Biology subjects

Vilar, J.

Publications and source records attributed to Vilar, J..

4 recordsLinked to original sources

bioRxiv2026

Cell-specific isolation and functional analysis of primary cardiomyocyte-derived large extracellular vesicles from infarcted mouse hearts

Extracellular vesicles (EVs) are membrane-bound particles secreted by all cells and serve as critical mediators of intercellular communication in both physiological and pathological contexts. However, elucidating the precise functions of endogenous EVs remains challenged by a fundamental technical bottleneck: current isolation methodologies depend on shared physical properties or generic surface markers, preventing the selective enrichment of cell-type-specific EVs within complex tissues. Consequently, EVs recovered from biofluids or tissue extracts represent heterogeneous populations, confounding the direct attribution of biochemical signatures and biological activities to distinct cellular origins. Here, we describe a versatile genetic-labeling and affinity-based purification strategy that enables the selective isolation of cell-specific primary EV. Using cardiomyocyte-derived EV (CM-EVs) as a proof-of-concept, we crossed Myh6-Cre mice with the ROSA26-mTmG reporter line to restrict membrane-targeted GFP expression to cardiomyocytes. Following myocardial infarction, cardiac EV were recovered and GFP-positive CM-EVs were selectively purified by anti-GFP nanobody-based magnetic pulldown. This approach yields highly enriched, functional cardiomyocyte-derived extracellular vesicles (CM-EVs). To accommodate diverse analytical needs, we established two distinct elution workflows: (i) a denaturing protocol optimized for downstream proteomics and immunoblotting, and (ii) a gentle, non-denaturing protocol that preserves vesicle integrity and biological activity for downstream in vitro and in vivo functional assays. Using this method, we characterized CM-EVs size distribution and proteomic signature, and demonstrated their capacity to transfer functional cargo to target cells in vitro. Because the purification approach relies solely on targeting a genetically restricted membrane tag rather than assumed surface markers, it is immediately adaptable to virtually any cell type for which a validated Cre driver exists. This method removes a long-standing bottleneck in EV research and provides a broadly applicable platform for deciphering the biochemical composition and in-vivo functions of cell-type-specific primary EV in physiological and pathological settings.

cell biology↗

bioRxiv2023

Chaperone-driven entropic separation of amyloid nanofilament bundles

The disassembly of misfolded protein aggregates is a requirement for the proper functioning of cells. It has implications in multiple neuropathologies, such as Alzheimers and Parkinsons diseases. The active unbundling of fibrillar aggregates has recently been identified as a key, rate-limiting step in the disassembly process. Yet, the nature of the underlying molecular mechanism remains an outstanding question. Here, we develop a coarse-grained computational approach from the atomistic structural information and show that the interactions of molecules tethered to fibrils lead to entropic forces consistent with the unbundling process observed in amyloid -synuclein disaggregation by Hsp70. We uncover two main types of entropic effects, categorized as intra- and inter-protofilament, which are differentially affected by the system parameters and conditions. Our results show that only highly efficient chaperone systems can overcome the free energy cost of the lateral association between two protofilaments. Through the analysis of cryo-electron tomography and high-speed atomic force microscopy data, we find that co-chaperone networks and ATP hydrolysis are needed to achieve the conditions for highly efficient entropic force generation. We highlight the implications of these results for the design of targeted therapies for the underlying neuropathologies.

biophysics↗

bioRxiv2023

The unreasonable effectiveness of equilibrium gene regulation through the cell cycle

Systems like the prototypical lac operon can reliably hold the repression of transcription upon DNA replication across cell cycles with just ten repressor molecules per cell and, in addition, behave as if they were at equilibrium. The origin of this type of phenomena is still an unresolved question of major implications. Here, we develop a general theory to analyze strong perturbations in quasi-equilibrium systems and use it to quantify the effects of DNA replication in gene regulation. We find a scaling law that connects actual transcription with its predicted equilibrium values in terms of a single kinetic parameter. We show that even the simplest, exceptionally reliable natural system functions beyond the physical limits of naive regulation through compensatory mechanisms that suppress nonequilibrium effects. We validate the approach with both in vivo cell-population and single-cell characterization of the lac operon. Analyses of synthetic systems without adjuvant activators, such as the cAMP receptor protein (CRP), do not show this reliability. Our results provide a rationale for the function of CRP, beyond just being a tunable activator, as a mitigator of cell cycle perturbations.

biophysics↗

bioRxiv2019

Time-resolved single-cell transcriptomics uncovers dynamics of cardiac neutrophil diversity in murine myocardial infarction

ObjectiveAfter myocardial infarction, neutrophils rapidly and massively infiltrate the heart, where they can promote both tissue healing and damage. Here, we investigated the dynamics of cardiac neutrophil heterogeneity after infarction.\n\nMethods and resultsWe employed single-cell transcriptomics (scRNA-seq) to investigate temporal neutrophil heterogeneity in the heart after murine myocardial infarction. At day 1, 3, and 5 after infarction, neutrophils could be delineated into six distinct clusters with specific time-dependent patterning and proportions. While the majority of neutrophils at day 1 were characterized by high expression of chemokines (e.g. Cxcl3, Ccl6), and putative activity of transcriptional regulators involved in hypoxic response (Hif1a) and emergency granulopoiesis (Cebpb), two major subsets of Siglecfhi (enriched for e.g. Icam1 and Tnf) and Siglecflow (Slpi, Ifitm1) neutrophils were found at 3 and 5 days. Flow cytometry analysis confirmed the presence of LY6G+SIGLECFhi and LY6G+SIGLECFlow neutrophils in the heart from 3 days after infarction onwards. LY6G+SIGLECFhi neutrophils were absent from the bone marrow, blood and spleen, suggesting local acquisition of surface SIGLECF. Acquisition of the SIGLECFhi state was paralleled by features of neutrophil ageing and activation (ICAM1hiCXCR4hiCD49dhiCD62Llow). scRNA-seq of atherosclerotic aortas revealed two neutrophil subsets with gene expression patterns reminiscent of the major Siglecfhi and Siglecflow cardiac neutrophil subpopulations, revealing that these populations may be present across distinct contexts of cardiovascular inflammation.\n\nConclusionAltogether, our data provide a time-resolved census of neutrophil diversity and gene expression dynamics in the mouse ischemic heart at the single-cell level, and suggests that temporal neutrophil heterogeneity is in part driven by local transition to a SIGLECFhi state.

immunology↗