Collaborative Research: Identifying and modeling the advantages of regulating protein abundance in Caulobacter crescentus

合作研究：识别和模拟调节新月柄杆菌蛋白质丰度的优势

• 批准号: 1615287
• 负责人: Kathleen Ryan
• 金额: $ 71.06万
• 依托单位: University of California-Berkeley
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-15 至 2021-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1615287&HistoricalAwards=false
• 关键词: Collaborative; Research; Identifying; modeling; advantages

The cell cycle is a series of cellular events leading to DNA duplication and cell division, and ultimately to the production of two daughter cells. This project will address broadly relevant questions about cell cycle regulation. Namely, what is the function of the regulated degradation of crucial cell cycle proteins and how does this regulatory mechanism integrate with other levels of regulation to control the cell cycle? By addressing these questions, this work has the potential to have a significant impact on our basic understanding of how the cell cycle is regulated. This project will expose students to interdisciplinary research and allow undergraduate students in summer laboratory courses to be active participants in research. The timed synthesis and degradation of specific proteins is a ubiquitous mechanism of cell regulation. However, some proteins can be regulated by covalent modification or binding to an allosteric effector. If a protein's activity can be controlled without the energetic cost of degradation and resynthesis, what circumstances would favor regulation by proteolysis? To address this question, the investigators are studying CtrA, a transcriptional regulator that promotes cell division in Caulobacter, yet also blocks chromosome replication. To reconcile these opposing functions, CtrA activity is temporarily eliminated from the cell just prior to chromosome replication by both dephosphorylation and proteolysis. Caulobacter strains expressing a non-degradable version of CtrA can still initiate chromosome replication, indicating that CtrA activity can be significantly reduced by dephosphorylation alone. However, cells with non-degradable CtrA have subtle defects that may compromise their competitive fitness. During each division cycle, ~9000 CtrA molecules are degraded and resynthesized. To understand the selective advantages conferred by this apparently wasteful strategy, experimental and modeling approaches will be used to predict and test the consequences of regulating CtrA only by phosphorylation. In particular, this study will investigate whether the periodic degradation and resynthesis of a neutral passenger protein confers a competitive disadvantage in Caulobacter, as in other bacteria. Caulobacter strains expressing steady levels of a non-degradable CtrA protein will be used to identify phenotypes that differ from cells with oscillating CtrA levels. Competitive fitness assays and quantitative microscopy approaches will test explicitly the predictions of existing mathematical models, e.g., that cells with non-degradable CtrA experience a delay in chromosome replication and a longer cell division cycle. Existing deterministic and stochastic models of the Caulobacter cell cycle will be revised, taking into account recent published findings. Stochastic spatiotemporal modeling will benefit from single-cell data on times of division, chromosome replication, and protein localization.

细胞周期是一系列导致DNA复制和细胞分裂的细胞事件，最终导致两个子细胞的产生。这个项目将解决与细胞周期调控相关的广泛问题。也就是说，关键细胞周期蛋白的受控降解的功能是什么，以及这种调控机制是如何与其他水平的调控相结合来控制细胞周期的？通过解决这些问题，这项工作有可能对我们对细胞周期如何调控的基本理解产生重大影响。这个项目将使学生接触到跨学科的研究，并允许暑期实验室课程的本科生积极参与研究。特定蛋白质的定时合成和降解是一种普遍存在的细胞调节机制。然而，一些蛋白质可以通过共价修饰或与变构效应器结合来调节。如果一种蛋白质的活性可以在没有降解和重新合成的能量成本的情况下被控制，那么在什么情况下有利于通过蛋白质分解来调节？为了解决这个问题，研究人员正在研究CtrA，这是一种转录调节因子，可以促进Caulbacter的细胞分裂，但也会阻止染色体复制。为了协调这些相反的功能，CtrA活性在染色体复制之前通过去磷酸化和蛋白分解暂时从细胞中消除。表达非降解型CtrA的Caulbacter菌株仍然可以启动染色体复制，这表明CtrA的活性可以通过单独去磷酸化而显著降低。然而，带有不可降解的CtrA的细胞有微妙的缺陷，可能会影响它们的竞争适应性。在每个分裂周期中，约有9000个CtrA分子被降解和重新合成。为了理解这一明显浪费的策略所带来的选择优势，将使用实验和建模方法来预测和测试仅通过磷酸化来调节CtrA的后果。特别是，这项研究将调查中性乘客蛋白的周期性降解和重新合成是否会像在其他细菌中一样，在硫杆菌中造成竞争劣势。表达稳定水平的不可降解的CtrA蛋白的Caulbacter菌株将被用于鉴定不同于CtrA水平振荡的细胞的表型。竞争性适合度分析和定量显微镜方法将明确测试现有数学模型的预测，例如，携带不可降解CtrA的细胞经历染色体复制延迟和较长的细胞分裂周期。考虑到最近发表的研究结果，现有的硫杆菌细胞周期的确定性和随机性模型将被修订。随机时空模型将受益于关于分裂时间、染色体复制和蛋白质定位的单细胞数据。