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 choleste...

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Autores principales:	Alice Dupont Juhl, Christian W. Heegaard, Stephan Werner, Gerd Schneider, Kathiresan Krishnan, Douglas F. Covey, Daniel Wüstner
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/9c8c613b5a6d43229d449d238c083ed0
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spelling	oai:doaj.org-article:9c8c613b5a6d43229d449d238c083ed02021-12-02T16:55:24ZQuantitative imaging of membrane contact sites for sterol transfer between endo-lysosomes and mitochondria in living cells10.1038/s41598-021-87876-72045-2322https://doaj.org/article/9c8c613b5a6d43229d449d238c083ed02021-04-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-87876-7https://doaj.org/toc/2045-2322Abstract 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.Alice Dupont JuhlChristian W. HeegaardStephan WernerGerd SchneiderKathiresan KrishnanDouglas F. CoveyDaniel WüstnerNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-22 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Alice Dupont Juhl Christian W. Heegaard Stephan Werner Gerd Schneider Kathiresan Krishnan Douglas F. Covey Daniel Wüstner Quantitative imaging of membrane contact sites for sterol transfer between endo-lysosomes and mitochondria in living cells
description	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.
format	article
author	Alice Dupont Juhl Christian W. Heegaard Stephan Werner Gerd Schneider Kathiresan Krishnan Douglas F. Covey Daniel Wüstner
author_facet	Alice Dupont Juhl Christian W. Heegaard Stephan Werner Gerd Schneider Kathiresan Krishnan Douglas F. Covey Daniel Wüstner
author_sort	Alice Dupont Juhl
title	Quantitative imaging of membrane contact sites for sterol transfer between endo-lysosomes and mitochondria in living cells
title_short	Quantitative imaging of membrane contact sites for sterol transfer between endo-lysosomes and mitochondria in living cells
title_full	Quantitative imaging of membrane contact sites for sterol transfer between endo-lysosomes and mitochondria in living cells
title_fullStr	Quantitative imaging of membrane contact sites for sterol transfer between endo-lysosomes and mitochondria in living cells
title_full_unstemmed	Quantitative imaging of membrane contact sites for sterol transfer between endo-lysosomes and mitochondria in living cells
title_sort	quantitative imaging of membrane contact sites for sterol transfer between endo-lysosomes and mitochondria in living cells
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/9c8c613b5a6d43229d449d238c083ed0
work_keys_str_mv	AT alicedupontjuhl quantitativeimagingofmembranecontactsitesforsteroltransferbetweenendolysosomesandmitochondriainlivingcells AT christianwheegaard quantitativeimagingofmembranecontactsitesforsteroltransferbetweenendolysosomesandmitochondriainlivingcells AT stephanwerner quantitativeimagingofmembranecontactsitesforsteroltransferbetweenendolysosomesandmitochondriainlivingcells AT gerdschneider quantitativeimagingofmembranecontactsitesforsteroltransferbetweenendolysosomesandmitochondriainlivingcells AT kathiresankrishnan quantitativeimagingofmembranecontactsitesforsteroltransferbetweenendolysosomesandmitochondriainlivingcells AT douglasfcovey quantitativeimagingofmembranecontactsitesforsteroltransferbetweenendolysosomesandmitochondriainlivingcells AT danielwustner quantitativeimagingofmembranecontactsitesforsteroltransferbetweenendolysosomesandmitochondriainlivingcells
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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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