Visualization of accessible cholesterol using a GRAM domain-based biosensor
Cholesterol is important for membrane integrity and cell signaling, and dysregulation of the distribution of cellular cholesterol is associated with numerous diseases, including neurodegenerative disorders. While regulated transport of a specific pool of cholesterol, known as "accessible choles...
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sg-ntu-dr.10356-1730102024-01-14T15:38:50Z Visualization of accessible cholesterol using a GRAM domain-based biosensor Koh, Dylan Hong Zheng Naito, Tomoki Na, Minyoung Yeap, Yee Jie Rozario, Pritisha Zhong, Franklin Lim, Kah-Leong Saheki, Yasunori Lee Kong Chian School of Medicine (LKCMedicine) Skin Research Institute of Singapore National Neuroscience Institute, Singapore Science::Medicine Cholesterol Lipid Transfer Protein Cholesterol is important for membrane integrity and cell signaling, and dysregulation of the distribution of cellular cholesterol is associated with numerous diseases, including neurodegenerative disorders. While regulated transport of a specific pool of cholesterol, known as "accessible cholesterol", contributes to the maintenance of cellular cholesterol distribution and homeostasis, tools to monitor accessible cholesterol in live cells remain limited. Here, we engineer a highly sensitive accessible cholesterol biosensor by taking advantage of the cholesterol-sensing element (the GRAM domain) of an evolutionarily conserved lipid transfer protein, GRAMD1b. Using this cholesterol biosensor, which we call GRAM-W, we successfully visualize in real time the distribution of accessible cholesterol in many different cell types, including human keratinocytes and iPSC-derived neurons, and show differential dependencies on cholesterol biosynthesis and uptake for maintaining levels of accessible cholesterol. Furthermore, we combine GRAM-W with a dimerization-dependent fluorescent protein (ddFP) and establish a strategy for the ultrasensitive detection of accessible plasma membrane cholesterol. These tools will allow us to obtain important insights into the molecular mechanisms by which the distribution of cellular cholesterol is regulated. Ministry of Education (MOE) Nanyang Technological University National Research Foundation (NRF) Published version This work was supported by the Singapore Ministry of Education Academic Research Fund Tier 2 (MOE-T2EP30120-0002 & MOE2017-T2-2-001), the Singapore Ministry of Education Academic Research Fund Tier 1 (RG20/ 21), and Grant-in-Aid for Scientific Research (B) (22H02620) from the Japan Society for Promotion of Science (JSPS), and a Nanyang Assistant Professorship (NAP) to Y.S., LKCMedicine LEARN grant to T.N., National Research Foundation Fellowship, Singapore (NRF-NRFF11-2019-0006) and Nanyang Assistant Professorship (NAP) to F.L.Z., MOH-000207, NMRC-LCG SPARKII to K.L.L. T.N. was supported by a fellowship from the JSPS. 2024-01-09T01:12:46Z 2024-01-09T01:12:46Z 2023 Journal Article Koh, D. H. Z., Naito, T., Na, M., Yeap, Y. J., Rozario, P., Zhong, F., Lim, K. & Saheki, Y. (2023). Visualization of accessible cholesterol using a GRAM domain-based biosensor. Nature Communications, 14(1), 6773-. https://dx.doi.org/10.1038/s41467-023-42498-7 2041-1723 https://hdl.handle.net/10356/173010 10.1038/s41467-023-42498-7 37880244 2-s2.0-85174935427 1 14 6773 en MOE-T2EP30120-0002 MOE2017-T2-2-001 RG20/21 NRF-NRFF11-2019-0006 MOH-000207 NMRC-LCG SPARKII Nature Communications © 2023 The Author(s). Open Access. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. application/pdf |
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Science::Medicine Cholesterol Lipid Transfer Protein Koh, Dylan Hong Zheng Naito, Tomoki Na, Minyoung Yeap, Yee Jie Rozario, Pritisha Zhong, Franklin Lim, Kah-Leong Saheki, Yasunori Visualization of accessible cholesterol using a GRAM domain-based biosensor |
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Cholesterol is important for membrane integrity and cell signaling, and dysregulation of the distribution of cellular cholesterol is associated with numerous diseases, including neurodegenerative disorders. While regulated transport of a specific pool of cholesterol, known as "accessible cholesterol", contributes to the maintenance of cellular cholesterol distribution and homeostasis, tools to monitor accessible cholesterol in live cells remain limited. Here, we engineer a highly sensitive accessible cholesterol biosensor by taking advantage of the cholesterol-sensing element (the GRAM domain) of an evolutionarily conserved lipid transfer protein, GRAMD1b. Using this cholesterol biosensor, which we call GRAM-W, we successfully visualize in real time the distribution of accessible cholesterol in many different cell types, including human keratinocytes and iPSC-derived neurons, and show differential dependencies on cholesterol biosynthesis and uptake for maintaining levels of accessible cholesterol. Furthermore, we combine GRAM-W with a dimerization-dependent fluorescent protein (ddFP) and establish a strategy for the ultrasensitive detection of accessible plasma membrane cholesterol. These tools will allow us to obtain important insights into the molecular mechanisms by which the distribution of cellular cholesterol is regulated. |
author2 |
Lee Kong Chian School of Medicine (LKCMedicine) |
author_facet |
Lee Kong Chian School of Medicine (LKCMedicine) Koh, Dylan Hong Zheng Naito, Tomoki Na, Minyoung Yeap, Yee Jie Rozario, Pritisha Zhong, Franklin Lim, Kah-Leong Saheki, Yasunori |
format |
Article |
author |
Koh, Dylan Hong Zheng Naito, Tomoki Na, Minyoung Yeap, Yee Jie Rozario, Pritisha Zhong, Franklin Lim, Kah-Leong Saheki, Yasunori |
author_sort |
Koh, Dylan Hong Zheng |
title |
Visualization of accessible cholesterol using a GRAM domain-based biosensor |
title_short |
Visualization of accessible cholesterol using a GRAM domain-based biosensor |
title_full |
Visualization of accessible cholesterol using a GRAM domain-based biosensor |
title_fullStr |
Visualization of accessible cholesterol using a GRAM domain-based biosensor |
title_full_unstemmed |
Visualization of accessible cholesterol using a GRAM domain-based biosensor |
title_sort |
visualization of accessible cholesterol using a gram domain-based biosensor |
publishDate |
2024 |
url |
https://hdl.handle.net/10356/173010 |
_version_ |
1789483105600405504 |