Here we use singe-molecule optical proteomics and computational analysis of live cell bacterial images, using millisecond super-resolved tracking and quantification of fluorescently labelled protein SpoIIE in single live Bacillus subtilis bacteria to understand its crucial role in cell development. Asymmetric cell division during sporulation in Bacillus subtilis presents a model system for studying cell development. SpoIIE is a key integral membrane protein phosphatase that couples morphological development to differential gene expression. However, the basic mechanisms behind its operation remain unclear due to limitations of traditional tools and technologies. We instead used advanced single-molecule imaging of fluorescently tagged SpoIIE in real time on living cells to reveal vital changes to the patterns of expression, localization, mobility and stoichiometry as cells undergo asymmetric cell division then engulfment of the smaller forespore by the larger mother cell. We find, unexpectedly, that SpoIIE forms tetramers capable of cell- and stage-dependent clustering, its copy number rising to ~ 700 molecules as sporulation progresses. We observed that slow moving SpoIIE clusters initially located at septa are released as mobile clusters at the forespore pole as phosphatase activity is manifested and compartment-specific RNA polymerase sigma factor, σ^F, becomes active. Our findings reveal that information captured in its quaternary organization enables one protein to perform multiple functions, extending an important paradigm for regulatory proteins in cells. Our findings more generally demonstrate the utility of rapid live cell single-molecule optical proteomics for enabling mechanistic insight into the complex processes of cell development during the cell cycle.

在这里，我们使用单分子光学蛋白质组学和活细胞细菌图像的计算分析，使用毫秒超分辨跟踪和量化单个活枯草芽孢杆菌细菌中的荧光标记蛋白SpoIIE，以了解其在细胞发育中的关键作用。枯草芽孢杆菌孢子形成过程中的不对称细胞分裂提出了用于研究细胞发育的模型系统。SpoIIE是关键的整合膜蛋白磷酸酶，可将形态学发育与差异基因表达相结合。但是，由于传统工具和技术的局限性，其运行背后的基本机制仍不清楚。我们取而代之的是在活细胞上实时使用先进的单分子荧光标记SpoIIE成像技术，以揭示表达，定位，迁移率和化学计量模式的重要变化，因为细胞经历了不对称的细胞分裂，然后被较大的母细胞吞噬了较小的前孢子。我们出乎意料地发现，SpoIIE形成了能够与细胞和阶段相关的簇的四聚体，随着孢子形成的进行，它的拷贝数增加到约700个分子。^F变为活动状态。我们的发现表明，在其四级组织中捕获的信息使一种蛋白质能够执行多种功能，从而扩展了细胞中调控蛋白质的重要​​范式。我们的发现更普遍地证明了快速活细胞单分子光学蛋白质组学的实用性，使人们能够深入了解细胞周期中细胞发育的复杂过程。