An Efficient Method for Isolating and Purifying Nuclei from Mice Brain for Single-Molecule Imaging Using High-Speed Atomic Force Microscopy.

Autor:	Qiu Y; Division of Nano Life Science, Graduate School of Frontier Science Initiative, Kanazawa University, Kanazawa 920-1192, Japan., Sajidah ES; WPI Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, Kanazawa 920-1192, Japan., Kondo S; Division of Nano Life Science, Graduate School of Frontier Science Initiative, Kanazawa University, Kanazawa 920-1192, Japan., Narimatsu S; Division of Nano Life Science, Graduate School of Frontier Science Initiative, Kanazawa University, Kanazawa 920-1192, Japan., Sandira MI; Division of Nano Life Science, Graduate School of Frontier Science Initiative, Kanazawa University, Kanazawa 920-1192, Japan., Higashiguchi Y; Division of Nano Life Science, Graduate School of Frontier Science Initiative, Kanazawa University, Kanazawa 920-1192, Japan., Nishide G; Division of Nano Life Science, Graduate School of Frontier Science Initiative, Kanazawa University, Kanazawa 920-1192, Japan., Taoka A; WPI Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, Kanazawa 920-1192, Japan., Hazawa M; WPI Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, Kanazawa 920-1192, Japan.; Cell-Bionomics Research Unit, Innovative Integrated Bio-Research Core, Institute for Frontier Science Initiative, Kanazawa University, Kanazawa 920-1192, Japan., Inaba Y; Metabolism and Nutrition Research Unit, Institute for Frontier Science Initiative, Kanazawa University, Kanazawa 920-8641, Japan., Inoue H; Metabolism and Nutrition Research Unit, Institute for Frontier Science Initiative, Kanazawa University, Kanazawa 920-8641, Japan., Matsushima A; Laboratory of Structure-Function Biochemistry, Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka 819-0395, Japan., Okada Y; Institute for Quantitative Biosciences, The University of Tokyo, Tokyo 113-0033, Japan., Nakada M; Department of Neurosurgery, Graduate School of Medical Science, Kanazawa University, Kanazawa 920-8641, Japan., Ando T; WPI Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, Kanazawa 920-1192, Japan., Lim K; WPI Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, Kanazawa 920-1192, Japan., Wong RW; Division of Nano Life Science, Graduate School of Frontier Science Initiative, Kanazawa University, Kanazawa 920-1192, Japan.; WPI Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, Kanazawa 920-1192, Japan.; Cell-Bionomics Research Unit, Innovative Integrated Bio-Research Core, Institute for Frontier Science Initiative, Kanazawa University, Kanazawa 920-1192, Japan.
Jazyk:	angličtina
Zdroj:	Cells [Cells] 2024 Feb 02; Vol. 13 (3). Date of Electronic Publication: 2024 Feb 02.
DOI:	10.3390/cells13030279
Abstrakt:	Nuclear pore complexes (NPCs) on the nuclear membrane surface have a crucial function in controlling the movement of small molecules and macromolecules between the cell nucleus and cytoplasm through their intricate core channel resembling a spiderweb with several layers. Currently, there are few methods available to accurately measure the dynamics of nuclear pores on the nuclear membranes at the nanoscale. The limitation of traditional optical imaging is due to diffraction, which prevents achieving the required resolution for observing a diverse array of organelles and proteins within cells. Super-resolution techniques have effectively addressed this constraint by enabling the observation of subcellular components on the nanoscale. Nevertheless, it is crucial to acknowledge that these methods often need the use of fixed samples. This also raises the question of how closely a static image represents the real intracellular dynamic system. High-speed atomic force microscopy (HS-AFM) is a unique technique used in the field of dynamic structural biology, enabling the study of individual molecules in motion close to their native states. Establishing a reliable and repeatable technique for imaging mammalian tissue at the nanoscale using HS-AFM remains challenging due to inadequate sample preparation. This study presents the rapid strainer microfiltration (RSM) protocol for directly preparing high-quality nuclei from the mouse brain. Subsequently, we promptly utilize HS-AFM real-time imaging and cinematography approaches to record the spatiotemporal of nuclear pore nano-dynamics from the mouse brain.
Databáze:	MEDLINE
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