Quantitative studies of cell size control and homeostasis in bacteria

 描述(申请人提供)：细胞如何决定其大小未知。最近描述的细胞大小稳态的“加法器”模型改变了以前教科书上的细胞大小控制模型(即“计时器”和“大小”模型，分别假设细胞主动监测细胞周期之间的时间或测量细胞的绝对大小)。加法器模型定量地跨越了10亿年的进化分歧，将革兰氏阴性大肠杆菌和革兰氏阳性枯草杆菌都包括在内。因此，任何驱动细胞分裂间质量增加的机制都必须对这些物种之间的遗传、分子和生理差异具有很强的抵抗力。这一机制的两个候选者是染色体和蛋白质组(细胞中所有蛋白质的集合)，了解两者在生长过程中是如何耦合的是很重要的。基因组在细胞材料中是独一无二的，因为在分裂时必须赋予每个后代细胞一个拷贝，基本上是一个单一分子。蛋白质组的组成必须使细胞的生长在给定的生长条件和细胞周期的控制下得到优化。该项目采用了多学科方法，从用于基因组工程的现代微生物基因技术到高通量测量蛋白质组的组成，并通过定量建模将它们联系起来。更具体地说，现代基因重组工程技术将被用来逐步增加大肠杆菌染色体的大小，使外来的非编码DNA使染色体的天然大小翻一番。利用以前用来发现加法器原理的微流体和成像技术，将在单个-细胞水平上测量分裂之间增加的质量，以建立基因组大小和细胞大小之间的直接关联和因果关系。将研究扩大基因组菌株的蛋白质组组成，以揭示一类用于细胞繁殖的蛋白质如何对各种生长抑制做出不同的反应。这项拟议的研究将进一步阐明细胞大小、细胞周期和增殖的潜在机制，这是对人类生命至关重要和临床上最重要的过程之一。

 DESCRIPTION (provided by applicant): How cells determine their size is unknown. A recently described "adder" model for cell size homeostasis transforms prior textbook models for cell size control (i.e. the "timer" and "sizer" models, which assumes cells actively monitor time between cell cycles or measure the absolute size of the cell, respectively). The adder model quantitatively spans over 1 billion years of evolutionary divergence to include both Gram‑negative Escherichia coli and Gram‑positive Bacillus subtilis. Therefore, any mechanism driving the quantity of mass added between cell divisions must be robust to the genetic, molecular, and physiologic difference between these species. The two candidates underlying the mechanism are the chromosome and the proteome (the set of all proteins in the cell), and it is important to understand how the two are coupled during growth. The genome is unique among cellular materials in that a single copy, essentially a single molecule, must be endowed to each descendent cell at division. The proteome must be composed so that the growth of the cell is optimized for a given growth condition and control of the cell cycle. The project employs multi‑disciplinary approach ranging from modern microbiological genetic techniques for genome engineering to high‑throughput measurements of the composition of the proteome, and connect them via quantitative modeling. More specifically, modern genetic recombineering techniques will be used to progressively increase the size of the E. coli chromosome with foreign, non‑coding DNA to double the native size of the chromosome. Using microfluidic and imaging techniques previously used to discover the adder principle, the mass added between divisions will be measured at the single--‐‑cell level to establish a direct correlative and causative lik between genome size and cell size. The proteome composition of the enlarged‑genome strains will be studied to unravel how the expression of a class of proteins for cellular reproduction differentially responds to various growth inhibitions. The proposed research will provide increased clarity of the mechanisms underlying cell size, the cell cycle and proliferation, one of the most basic processes critically and clinically important to human life.