Application of image cytometry to characterize heterologous lipid flippases in yeast

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Maria Stumph Jensen, Sara Costa, Lisa Theorin, Jan Pravsgaard Christensen, Thomas Günther-Pomorski, Rosa Laura Lopez Marques

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.

OriginalsprogEngelsk
TidsskriftCytometry. Part A
Vol/bind89
Udgave nummer7
Sider (fra-til)673–680
Antal sider8
ISSN1552-4922
DOI
StatusUdgivet - 2016

ID: 163776869