Biology subjects

Arribas Perez, M.

Publications and source records attributed to Arribas Perez, M..

3 recordsLinked to original sources

bioRxiv2025

Quantification of Cholesterol Incorporation in Giant Unilamellar Vesicles Produced by a Modified cDICE Method

AbstractCholesterol is an essential component of eukaryotic cell membranes, influencing membrane packing, fluidity, and domain formation. Replicating these properties in model membranes is critical for reconstitution studies, but common emulsion-based methods for producing giant unilamellar vesicles (GUVs) fail to incorporate cholesterol efficiently. Here, we use methyl-{beta}- cyclodextrin-cholesterol (M{beta}CD-CL) complexes to deliver cholesterol into GUVs produced by the emulsion droplet interface crossing encapsulation (eDICE) method and demonstrate a convenient way to quantify the degree of cholesterol incorporation using fluorescent membrane biosensors. Spectral imaging of NR12A as well as fluorescence lifetime imaging of Flipper-TR revealed dose- dependent increases in cholesterol content for DOPC GUVs upon M{beta}CD-CL addition, consistent with increased membrane order. By calibrating these effects against GUVs with defined cholesterol contents prepared via gel-assisted swelling, we found that the cholesterol content of eDICE vesicles can be increased to at least 40 mol%. Binary mixtures of DOPC with saturated lipids (DMPC and PC(18:0-14:0)) showed a similar trend as pure DOPC GUVs. Interestingly, we could trigger liquid-ordered domain formation by adding cholesterol to DOPC:DMPC vesicles. Our findings provide a quantitative and non-disruptive method to modulate and assess cholesterol content in emulsion-based GUVs, advancing their use in bottom-up synthetic biology and membrane biophysics.

biophysics↗

bioRxiv2022

Reconstituted branched actin networks sense and generate micron-scale membrane curvature

The actin cortex is a complex cytoskeletal machinery which drives and responds to changes in cell shape. It must generate or adapt to plasma membrane curvature to facilitate diverse functions such as cell division, migration and phagocytosis. Due to the complex molecular makeup of the actin cortex, it remains unclear whether actin networks are inherently able to sense and generate membrane curvature, or whether they rely on their diverse binding partners to accomplish this. Here, we show that curvature sensing and generation is an inherent capability of branched actin networks nucleated by Arp2/3 and VCA. We develop a robust method to encapsulate actin inside giant unilamellar vesicles (GUVs) and assemble an actin cortex at the inner surface of the GUV membrane. We show that actin forms a uniform and thin cortical layer when present at high concentration and distinct patches that generate negative membrane curvature at low concentration. Serendipitously, we find that the GUV production method also produces dumbbell-shaped GUVs, which we explain using mathematical modelling in terms of membrane hemifusion of nested GUVs. We find that dendritic actin networks preferentially assemble at the neck of the dumbbells, which possess a micron-range convex curvature that matches the curvature generated by actin patches in spherical GUVs. Minimal dendritic actin networks can thus both generate and sense membrane curvatures, which may help mammalian cells to robustly recruit actin to curved membranes in order to facilitate diverse cellular functions such as cytokinesis and migration. SIGNIFICANCEAnimal cells move, deform and divide using their actin cortex, a thin layer of filamentous proteins that supports the plasma membrane. For these actions, actin must often assemble at curved sections of the membrane, which is widely believed to require the action of dedicated actin- or membrane-bending proteins. Here, we use a bottom-up reconstitution approach to ask whether actin networks are intrinsically able to generate and sense membrane curvature. We show that membrane-nucleated actin cortices can indeed preferentially self-assemble at concave membranes generated by hemifusion of lipid vesicles. This raises intriguing questions about how such curvature recognition works, and whether cells exploit this intrinsic capability of branched actin networks to concentrate actin in specific cortical regions.

biophysics↗

bioRxiv2019

Isolated occurrences of membrane perturbation by mechanosensing from weakly aggregating silver nanoparticles

Silver nanoparticles (AgNPs) have wide-ranging applications, including as additives in consumer products and in medical diagnostics and therapy. Therefore understanding how AgNPs interact with biological systems is important for ascertaining any potential health risks due to the likelihood of high levels of human exposure. Besides any severe, acute effects, it is desirable to understand more subtle interactions that could lead to milder, chronic health impacts. Nanoparticles are small enough to be able to enter biological cells and interfere with their internal biochemistry. The initial contact between nanoparticle and cell is at the plasma membrane. To gain fundamental mechanistic insight into AgNP-membrane interactions, we investigate these phenomena in minimal model systems using a wide-range of biophysical approaches applied to lipid vesicles. We find a strong dependence on the medium composition, where colloidally stable AgNPs in a glucose buffer have negligible effect on the membrane. However, at a physiological salt concentrations, the AgNPs start to weakly aggregate and sporadic but significant membrane perturbation events are observed. Under these latter conditions, transient poration and structural remodelling of some vesicle membranes is observed. We observe that the fluidity of giant vesicle membranes universally decreases by an average of 16% across all vesicles. However, we observe a small population of vesicles display a significant change in mechanical properties with lower bending rigidity and higher membrane tension. Therefore we argue that the isolated occurrences of membrane perturbation by AgNPs are due to low probability mechanosensing events of AgNP aggregation at the membrane.\n\nGRAPHICAL ABSTRACT\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC=\"FIGDIR/small/623678v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (54K):\norg.highwire.dtl.DTLVardef@196aa51org.highwire.dtl.DTLVardef@87c12borg.highwire.dtl.DTLVardef@9b2096org.highwire.dtl.DTLVardef@7877f6_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗