Intellectual merit. This project aims to understand the molecular machinery that controls endosomal-mediated degradation of plasma membrane proteins in plants as well as the cellular and developmental processes that depend on endosomal trafficking pathways. Endosomal compartments play a fundamental role in the sorting, recycling, and turnover of membrane proteins, the down-regulation of receptors, and the trafficking of proteins to vacuoles and lysosomes in all eukaryotic cells. This project focuses on two important plant endosomal proteins, SKD1 and CHMP1, which work in coordination with other endosomal protein complexes to regulate sorting of membrane proteins for degradation. Endosomal trafficking plays a role in various aspects of the plant life, such as development, cell type specification, cell wall remodeling, gravitropic response, signal transduction, and hormone transport. The research proposed here will provide valuable information in this plant cell biology area.
Broader impacts. The project will have important impacts on education and outreach. The Principal Investigator is currently teaching undergraduate courses at the University of Wisconsin. An additional part of the plan is to recruit one or more graduate students, in addition one already in the laboratory, to work on the central topic of this research proposal. Due consideration will be given to recruitment of individuals from under-represented groups. The development of new imaging techniques will also be important for the educational plan, since the PI is committed to develop workshops on cell biology and cell imaging for undergraduate and graduate students across campus as part of the educational activities of the new Imaging Center in the Department of Botany-University of Wisconsin. The Center was initially supported by NSF funds.
Scientific Merits This project aimed to understand how plant cell control de composition of their cell surface by modulating the degradation of plasma membrane proteins. Cells are able to interact with other cells and their environment through molecules located in their surface. At the plasma membrane, there are a number of different types of proteins and protein complexes that mediate cell-to-cell communication and exchange of substances with the cell environment. It is critical for a cell to be able to control the protein composition of its plasma membrane. This is achieved by several mechanisms, most of which depend on endosomal trafficking pathways. Plasma membrane proteins are internalized by endocytosis (formation of vesicles at the plasma membrane), and delivered to endosomes. At endosomes, these proteins are either recycled back to the plasma membrane or sorted for degradation. The recycling of plasma membrane proteins mostly occurs at early and recycling endosomes whereas their degradative sorting is achieved in intermediate/late endosomes also called multivesicular bodies (MVBs) or multivesicular endosomes. We have analyzed the function of different ESCRT (Endosomal Sorting Complex Required for Transport Proteins) proteins in plants that control the degradation of plasma membrane proteins at multivesicular bodies. In plants, ESCRT proteins are essential for development by controlling the turnover of proteins that control the transport of the phytohormone auxin, and the degradation rate of signaling receptors. We have also found that ESCRT function is important for embryo and seed differentiation, leaf and root growth, and sexual reproduction in all plants. We have analyzed biochemical properties of these proteins to establish their interactions and enzymatic activities. We have also isolated mutants that fail to express ESCRT genes to understand how the absence of ESCRT function affects plant development. Nine scientific publications have resulted from this grant. Broader Impacts Education Activities and Training Three postdoctoral fellows, two graduate students, and five undergraduate students have been working in this project: Dr Christoph Spitzer worked on endosomal and non-endosomal functions of ESCRT components in plants. Christoph has given talks for the plant community on campus, a poster in the 2011 International Conference of Arabidopsis Research (ICAR), and in the 2012 Panamerican Workshop of Plant Membrane Biology. He has also been training undergraduate students in the laboratory. Dr Guang Wu worked on the downregulation of signaling receptors in plants. He is now started his own laboratory in Shaanxi, China. He actively participated in laboratory activities and discussions and was involved in the training of a graduate student. Dr Francisca Reyes worked part time in 2010 in the characterization of a plant-specific ESCRT component and its function in plants. She has been presenting her work in seminars on campus and also in the American Society for Biochemistry and Molecular Biology meeting on Biochemistry and Cell Biology of ESCRTs in Health and Disease (2010, Snowbird, Utah). She also gave oral presentations in the 2011 International Conference of Arabidopsis Research (ICAR), 2012 Panamerican Workshop of Plant Membrane Biology, and in the 2012 Midwest Plant Cell Dynamics Meeting. Francisca has trained Rafael Buono. Rafael Buono is a graduate student in the laboratory. He is working in the characterization of several ESCRT proteins in plants. Rafael has been trained in molecular biology, protein purification and analysis, and confocal imaging. He presented a poster in the 2010 ASBMB meeting on Biochemistry and Cell Biology of ESCRTs in Health and Disease and his abstract was selected for an oral presentation in the 2011 Annual Meeting of the American Society of Plant Biologists. Maren Roe was another graduate student in the lab working on an ESCRT component that affects root growth. Maren graduated last summer 2010 with a Masters degree and she has now moved to Pioneer Hi-Bred in Iowa where she holds a research position. Alexandra Musial was an undergraduate researcher who worked together with Rafael Buono. During Fall 2009 she completed with us her research project for Biology 152 (Introductory Biology) and she kept on working on the same project as part of a Directed Study for the following two semesters. Jesse Judkins was an undergraduate researcher who worked under the supervision of Dr Spitzer. During Spring 2011 her completed his research project for Introductory Biology 152 on this topic. Curtis Frank was an undergraduate student in the lab. He helped with general lab maintenance tasks and plant growth. David Charles completed his research project for Introductory Biology 152 in our lab during Fall 2011. He worked with Rafael Buono in the characterization of ESCRT mutants. He continued working with us for the following two semesters. Jean Mutambuze participated as an Summer Research Opportunity Program for undergraduates in 2012. He has been working with Rafael Buono in the characterization ESCRT mutants in plants. His project also involved cloning, transformation of bacterial cells, and statistical analysis of root growth data.
