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Comment on ‘Orthogonal lipid sensors identify transbilayer asymmetry of plasma membrane cholesterol’

  1. Kevin C Courtney
  2. Karen YY Fung
  3. Frederick R Maxfield
  4. Gregory D Fairn  Is a corresponding author
  5. Xiaohui Zha  Is a corresponding author
  1. University of Ottawa, Canada
  2. St. Michael’s Hospital, Canada
  3. Weill Cornell Medical College, United States
  4. Ottawa Hospital Research Institute, Canada
Cite this article as: eLife 2018;7:e38493 doi: 10.7554/eLife.38493
2 figures and 1 additional file


D4 binding is influenced by phospholipid composition and is subject to competition from proteins.

(A) Purified DAN-D4 (0.5 µM) was incubated with 100 µM large unilamellar vesicles (LUVs) composed of POPC/egg SM/cholesterol (36:24:40) and POPC/POPE/POPS/soy PI/cholesterol (18:18:18:6:40). The change in fluorescence emission (ΔF) at 450 nm is used to approximate cholesterol-dependent liposome binding and is corrected for non-specific binding to a cholesterol-free liposome. The results were normalized to the maximal ΔF (ΔFmax). (B) DAN-D4 (0.5 µM) binding to increasing concentrations of phosphatidylcholine/cholesterol (60:40) LUVs with various phosphatidylcholine acyl chain saturation. (C) DAN-D4 (0.5 µM) binding to 100 µM DOPC/cholesterol (60:40) LUVs in the presence of increasing concentrations of rat liver cytosol. The change in fluorescence was determined relative to cholesterol-free liposomes at 450 nm and then normalized to the control (ΔF/F). All data were acquired with a PTI scanning spectrofluorometer (ex. 380 nm and em. 420–560 nm). Each experiment was repeated at least three times and error bars represent standard error of the mean.

D4 variants can bind to the cytoplasmic leaflet of the PM in a cholesterol-dependent manner.

(A) CHO cells transiently transfected with mCherry-D4D434A and D4D434A, A463W and the plasma membrane marker, Pleckstrin homology domain of phospholipase C δ (PH-PLC δ) were examined using spinning-disc confocal microscopy. (B) Live-cell images were acquired of cells expressing the same probes as in (A) following incubation with 10 mM methyl-β-cyclodextrin (mβCD) for 20 min to extract plasmalemmal cholesterol. Scale bar, 10 µm. (C) Quantitation of the plasmalemmal enrichment of the mCherry signal seen in (A) and (B). means ± std. dev. n = 20.


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