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Quantitative imaging of membrane contact sites for sterol transfer between endo-lysosomes and mitochondria in living cells.
Juhl, Alice Dupont; Heegaard, Christian W; Werner, Stephan; Schneider, Gerd; Krishnan, Kathiresan; Covey, Douglas F; Wüstner, Daniel.
Afiliação
  • Juhl AD; Department of Biochemistry and Molecular Biology, VILLUM Center for Bioanalytical Sciences, University of Southern Denmark, Campusvej 55, 5230, Odense M, Denmark.
  • Heegaard CW; Department of Molecular Biology and Genetics, University of Aarhus, 8000, Aarhus C, Denmark.
  • Werner S; Department of X-Ray Microscopy, Helmholtz-Zentrum Berlin, Albert-Einstein-Str. 15, 12489, Berlin, Germany.
  • Schneider G; Department of X-Ray Microscopy, Helmholtz-Zentrum Berlin, Albert-Einstein-Str. 15, 12489, Berlin, Germany.
  • Krishnan K; Department of Developmental Biology, Washington University, St. Louis, MO, 63110, USA.
  • Covey DF; Department of Developmental Biology, Washington University, St. Louis, MO, 63110, USA.
  • Wüstner D; Department of Biochemistry and Molecular Biology, VILLUM Center for Bioanalytical Sciences, University of Southern Denmark, Campusvej 55, 5230, Odense M, Denmark. wuestner@bmb.sdu.dk.
Sci Rep ; 11(1): 8927, 2021 04 26.
Article em En | MEDLINE | ID: mdl-33903617
ABSTRACT
Mitochondria receive cholesterol from late endosomes and lysosomes (LE/LYSs) or from the plasma membrane for production of oxysterols and steroid hormones. This process depends on the endo-lysosomal sterol transfer protein Niemann Pick C2 (NPC2). Using the intrinsically fluorescent cholesterol analog, cholestatrienol, we directly observe sterol transport to mitochondria in fibroblasts upon treating NPC2 deficient human fibroblasts with NPC2 protein. Soft X-ray tomography reveals the ultrastructure of mitochondria and discloses close contact to endosome-like organelles. Using fluorescence microscopy, we localize endo-lysosomes containing NPC2 relative to mitochondria based on the Euclidian distance transform and use statistical inference to show that about 30% of such LE/LYSs are in contact to mitochondria in human fibroblasts. Using Markov Chain Monte Carlo image simulations, we show that interaction between both organelle types, a defining feature of membrane contact sites (MCSs) can give rise to the observed spatial organelle distribution. We devise a protocol to determine the surface fraction of endo-lysosomes in contact with mitochondria and show that this fraction does not depend on functional NPC1 or NPC2 proteins. Finally, we localize MCSs between LE/LYSs containing NPC2 and mitochondria in time-lapse image sequences and show that they either form transiently or remain stable for tens of seconds. Lasting MCSs between endo-lysosomes containing NPC2 and mitochondria move by slow anomalous sub-diffusion, providing location and time for sterol transport between both organelles. Our quantitative imaging strategy will be of high value for characterizing the dynamics and function of MCSs between various organelles in living cells.
Assuntos

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Esteróis / Membranas Mitocondriais / Fibroblastos / Lisossomos / Mitocôndrias Limite: Humans / Male Idioma: En Ano de publicação: 2021 Tipo de documento: Article

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Esteróis / Membranas Mitocondriais / Fibroblastos / Lisossomos / Mitocôndrias Limite: Humans / Male Idioma: En Ano de publicação: 2021 Tipo de documento: Article