Assembly, Dynamics and Regulation of Chloroplast FtsZ

智力价值叶绿体的分裂是光合作用真核生物的关键过程。这些细胞器是由自由生活的蓝藻通过内共生产生的，并从它们的原核祖先的细胞分裂机制中继承了它们的几个分裂成分。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自组装成一个中心的“Z环”，启动细胞或细胞器的分裂，并为膜收缩产生力量。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，它以均聚物的形式组装，而叶绿体Z环由两种不同形式的FtsZ组成，称为FtsZ1和FtsZ2，它们以杂聚体的形式共同组装，共同作用于细胞器的分裂。这项资助的研究将结合一系列互补的方法来探索FtsZ1和FtsZ2组装的机制、动态特性和调控，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。实验目标如下：1)分析FtsZ1和FtsZ2组装的动力学，以确定FtsZ1和FtsZ2组装是如何成核的，以及组装亚单位是否为FtsZ1/FtsZ2杂二聚体。这些分析和其他分析还将扩展到来自一个红藻和一个绿藻物种的相关叶绿体FtsZ对，以扩大这些研究的进化范围。2)使用最近针对细菌FtsZ开发的一种新的脂质体变形分析方法来分析不同FtsZ1：FtsZ2比率下的力产生，并使用类似的系统来重建脂质体内的叶绿体Z环。这些实验将揭示杂聚体的形成如何影响叶绿体Z环的组装和紧缩力。3)利用光漂白后的荧光恢复来分析FtsZ1和FtsZ2在异源裂解酵母系统中的组装行为和动力学，作为体外和体内研究的有力补充。4)利用荧光显微镜和标记的功能融合蛋白在拟南芥体内研究Z环的组成和动态，包括FtsZ1/FtsZ2的比率在Z环收缩过程中是动态变化的，还是与叶绿体分裂活动的发育变化相一致地调节的。5)启动对叶绿体Z环组装的植物特有调控因子的功能分析，以了解FtsZ自组装是如何在叶绿体中受到控制的。密歇根州立大学的一个研究小组利用生化、遗传和细胞生物学方法，在模式植物拟南芥中研究了叶绿体中的FtsZ。杜克大学的另一个小组使用体外生化和生物物理方法研究来自大肠杆菌和其他细菌的FtsZ。项目人员之间通过电子邮件、视频通话和互访的互动将提供一个跨学科的视角，有可能催化新的想法和研究方向。对藻类FtsZ的新研究将为理解叶绿体FtsZ如何在所有光合作用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，一些生物保留FtsZ进行线粒体分裂，因此这项研究将有助于理解线粒体和叶绿体中FtsZ的进化。此外，由于微管α-和β-微管蛋白亚基是由FtsZ进化而来的，因此对叶绿体FtsZ异聚体的研究可能有助于理解微管细胞骨架的进化。最后，将利用拟南芥FtsZ等叶绿体分裂突变体，为本科生态学专业学生开发一个教学模块，探索关于叶绿体分裂和形态的自然选择。这将鼓励人们对分子事件、细胞结构和种群过程之间的联系进行批判性思考。这项研究还将为本科生提供生物学研究方面的培训。

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.