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

细胞周期是一系列细胞事件，导致 DNA 复制和细胞分裂，并最终产生两个子细胞。该项目将解决有关细胞周期调控的广泛相关问题。即，关键细胞周期蛋白的受调节降解的功能是什么？这种调节机制如何与其他水平的调节相结合以控制细胞周期？ 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。 该项目将使学生接触跨学科研究，并让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细胞调节的普遍机制。然而，一些蛋白质可以通过共价修饰或与变构效应物结合来调节。如果可以控制蛋白质的活性，而无需降解和再合成的能量消耗，那么什么情况下有利于通过蛋白水解进行调节？为了解决这个问题，研究人员正在研究 CtrA，它是一种转录调节因子，可促进柄杆菌细胞分裂，但也能阻止染色体复制。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水解暂时从细胞中消除。表达不可降解版本的 CtrA 的茎杆菌菌株仍然可以启动染色体复制，表明单独去磷酸化可以显着降低 CtrA 活性。然而，具有不可降解 CtrA 的细胞具有微妙的缺陷，可能会损害其竞争适应性。在每个分裂周期中，约 9000 个 CtrA 分子被降解并重新合成。为了了解这种明显浪费的策略所带来的选择性优势，将使用实验和建模方法来预测和测试仅通过磷酸化调节 CtrA 的后果。特别是，这项研究将调查中性过客蛋白的周期性降解和重新合成是否会像其他细菌一样给柄杆菌带来竞争劣势。表达稳定水平的不可降解 CtrA 蛋白的茎杆菌菌株将用于识别与具有振荡 CtrA 水平的细胞不同的表型。竞争性适应性测定和定量显微镜方法将明确测试现有数学模型的预测，例如，具有不可降解 CtrA 的细胞会经历染色体复制延迟和更长的细胞分裂周期。考虑到最近发表的研究结果，将对柄杆菌细胞周期的现有确定性和随机模型进行修订。随机时空模型将受益于有关分裂时间、染色体复制和蛋白质定位的单细胞数据。

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.