Yamamoto Johtaro, Matsui Akito, Gan Fusako, Oura Makoto, Ando Riku, Matsuda Takahiro, Gong Jian Ping, Kinjo Masataka
Bioimaging Research Group, Health and Medical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, 305-8566, Japan.
Laboratory of Molecular Cell Dynamics, Faculty of Advanced Life Science, Hokkaido University, Sapporo, 001-0021, Japan.
Sci Rep. 2021 May 19;11(1):10594. doi: 10.1038/s41598-021-89987-7.
Macromolecular crowding (MMC) in cells is a hot topic in biology; therefore, well-characterized measurement standards for the evaluation of the nano-environment in MMC solutions are necessary. We propose to use polarization-dependent fluorescence correlation spectroscopy (Pol-FCS) for evaluation of macromolecular crowding in solutions. Pol-FCS can simultaneously measure the relaxation times of rotational and translational diffusion of fluorescent molecules at the same position, even in living cells with low damage. In this report, the differences in the nano-environment among solutions of small molecules, gels, and MMC solutions were evaluated by comparing their rotational and translational diffusion using Pol-FCS. Moreover, this method could distinguish the phase shift in the polyethylene glycol solution. Finally, we separately evaluated the nano-environment in the cytosol and nucleus of living cells in different cell lines and cell cycles. We expect this evaluation method to be useful in characterizing the nano-environment in MMC studies. In addition, the proposed method may be useful for other nano-environments such as liquid-liquid phase separation.
细胞中的大分子拥挤现象（MMC）是生物学中的一个热门话题；因此，需要有特征明确的测量标准来评估MMC溶液中的纳米环境。我们建议使用偏振相关荧光相关光谱法（Pol-FCS）来评估溶液中的大分子拥挤情况。Pol-FCS能够在同一位置同时测量荧光分子旋转和平动扩散的弛豫时间，即使在对活细胞损伤较小的情况下也是如此。在本报告中，通过使用Pol-FCS比较小分子溶液、凝胶溶液和MMC溶液的旋转和平动扩散，评估了它们之间纳米环境的差异。此外，该方法能够区分聚乙二醇溶液中的相移。最后，我们分别评估了不同细胞系和细胞周期中活细胞胞质溶胶和细胞核内的纳米环境。我们期望这种评估方法在表征MMC研究中的纳米环境方面有用。此外，所提出的方法可能对其他纳米环境（如液-液相分离）也有用。
