Temporal and Spatial Control of the Cell Cycle in Caulobacter Crescentus

描述(由申请人提供)：细菌以其快速的复制速度而臭名昭著，这在一定程度上是因为强大的细胞周期机制，确保几乎每个分裂产生的后代都有完整的遗传物质。在这个项目中，我们试图了解DNA分割和细胞周期控制所涉及的机制，长期目标是产生新的策略来控制细菌生长，因为细菌仍然严重威胁人类健康。我们选择的细菌模型是遗传易驯化的新月弯杆菌，这是一个成熟的细胞周期模型，很容易获得同步的细胞周期种群，其细胞周期与形态转变方便地联系在一起。在Crescentus中，染色体分离是由Pars DNA序列在复制起点附近的运动启动的。这种活跃的运动依赖于广泛保守的蛋白质PARA和PARB。我们的第一个目标是以我们之前的发现为基础，剖析ParABS依赖的分离机制背后的机制。为此，我们将使用一组体外生化分析来检测野生型和突变型副蛋白在不同浓度的DNA、PARB和核苷酸存在下的二聚化和ATPase活性。此外，我们将使用超分辨率显微镜技术来成像细胞内DNA分离的过程。由于细胞极化因子TipN和POPZ影响ParABS系统，我们的第二个目的是阐明这两个蛋白的功能。我们最近的发现表明，TipN通过隔离对位单体来影响DNA分离的方向性和速度。我们将使用不同的副突变体和已建立的生化、细胞学和遗传分析的组合来测试这一模型。已知POPZ在细胞极点组装成高阶结构，在那里它拴住PARB/PARS分割复合体，并招募细胞周期信号蛋白。使用突变方法，我们将解决POPZ如何实现这些活动，以及一个活动(例如，组装成高阶结构)是否需要另一个活动(例如，极性定位和/或与PARB/PAR的相互作用)。我们的第三个目标是解决调节细胞大小的内在细胞周期机制 在C.crescentus。这与前两个目标直接相关，因为我们实验室以前的工作表明，PARB/PARS复合体的分离是由细胞大小而不是时间调节的。由于DNA分离、生长和分裂之间的耦合，TipN、POPZ或ParABS系统的缺陷会导致细胞大小异常。我们建议使用我们开发的强大的图像分析软件MicrobeTracker对不同细胞大小分布的各种菌株进行全面的单细胞研究。这项研究将考察每个细胞周期事件在细胞大小补偿中的贡献，并为细胞周期控制提供新的视角。

DESCRIPTION (provided by applicant): Bacteria are infamous for their fast replication rates, which are in part possible because of robust cell cycle mechanisms that ensure that virtually every division produces progeny with a full complement of the genetic material. In this project, we seek to understand the mechanisms involved in DNA partitioning and cell cycle control with the long-term goal of generating new strategies to control bacterial growth as bacteria remain a serious threat to human health. Our bacterial model of choice is the genetically tractable bacterium Caulobacter crescentus, a well-established cell cycle model for which synchronized cell cycle populations are easily obtained and whose cell cycle is conveniently associated with morphological transitions. In C. crescentus, chromosome segregation is initiated by the motion of the parS DNA sequence near the origin of replication. This active motion is dependent on the broadly conserved proteins ParA and ParB. Our first aim is to build on our previous findings to dissect the mechanism underlying the ParABS-dependent segregation mechanism. For this, we will use a battery of in vitro biochemical assays to examine the dimerization and ATPase activity of wild-type and mutant ParA proteins in the presence of varying concentrations of DNA, ParB and nucleotides. In addition, we will use super-resolution microscopy techniques to image the process of DNA segregation inside cells. Since the cell polarization factors TipN and PopZ affect the ParABS system, our second aim is to elucidate the function of these two proteins. Our recent findings suggest that TipN affects the directionality and speed of DNA segregation by sequestering ParA monomers. We will test this model using different ParA mutants and a combination of established biochemical, cytological and genetic assays. PopZ is known to assemble into a high-order structure at the cell poles where it tethers the ParB/parS partition complex and recruits cell cycle signaling proteins. Using a mutagenesis approach, we will address how PopZ achieves these activities and whether one activity (e.g., assembly into a high-order structure) is required for another (e.g., polar localization and/or interaction with ParB/parS). Our third aim is to address the intrinsic cell cycle mechanisms that regulate cell size in C. crescentus. This directly relates to the first two aims as previous work from our lab shows that the segregation of the ParB/parS complex is regulated by cell size and not time. Because of the coupling between DNA segregation, growth and division, defects in TipN, PopZ or the ParABS system result in cell size aberrations. We propose to perform a comprehensive single-cell study of various strains with different cell size distributions using the powerful image analysis software MicrobeTracker that we developed. This study will examine the contribution of each cell cycle event in cell size compensation, and provide new insight into cell cycle control.