Application of image cytometry to characterize heterologous lipid flippases in yeast. Issue 7 (6th June 2016)

• Record Type: Journal Article
• Authors: Jensen, Maria S.; Costa, Sara R.; Theorin, Lisa; Christensen, Jan Pravsgaard; Pomorski, Thomas Günther; López‐Marqués, Rosa L.
• Is Part Of: Cytometry. Volume 89:Issue 7(2016)
• Page Start: 673
• Page End: 680
• Publication Date: 2016-06-06
• Subjects: Key terms: yeast -- flippases -- NBD‐lipid uptake -- cytometry -- flow cytometer -- automated cell counter; Flow cytometry -- Periodicals; Imaging systems in biology -- Periodicals; Imaging systems in medicine -- Periodicals; Diagnostic imaging -- Periodicals; 571.605
• DOI: 10.1002/cyto.a.22886
• Languages: English
• ISSNs: 1552-4922

Abstract

Abstract: Lipid flippases are integral membrane proteins that play a central role in moving lipids across cellular membranes. Some of these transporters are ATPases that couple lipid translocation to ATP hydrolysis, whereas others function without any discernible metabolic energy input. A growing number of lipid flippases has been identified but key features of their activity remain to be elucidated. A well‐established method to characterize ATP‐driven flippases is based on their heterologous expression in yeast, followed by incubation of the cells with fluorescent lipids. Internalization of these probes is typically monitored by flow cytometry, a costly and maintenance‐intensive method. Here, we have optimized a protocol to use an automated image‐based cell counter to accurately measure lipid uptake by heterologous lipid flippases expressed in yeast. The method was validated by comparison with the classical flow cytometric evaluation of lipid‐labeled cells. In addition, we demonstrated that expression of fluorescently tagged flippase complexes can be directly co‐related with fluorescent lipid uptake using the image‐based cell counter system. The method extends the number of techniques available for characterization of lipid flippase activity, and should be readily adaptable to analyze a variety of other transport systems in yeast, parasites, and mammalian cells. © 2016 International Society for Advancement of Cytometry