Cell Biology


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0 Q&A 379 Views Oct 5, 2023

Disruptions and perturbations of the cellular plasma membrane by peptides have garnered significant interest in the elucidation of biological phenomena. Typically, these complex processes are studied using liposomes as model membranes—either by encapsulating a fluorescent dye or by other spectroscopic approaches, such as nuclear magnetic resonance. Despite incorporating physiologically relevant lipids, no synthetic model truly recapitulates the full complexity and molecular diversity of the plasma membrane. Here, biologically representative membrane models, giant plasma membrane vesicles (GPMVs), are prepared from eukaryotic cells by inducing a budding event with a chemical stressor. The GPMVs are then isolated, and bilayers are labelled with fluorescent lipophilic tracers and incubated in a microplate with a membrane-active peptide. As the membranes become damaged and/or aggregate, the resulting fluorescence resonance energy transfer (FRET) between the two tracers increases and is measured periodically in a microplate. This approach offers a particularly useful way to detect perturbations when the membrane complexity is an important variable to consider. Additionally, it provides a way to kinetically detect damage to the plasma membrane, which can be correlated with the kinetics of peptide self-assembly or structural rearrangements.

Key features

• Allows testing of various peptide–membrane interaction conditions (peptide:phospholipid ratio, ionic strength, buffer, etc.) at once.

• Uses intact plasma membrane vesicles that can be prepared from a variety of cell lines.

• Can offer comparable throughput as with traditional synthetic lipid models (e.g., dye-encapsulated liposomes).

Graphical overview

0 Q&A 546 Views Jul 5, 2023

Eukaryotic cells use a series of membrane transporters to control the movement of lipids across their plasma membrane. Several tools and techniques have been developed to analyze the activity of these transporters in the plasma membrane of mammalian cells. Among them, assays based on fluorescence microscopy in combination with fluorescent lipid probes are particularly suitable, allowing visualization of lipid internalization in living cells. Here, we provide a step-by-step protocol for mammalian cell culture, lipid probe preparation, cell labeling, and confocal imaging to monitor lipid internalization by lipid flippases at the plasma membrane based on lipid probes carrying a fluorophore at a short-chain fatty acid. The protocol allows studying a wide range of mammalian cell lines, to test the impact of gene knockouts on lipid internalization at the plasma membrane and changes in lipid uptake during cell differentiation.

Key features

• Visualization and quantification of lipid internalization by lipid flippases at the plasma membrane based on confocal microscopy.

• Assay is performed on living adherent mammalian cells in culture.

• The protocol can be easily modified to a wide variety of mammalian cell lines.

Graphical overview

Analysis of NBD-lipid uptake in adherent mammalian cells by confocal microscopy. Scale bar, 30 μm.

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