细胞核中的三维蛋白质动力学
3D Protein Dynamics in the Cell Nucleus.
作者信息
Singh Anand P, Galland Rémi, Finch-Edmondson Megan L, Grenci Gianluca, Sibarita Jean-Baptiste, Studer Vincent, Viasnoff Virgile, Saunders Timothy E
机构信息
Mechanobiology Institute, National University of Singapore, Singapore.
Institut Interdisciplinaire de Neurosciences, University of Bordeaux, France; Centre National de la Recherche Scientifique UMR 5297, University of Bordeaux, France.
出版信息
Biophys J. 2017 Jan 10;112(1):133-142. doi: 10.1016/j.bpj.2016.11.3196.
Abstract
The three-dimensional (3D) architecture of the cell nucleus plays an important role in protein dynamics and in regulating gene expression. However, protein dynamics within the 3D nucleus are poorly understood. Here, we present, to our knowledge, a novel combination of 1) single-objective based light-sheet microscopy, 2) photoconvertible proteins, and 3) fluorescence correlation microscopy, to quantitatively measure 3D protein dynamics in the nucleus. We are able to acquire >3400 autocorrelation functions at multiple spatial positions within a nucleus, without significant photobleaching, allowing us to make reliable estimates of diffusion dynamics. Using this tool, we demonstrate spatial heterogeneity in Polymerase II dynamics in live U2OS cells. Further, we provide detailed measurements of human-Yes-associated protein diffusion dynamics in a human gastric cancer epithelial cell line.
摘要
细胞核的三维（3D）结构在蛋白质动力学和基因表达调控中起着重要作用。然而，三维细胞核内的蛋白质动力学仍知之甚少。在此，据我们所知，我们提出了一种新颖的组合方法，包括1）基于单目标的光片显微镜、2）光转换蛋白和3）荧光相关显微镜，用于定量测量细胞核内的三维蛋白质动力学。我们能够在细胞核内的多个空间位置获取超过3400个自相关函数，且无明显光漂白现象，从而使我们能够对扩散动力学做出可靠估计。使用该工具，我们证明了活U2OS细胞中聚合酶II动力学的空间异质性。此外，我们还提供了人胃癌上皮细胞系中人类Yes相关蛋白扩散动力学的详细测量结果。
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