Super-Resolution Microscopy Using a Bioorthogonal-Based Cholesterol Probe Provides Unprecedented Capabilities for Imaging Nanoscale Lipid Heterogeneity in Living Cells.

Autor: Lorizate M; Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU) and Instituto Biofisika (UPV/EHU, CSIC), Barrio Sarriena s/n, Leioa, 48940, Spain., Terrones O; Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU), Barrio Sarriena s/n, Leioa, 48940, Spain., Nieto-Garai JA; Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU) and Instituto Biofisika (UPV/EHU, CSIC), Barrio Sarriena s/n, Leioa, 48940, Spain.; Fundación Biofísica Bizkaia/Biofisika Bizkaia Fundazioa (FBB), Barrio Sarriena s/n, Leioa, 48940, Spain., Rojo-Bartolomé I; Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU) and Instituto Biofisika (UPV/EHU, CSIC), Barrio Sarriena s/n, Leioa, 48940, Spain.; Fundación Biofísica Bizkaia/Biofisika Bizkaia Fundazioa (FBB), Barrio Sarriena s/n, Leioa, 48940, Spain., Ciceri D; Fundación Biofísica Bizkaia/Biofisika Bizkaia Fundazioa (FBB), Barrio Sarriena s/n, Leioa, 48940, Spain., Morana O; Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU) and Instituto Biofisika (UPV/EHU, CSIC), Barrio Sarriena s/n, Leioa, 48940, Spain.; Fundación Biofísica Bizkaia/Biofisika Bizkaia Fundazioa (FBB), Barrio Sarriena s/n, Leioa, 48940, Spain., Olazar-Intxausti J; Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU), Barrio Sarriena s/n, Leioa, 48940, Spain., Arboleya A; Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU) and Instituto Biofisika (UPV/EHU, CSIC), Barrio Sarriena s/n, Leioa, 48940, Spain.; Fundación Biofísica Bizkaia/Biofisika Bizkaia Fundazioa (FBB), Barrio Sarriena s/n, Leioa, 48940, Spain., Martin A; National Heart and Lung Institute, Imperial College London, Sir Alexander Fleming Building, London, SW7 2AZ, UK., Szynkiewicz M; Central Laser Facility, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Research Complex at Harwell, Oxford, OX11 0FA, UK., Calleja-Felipe M; Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU) and Instituto Biofisika (UPV/EHU, CSIC), Barrio Sarriena s/n, Leioa, 48940, Spain., Bernardino de la Serna J; National Heart and Lung Institute, Imperial College London, Sir Alexander Fleming Building, London, SW7 2AZ, UK.; Central Laser Facility, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Research Complex at Harwell, Oxford, OX11 0FA, UK.; NIHR Imperial Biomedical Research Centre, London, SW7 2AZ, UK., Contreras FX; Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU) and Instituto Biofisika (UPV/EHU, CSIC), Barrio Sarriena s/n, Leioa, 48940, Spain.; IKERBASQUE, Basque Foundation for Science, Bilbao, 48011, Spain.
Jazyk: angličtina
Zdroj: Small methods [Small Methods] 2021 Sep; Vol. 5 (9), pp. e2100430. Date of Electronic Publication: 2021 Jul 29.
DOI: 10.1002/smtd.202100430
Abstrakt: Despite more than 20 years of work since the lipid raft concept was proposed, the existence of these nanostructures remains highly controversial due to the lack of noninvasive methods to investigate their native nanorganization in living unperturbed cells. There is an unmet need for probes for direct imaging of nanoscale membrane dynamics with high spatial and temporal resolution in living cells. In this paper, a bioorthogonal-based cholesterol probe (chol-N 3 ) is developed that, combined with nanoscopy, becomes a new powerful method for direct visualization and characterization of lipid raft at unprecedented resolution in living cells. The chol-N 3 probe mimics cholesterol in synthetic and cellular membranes without perturbation. When combined with live-cell super-resolution microscopy, chol-N 3 demonstrates the existence of cholesterol-rich nanodomains of <50 nm at the plasma membrane of resting living cells. Using this tool, the lipid membrane structure of such subdiffraction limit domains is identified, and the nanoscale spatiotemporal organization of cholesterol in the plasma membrane of living cells reveals multiple cholesterol diffusion modes at different spatial localizations. Finally, imaging across thick organ samples outlines the potential of this new method to address essential biological questions that were previously beyond reach.
(© 2021 The Authors. Small Methods published by Wiley-VCH GmbH.)
Databáze: MEDLINE
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