Temporal and Spatial Control of the Cell Cycle in Caulobacter Crescentus

新月柄杆菌细胞周期的时空控制

基本信息

批准号：
9060382
负责人：
Christine Jacobs-Wagner
金额：
$ 29.97万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-05-01 至 2018-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9060382
关键词：
ATP phosphohydrolase Address Affect Anti-Bacterial Agents Bacteria Bacterial Genome Bacterial Infections Bacterial Model Biochemical Biological Assay Biology Caulobacter crescentus Cell Cycle Cell Cycle Progression Cell Cycle Proteins Cell Cycle Regulation Cell Growth Processes Cell Size Cell division Cells Chromosome Segregation Chromosomes Complement Complex Computer software Coupling DNA DNA Sequence Defect Development Dimensions Dimerization Electron Microscopy Ensure Event Financial compensation Fluorescence Microscopy Funding Genetic Genetic Materials Goals Grant Growth Health Higher Order Chromatin Structure Human Image Analysis In Vitro Inherited Knowledge Lead Length Measurement Measures Mediating Microscopy Modeling Molecular Motion Mutagenesis Nature Nucleotides Population Process Progress Reports Proteins Quantitative Microscopy Recruitment Activity Replication Origin Research Resolution Signaling Protein Speed System Techniques Testing Therapeutic Time Work base cell growth daughter cell design image processing insight monomer mutant novel prevent segregation tool development

项目摘要

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.

描述（由申请人提供）：细菌以其快速复制率而臭名昭著，这部分可能是由于强大的细胞周期机制，这些机制可确保几乎每个分裂都会产生后代，并完全补充遗传物质。在该项目中，我们试图了解DNA分配和细胞周期控制所涉及的机制，其长期目标是生成新的策略来控制细菌生长，因为细菌仍然对人类健康构成严重威胁。我们选择的细菌模型是可遗传处理的细菌Caulobacter Crescentus，这是一个公认的细胞周期模型，可轻松获得同步的细胞周期群体，其细胞周期与形态过渡方便相关。在C. crescentus中，染色体分离是由复制起源附近的PARS DNA序列的运动引发的。这种主动运动取决于广泛保守的蛋白质和PARB。我们的第一个目的是建立我们以前的发现，以剖析依赖于parabs的隔离机制的机制。为此，我们将使用一系列体外生化测定来检查在存在不同浓度的DNA，PARB和核苷酸的情况下，野生型和突变体Para蛋白的二聚化和ATPase活性。此外，我们将使用超分辨率显微镜技术来对细胞内的DNA分离过程进行成像。由于细胞极化因子TIPN和POPZ会影响Parabs系统，因此我们的第二个目的是阐明这两种蛋白质的功能。我们最近的发现表明，TIPN通过隔离Para单体影响DNA分离的方向性和速度。我们将使用不同的PARA突变体以及已建立的生化，细胞学和遗传测定的组合测试该模型。已知Popz会在细胞极点组装成高阶结构，在该细胞极中，它可以构成PARB/PARS分区复合物并募集细胞周期信号传导蛋白。使用诱变方法，我们将解决POPZ如何实现这些活动，以及另一个活动（例如，将组装成高阶结构）是否需要另一个活动（例如，极性定位和/或与PARB/PARS的相互作用）。我们的第三个目标是解决调节细胞大小的固有细胞周期机制在C. crescentus中。这直接与前两个目标有关，因为我们实验室的先前工作表明，PARB/PARS复合物的隔离受细胞大小而不是时间调节。由于DNA分离，生长和分裂之间的耦合，TIPN，POPZ或Parabs系统的缺陷导致细胞大小的畸变。我们建议使用我们开发的功能强大的图像分析软件微处机架对各种菌株进行各种菌株的全面单细胞研究。这项研究将研究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见解。