TY - JOUR
T1 - Application of image cytometry to characterize heterologous lipid flippases in yeast
AU - Jensen, Maria Stumph
AU - Costa, Sara
AU - Theorin, Lisa
AU - Christensen, Jan Pravsgaard
AU - Günther-Pomorski, Thomas
AU - Lopez Marques, Rosa Laura
N1 - © 2016 International Society for Advancement of Cytometry.
PY - 2016/7/1
Y1 - 2016/7/1
N2 - 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.
AB - 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.
U2 - 10.1002/cyto.a.22886
DO - 10.1002/cyto.a.22886
M3 - Journal article
C2 - 27272389
SN - 1046-7386
VL - 89
SP - 673
EP - 680
JO - Cytometry. Supplement : the journal of the Society for Analytical Cytology
JF - Cytometry. Supplement : the journal of the Society for Analytical Cytology
IS - 7
ER -