Search Results (7)

Membrane solubilization induced by Triton X-100 (TX-100) was investigated. Different membrane compositions and phase states were studied along the detergent titration. Expected solubilization profiles were obtained but new information is provided. The fluorescence of nitrobenzoxadiazole (NBD)-labeled lipids indicates that the liquid-ordered (Lo)/liquid-disordered (Ld) phase coexistence is barely unaffected at sub-solubilizing detergent concentrations and highlights the vesicle-to-micelle transition. Moreover, the location of the NBD group in the bilayer emphasizes a detergent–membrane interaction in the case of the insoluble Lo phase membrane. It has also been shown that the molecular packing of the membrane loosens in the presence of TX-100, regardless of the solubilization profile. Motivated by studies on GPMVs, the solubilization of less ordered Lo phase membranes was considered in order to improve the effect of molecular packing on the extent of solubilization. Membranes composed of SM and Chol in an equimolar ratio doped with different amounts of PC were studied. The more ordered the Lo phase membrane is in the absence of detergent, the less likely it is to be solubilized. Furthermore, and in contrast to what is observed for membranes exhibiting an Lo/Ld phase coexistence, a very small decrease in the molecular packing of the Lo phase membrane radically modifies the extent of solubilization. These results have implications for the reliability of TX-100 insolubility as a method to detect ordered domains. Full article

Recently, self-supervised multi-view stereo (MVS) methods, which are dependent primarily on optimizing networks using photometric consistency, have made clear progress. However, the difference in lighting between different views and reflective objects in the scene can make photometric consistency unreliable. To address this issue, a geometric prior-guided multi-view stereo (GP-MVS) for self-supervised learning is proposed, which exploits the geometric prior from the input data to obtain high-quality depth pseudo-labels. Specifically, two types of pseudo-labels for self-supervised MVS are proposed, based on the structure-from-motion (SfM) and traditional MVS methods. One converts the sparse points of SfM into sparse depth maps and combines the depth maps with spatial smoothness constraints to obtain a sparse prior loss. The other generates initial depth maps for semi-dense depth pseudo-labels using the traditional MVS, and applies a geometric consistency check to filter the wrong depth in the initial depth maps. We conducted extensive experiments on the DTU and Tanks and Temples datasets, which demonstrate that our method achieves state-of-the-art performance compared to existing unsupervised/self-supervised approaches, and even performs on par with traditional and supervised approaches. Full article

Continuous progress has been made in the development of new molecules for therapeutic purposes. This is driven by the need to address several challenges such as molecular instability and biocompatibility, difficulties in crossing the plasma membrane, and the development of host resistance. In this context, cell-penetrating peptides (CPPs) constitute a promising tool for the development of new therapies due to their intrinsic ability to deliver therapeutic molecules to cells and tissues. These short peptides have gained increasing attention for applications in drug delivery as well as for their antimicrobial and anticancer activity but the general rules regulating the events involved in cellular uptake and in the following processes are still unclear. Here, we use fluorescence microscopy methods to analyze the interactions between the multifunctional peptide Transportan 10 (TP10) and the giant plasma membrane vesicles (GPMVs) derived from cancer cells. This aims to highlight the molecular mechanisms underlying functional interactions which bring its translocation across the membrane or cytotoxic mechanisms leading to membrane collapse and disruption. The Fluorescence Lifetime Imaging Microscopy (FLIM) method coupled with the phasor approach analysis proved to be the winning choice for following highly dynamic spatially heterogeneous events in real-time and highlighting aspects of such complex phenomena. Thanks to the presented approach, we were able to identify and monitor TP10 translocation into the lumen, internalization, and membrane-induced modifications depending on the peptide concentration regime. Full article

Although liquid–liquid phase separation of cytoplasmic or nuclear components in cells has been a major focus in cell biology, it is only recently that the principle of phase separation has been a long-standing concept and extensively studied in biomembranes. Membrane phase separation has been reconstituted in simplified model systems, and its detailed physicochemical principles, including essential phase diagrams, have been extensively explored. These model membrane systems have proven very useful to study the heterogeneity in cellular membranes, however, concerns have been raised about how reliably they can represent native membranes. In this review, we will discuss how phase-separated membrane systems can mimic cellular membranes and where they fail to reflect the native cell membrane heterogeneity. We also include a few humble suggestions on which phase-separated systems should be used for certain applications, and which interpretations should be avoided to prevent unreliable conclusions. Full article

Due to the global rise of type 2 diabetes mellitus (T2DM) in combination with insulin resistance, novel compounds to efficiently treat this pandemic disease are needed. Screening for compounds that induce the translocation of glucose transporter 4 (GLUT4) from the intracellular compartments to the plasma membrane in insulin-sensitive tissues is an innovative strategy. Here, we compared the applicability of three fluorescence microscopy-based assays optimized for the quantitation of GLUT4 translocation in simple cell systems. An objective-type scanning total internal reflection fluorescence (TIRF) microscopy approach was shown to have high sensitivity but only moderate throughput. Therefore, we implemented a prism-type TIR reader for the simultaneous analysis of large cell populations grown in adapted microtiter plates. This approach was found to be high throughput and have sufficient sensitivity for the characterization of insulin mimetic compounds in live cells. Finally, we applied confocal microscopy to giant plasma membrane vesicles (GPMVs) formed from GLUT4-expressing cells. While this assay has only limited throughput, it offers the advantage of being less sensitive to insulin mimetic compounds with high autofluorescence. In summary, the combined implementation of different fluorescence microscopy-based approaches enables the quantitation of GLUT4 translocation with high throughput and high content. Full article

This study was aimed at preparing and characterizing plasma membranes (PM) from Chinese Hamster Ovary (CHO) cells. Two methods of PM preparation were applied, one based on adhering cells to a poly-lysine-coated surface, followed by hypotonic lysis and removal of intracellular components, so that PM patches remain adhered to each other, and a second one consisting of bleb induction in cells, followed by separation of giant plasma membrane vesicles (GPMV). Both methods gave rise to PM in sufficient amounts to allow biophysical and biochemical characterization. Laurdan generalized polarization was used to measure molecular order in membranes, PM preparations were clearly more ordered than the average cell membranes (GP ≈0.450 vs. ≈0.20 respectively). Atomic force microscopy was used in the force spectroscopy mode to measure breakthrough forces of PM, both PM preparations provided values in the 4–6 nN range, while the corresponding value for whole cell lipid extracts was ≈2 nN. Lipidomic analysis of the PM preparations revealed that, as compared to the average cell membranes, PM were enriched in phospholipids containing 30–32 C atoms in their acyl chains but were relatively poor in those containing 34–40 C atoms. PM contained more saturated and less polyunsaturated fatty acids than the average cell membranes. Blebs (GPMV) and patches were very similar in their lipid composition, except that blebs contained four-fold the amount of cholesterol of patches (≈23 vs. ≈6 mol% total membrane lipids) while the average cell lipids contained 3 mol%. The differences in lipid composition are in agreement with the observed variations in physical properties between PM and whole cell membranes. Full article

Model membrane systems are essential tools for the study of biological processes in a simplified setting to reveal the underlying physicochemical principles. As cell-derived membrane systems, giant plasma membrane vesicles (GPMVs) constitute an intermediate model between live cells and fully artificial structures. Certain applications, however, require planar membrane surfaces. Here, we report a new approach for creating supported plasma membrane bilayers (SPMBs) by bursting cell-derived GPMVs using ultrasound within a microfluidic device. We show that the mobility of outer leaflet molecules is preserved in SPMBs, suggesting that they are accessible on the surface of the bilayers. Such model membrane systems are potentially useful in many applications requiring detailed characterization of plasma membrane dynamics. Full article