Assembly, Dynamics and Regulation of Chloroplast FtsZ

在光合作用的真核生物中，叶绿体的分裂是一个关键的过程。这些细胞器由自由生活的蓝藻内共生产生，并从其原核祖先的细胞分裂机制中继承了一些分裂成分。其中最重要的是FtsZ，它是FtsZ/微管蛋白超家族的成员。FtsZ自组装成一个中央“Z环”，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，细菌的Z环是由单一形式的FtsZ组成的，而叶绿体的Z环是由两种不同形式的FtsZ组成的，称为FtsZ1和FtsZ2，它们以异聚物的形式聚集在一起，共同作用于细胞器的分裂。该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，总体目标是推进对两种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)分别分析FtsZ1和FtsZ2组装动力学，确定组装是如何成核的，以及组装亚基是否是FtsZ1/FtsZ2异源二聚体。这些分析和其他分析还将扩展到一种红藻和一种绿藻的相关叶绿体FtsZ对，以扩大这些研究的进化范围。2)利用最近为细菌FtsZ开发的新型脂质体变形法分析不同FtsZ1:FtsZ2比例下的力产生，并使用类似的系统重建脂质体内的叶绿体Z环。这些实验将揭示异聚物的形成如何影响叶绿体z环的组装和收缩力。3)利用光漂白后的荧光恢复作为体外和体内研究的有力补充，分析FtsZ1和FtsZ2在异源裂变酵母系统中的组装行为和动力学。4)利用带标记的功能性融合蛋白的定量荧光显微镜研究拟南芥体内z环的组成和动态，包括FtsZ1:FtsZ2比值是否在z环收缩过程中动态变化，还是与叶绿体分裂活性的发育变化相一致。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，以了解叶绿体中FtsZ自组装是如何被控制的。更广泛的影响该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模式植物拟南芥的生化、遗传和细胞生物学方法，从叶绿体中研究FtsZ。杜克大学（Duke University）的另一个研究小组利用体外生化和生物物理方法，从大肠杆菌和其他细菌中研究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。藻类ftsz的新研究将为理解所有光合作用真核生物叶绿体ftsz的进化过程奠定基础。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，因此该研究将有助于了解FtsZ在线粒体和叶绿体中的进化。此外，由于微管亚基α -微管蛋白和β -微管蛋白是由FtsZ进化而来的，因此对叶绿体FtsZ异聚合物的研究可能与了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，开发生态学本科学生的教学模块，探索叶绿体分裂和形态的自然选择。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。该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.