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bioRxiv · 10.1101/2025.06.25.661602

A cell-based scrambling assay reveals phospholipid headgroup preference of TMEM16F on the plasma membrane

Teo, C. F.·Tuomivaara, S. T.·van Hilten, N.·Crottes, D.·Jan, Y. N.·Grabe, M.·Jan, L. Y.
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Abstract

The asymmetric resting distribution of the three major phospholipid classes on the mammalian plasma membrane, with phosphatidylserine and phosphatidylethanolamine mostly on the inner leaflet, and phosphatidylcholine mostly on the outer leaflet, is maintained by ATP-dependent flippases and floppases that exhibit headgroup selectivity. Upon signaling cues, this asymmetry can be dissipated by various phospholipid scramblases, allowing cells to respond to stimuli and adapt to different physiological contexts. The prevailing view in the field is that phospholipid scramblases on the plasma membrane act without headgroup preference. Here we report contrary experimental evidence based on a phospholipid scrambling assay that quantifies the fluorescence polarization of nitrobenzoxadiazole-labeled phospholipids for kinetic monitoring of phospholipid scrambling on the plasma membrane of living cells. Our experiments reveal that the plasma membrane-residing calcium-activated phospholipid scramblase TMEM16F preferentially acts on phosphatidylserine and phosphatidylcholine over phosphatidylethanolamine. Significance StatementPhospholipid scramblases on the mammalian plasma membrane are thought to act promiscuously without preference for headgroup. Thoroughly addressing this question, however, requires the development of new methodologies. We devised a cell-based phospholipid scrambling assay that utilizes the fluorescence polarization of nitrobenzoxadiazole (NBD)-labeled phospholipids, allowing the monitoring of their scrambling in a native environment. We discovered that the plasma membrane-residing calcium-activated phospholipid scramblase TMEM16F preferentially acts on phosphatidylserine and phosphatidylcholine over phosphatidylethanolamine. Major Classification: Biological Sciences; Minor Classification: Cell biology, Biophysics and Computational Biology

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Teo, C. F., Tuomivaara, S. T., van Hilten, N., Crottes, D., Jan, Y. N., Grabe, M., Jan, L. Y.. 2025-06-27. A cell-based scrambling assay reveals phospholipid headgroup preference of TMEM16F on the plasma membrane. https://doi.org/10.1101/2025.06.25.661602

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