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Imaging cholesterol depletion at the plasma membrane by methyl-β-cyclodextrin

  • Mitsuhiro Abe
    Affiliations
    Lipid Biology Laboratory, RIKEN, Wako, Saitama, Japan

    Cellular Informatics Laboratory, RIKEN, Wako, Saitama, Japan
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  • Toshihide Kobayashi
    Correspondence
    For correspondence: Toshihide Kobayashi
    Affiliations
    Lipid Biology Laboratory, RIKEN, Wako, Saitama, Japan

    Cellular Informatics Laboratory, RIKEN, Wako, Saitama, Japan

    Laboratoire de Bioimagerie et Pathologies, UMR 7021 CNRS, Université de Strasbourg, Faculté de Pharmacie, Illkirch, France
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Open AccessPublished:April 20, 2021DOI:https://doi.org/10.1016/j.jlr.2021.100077
      The most common method to modify cellular cholesterol (Chol) content in vitro is to incubate cells with cyclodextrin (CD). However, it is not well understood how Chol is removed by CD. Recent development of protein probes that bind Chol allowed to visualize and to follow cellular Chol semi-quantitatively. In this image, non-toxic Chol-binding D4 fragment of bacterial toxin, perfringolysin O was conjugated with mCherry (mCherry-D4) (
      • Abe M.
      • Makino A.
      • Hullin-Matsuda F.
      • Kamijo K.
      • Ohno-Iwashita Y.
      • Hanada K.
      • Mizuno H.
      • Miyawaki A.
      • Kobayashi T.
      A role for sphingomyelin-rich lipid domains in the accumulation of phosphatidylinositol 4,5-bisphosphate to the cleavage furrow during cytokinesis.
      ) and the protein was transiently expressed in HeLa cells for 48 h. Cells were synchronized to mitosis and incubated with and without 1 mM methyl-b-CD (MbCD) in the presence of EGFP-D4 in the medium. Selected images are shown. Accumulated images are shown in Supplemental videos 1 and 2. Average fluorescence intensities of EGFP-D4 (green) and mCherry-D4 (magenta) were quantified in the area surrounded by the white rectangle and schematically shown in the kymographs. The quantitated data are shown as graphs in Supplemental Fig. 1. Supplemental Fig. 2 shows that equivalent amount of EGFP-D4 and mCherry-D4 displayed similar Chol concentration dependent binding to phosphatidylcholine/Chol membranes. Under this condition, mCherry-D4 and EGFP-D4 bind accessible Chol (
      • Das A.
      • Brown M.S.
      • Anderson D.D.
      • Goldstein J.L.
      • Radhakrishnan A.
      Three pools of plasma membrane cholesterol and their relation to cholesterol homeostasis.
      ) in the inner and outer leaflet of the plasma membrane, respectively (
      • Abe M.
      • Makino A.
      • Hullin-Matsuda F.
      • Kamijo K.
      • Ohno-Iwashita Y.
      • Hanada K.
      • Mizuno H.
      • Miyawaki A.
      • Kobayashi T.
      A role for sphingomyelin-rich lipid domains in the accumulation of phosphatidylinositol 4,5-bisphosphate to the cleavage furrow during cytokinesis.
      ). Due to high threshold of Chol-detection by D4, intracellular organelles were not labeled with mCherry-D4 and depletion of Chol was accompanied by the intracellular accumulation of the protein. MbCD induced the rapid decrease (5-15 min) of inner leaflet Chol below the D4-binding threshold followed by the slow depletion phase in both outer and inner leaflets. The interaction between D4 or MbCD and cultured cells is not fully elucidated. Although MbCD is endocytosed, endocytosis is inhibited in mitotic cells (
      • Kobayashi T.
      • Pagano R.E.
      Lipid transport during mitosis. Alternative pathways for delivery of newly synthesized lipids to the cell surface.
      ). Binding of acrylodan-labeled D4 to PC/Chol membrane is inhibited by cytosol (
      • Courtney K.C.
      • Fung K.Y.
      • Maxfield F.R.
      • Fairn G.D.
      • Zha X.
      Comment on 'Orthogonal lipid sensors identify transbilayer asymmetry of plasma membrane cholesterol'.
      ). In addition to Chol, MbCD is reported to interact with other lipids and protein components of the membrane. However, extraction of cytosolic protein by MbCD has not been reported. Our result suggests that the removal of outer leaflet Chol by MbCD is accompanied by the rapid flip of Chol from the inner leaflet to the outer leaflet. However, we cannot exclude the possibility that MbCD preferentially removed Chol from the inner leaflet.
      EQUIPMENT: FV 1000 confocal microscope with a 60x 1.1 NA Plan Apo objective lens (Olympus) equipped with an environmental chamber maintained with humidity, 37°C, and 5% CO2. ImageQuant LAS 500 (Cytiva).
      REAGENTS: DMEM supplemented with 10% fetal bovine serum, Lipofectamine 3000 (Thermo Fisher Scientific), pET28/His6-EGFP-D4 (RIKEN BRC, Gene Engineering Division, Catalog # RDB13961), pET28/His6-mCherry-D4 (RIKEN BRC, Catalog # RDB14300), pDEST/mCherry-D4 (1), nocodazole (Sigma-Aldrich), MβCD (Cyclolab), SYPRO Ruby (Thermo Fisher Scientific).

      Supplemental data

      This article contains supplemental data.

      Conflict of interest

      The authors declare that they have no conflicts of interest with the contents of this article.

      Acknowledgments

      We are grateful to Dr. Françoise Hullin-Matsuda from INSA Lyon for critically reading the manuscript.

      Author contributions

      M. A. conceptualization, methodology, investigation, data curation, writing-original draft preparation, reviewing, and editing. T. K. conceptualization, writing-original draft preparation, reviewing, and editing.

      Funding and additional information

      This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (18K06677 to M. A. and 22390018 and 25293015 to T. K.) and the Lipid Dynamics Program and Glycolipidologue Program of RIKEN (to T. K). T. K. also acknowledges Grants from Agence Nationale pour la Recherche (A20R417C), Agence Nationale de Recherche sur le Sida et les Hépatites virale (18365), Ligue Contre le Cancer, and Vaincre les Maladies Lysosomales (19/LBPH/S44).

      Supplemental Data

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