Biochemistry

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.

Nucleotide-sugar transporters (NSTs) facilitate eukaryotic cellular glycosylation by transporting nucleotide-sugar conjugates into the Golgi lumen and endoplasmic reticulum for use by glycosyltransferases, while also transferring nucleotide monophosphate byproducts to the cytoplasm. Mutations in this family of proteins can cause a number of significant cellular pathologies, and wild type members can act as virulence factors for many parasites and fungi. Here, we describe an in vitro assay to measure the transport activity of the CMP-sialic acid transporter (CST), one of seven NSTs found in mammals. While in vitro transport assays have been previously described for CST, these studies failed to account for the fact that 1) commercially available stocks of CMP-sialic acid (CMP-Sia) are composed of ~10% of the higher-affinity CMP and 2) CMP-Sia is hydrolyzed into CMP and sialic acid in aqueous solutions. Herein we describe a method for treating CMP-Sia with a nonselective phosphatase, Antarctic phosphatase, to convert all free CMP to cytidine. This allows us to accurately measure substrate affinities and transport kinetics for purified CST reconstituted into proteoliposomes.

The here described method can be used to estimate the uptake of orally provided cholesterol in mice. Briefly, mice are gavaged with radiolabeled cholesterol and 4 h later, organ distribution of the radiolabel is determined by liquid scintillation counting. The method has been applied successfully to determine dietary cholesterol handling of mice housed at different ambient temperatures

This is a protocol to detect lipid transfer activity of NRF-5, a member of the LPS binding/lipid transfer protein family. The lipid transfer activity is examined by using isotope-labeled cholesterol and liposomes, and tested in two directions (Figure 1): from proteins to liposomes and from liposomes to proteins.