Assembly, Dynamics and Regulation of Chloroplast FtsZ

叶绿体 FtsZ 的组装、动力学和调控

• 批准号: 1121943
• 负责人: Katherine Osteryoung
• 金额: $ 100.94万
• 依托单位: Michigan State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-15 至 2017-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1121943&HistoricalAwards=false
• 关键词: Assembly; Dynamics; Regulation; Chloroplast; FtsZ

Intellectual MeritThe division of chloroplasts is a critical process in photosynthetic eukaryotes. These organelles arose by endosymbiosis from a free-living cyanobacterium and inherited several of their division components from the cell division machinery in their prokaryotic ancestor. Foremost among these is FtsZ, a member of the FtsZ/tubulin superfamily of cytoskeletal proteins. FtsZ self-assembles into a central "Z ring" to initiate division of the cell or organelle and generate force for membrane constriction. A crucial difference between bacterial cell and chloroplast division systems is that the bacterial Z ring is composed of a single form of FtsZ that assembles as a homopolymer whereas the chloroplast Z ring is composed of two distinct forms of FtsZ called FtsZ1 and FtsZ2 that coassemble as a heteropolymer and function together in division of the organelle. The funded research will combine a series of complementary approaches to probe the mechanisms, dynamic properties and regulation of FtsZ1 and FtsZ2 assembly with the overall goal of advancing understanding of the functional and evolutionary significance of the involvement of two FtsZ types in chloroplast division. The experimental objectives are as follows: 1) Analyze the kinetics of FtsZ1 and FtsZ2 assembly separately and together to establish how assembly is nucleated and whether the assembly subunit is an FtsZ1/FtsZ2 heterodimer. These and other analyses will also be extended to related chloroplast FtsZ pairs from one red algal and one green algal species to expand the evolutionary scope of these studies. 2) Analyze force generation at different FtsZ1:FtsZ2 ratios using a novel liposome deformation assay recently developed for bacterial FtsZ, and use a similar system to reconstitute chloroplast Z rings inside liposomes. These experiments will reveal how heteropolymer formation affects chloroplast Z-ring assembly and constrictive force. 3) Analyze FtsZ1 and FtsZ2 assembly behavior and dynamics in a heterologous fission yeast system using fluorescence recovery after photobleaching as a powerful complement to in vitro and in vivo studies. 4) Use quantitative fluorescence microscopy with tagged, functional fusion proteins in Arabidopsis to investigate Z-ring composition and dynamics in vivo, including whether the FtsZ1:FtsZ2 ratio changes dynamically during Z-ring constriction or is regulated in concert with developmental changes in chloroplast division activity. 5) Initiate functional analysis of plant-specific regulators of chloroplast Z-ring assembly towards understanding how FtsZ self-assembly is controlled in chloroplasts.Broader impactsThe research brings together the complementary expertise of the project leaders and their lab members. One group, at Michigan State University, studies FtsZ from chloroplasts using biochemical, genetic, and cell biological approaches in the model plant Arabidopsis thaliana. The other group, at Duke University, studies FtsZ from E. coli and other bacteria using in vitro biochemical and biophysical approaches. Interaction among project personnel by email, video calling, and exchange visits will provide an interdisciplinary perspective that has the potential to catalyze new ideas and research directions. The new studies on algal FtsZs will lay the groundwork for understanding how chloroplast FtsZs have evolved in all photosynthetic eukaryotes. Since mitochondria, like chloroplasts, originated from a bacterial endosymbiont and some organisms retain FtsZ for mitochondrial division, the research will be relevant to understanding FtsZ evolution in mitochondria as well as chloroplasts. In addition, because the microtubule subunits alpha- and beta-tubulin evolved from FtsZ, the research on chloroplast FtsZ heteropolymers may be relevant to understanding evolution of the microtubule cytoskeleton. Finally, Arabidopsis ftsZ and other chloroplast division mutants will be exploited to develop a teaching module for undergraduate ecology students to explore natural selection on chloroplast division and morphology. This will encourage critical thinking on the connection between molecular events, cellular structure and population processes. The research will also provide training for undergraduates in biological research.

叶绿体的分裂是真核生物进行光合作用的关键过程。这些细胞器是由自由生活的蓝藻的内共生作用产生的，并从其原核祖先的细胞分裂机制中继承了几种分裂成分。其中最重要的是FtsZ，它是细胞骨架蛋白FtsZ/微管蛋白超家族的成员。FtsZ自组装成中心“Z环”以启动细胞或细胞器的分裂并产生膜收缩的力。细菌细胞和叶绿体分裂系统之间的一个关键区别是，细菌Z环由单一形式的FtsZ组成，其组装为均聚物，而叶绿体Z环由两种不同形式的FtsZ组成，称为FtsZ 1和FtsZ 2，它们共同组装为杂聚物，并在细胞器的分裂中共同发挥作用。资助的研究将结合联合收割机一系列互补的方法来探索FtsZ 1和FtsZ 2组装的机制，动态特性和调节，其总体目标是促进对两种FtsZ类型参与叶绿体分裂的功能和进化意义的理解。实验目的如下：1）分别和一起分析FtsZ 1和FtsZ 2组装的动力学，以确定组装如何成核以及组装亚基是否为FtsZ 1/FtsZ 2异二聚体。这些和其他分析也将扩展到相关的叶绿体FtsZ对从一个红藻和一个绿色藻类物种，以扩大这些研究的进化范围。2)使用最近开发的用于细菌FtsZ的新型脂质体变形测定法分析不同FtsZ 1：FtsZ 2比率下的力产生，并使用类似的系统在脂质体内重构叶绿体Z环。这些实验将揭示杂聚物的形成如何影响叶绿体Z环组装和收缩力。3)分析FtsZ 1和FtsZ 2组装行为和动力学在异源裂变酵母系统中使用荧光恢复后光漂白作为一个强有力的补充，在体外和体内研究。4)使用定量荧光显微镜标记，功能融合蛋白在拟南芥中调查Z-环的组成和动态在体内，包括是否FtsZ 1：FtsZ 2的比例动态变化在Z-环收缩或调节与叶绿体分裂活动的发展变化。5)启动叶绿体Z环组装的植物特异性调节因子的功能分析，以了解FtsZ自组装在叶绿体中是如何控制的。更广泛的影响这项研究汇集了项目负责人及其实验室成员的互补专业知识。 密歇根州立大学的一个研究小组利用生物化学、遗传学和细胞生物学方法，在模式植物拟南芥中研究了叶绿体中的FtsZ。另一组来自杜克大学，研究E.大肠杆菌和其他细菌的体外生物化学和生物物理方法。项目人员之间通过电子邮件、视频通话和互访进行的互动将提供一个跨学科的视角，有可能促进新的想法和研究方向。对藻类FtsZs的新研究将为理解叶绿体FtsZs如何在所有光合真核生物中进化奠定基础。由于线粒体和叶绿体一样，起源于细菌内共生体，并且一些生物体保留FtsZ用于线粒体分裂，因此该研究将与理解线粒体和叶绿体中的FtsZ进化有关。此外，由于微管亚基α-和β-微管蛋白从FtsZ进化而来，叶绿体FtsZ杂聚体的研究可能与理解微管细胞骨架的进化有关。最后，利用拟南芥ftsZ和其他叶绿体分裂突变体开发一个教学模块，为本科生态学学生探索自然选择对叶绿体分裂和形态的影响。这将鼓励对分子事件，细胞结构和人口过程之间的联系进行批判性思考。这项研究还将为生物研究的本科生提供培训。