A simple method to determine the phase preference of proteins on the membranes of living cells

Scientists from the National University of Singapore have demonstrated a quick and simple method to determine whether a biomolecule divides into lipid domains on the membranes of living cells. Their work has been published in the Lipid Research Journal.

Cell membranes are dynamic assemblies of lipids and proteins, some components of which are organized into domains. Proper cellular function requires partitioning of lipids and proteins into these areas, which are often rich in cholesterol and sphingolipids. However, they are too small (10–200 nm in size) and dynamic (perhaps only a few tens of milliseconds in lifetime) to be observed even with modern super-resolution microscopy techniques.

Traditional methods for determining domain localization involve biochemical assays that require many cells, are prone to artifacts because they are performed in vitro, and are slow. Although fluorescence-based techniques can probe these domains in living cells, they require specialized instrumentation and are often difficult to interpret.

The NUS research team has developed a simple fluidizer-based method to determine whether a molecule prefers to partition into lipid domains on cell membranes.

Figure: (a) Schematic shows heptanol-induced membrane fluidization that results in clustering of domains. (b) Control: A resting-state SH-SY5Y cell showing homogeneous distribution of green fluorescent protein glycosylphosphatidylinositol (GFP-GPI) on the cell membrane. (c) An SH-SY5Y cell after heptanol treatment showing clustering of GFP-GPI in cell membrane lipid domains.

The team added heptanol to live cells and showed that within 15 minutes it induces clustering of nanometer-sized lipid domains into larger micrometer domains that are easily detected by standard fluorescence microscopes. The method works both with molecules genetically marked with fluorescent proteins and with those marked with extrinsic markers, for example antibodies.

The work was conducted in the lab of Thorsten Wohland and led by first author Anjali Gupta, who is now a research fellow at Harvard Medical School and Boston Children’s Hospital.

Gupta explained the importance of studying the phase preference of molecules in membranes.

“The phase preference of molecules in membranes is fundamentally crucial for essential biological processes originating in membranes, such as T-cell activation, a critical step during an immune response,” she said. “Knowledge of the phase preference of molecules will support therapeutic development based on the modulation of lipid domains.”

Wohland said: “The phase preference of molecules used to be difficult and time-consuming to establish. This new method, detected by chance, provides results in up to 15 minutes on live cells and can essentially be seen with the naked eye in a simple microscope.

The team hopes this technique will allow for quick and easy identification of the domain’s location and help the wider research community.

Source link

About Donald P. Hooten

Check Also

The 369 Manifestations Method: How to Make Your Therapy Sessions Work

Psychologist Nancy Sokarno walks us through the 369 Manifestations Method and how to get the …