NBD-lipid Uptake Assay for Mammalian Cell Lines

Background

A remarkable feature of many biological membranes is that their phospholipids are asymmetrically distributed across the lipid bilayer, a phenomenon known as transbilayer lipid asymmetry. This lipid asymmetry is essential for several vital cellular functions, including regulation of membrane protein activity, signaling, and vesicle formation in the secretory and endocytic pathways. Thus, establishing and regulating lipid asymmetry is crucial for cells, and a number of membrane proteins have evolved to fulfill the function of cross-bilayer lipid transporters. These transporters include ATP-dependent flippases and floppases—which catalyze the inward movement of lipids from the extracellular/luminal leaflet to the cytoplasmic leaflet, and the outward movement of lipids from the intracellular leaflet to the extracellular/ luminal leaflet, respectively, and ATP-independent scramblases (Holthuis and Levine, 2005; Contreras et al., 2010). Despite their fundamental cellular importance, key aspects of how these lipid transporters operate await elucidation.

A subgroup of P-type ATPases, the P4-ATPases, emerged as a major group of lipid flippases that form heterodimeric complexes with members of the Cdc50 (cell division control 50) protein family (Lopez-Marques et al., 2014). Mutations in these transporters generate impairments in physiological processes and, in humans they have been linked to diseases such as intrahepatic cholestasis and cerebellar ataxia (van der Mark et al., 2013). While initially characterized as aminophospholipid flippases, recent studies of individual family members from yeast, parasites such as Leishmania, plants, and mammalian cells show that P4-ATPases differ in their substrate specificities and mediate transport of a broader range of lipid substrates, including lysophospholipids, synthetic alkylphospholipids, and glycolipids (Roland et al., 2019; Shin and Takatsu, 2019).

Characterization of phospholipid movement at a quantitative level in the plasma membrane of eukaryotic cells is frequently based on fluorescent lipids with a covalently linked 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group in the sn-2 position. These lipid analogs have a NBD group attached to a short fatty acid chain (C6) and maintain most of the properties of endogenous phospholipids, except that they are more water-soluble, which facilitates incorporation from the medium into the outer monolayer of the plasma membrane. Transport of these probes is usually monitored by extracting the residual fraction of analogs not transported across the membrane with bovine serum albumin (BSA). As BSA extracts all analogs from the exoplasmic monolayer of the plasma membrane, the inaccessible fraction reflects analogs that have been redistributed across the plasma membrane into cells.

The protocol presented here utilizes flow cytometry to study NBD-lipid internalization at a quantitative level in mammalian cells, exemplified on Chinese hamster ovary-K1 (CHO-K1) cells, and has been applied by us to fibroblasts (Pomorski et al., 1996), lymphocytes (Fischer et al., 2006), and myoblasts (Grifell-Junyent et al., 2022). For lipid uptake assays optimized for fungi and plants, the reader is referred to previously published protocols (Jensen et al., 2016; López-Marqués and Pomorski, 2021). The protocol includes the preparation of NBD-lipids, labelling of cells with NBD-lipids, flow cytometry measurements, and data analysis. Additionally, cells are subjected to lipid analysis via thin-layer chromatography (TLC) to assess metabolic conversion of the NBD-lipids. This protocol can be easily adapted to parasites such as Toxoplasma and Leishmania (Weingartner et al., 2011; dos Santos et al., 2013; Chen et al., 2021), and has a broad range of applications, including: i) screening of mammalian cell lines for their lipid uptake profile, ii) testing the impact of gene knockouts on lipid internalization at the plasma membrane, and iii) uncovering the dynamics of lipid transport at the plasma membrane.

Things to consider before starting

1. Choice of NBD-lipid

   The NBD-lipids need to fulfill three important requirements: (i) they cannot be modified at their polar head group, as this is the key structural element for substrate recognition by ATP-dependent flippases and floppases (Theorin et al., 2019); (ii) they have to incorporate readily into the outer plasma membrane, to get the time zero for the uptake kinetic; and (iii) they have to be extractable by BSA. These requirements are best met by lipid analogs in which one fatty acid has been replaced by a short chain of six carbons (C6) carrying the fluorescent NBD moiety in the sn-2 position of the glycerophospholipids, or linked by an amide bond to the ceramide backbone, as well as lyso-glycerophospholipid derivates labeled at the sn-1 position (Figure 1).
   

   
   
   Figure 1. Chemical structure of NBD-lipids used in lipid uptake assays. Fluorescent analogs of phosphatidylserine with different acyl chain lengths and positions of the NBD moiety. A) 1-palmitoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphoserine (C16:0-C6:0 NBD-PS). In these analogs, one fatty acid has been replaced by a short chain of six carbons (C6) carrying the fluorescent NBD moiety in the sn-2 position, whereas the sn-1 chain is composed of sixteen carbons (C16). B) 1-myristoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphoserine (C14:0-C6:0 NBD-PS); similar to the analog in (A), but the sn-1 chain is composed of fourteen carbons (C14). C) 1-NBD-dodecanoyl-2-hydroxy-sn-glycero-3-phospho-serine (NBD-lyso-PS). In these analogs, the C12 sn-1 acyl chain carries the NBD moiety. D) The NBD moiety is attached to the carbon twelve of a C18 sn-1 acyl chain, whereas the sn-2 chain is composed of four carbons (Colleau et al., 1991).
   
   

2. Metabolic conversion of the NBD-lipids

   In nucleated cells, lipid internalization is not only affected by transbilayer movement and intracellular membrane trafficking, but also by lipid metabolism. Some lipid analogs are known to be actively metabolized by phospholipase activities at the cell surface. Examples are the hydrolysis of NBD- phosphatidic acid to NBD-diacylglycerol (Pagano and Longmuir, 1985), and the hydrolysis of NBD-sphingomyelin to NBD-ceramide, both occurring at the plasma membrane. The fluorescent analogs of diacylglycerol and ceramide undergo rapid spontaneous transbilayer movement, and therefore label intracellular membranes (Pagano and Sleight, 1985).
   

   
   Thus, the conversion of an NBD-lipid may be followed by rapid spontaneous movement of its metabolic products and should always be critically evaluated. Another frequent modification is the hydrolysis of the NBD-lipid analogs into lyso-derivates by phospholipase A₂ activities, which results in the removal of the fatty acid attached to the sn-2 position. The labeled C6 fatty acids liberated are released into the medium, hampering quantitative analysis of NBD-lipid internalization. To block this conversion, the assay is typically performed in the presence of phospholipase inhibitors (Pomorski et al., 1996; Fischer et al., 2006; Grifell-Junyent et al., 2022).
   

   
   Therefore, it is highly recommended to analyze the degree of NBD-lipid conversion using, for example, lipid extraction in organic solvents followed by thin layer chromatography (see under Procedure, section D).
   
   

3. Lipid internalization by endocytosis

   Lipid analogs inserted into the outer plasma membrane leaflet can also be internalized by endocytosis. To suppress uptake by endocytosis, the assay is typically performed at 20°C or below.