Quantitative methods to analyze subnuclear protein organization in cell populations with varying degrees of protein expression.
Quantitative methods to analyze subnuclear protein organization in cell populations with varying degrees of protein expression.
复制标题
分析具有不同蛋白质表达程度的细胞群中亚核蛋白质组织的定量方法。
DOI：
10.1117/1.1891085
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发表时间：
2005
影响因子：
3.5
通讯作者：
Day,RichardN
中科院分区：
文献类型：
--
作者：
Voss,TyC;Demarco,IgnacioA;Booker,CynthiaF;Day,RichardN
基因转录的控制依赖于在细胞核不同区域组装的 DNA 结合蛋白和共调节蛋白。我们使用多光谱宽视野显微镜对表达荧光蛋白（FP）标记的转录共调节因子的细胞进行研究，以研究这些因子在活细胞中的亚核定位和功能。在共表达研究中，糖皮质激素受体相互作用蛋白（GRIP）共激活蛋白和类维生素A和甲状腺沉默介导物（SMRT）辅阻遏蛋白形成空间上不同的球形亚核焦点体，表明特定的蛋白质相互作用将这些不同的蛋白质集中在不同的亚核区域。然而，群体内细胞之间这些亚核体的变异性使得基于“代表性图像”的分析变得困难，甚至不可能。为了解决这个问题，我们开发了一种协议，用于从群体中公正地选择细胞，然后对标记蛋白质的亚核组织进行自动定量。统计方法确定了 FP-GRIP 和 FP-SMRT 的 FP 共调节子表达水平与亚核焦体形成之间存在显着的线性相关性。重要的是，我们确认亚核组织的这些变化可以根据共调节子表达水平的差异进行统计标准化。这种集成的定量图像分析方法将允许对表达不同水平的 FP 融合蛋白的不同实验细胞群进行严格比较。 ©2005 光电仪器工程师协会版权所有。
The control of gene transcription is dependent on DNA-binding and coregulatory proteins that assemble in distinct regions of the cell nucleus. We use multispectral wide-field microscopy of cells expressing transcriptional coregulators labeled with fluorescent proteins (FP) to study the subnuclear localization and function of these factors in living cells. In coexpression studies, the glucocorticoid receptor interacting protein (GRIP) coactivator protein and the silencing mediator of retinoid and thyroid (SMRT) corepressor protein form spherical subnuclear focal bodies that are spatially distinct, suggesting that specific protein interactions concentrate these divergent proteins in separate subnuclear regions. However, the variability of these subnuclear bodies between cells within the population makes analysis based on “representative images” difficult, if not impossible. To address this issue, we develop a protocol for unbiased selection of cells from the population, followed by the automated quantification of the subnuclear organization of the labeled proteins. Statistical methods identify a significant linear correlation between the FP-coregulator expression level and subnuclear focal body formation for both FP-GRIP and FP-SMRT. Importantly, we confirm that these changes in subnuclear organization could be statistically normalized for differences in coregulator expression level. This integrated quantitative image analysis method will allow the rigorous comparison of different experimental cell populations that express variable levels of FP fusion proteins. ©2005 Society of Photo-Optical Instrumentation Engineers.