Towards a Model for FtsZ Structure and Dynamics in Chloroplast Division

智力优点：叶绿体是植物和藻类中的亚细胞区室，其利用阳光的能量并通过光合作用过程将该能量转化为糖（葡萄糖）形式的化学能。 在进化上，它们源自内共生光合细菌;它们在结构上高度复杂，具有两个包膜和一组称为“类囊体”的内部膜，光合酶位于其中。 当植物和藻类细胞生长和分裂时，每个子细胞获得其叶绿体份额至关重要。 真核细胞中叶绿体的分裂由一个拓扑结构复杂的大分子机器协调，该机器驱动围绕细胞器的两个包膜膜的收缩和分裂。Osteryoung博士实验室的长期目标是阐明植物细胞中协调叶绿体分裂的分子途径和生化机制。在该奖项的支持下进行的研究将集中在与关键细菌细胞分裂蛋白FtsZ密切相关的两种叶绿体分裂蛋白上。FtsZ是一种微管蛋白样GT3，是细菌细胞分裂器的必要成环成分，在原核生物中通常由单个基因编码。Osteryoung博士以前的工作已经确定，植物中两种不同形式的FtsZ，FtsZ 1和FtsZ 2，是叶绿体分裂机制的核心组成部分，并且它们共同定位于一个动态的中叶绿体环，该环在整个细胞器的分裂过程中发挥作用。FtsZ 1和FtsZ 2在体内紧密共定位，在可溶性复合物中共纯化，可以共免疫沉淀，并在酵母双杂交测定中直接相互作用。此外，Osteryoung博士还证明，在模式植物拟南芥中，FtsZ 1和FtsZ 2在整个植物生长和发育过程中保持1：2的恒定摩尔比，并且拟南芥中FtsZ 1或FtsZ 2水平的改变会导致叶绿体分裂的剂量依赖性缺陷。Osteryoung博士现在将在这些发现的基础上研究FtsZ 1和FtsZ 2之间的生化关系。 具体而言，该奖项将支持以下目标：通过体内水平和比例的遗传操作，测试FtsZ 1：FtsZ 2化学计量和总FtsZ丰度对拟南芥叶绿体分裂的重要性;分析重组FtsZ 1和FtsZ 2单独和组合的体外GTclase活性，并使用最近开发的荧光测定法研究其聚合物组装特性;并研究FtsZ 1和FtsZ 2的功能意义和结构特征。 这些研究将奠定基础的发展模型描述的结构组织和动态特性的叶绿体FtsZ环的活动在分裂的细胞器。更广泛的影响：叶绿体分裂与线粒体和细菌细胞分裂的功能和进化相关过程有一些相似之处。因此，这项研究将加深对真核和原核细胞生物学这些基本方面的理解。该项目将提供蛋白质复合物结构和动力学分析的研究生培训，并为研究生，博士后学者和本科生提供额外的培训机会。

Intellectual merit: Chloroplasts are the subcellular compartments in plants and algae that harness the energy of sunlight and convert that energy into chemical energy in the form of sugar (glucose) via the process of photosynthesis. Evolutionarily, they are derived from endosymbiotic photosynthetic bacteria; they are structurally highly complex, with two enveloping membranes and an internal set of membranes termed 'thylakoids' where the photosynthetic enzymes reside. When plant and algal cells grow and divide, it is critically important that each daughter cell receive its share of chloroplasts. The division of chloroplasts in eukaryotic cells is coordinated by a topologically complex macromolecular machine that drives the constriction and fission of the two envelope membranes surrounding the organelle. The long-term goal of Dr. Osteryoung's laboratory is to elucidate the molecular pathway and biochemical mechanisms that orchestrate chloroplast division in plant cells. The studies that will be carried out with support from this award will focus on two chloroplast division proteins closely related to the key bacterial cell division protein FtsZ. FtsZ is a tubulin-like GTPase and essential ring-forming component of the bacterial cell division apparatus, and is usually encoded by a single gene in prokaryotes. Dr. Osteryoung's previous work has established that two distinct forms of FtsZ in plants, FtsZ1 and FtsZ2, are core components of the chloroplast division machinery, and that they co-localize to a dynamic mid-chloroplast ring that functions throughout division of the organelle. FtsZ1 and FtsZ2 are tightly colocalized in vivo, copurify in a soluble complex and can be co-immunoprecipitated, and interact directly in yeast two-hybrid assays. In addition, Dr. Osteryoung has demonstrated that, in the model plant Arabidopsis, FtsZ1 and FtsZ2 are maintained at a constant molar ratio of 1:2 throughout the plant's growth and development and that alterations in FtsZ1 or FtsZ2 levels in Arabidopsis confer dose-dependent defects in chloroplast division. Dr. Osteryoung will now build on these findings to investigate the biochemical relationship between FtsZ1 and FtsZ2. Specifically, this award will support the following aims: test the importance of FtsZ1:FtsZ2 stoichiometry and total FtsZ abundance for chloroplast division in Arabidopsis via genetic manipulation of their in vivo levels and ratios; analyze the in vitro GTPase activities of recombinant FtsZ1 and FtsZ2 separately and in combination, and investigate their polymer-assembly properties using recently developed fluorescence assays; and investigate the functional significance structural features that distinguish FtsZ1 and FtsZ2. These studies will lay the groundwork for the development of models describing the structural organization and dynamic properties underlying the activity of the chloroplast FtsZ ring during division of the organelle. Broader impacts: Chloroplast division shares some similarities with the functionally and evolutionarily related processes of mitochondrial and bacterial cell division. Therefore, this research will deepen understanding of these fundamental aspects of eukaryotic and prokaryotic cell biology. The project will provide graduate student training in structural and kinetic analysis of protein complexes, and provide additional training opportunities for graduate students, postdoctoral scholars, and undergraduates.