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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

Figures

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.

https://doi.org/10.7554/eLife.38493.002
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.

https://doi.org/10.7554/eLife.38493.003

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