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Quantitative imaging of membrane contact sites for sterol transfer between endo‑lysosomes and mitochondria in living cells

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Quantitative imaging of membrane contact sites for sterol transfer between endo‑lysosomes and mitochondria in living cells. / Dupont Juhl, Alice ; Würtz Heegaard, Christian; Werner, Stephan ; Schneider, Gerd ; Covey, Douglas F.; Wüstner, Daniel.

I: Scientific Reports, Bind 11, 8927 , 04.2021.

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avisTidsskriftartikelForskningpeer review

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Dupont Juhl, Alice ; Würtz Heegaard, Christian ; Werner, Stephan ; Schneider, Gerd ; Covey, Douglas F. ; Wüstner, Daniel. / Quantitative imaging of membrane contact sites for sterol transfer between endo‑lysosomes and mitochondria in living cells. I: Scientific Reports. 2021 ; Bind 11.

Bibtex

@article{8891928a4d2a4422b49671eaa00f2e99,
title = "Quantitative imaging of membrane contact sites for sterol transfer between endo‑lysosomes and mitochondria in living cells",
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.",
author = "{Dupont Juhl}, Alice and {W{\"u}rtz Heegaard}, Christian and Stephan Werner and Gerd Schneider and Covey, {Douglas F.} and Daniel W{\"u}stner",
year = "2021",
month = apr,
doi = "10.1038/s41598-021-87876-7",
language = "English",
volume = "11",
journal = "Scientific Reports",
issn = "2045-2322",
publisher = "Nature Publishing Group",

}

RIS

TY - JOUR

T1 - Quantitative imaging of membrane contact sites for sterol transfer between endo‑lysosomes and mitochondria in living cells

AU - Dupont Juhl, Alice

AU - Würtz Heegaard, Christian

AU - Werner, Stephan

AU - Schneider, Gerd

AU - Covey, Douglas F.

AU - Wüstner, Daniel

PY - 2021/4

Y1 - 2021/4

N2 - 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.

AB - 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.

U2 - 10.1038/s41598-021-87876-7

DO - 10.1038/s41598-021-87876-7

M3 - Journal article

VL - 11

JO - Scientific Reports

JF - Scientific Reports

SN - 2045-2322

M1 - 8927

ER -