Computer-based fluorescence quantification: a novel approach to study nucleolar biology.
Computer-based fluorescence quantification: a novel approach to study nucleolar biology.
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DOI：
10.1186/1471-2121-12-25
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发表时间：
2011-06-03
期刊：
影响因子：
--
通讯作者：
Stochaj U
中科院分区：
文献类型：
--
作者：
Kodiha M;Bański P;Stochaj U
核仁可能由数千种不同的蛋白质组成，是细胞核中最显眼的隔室；它们在许多细胞过程的正常执行中起着至关重要的作用。因此，核仁进行核糖体的生物生成和隔离，或与调节细胞周期进程、肿瘤发生、细胞凋亡和应激反应的关键分子相关联。核仁是动态的隔室，其特征是大分子的不断流动。鉴于核仁蛋白质组的复杂和动态组成，将核仁组成的修饰与下游效应联系起来是具有挑战性的。在这篇文章中，我们提出了依赖于基于计算机的图像分析的定量免疫荧光方法。我们通过监测不同生理条件下蛋白质和RNA与核仁的动态关联来证明这些技术的有效性。因此，我们所描述的方案被用于研究内源性和gfp标记蛋白的核仁浓度的应力依赖性变化。此外，我们的方法被应用于测量与核仁相关的从头RNA合成。我们表明，这里描述的技术可以很容易地与自动化高通量筛选（HTS）平台相结合，从而可以获得大型数据集并分析位于核仁中的许多生物过程。我们的方案为在单细胞水平和大量细胞水平上以定量方式分析穿梭核仁蛋白的动力学奠定了基础。此外，本文描述的程序与使用HTS自动化平台的高通量图像采集和分析相兼容，从而为量化众多样品和实验条件下的核仁成分和活性提供了基础。随着核仁蛋白质组获得的信息越来越多，定量显微镜的改进，正如它们在这里所描述的，可以期望对核仁精心安排的复杂生物功能产生新的见解。
Nucleoli are composed of possibly several thousand different proteins and represent the most conspicuous compartments in the nucleus; they play a crucial role in the proper execution of many cellular processes. As such, nucleoli carry out ribosome biogenesis and sequester or associate with key molecules that regulate cell cycle progression, tumorigenesis, apoptosis and the stress response. Nucleoli are dynamic compartments that are characterized by a constant flux of macromolecules. Given the complex and dynamic composition of the nucleolar proteome, it is challenging to link modifications in nucleolar composition to downstream effects. In this contribution, we present quantitative immunofluorescence methods that rely on computer-based image analysis. We demonstrate the effectiveness of these techniques by monitoring the dynamic association of proteins and RNA with nucleoli under different physiological conditions. Thus, the protocols described by us were employed to study stress-dependent changes in the nucleolar concentration of endogenous and GFP-tagged proteins. Furthermore, our methods were applied to measure de novo RNA synthesis that is associated with nucleoli. We show that the techniques described here can be easily combined with automated high throughput screening (HTS) platforms, making it possible to obtain large data sets and analyze many of the biological processes that are located in nucleoli. Our protocols set the stage to analyze in a quantitative fashion the kinetics of shuttling nucleolar proteins, both at the single cell level as well as for a large number of cells. Moreover, the procedures described here are compatible with high throughput image acquisition and analysis using HTS automated platforms, thereby providing the basis to quantify nucleolar components and activities for numerous samples and experimental conditions. Together with the growing amount of information obtained for the nucleolar proteome, improvements in quantitative microscopy as they are described here can be expected to produce new insights into the complex biological functions that are orchestrated by the nucleolus.
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影响因子：
8
作者：
Kodiha，M.;Banski，P.;Stochaj，U.
通讯作者：
Stochaj，U.
影响因子：
64.8
作者：
Phair, RD;Misteli, T
通讯作者：
Misteli, T
影响因子：
3.3
作者：
Pendle, AF;Clark, GP;Shaw, PJ
通讯作者：
Shaw, PJ
影响因子：
7.3
作者：
Kodiha, Mohamed;Brown, Claire M.;Stochaj, Ursula
通讯作者：
Stochaj, Ursula
DOI：
10.1073/pnas.0808480105
发表时间：
2008-10-14
影响因子：
11.1
作者：
Jao, Cindy Y.;Salic, Adrian
通讯作者：
Salic, Adrian