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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| Format: | Article |
| Language: | English |
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Nature Portfolio
2021-04-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-021-87876-7 |
| _version_ | 1818432940243156992 |
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| 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 |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-14T16:13:11Z |
| format | Article |
| id | doaj.art-9c8c613b5a6d43229d449d238c083ed0 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-12-14T16:13:11Z |
| publishDate | 2021-04-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-9c8c613b5a6d43229d449d238c083ed02022-12-21T22:54:57ZengNature PortfolioScientific Reports2045-23222021-04-0111112210.1038/s41598-021-87876-7Quantitative imaging of membrane contact sites for sterol transfer between endo-lysosomes and mitochondria in living cellsAlice Dupont Juhl0Christian W. Heegaard1Stephan Werner2Gerd Schneider3Kathiresan Krishnan4Douglas F. Covey5Daniel Wüstner6Department of Biochemistry and Molecular Biology, VILLUM Center for Bioanalytical Sciences, University of Southern DenmarkDepartment of Molecular Biology and Genetics, University of AarhusDepartment of X-Ray Microscopy, Helmholtz-Zentrum BerlinDepartment of X-Ray Microscopy, Helmholtz-Zentrum BerlinDepartment of Developmental Biology, Washington UniversityDepartment of Developmental Biology, Washington UniversityDepartment of Biochemistry and Molecular Biology, VILLUM Center for Bioanalytical Sciences, University of Southern DenmarkAbstract 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.https://doi.org/10.1038/s41598-021-87876-7 |
| spellingShingle | 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 Scientific Reports |
| title | 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_short | 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 |
| url | https://doi.org/10.1038/s41598-021-87876-7 |
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