Asan Institute for Life Sciences, Asan Medical Center, Seoul 05505, Korea.
Division of Physical Pharmacy, Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Kobe 650-8586, Japan.
Cells. 2019 Jul 10;8(7):699. doi: 10.3390/cells8070699.
The cell nucleus is three-dimensionally and dynamically organized by nuclear components with high molecular density, such as chromatin and nuclear bodies. The structure and functions of these components are represented by the diffusion and interaction of related factors. Recent studies suggest that the nucleolus can be assessed using various protein probes, as the probes are highly mobile in this organelle, although it is known that they have a densely packed structure. However, physicochemical properties of the nucleolus itself, such as molecular density and volume when cellular conditions are changed, are not yet fully understood. In this study, physical parameters such as the refractive index (RI) and volume of the nucleoli in addition to the diffusion coefficient () of fluorescent probe protein inside the nucleolus are quantified and compared by combining label-free optical diffraction tomography (ODT) with confocal laser scanning microscopy (CLSM)-based fluorescence correlation spectroscopy (FCS). 3D evaluation of RI values and corresponding RI images of nucleoli in live HeLa cells successfully demonstrated varying various physiological conditions. Our complimentary method suggests that physical property of the nucleolus in live cell is sensitive to ATP depletion and transcriptional inhibition, while it is insensitive to hyper osmotic pressure when compared with the cytoplasm and nucleoplasm. The result demonstrates that the nucleolus has unique physicochemical properties when compared with other cellular components.
细胞核由具有高分子密度的核成分（如染色质和核体）在三维和动态上进行组织。这些成分的结构和功能通过相关因素的扩散和相互作用来表示。最近的研究表明，可以使用各种蛋白质探针来评估核仁，因为这些探针在该细胞器中具有高度的流动性，尽管已知其具有致密的结构。然而，核仁本身的物理化学性质，例如在细胞条件改变时的分子密度和体积，尚未得到充分理解。在这项研究中，通过将无标记光衍射断层扫描（ODT）与基于共聚焦激光扫描显微镜（CLSM）的荧光相关光谱（FCS）相结合，对荧光探针蛋白在核仁内的扩散系数（）以及核仁的折射率（RI）和体积等物理参数进行了量化和比较。成功地对活 HeLa 细胞中核仁的 RI 值和相应的 RI 图像进行了 3D 评估，展示了各种生理条件的变化。我们的补充方法表明，与细胞质和核质相比，当核仁的物理性质对细胞内的三磷酸腺苷（ATP）耗竭和转录抑制敏感，而对高渗压不敏感。该结果表明，与其他细胞成分相比，核仁具有独特的物理化学性质。
