and the temporal information, a method that simultaneously measures the diffusion in confocal and STED modes is needed. Here, to overcome this problem, we establish an advanced STED-FCS measurement method; line interleaved excitation scanning STED-FCS (LIESS-FCS) which discloses the molecular diffusion modes at different spatial positions with a single measurement. It relies on fast beam-scanning along a line with alternating laser illumination that yields, for each pixel, the apparent diffusion coefficients for two different observation spot sizes (conventional confocal and super-resolved STED). We demonstrate the potential of the LIESS-FCS approach with simulations and experiments on lipid diffusion in model and live cell plasma membranes. We also apply LIESS-FCS to investigate the spatio-temporal organization of GPI-anchored proteins in the plasma membrane of live cells which interestingly show multiple diffusion modes at different spatial positions.

biophysics↗

bioRxiv2017

Dissecting single cell molecular spatiotemporal mobility and clustering at Focal Adhesions in polarised cells by fluorescence fluctuation spectroscopy methods

Quantitative fluorescence fluctuation spectroscopy from optical microscopy datasets is a very powerful tool to resolve multiple spatiotemporal cellular and subcellular processes at the molecular level. In particular, raster image correlation spectroscopy (RICS) and number and brightness analyses (N&B) yield molecular mobility and clustering dynamic information extracted from real-time cellular processes. This quantitative information can be inferred in a highly flexibly and detailed manner, i.e. 1) at the localisation level: from full-frame datasets and multiple regions of interest within; and 2) at the temporal level: not only from full-frame and multiple regions, but also intermediate temporal events. Here we build on previous research in deciphering the molecular dynamics of paxillin, a main component of focal adhesions. Cells use focal adhesions to attach to the extracellular matrix and interact with their local environment. Through focal adhesions and other adhesion structures, cells sense their local environment and respond accordingly; due to this continuous communication, these structures can be highly dynamic depending on the extracellular characteristics. By using a previously well-characterised model like paxillin, we examine powerful sensitivity characteristics and some limitations of RICS and N&B analyses. We show that cells upon contact to different surfaces show differential self-assembly dynamics in terms of molecular diffusion and oligomerisation. In addition, single-cell studies show that these dynamics change gradually following an antero-posterior gradient.

biophysics↗

bioRxiv2017

Advanced processing and analysis of conventional confocal microscopy generated scanning FCS data

Scanning Fluorescence Correlation Spectroscopy (scanning FCS) is a variant of conventional point FCS that allows molecular diffusion at multiple locations to be measured simultaneously. It enables disclosure of potential spatial heterogeneity in molecular diffusion dynamics and also the acquisition of a large amount of FCS data at the same time, providing large statistical accuracy. Here, we optimize the processing and analysis of these large-scale acquired sets of FCS data. On one hand we present FoCuS-scan, scanning FCS software that provides an end-to-end solution for processing and analysing scanning data acquired on commercial turnkey confocal systems. On the other hand, we provide a thorough characterisation of large-scale scanning FCS data over its intended time-scales and applications and propose a unique solution for the bias and variance observed when studying slowly diffusing species. Our manuscript enables researchers to straightforwardly utilise scanning FCS as a powerful technique for measuring diffusion across a broad range of physiologically relevant length scales without specialised hardware or expensive software.

biophysics↗