NBD-lipid Uptake Assay for Mammalian Cell Lines.

All eukaryotic cells are equipped with transmembrane lipid transporters, which are key players in membrane lipid asymmetry, vesicular trafficking, and membrane fusion. The link between mutations in these transporters and disease in humans highlights their essential role in cell homeostasis. Yet, many key features of their activities, their substrate specificity, and their regulation remain to be elucidated. Here, we describe an optimized quantitative flow cytometry-based lipid uptake assay utilizing nitrobenzoxadiazolyl (NBD) fluorescent lipids to study lipid internalization in mammalian cell lines, which allows characterizing lipid transporter activities at the plasma membrane. This approach allows for a rapid analysis of large cell populations, thereby greatly reducing sampling variability. The protocol can be applied to study a wide range of mammalian cell lines, to test the impact of gene knockouts on lipid internalization at the plasma membrane, and to uncover the dynamics of lipid transport at the plasma membrane. Graphic abstract: Internalization of NBD-labeled lipids from the plasma membrane of CHO-K1 cells.

CDC50A is required for aminophospholipid transport and cell fusion in mouse C2C12 myoblasts.

Myoblast fusion is essential for the formation of multinucleated muscle fibers and is promoted by transient changes in the plasma membrane lipid distribution. However, little is known about the lipid transporters regulating these dynamic changes. Here, we show that proliferating myoblasts exhibit an aminophospholipid flippase activity that is downregulated during differentiation. Deletion of the P4-ATPase flippase subunit CDC50A (also known as TMEM30A) results in loss of the aminophospholipid flippase activity and compromises actin remodeling, RAC1 GTPase membrane targeting and cell fusion. In contrast, deletion of the P4-ATPase ATP11A affects aminophospholipid uptake without having a strong impact on cell fusion. Our results demonstrate that myoblast fusion depends on CDC50A and may involve multiple CDC50A-dependent P4-ATPases that help to regulate actin remodeling.

Short-chain lipid-conjugated pH sensors for imaging of transporter activities in reconstituted systems and living cells.

The design of ion sensors has gained importance for the study of ion dynamics in cells, with fluorescent proton nanosensors attracting particular interest because of their applicability in monitoring pH gradients in biological microcompartments and reconstituted membrane systems. In this work, we describe the improved synthesis, photophysical properties and applications of pH sensors based on amine-reactive pHrodo esters and short-chain lipid derivatives of phosphoethanolamine. The major features of these novel probes include strong fluorescence under acidic conditions, efficient partitioning into membranes, and extractability by back exchange to albumin. These features allow for the selective labeling of the inner liposomal leaflet in reconstituted membrane systems for studying proton pumping activities in a quantitative fashion, as demonstrated by assaying the activity of a plant plasma membrane H+-ATPase. Furthermore, the short-chain lipid-conjugated pH sensors enable the monitoring of pH changes from neutral to acidic conditions in the endocytic pathway of living cells. Collectively, our results demonstrate the applicability of short-chain lipid-conjugated sensors for in vivo and in vitro studies and thus pave the way for the design of lipid-conjugated sensors selective to other biologically relevant ions, e.g. calcium and sodium.