Biology subjects

Garcia-Arcos, J. M.

Publications and source records attributed to Garcia-Arcos, J. M..

6 recordsLinked to original sources

bioRxiv2026

Agile manufacturing of PDMS microfluidic devices for cell culture without a clean room

Microfluidic devices control fluid flow and the cellular microenvironment, and their fabrication usually relies on photolithography in a clean room. For devices with features of hundreds of micrometers to millimeters, this route is slow and costly, especially during prototyping, when the design changes after each test. Here, we describe and benchmark a workflow to fabricate polydimethylsiloxane (PDMS) devices from laser-cut master molds, with equipment accessible to most biology laboratories. We cut three inexpensive materials, poly(methyl methacrylate) (PMMA), polystyrene and polyvinyl chloride (PVC) tape, over a grid of laser power and speed and measured each cut by scanning electron microscopy. Cut depth and width increased line-arly with the laser energy delivered per unit length, which gives a simple rule to choose the settings for each material. We then bonded the PDMS replicas to glass without a plasma cleaner, by stopping curing early, once the replica could be demolded, and completing it in contact with the glass. Without any surface treatment, these devices held up to about 400 mbar; activating the glass with a handheld corona device raised this to about 900 mbar, enough for perfusion. The master material set the surface of the device: PMMA gave smooth replicas, whereas PVC tape gave rough walls. A three-channel device molded on PMMA held two cell-laden gels in place with phase guides and supported the co-culture of cancer cells and fibroblasts under perfusion. Further analysis of flow laminarity and different mixer designs confirmed the robustness of this method. This workflow takes a digital design to a working de-vice within a day and lets biology laboratories adapt microfluidic devices to their experiments without the need of a clean room.

bioengineering↗

bioRxiv2026

Analysis of 3D epithelial tissue packing reveals correlated patterns of cell height and skewing

Epithelia are tissues which envelop organs, exchange with and protect from their environment. The tight packing of cells within an epithelium guarantees its cohesiveness and impermeability, essential to its functions. Yet, how cells spatially arrange in three dimensions within an epithelium, and how the three-dimensional cell packing responds to geometrical constraints, is not fully understood. In a proliferating epithelium, cellular volumes vary from cell to cell, notably due to cell growth. At the same time, epithelia tend to display a smooth, continuous apical surface, indicating that cell shape determinants are spatially coupled. It is unclear how these and other factors modulate cell shape variability within the epithelium. Here, we segmented three-dimensional cell shapes of MDCK epithelia, grown in hollow spheres of alginate. With this assay, we could modulate the substrate adhesive properties, tissue curvature, and cell area density. We observed that as the tissue proliferates and cell density increases, average cell volume decreases. In contrast, cell height is relatively conserved over time, with an average value sensitive to the rigidity, curvature and adhesive strength of the substrate. We identified large and spatially correlated fluctuations in cell skewing, defined as the relative difference between apical and basal area that do not arise from curvature. Skewing is associated to spatial patterns of tilted cells whose apico-basal axis deviates from orthogonality to the substrate. Surprisingly, cell skewing and cell height are correlated, indicating internal rules for three-dimensional cell shapes. Altogether, our study identifies unexpected patterns of three-dimensional cell shape variation within a proliferative epithelium.

biophysics↗

bioRxiv2025

Molecular basis for cellular compartmentalization by an ancient membrane fission mechanism

The emergence of cell compartmentalization depends on membrane fission to create the endomembrane compartments. In eukaryotes, membrane fission is commonly executed by ESCRT-III, a protein complex conserved in all domains of life. However, whether membrane fission was an ancestral ESCRT-III activity predating eukaryogenesis remains unknown. Here we show that ESCRT-IIIA from Asgard Heimdallarchaeota, the closest archaeal relatives of eukaryotes, performs membrane fission through an N-terminal amphipathic helix, which we term Hofund. In eukaryotes, Hofund is fragmented across ESCRT-IIIA paralogs, and disrupting these regions causes severe fission defect in yeast. Remarkably, Heimdallarchaeota Hofund restores fission when fused to defective eukaryotic paralogs. These findings suggest that ESCRT-III-mediated fission arose before eukaryogenesis and later diversified to support the regulatory complexity of eukaryotic compartmentalization.

cell biology↗

bioRxiv2025

Fluorescence lifetime estimation: a practical approach using Flipper-TR FLIM

Flipper-TR is a membrane dye sensitive to lipid packing widely used to probe membrane tension in live cells via fluorescence lifetime imaging microscopy (FLIM). However, no consensus currently exists on the optimal strategy for extracting lifetime values, particularly across varying experimental setups and biological systems. Here, we systematically compare multiple approaches to estimate Flipper-TR lifetime, including multi-exponential reconvolution fitting, tail fitting, mean photon arrival time (first moment), and phasor analysis. These estimators are tested against changes in photon budget, sample characteristics, microscope manufacturer, and laser frequency. This offers a comprehensive benchmark and decision-making framework for quantitative FLIM analysis of Flipper dyes in various contexts.

biophysics↗

bioRxiv2024

Actin dynamics sustains spatial gradients of membrane tension in adherent cells

Tension propagates in lipid bilayers over hundreds of microns within milliseconds, precluding the formation of tension gradients. Nevertheless, plasma membrane tension gradients have been evidenced in migrating cells and along axons. Here, using a fluorescent membrane tension probe, we show that membrane tension gradients exist in all adherent cells, whether they migrate or not. Non-adhering cells do not display tension gradients. We further show that branched actin increases tension, while membrane-to-cortex attachments facilitate its propagation. Tension is the lowest at the edge of adhesion sites and highest at protrusions, setting the boundaries of the tension gradients. By providing a quantitative and mechanistic basis behind the organization of membrane tension gradients, our work explains how they are actively sustained in adherent cells.

cell biology↗

bioRxiv2022

Advected percolation in the actomyosin cortex drives amoeboid cell motility

Spontaneous locomotion is a common feature of most metazoan cells, generally attributed to the fundamental properties of the actomyosin network. This force-producing machinery has been studied down to the most minute molecular details, especially in lamellipodium-driven migration. Nevertheless, how actomyosin networks work inside contraction-driven amoeboid cells still lacks unifying principles. Here, using stable motile blebs as a model amoeboid motile system, we image the dynamics of the actin cortex at the single filament level and reveal the co-existence of three phases of the actin network with distinct rheological properties. Physical modelling shows that these three phases organize spontaneously due to a rigidity percolation transition combined with an active advection of the percolated network. This spontaneous spatial organization of the mechanical properties of the actin network, which we propose to call advected percolation, constitutes a minimal and generic locomotion mechanism. It explains, down to the single actin filament level and up to the scale of the entire cell, how amoeboid cells can propel efficiently through complex 3D environments, a feature shared by immune and cancer cells.

biophysics↗