Measuring In Vivo Protein Dynamics Throughout the Cell Cycle Using Microfluidics
Measuring In Vivo Protein Dynamics Throughout the Cell Cycle Using Microfluidics
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DOI：
10.1007/978-1-4939-7098-8_18
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发表时间：
2017-01-01
期刊：
影响因子：
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通讯作者：
Dekker, Nynke H.
中科院分区：
文献类型：
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作者：
de Leeuw, Roy;Brazda, Peter;Dekker, Nynke H.
研究细胞内过程的动力学和研究活细胞中单个大分子的相互作用是细胞生物学的主要目标之一。这些大分子在整个细胞周期的特定生理条件下以不同的方式移动、组装、分解和重组。因此，在体内实验方法，使细胞内的单个分子在受控的培养条件下的研究已被证明是强大的工具，以获得洞察这些大分子的分子作用，以及他们的个人行为如何影响细胞生理学。当研究的现象涵盖很长的时间段，或者可能是多个细胞周期时，控制实验条件的重要性就增强了。一个例子是细菌DNA复制过程中蛋白质的检测和定量。与微流体相结合的宽视场显微镜是一种合适的技术。在荧光实验中，微流体提供了明确的细胞定向和固定，流动和介质可重复性，以及细胞的高通量长期实验。在这里，我们提出了一个协议，结合使用宽视场显微镜和微流体的蛋白质的大肠杆菌DNA复制过程的研究。我们讨论了微流控装置的制备和应用，数据采集步骤和图像分析程序，以确定活细菌细胞的整个细胞周期的复制体组分的化学计量和动力学。
Studying the dynamics of intracellular processes and investigating the interaction of individual macromolecules in live cells is one of the main objectives of cell biology. These macromolecules move, assemble, disassemble, and reorganize themselves in distinct manners under specific physiological conditions throughout the cell cycle. Therefore, in vivo experimental methods that enable the study of individual molecules inside cells at controlled culturing conditions have proved to be powerful tools to obtain insights into the molecular roles of these macromolecules and how their individual behavior influence cell physiology. The importance of controlled experimental conditions is enhanced when the investigated phenomenon covers long time periods, or perhaps multiple cell cycles. An example is the detection and quantification of proteins during bacterial DNA replication. Wide-field microscopy combined with microfluidics is a suitable technique for this. During fluorescence experiments, microfluidics offer well-defined cellular orientation and immobilization, flow and medium interchangeability, and high-throughput long-term experimentation of cells. Here we present a protocol for the combined use of wide-field microscopy and microfluidics for the study of proteins of the Escherichia coli DNA replication process. We discuss the preparation and application of a microfluidic device, data acquisition steps, and image analysis procedures to determine the stoichiometry and dynamics of a replisome component throughout the cell cycle of live bacterial cells.