High-throughput identification of protein localization dependency networks
High-throughput identification of protein localization dependency networks
复制标题
DOI：
10.1073/pnas.1000846107
复制
发表时间：
2010-03-09
影响因子：
11.1
通讯作者：
McAdams, Harley H.
中科院分区：
文献类型：
--
作者：
Christen, Beat;Fero, Michael J.;McAdams, Harley H.
细菌细胞是高度组织化的，在细胞周期的过程中，许多蛋白质复合体和DNA位点动态定位于不同的亚细胞位置。这种动态的蛋白质定位对于新月藻细胞周期中极细胞器的发育、不对称性的建立和染色体复制是必不可少的。我们使用高通量优化的荧光显微镜筛选，在转座子产生的突变体库中寻找具有异常时间或空间蛋白质定位模式的菌株。自动图像采集和分析使我们能够识别影响两个极性细胞周期组氨酸激酶Plec和DivJ以及极特异性菌毛蛋白CPAE定位的基因，每一个都在单个菌株中标记了不同的荧光标记。从854个定位突变菌株的数百个细胞图像中提取了表征观察到的三个标记蛋白质中每一个的定位模式的四个指标。通过对每个菌株的12元组载体进行聚类分析，我们鉴定了52个突变株，这些突变影响了三个标记蛋白的定位模式。这一信息，结合来自表皮试验的定量定位数据，也确定了所有先前已知的影响这种定位的蛋白质。这些研究为深入了解影响Plec/DivJ定位网络的因素以及菌毛组装蛋白CPAE和激酶定位途径之间的调控联系提供了见解。我们的高通量筛选方法可以很容易地适应任何已测序的细菌物种，为跨物种的定位调控网络数据库和定位网络系统发育调查打开了可能性。
Bacterial cells are highly organized with many protein complexes and DNA loci dynamically positioned to distinct subcellular sites over the course of a cell cycle. Such dynamic protein localization is essential for polar organelle development, establishment of asymmetry, and chromosome replication during the Caulobacter crescentus cell cycle. We used a fluorescence microscopy screen optimized for high-throughput to find strains with anomalous temporal or spatial protein localization patterns in transposon-generated mutant libraries. Automated image acquisition and analysis allowed us to identify genes that affect the localization of two polar cell cycle histidine kinases, PleC and DivJ, and the pole-specific pili protein CpaE, each tagged with a different fluorescent marker in a single strain. Four metrics characterizing the observed localization patterns of each of the three labeled proteins were extracted for hundreds of cell images from each of 854 mapped mutant strains. Using cluster analysis of the resulting set of 12-element vectors for each of these strains, we identified 52 strains with mutations that affected the localization pattern of the three tagged proteins. This information, combined with quantitative localization data from epitasis experiments, also identified all previously known proteins affecting such localization. These studies provide insights into factors affecting the PleC/DivJ localization network and into regulatory links between the localization of the pili assembly protein CpaE and the kinase localization pathway. Our high-throughput screening methodology can be adapted readily to any sequenced bacterial species, opening the potential for databases of localization regulatory networks across species, and investigation of localization network phylogenies.