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Visualization of accessible cholesterol using a GRAM domain-based biosensor

Author

Listed:
  • Dylan Hong Zheng Koh

    (Nanyang Technological University)

  • Tomoki Naito

    (Nanyang Technological University)

  • Minyoung Na

    (Nanyang Technological University)

  • Yee Jie Yeap

    (Nanyang Technological University)

  • Pritisha Rozario

    (Nanyang Technological University)

  • Franklin L. Zhong

    (Nanyang Technological University
    Skin Research Institute of Singapore (SRIS))

  • Kah-Leong Lim

    (Nanyang Technological University
    National Neuroscience Institute)

  • Yasunori Saheki

    (Nanyang Technological University
    Kumamoto University)

Abstract

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.

Suggested Citation

  • Dylan Hong Zheng Koh & Tomoki Naito & Minyoung Na & Yee Jie Yeap & Pritisha Rozario & Franklin L. Zhong & Kah-Leong Lim & Yasunori Saheki, 2023. "Visualization of accessible cholesterol using a GRAM domain-based biosensor," Nature Communications, Nature, vol. 14(1), pages 1-20, December.
    • Handle: RePEc:nat:natcom:v:14:y:2023:i:1:d:10.1038_s41467-023-42498-7
    DOI: 10.1038/s41467-023-42498-7
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    References listed on IDEAS

    as
    1. Tomoki Naito & Haoning Yang & Dylan Hong Zheng Koh & Divyanshu Mahajan & Lei Lu & Yasunori Saheki, 2023. "Regulation of cellular cholesterol distribution via non-vesicular lipid transport at ER-Golgi contact sites," Nature Communications, Nature, vol. 14(1), pages 1-25, December.
    2. Jay Xiaojun Tan & Toren Finkel, 2022. "A phosphoinositide signalling pathway mediates rapid lysosomal repair," Nature, Nature, vol. 609(7928), pages 815-821, September.
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