Adaptation of RanGAP localization and function for plant-specific development

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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.