Adaptation of RanGAP localization and function for plant-specific development

学术价值这个项目的长期目标是了解植物和动物中信号分子亚细胞定位的分子机制，生物功能和进化历史。 特别是，这里的项目是表征RanGAP在植物细胞中的亚细胞位置和功能。 Ran是一种小的GTdR，控制多个细胞过程，并在脊椎动物细胞的空间和时间组织中发挥核心作用。 这一作用的关键是两种形式的Ran，RanGTP和RanGDP之间的动态梯度，充当蜂窝定位系统。 在动物细胞中，RanGAP在核质运输、纺锤体组织和有丝分裂后核组装中起作用。 在先前的研究中，PI表明，在植物中，RanGAP在基于成膜体的胞质分裂中起作用，这表明植物中RanGAP的空间组织和功能的进化历史与动物中的不同。 本项目将以拟南芥为模型系统，重点研究RanGAP在植物中的具体作用。 有三个具体目标。 从动物系统中的应用程序改编的分子内荧光共振能量转移（FRET）的体内方法将用于测试假设一，即在植物细胞有丝分裂过程中建立了RanGTP：：RanGDP的梯度。 具体目标二将使用双分子荧光互补来确定植物核孔处的最小RanGAP锚。 为了特定目的，将测试三种突变RanGAP衍生物对RanGAP耗尽的植物系的功能互补，以剖析RanGAP的不同区域并确定是否存在具有不同功能的不同蛋白质结构域。 这项工作具有进化的意义，因为它表明，多种方法已经发展到空间组织高度保守的Ran蛋白，无论是在间期细胞和有丝分裂期间。 这表明植物和动物核孔结构之间存在更大的潜在差异，并确定了植物核孔，Ran和胞质分裂之间以前未被认识到的联系。 更广泛的影响博士后，研究生和本科生将在这个项目的培训。基于这一正在进行的研究项目，PI已经开发了一个新的植物细胞生物学课程的高级本科生和研究生，目前唯一的植物细胞生物学课程，这种类型的俄亥俄州州立大学提供。通过先前的NSF资助，PI已经开发了一个可搜索的拟南芥卷曲螺旋蛋白数据库，并将其用于当地功能基因组学夏季课程的计算模块。 学生创建的数据将被用作水稻和衣原体的平行卷曲螺旋数据库的基础。 这些数据目前正在与两个本科院校合作进行改进和验证，将其用作计算生物学课程的教学资源。 这里提出的研究有助于计算机识别的卷曲螺旋蛋白质的功能注释，并在本科院校的活动中分享新的数据。PI参与了一项推广活动，为公众提供了一个关于植物生物技术的10个模块的在线课程。这又导致出版了一份关于科学外联的文件，向其他研究人员介绍学术外联活动的必要性和方法。 这些活动和类似活动正在进行中，预计将在本授标期内继续进行。

Intellectual meritThe long-term goal of this project is to understand the molecular mechanism, biological function, and evolutionary history of subcellular positioning of signaling molecules in plants and animals. In particular the project here is to characterize subcellular position and function of RanGAP in plant cells. Ran is a small GTPase that controls multiple cellular processes and plays a central role in the spatial and temporal organization of vertebrate cells. Key to this role is a dynamic gradient between the two forms of Ran, RanGTP and RanGDP, acting as a cellular positioning system. In animal cells RanGAP functions in nucleocytoplasmic transport, spindle organization, and post-mitotic nuclear assembly. In a previous study the PI showed that in plants RanGAP functions in phragmoplast-based cytokinesis which suggests a separate evolutionary history for the spatial organization and function of RanGAP in plants than that in animals. This project will focus on the specific role of RanGAP in plants using Arabidopsis as the model system. There are three specific aims. In vivo methods for intramolecular fluorescence resonance energy transfer (FRET) adapted from applications in animal systems will be used to test hypothesis one that a gradient of RanGTP::RanGDP is established during mitosis in plant cells. Bimolecular fluorescence complementation will be use in specific aim two to define the minimal RanGAP anchor at the plant nuclear pore. For specific aim three mutant RanGAP derivatives will be tested for functional complementation of RanGAP depleted plant lines to dissect the different regions of RanGAP and determine if there are separate protein domains with separate functions. This work has evolutionary implications in that it suggests that multiple methods have evolved to spatially organize the highly conserved Ran protein, both in interphase cells and during mitosis. This suggests larger underlying differences between plant and animal nuclear pore architecture, and identifies a previously unrecognized connection in plants between the nuclear pore, Ran and cytokinesis. Broader impactPostdocs, graduate-students and undergraduate students will be trained on this project. Based on this ongoing research project, the PI has developed a new Plant Cell Biology course for advanced undergraduate and graduate students, currently the only Plant Cell Biology course of this type offered at Ohio State University. Through prior NSF funding, the PI has developed a searchable database for Arabidopsis coiled-coil proteins and has used it for a computational module in a local Functional Genomics Summer Course. Student-created data will be used as the basis for parallel coiled-coil databases for rice and Chlamydomonas. These data are currently improved and validated in collaboration with two undergraduate institutions to use them as a teaching resource for computational biology courses. The research proposed here aids in the functional annotation of the computationally identified coiled-coil proteins and in sharing new data for the activities at the undergraduate institutions. The PI is involved in an outreach activity, offering a 10-module, online course for the general public on Plant Biotechnology. This in turn resulted in the publication of a paper on outreach in science, to communicate to other researchers the need for and approaches to academic outreach activities. These and comparable activities are ongoing and expected to continue for the duration of the current award period.