Naohiro Kato
Assistant Professor
Ph.D. Hiroshima
University, 1998
molecular genetics and bioimaging
I am interested in visualizing the functions of proteins in live cells. Over 25,000 different proteins are available to build a single functional cell in multicellular systems. When, Where, and for What purpose, these proteins are used in the cells? My laboratory addresses these questions by combining molecular genetic and bioimaging techniques, using Arabidopsis thaliana plant as a model organism. Some of our projects currently conducted are described below.
1. Mapping dynamic protein-interaction networks in plants We developed a split luciferase complementation assay to study protein-protein interactions in plant protoplasts. In this assay, the N- and C-terminal fragments of Renilla reniforms luciferase are translationally fused to bait and prey proteins, respectively. When the proteins interact, split luciferase becomes activated and emits luminescence that can be measured by a microplate luminometer. Split luciferase activity was measured by first transforming protoplasts with a DNA vector in a 96-well plate. DNA vector expressing both bait and prey genes was constructed through two independent in vitro DNA recombinant reactions, Gateway and Cre-loxP. We wish this system will be an alternative assay method for investigators who are interested in studying dynamics of protein-protein interactions in living plant cells.This project is currently supported by National Research Initiative Competitive Grant 2006-35640-16627 from the USDA Cooperative State Research, Education, and Extension Service.
2. A systems approach to measure kinetics of protein-protein Interaction cascades in living organisms The function of many proteins depends on their ability to bind other proteins with a high degree of specificity. Current molecular tools and the instruments used to detect the specific interactions in living organisms allow the analysis between two proteins of interest. Proteins in the organisms, however, can change their interaction partner as often seen in signaling pathways. The goal of this project is to establish a novel method to track multiple protein interactions in cells and living organisms. We have identified paralogous luciferase proteins that can functionally complement one another when the two fragments are closely positioned. Depending on the combinations, the luciferase activity can emit luminescence in distinct colors by catalyzing a same substrate. We are optimizing the amino acid compositions of the proteins and 2) measureing kinetics of a protein-protein interaction cascade involved in transducing a specific signaling pathway in living plant cells. The project should open up a systems approach to measure kinetics of multiple protein interactions in various signaling cascades in living organisms. This project is currently supported by LA R&D Board of Regents Fund, LEQSF (2007-09)-RD-A-11.
3. Endoreplication-dependent genes and nuclear proteins Endoreplication is a phenomenon in which cells of higher eukaryotes replicate the chromosomes without intervening mitosis. The cells, as the result of endoreplication, contain several sets of chromosomes in the single nuclei. The tissue-specific patterns of endoreplication in several higher eukaryotes suggest the essential role in the normal development programs. Although many of mammalian cancer cells also replicate the chromosomes without mitosis, the function is different from endoreplication. We are specifically interested in the function of endoreplication in plants. The goal of this project is to identify genes and proteins specifically accumulated in endoreplicated nuclei using genome-wide screening methods. This project is currently supported by Louisiana EPSCoR, NSF(2007)-LINK-21.
4. Dynamics of nucleus-plastid and nucleus-mitochondria interactions in Arabidopsis thaliana Nuclei, plastids, and mitochondria move dynamically to maintain proper cellular function. The interactions among these organelles can be identified and analyzed by tracking their movements simultaneously. To study the dynamics of these organelle-organelle interactions in plant cells, transgenic Arabidopsis, designated Kaleidocell, was engineered. Kaleidocell plant cells contain nuclei, plastids, mitochondria, and plasma membranes that are genetically tagged with cyan, red, yellow, and green fluorescent proteins, respectively. Optically sectioned images of Kaleidocell root epidermis are obtained at a submicron resolution to reveal in living cells the interaction between nuclei, plastids and mitochondria. A three-dimensional model is generated to understand localization of these organelles in the cells. Furthermore, time-lapse observations of three-dimensional projections of the root epidermal cells are generated to track movements of these organelles.
Publications since 2005
Publications 2001 - 2004
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