Biology subjects

Carravilla, P.

Publications and source records attributed to Carravilla, P..

8 recordsLinked to original sources

bioRxiv2025

Prodan-based solvatochromic probes for polarity imaging of organelles

Solvatochromic probes provide microscopic, structural and functional information on their targeted cellular compartments. In this field, the challenge lies in designing probes that are both sufficiently sensitive to environment, and specific in their subcellular localization. Here, we design Prodan-based polarity probes targeting the following organelles: mitochondria, endoplasmic reticulum, Golgi apparatus, lysosomes and lipid droplets. The new probes provide robust organelle targeting, except for the mitochondrial probe, whose targeting ability is cell type dependent. Due to operating range of Prodan fluorophore in the UV-Blue region, our probes can be easily combined with other fluorescent tags in the visible range. Therefore, polarity of sub-cellular compartments can be studied together with additional fluorescent reporters. We used these probes to show the polarity of organelle membranes in healthy cells and under starvation condition where we observed organelle-specific polarity remodeling. These probes are important addition to the repertoire of smart cellular probes and will find critical use in understanding spatiotemporal regulation of cellular physiology.

biophysics↗

bioRxiv2025

NanTex enables computational multiplexing and phenotyping of organelles across super-resolution modalities

Super-resolution microscopy (SRM) enables nanoscale visualization of cellular organelles but remains constrained by spectral overlap, labeling requirements, and temporal offsets in multicolor imaging. We present NanTex, a deep learning framework that introduces nanotexture as a universal descriptor of subcellular organization and achieves probabilistic demixing of multiple organelles from single-channel SRM images. Unlike segmentation methods that enforce exclusivity, NanTex preserves overlapping morphologies, enabling faithful reconstruction even in crowded regions. Trained on small curated datasets with augmentation, NanTex generalized across SMLM, MINFLUX, STED, SIM, and live-cell Airyscan, with modality-specific retraining where necessary. Demonstrated on cytoskeletal, endomembrane, and metabolic organelles, NanTex achieved high-fidelity reconstructions and extended uniquely to live-cell imaging, where it eliminated temporal misalignment and enabled quantitative tracking of vesicle-like ER subdomains. Notably, NanTex enables quantitative dynamic readouts directly from single-channel live-cell data, revealing and tracking hidden subdomains in real time without the need for additional labels. Beyond multiplexing, NanTex supported computational phenotyping by distinguishing structurally distinct yet molecularly identical populations, exemplified by nocodazole-induced microtubule depolymerization and its glyoxal-mediated modulation. Together, these results establish nanotexture as a paradigm-shifting descriptor for organelle identity and position NanTex as a modality-agnostic, label-efficient, and live-cell-ready strategy for quantitative nanoscale biology. Significance StatementNanTex reframes multiplexing as texture-based demixing, introducing nanotexture as a universal fingerprint of organelle identity. By enabling quantitative, label-efficient reconstructions across SRM modalities and live-cell phenotyping, including vesicle tracking from single-channel data, NanTex opens a path to studying organelle remodeling and disease progression with unprecedented fidelity across SRM modalities.

biophysics↗

bioRxiv2023

Estrogen receptor activation remodels TEAD1 gene expression to alleviate nonalcoholic fatty liver disease

IntroductionThe occurrence of obesity-related hepatic malignancies differs between sexes, suggesting the involvement of sex hormones. Female sex hormones maintain cell homeostasis through estrogen receptor (ER) signaling and protect from developing nonalcoholic fatty liver disease (NAFLD) in mice and humans. RationaleTo understand recovery from high-fat diet (HFD)-induced liver disease in males upon estrogen treatment, we comprehensively characterized molecular changes in the liver upon selective activation of estrogen receptors (ERs) to identify novel therapeutic targets downstream of estrogen signaling. MethodsTo dissect hepatic ER isoform-driven responses, we integrated liver transcriptomes from female and male HFD mice treated with or without four different estrogen agonists, along with multiomics data, including bulk, single-cell and spatial transcriptomics, chromatin profiling, machine learning models and advanced microscopy. Patient cohorts and primary human hepatocyte spheroids datasets were included. ResultsOnly males developed liver steatosis. We found that selective activation of either ER or ER{beta} reduced HFD-induced hepatic steatosis in male mice. Systemic ER activation restored HFD-induced aberrant gene expression of cellular processes across liver cell types, including hepatocytes. Profiling of marked histones revealed that ER activation modulated promoter and enhancer sites and identified 68 estrogen-sensitive enhancer-gene pairs. Most of these genes were similarly deregulated in human nonalcoholic fatty liver disease (NAFLD) patients, including the transcription factor TEAD1. TEAD1 expression increased in NAFLD patients, and inhibiting TEAD ameliorated steatosis in spheroids by suppressing lipogenic pathways. ConclusionsSystemic activation of ER or ER{beta} modulates molecular pathways in the liver to counteract NAFLD. Our study identified TEAD1 as a key ER-sensitive gene and suggested that its inhibition poses a therapeutic strategy to combat NAFLD without the undesired side effects elicited by estrogen signaling. Clinical research relevanceWe identified drug targets downstream of estrogen signaling, including TEAD1, and demonstrate that TEAD inhibition improves steatosis by suppressing lipogenic pathways. Basic research relevanceThe targeted activation of nuclear ERs recovers high-fat diet-induced molecular and physiological liver phenotypes by remodeling core pathways beyond lipid metabolism. ER-responsive enhancers regulate central metabolic genes of clinical significance in NAFLD patients, highlighting the potential impact of this research on understanding liver cell plasticity. HIGHLIGHTSO_LIsteatosis in livers of high-fat diet (HFD) male mice was effectively reduced by selective activation of estrogen receptors (ER and ER{beta}) with four different agonists. C_LIO_LIER agonist treatments successfully reversed HFD-induced changes in gene regulation and expression, revealing new treatment targets involving previously unconnected molecular pathways. C_LIO_LIestrogen-sensitive enhancers regulated important genes, including TEAD1, emerging as pivotal NAFLD regulators significantly impacting metabolic processes. C_LIO_LIhigh TEAD1 gene expression in NAFLD patients correlated with disease severity, underscoring its clinical significance in disease progression. C_LIO_LIinhibiting TEAD with small molecules alleviated steatosis by suppressing lipogenic pathways, resembling some of the same beneficial effects as estrogen treatment. C_LI

genomics↗

bioRxiv2023

Neural Network Informed Photon Filtering Reduces Artifacts in Fluorescence Correlation Spectroscopy Data

Fluorescence Correlation Spectroscopy (FCS) techniques are well-established tools to investigate molecular dynamics in confocal and super-resolution microscopy. In practice, users often need to handle a variety of sample or hardware-related artifacts, an example being peak artifacts created by bright, slow-moving clusters. Approaches to address peak artifacts exist, but measurements suffering from severe artifacts are typically non-analyzable. Here, we trained a 1-dimensional U-Net to automatically identify peak artifacts in fluorescence time-series and then analyzed the purified, non-artifactual fluctuations by time-series editing. We show that in samples with peak artifacts, the transit time and particle number distributions can be restored in simulations and validated the approach in two independent biological experiments. We propose that it is adaptable for other FCS artifacts, such as detector dropout, membrane movement, or photobleaching. In conclusion, this simulation-based, automated, open-source pipeline makes measurements analyzable which previously had to be discarded and extends every FCS users experimental toolbox.

biophysics↗

bioRxiv2023

Deep learning enables fast, gentle STED microscopy

STED microscopy is widely used to image subcellular structures with super-resolution. Here, we report that denoising STED images with deep learning can mitigate photobleaching and photodamage by reducing the pixel dwell time by one or two orders of magnitude. Our method allows for efficient and robust restoration of noisy 2D and 3D STED images with multiple targets and facilitates long-term imaging of mitochondrial dynamics.

bioengineering↗

bioRxiv2021

STED super-resolution imaging of membrane packing and dynamics by exchangeable polarity-sensitive dyes

Understanding the plasma membrane nano-scale organisation and dynamics in living cells requires microscopy techniques with high spatial and temporal resolution, permitting for long acquisition times, and that allow for the quantification of membrane biophysical properties such as lipid ordering. Among the most popular super-resolution techniques, stimulated emission depletion (STED) microscopy offers one of the highest temporal resolutions, ultimately defined by the scanning speed. However, monitoring live processes using STED microscopy is significantly limited by photobleaching, which recently has been circumvented by exchangeable membrane dyes that only temporarily reside in the membrane. Here, we show that NR4A, a polarity-sensitive exchangeable plasma membrane probe based on Nile Red, permits the super-resolved quantification of membrane biophysical parameters in real time with high temporal and spatial resolution as well as long acquisition times. The potential of this polarity-sensitive exchangeable dye is showcased by live-cell real-time 3D-STED recordings of bleb formation and lipid exchange during membrane fusion, as well as by STED-fluorescence correlation spectroscopy (STED-FCS) experiments for the simultaneous quantification of membrane dynamics and lipid packing, which correlate in model and live-cell membranes.

biophysics↗

bioRxiv2021

Diffusion and interaction dynamics of the cytosolic peroxisomal import receptor PEX5

Measuring diffusion dynamics in living cells is essential for the understanding of molecular interactions. While various techniques have been used to explore such characteristics in the plasma membrane, this is less developed for measurements inside the cytosol. An example of cytosolic action is the import of proteins into peroxisomes, via the peroxisomal import receptor PEX5. Here, we combined advanced microscopy and spectroscopy techniques such as fluorescence correlation spectroscopy (FCS) and super-resolution STED microscopy to present a detailed characterization of the diffusion and interaction dynamics of PEX5. Among other features, we disclose a slow diffusion of PEX5, independent of aggregation or target binding, but associated with cytosolic interaction partners via its N-terminal domain. This sheds new light on the functionality of the receptor in the cytosol. Besides specific insights, our study highlights the potential of using complementary microscopy tools to decipher molecular interactions in the cytosol via studying their diffusion dynamics. SummaryThe peroxisomal import receptor PEX5 transports newly synthesized proteins from the cytosol to the peroxisomal matrix. Here the cytosolic diffusion and interaction dynamics of PEX5 are characterized by advanced microscopic spectroscopy methods, revealing a so far unknown interaction partner.

biophysics↗

bioRxiv2020

Nanoscopy on the Chea(i)p

Super-resolution microscopy allows for stunning images with a resolution well beyond the optical diffraction limit, but the imaging techniques are demanding in terms of instrumentation and software. Using scientific-grade cameras, solid-state lasers and top-shelf microscopy objective lenses drives the price and complexity of the system, limiting its use to well-funded institutions. However, by harnessing recent developments in CMOS image sensor technology and low-cost illumination strategies, super-resolution microscopy can be made available to the mass-markets for a fraction of the price. Here, we present a 3D printed, self-contained super-resolution microscope with a price tag below 1000 $ including the objective and a cellphone. The system relies on a cellphone to both acquire and process images as well as control the hardware, and a photonic-chip enabled illumination. The system exhibits 100nm optical resolution using single-molecule localization microscopy and can provide live super-resolution imaging using light intensity fluctuation methods. Furthermore, due to its compactness, we demonstrate its potential use inside bench-top incubators and high biological safety level environments imaging SARS-CoV-2 viroids. By the development of low-cost instrumentation and by sharing the designs and manuals, the stage for democratizing super-resolution imaging is set.

microbiology↗