Cellular Membrane-Derived Nanovesicles Expressing hCD64 for Targeting Prostate Cancer.

Extracellular vesicles are ideal therapeutic potentiators for various diseases. However, they commonly lack targeting capability and are rapidly cleared by phagocytes. This requires appropriate administration at high doses, which can lead to toxic and adverse reactions. To overcome these limitations, we developed bleb nanovesicles containing human Fcγ receptor I (hCD64), known for their strong affinity to monomeric IgG. In this study, we focused on prostate cancer, which has a specific membrane antigen. We have utilized the hCD64-expressing bleb nanovesicles attaching anti-prostate-specific membrane antigen (PSMA) antibodies and confirmed their targeting ability in PSMA-related cell lines and prostate cancer xenograft models. Our findings underscore the promising potential of nanovesicle Fcγ receptor-IgG as a platform for cancer diagnosis and therapy systems, inspiring further research.

Pore-Spanning Plasma Membranes Derived from Giant Plasma Membrane Vesicles.

Giant plasma membrane vesicles (GPMVs) are a highly promising model system for the eukaryotic plasma membrane. The unresolved challenge, however, is a path to surface-based structures that allows accessibility to both sides of the plasma membrane through high-resolution techniques. Such an approach would pave the way to advanced chip-based technologies for the analysis of complex cell surfaces to study the roles of membrane proteins, host-pathogen interactions, and many other bioanalytical and sensing applications. This study reports the generation of planar supported plasma membranes and for the first-time pore-spanning plasma membranes (PSPMs) derived from pure GPMVs that are spread on activated solid and highly ordered porous silicon substrates. GPMVs were produced by two different vesiculation agents and were first investigated with respect to their growth behavior and phase separation. Second, these GPMVs were spread onto silicon substrates to form planar supported plasma membrane patches. PSPMs were obtained by spreading of pure GPMVs on oxygen-plasma activated porous substrates with pore diameters of 3.5 µm. Fluorescence micrographs unambiguously showed that the PSPMs partially phase separate in a mobile ordered phase surrounded by a disordered phase, which was supported by cholesterol extraction using methyl-ß-cyclodextrin.

How Does Liquid-Liquid Phase Separation in Model Membranes Reflect Cell Membrane Heterogeneity?

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.

Direct label-free imaging of nanodomains in biomimetic and biological membranes by cryogenic electron microscopy.

The nanoscale organization of biological membranes into structurally and compositionally distinct lateral domains is believed to be central to membrane function. The nature of this organization has remained elusive due to a lack of methods to directly probe nanoscopic membrane features. We show here that cryogenic electron microscopy (cryo-EM) can be used to directly image coexisting nanoscopic domains in synthetic and bioderived membranes without extrinsic probes. Analyzing a series of single-component liposomes composed of synthetic lipids of varying chain lengths, we demonstrate that cryo-EM can distinguish bilayer thickness differences as small as 0.5 Å, comparable to the resolution of small-angle scattering methods. Simulated images from computational models reveal that features in cryo-EM images result from a complex interplay between the atomic distribution normal to the plane of the bilayer and imaging parameters. Simulations of phase-separated bilayers were used to predict two sources of contrast between coexisting ordered and disordered phases within a single liposome, namely differences in membrane thickness and molecular density. We observe both sources of contrast in biomimetic membranes composed of saturated lipids, unsaturated lipids, and cholesterol. When extended to isolated mammalian plasma membranes, cryo-EM reveals similar nanoscale lateral heterogeneities. The methods reported here for direct, probe-free imaging of nanodomains in unperturbed membranes open new avenues for investigation of nanoscopic membrane organization.

Creating Supported Plasma Membrane Bilayers Using Acoustic Pressure.

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.

Laurdan and Di-4-ANEPPDHQ probe different properties of the membrane.

Lipid packing is a crucial feature of cellular membranes. Quantitative analysis of membrane lipid packing can be achieved using polarity sensitive probes whose emission spectrum depends on the lipid packing. However, detailed insights into the exact mechanisms that cause the changes in the spectra are necessary to interpret experimental fluorescence emission data correctly. Here, we analysed frequently used polarity sensitive probes, Laurdan and di-4-ANEPPDHQ, to test whether the underlying physical mechanisms of their spectral changes are the same and, thus, whether they report on the same physico-chemical properties of the cell membrane. Steady-state spectra as well as time-resolved emission spectra of the probes in solvents and model membranes revealed that they probe different properties of the lipid membrane. Our findings are important for the application of these dyes in cell biology.