Cell age dependent concentration of Escherichia coli divisome proteins analyzed with ImageJ and ObjectJ.
Cell age dependent concentration of Escherichia coli divisome proteins analyzed with ImageJ and ObjectJ.
复制标题
DOI：
10.3389/fmicb.2015.00586
复制
发表时间：
2015
影响因子：
5.2
通讯作者：
den Blaauwen T
中科院分区：
文献类型：
--
作者：
Vischer NO;Verheul J;Postma M;van den Berg van Saparoea B;Galli E;Natale P;Gerdes K;Luirink J;Vollmer W;Vicente M;den Blaauwen T
杆状革兰氏阴性细菌大肠杆菌通过伸长繁殖，然后进行二元分裂。细胞膜的纵向生长和新极点的合成分别由两个蛋白质复合体组成，分别称为细长体和分裂体。我们通过免疫标记野生型菌株MC4100在28°C的微量葡萄糖介质中生长到稳定状态，分析了许多这些形态发生蛋白的时空定位模式，这使得通过相差成像和荧光广场显微镜成像的形态发生蛋白定位模式与细胞年龄的函数的直接比较成为可能。在稳态条件下，细胞的年龄分布是恒定的，并与细胞长度直接相关。为了量化数千个标记细胞中的细胞大小和蛋白质定位参数，我们开发了一个名为Coli-Inspector的项目，这是一个在ImageJ下运行的项目，插件为“ObjectJ”。ObjectJ使用一种集成的方法组织图像分析任务，可以灵活地从现有标记(如强度数据和几何参数)生成不同的输出格式。ObjectJ支持自动和交互方法的组合，使用户能够完全控制图像分析和数据收集的方法，并使用视觉检查工具快速消除伪影。Coli-Inspector用于根据分裂周期细胞年龄对细胞进行分类，并分析每个蛋白质的时空定位模式。关于细胞周期中蛋白质的浓度和位置已经创建了一个独特的数据集。我们首次发现，形态发生蛋白的一个子集在细胞分裂周期中具有恒定的细胞浓度，而另一子集则显示出依赖于细胞分裂周期的浓度变化。利用存在于中细胞的蛋白质数量，讨论了分裂体的化学计量。
The rod-shaped Gram-negative bacterium Escherichia coli multiplies by elongation followed by binary fission. Longitudinal growth of the cell envelope and synthesis of the new poles are organized by two protein complexes called elongasome and divisome, respectively. We have analyzed the spatio-temporal localization patterns of many of these morphogenetic proteins by immunolabeling the wild type strain MC4100 grown to steady state in minimal glucose medium at 28°C. This allowed the direct comparison of morphogenetic protein localization patterns as a function of cell age as imaged by phase contrast and fluorescence wide field microscopy. Under steady state conditions the age distribution of the cells is constant and is directly correlated to cell length. To quantify cell size and protein localization parameters in 1000s of labeled cells, we developed ‘Coli-Inspector,’ which is a project running under ImageJ with the plugin ‘ObjectJ.’ ObjectJ organizes image-analysis tasks using an integrated approach with the flexibility to produce different output formats from existing markers such as intensity data and geometrical parameters. ObjectJ supports the combination of automatic and interactive methods giving the user complete control over the method of image analysis and data collection, with visual inspection tools for quick elimination of artifacts. Coli-inspector was used to sort the cells according to division cycle cell age and to analyze the spatio-temporal localization pattern of each protein. A unique dataset has been created on the concentration and position of the proteins during the cell cycle. We show for the first time that a subset of morphogenetic proteins have a constant cellular concentration during the cell division cycle whereas another set exhibits a cell division cycle dependent concentration variation. Using the number of proteins present at midcell, the stoichiometry of the divisome is discussed.