DESCRIPTION (provided by applicant): This work aims to explore the developmental assembly and molecular mechanisms associated with two dynamic organelles within a unicellular model organism: Tetrahymena thermophila. The nuclear exchange junction represents a molecular theater for studying membrane trafficking and cytoskeletal dynamics as they pertain to developmentally programmed remodeling of the cell cortex. Membrane trafficking pathways have been implicated in numerous neuro-degenerative diseases (among others), and are critical to the proper functioning of the immune system during inflammation. The "conjusome" is a non-membrane bound electron-dense organelle resembling germ-plasm or nuage from metazoan embryos. As such, it represents a potentially tractable system for exploring mechanisms of small RNA-mediated remodeling of an organism's genome in service of genome defense. The use of small RNA molecules (the RNAi phenomenon) has become a leading area for therapeutic research. Our long term objectives include: 1) Understanding the dynamics of cytoskeletal and membrane trafficking that remodel the cell cortex during nuclear exchange and mating in Tetrahymena. 2) Understanding the role of the Tetrahymena "conjusome", a germ-plasm-like organelle assembled during a period of genome-reorganization in this ciliate model, and its potential evolutionary relationship to such structures in higher eukaryote embryos, and 3) providing interdisciplinary training for undergraduates conducting research into the develop- mental genetics of Tetrahymena thermophila. Our specific goals include: 1) Gene discovery. We plan to conduct protein purification and mass spectrometry to identify genes involved in the cortical remodeling that accompanies nuclear exchange during mating, and the assembly of a germ-plasm-like organelle during late stages of mating in Tetrahymena. 2) Functional analysis. Investigate these candidate genes through functional genomics by generating loss of function mutations and analyzing their phenotypes. 3) Protein localization. Determine the intracellular localization of candidate gene products identified via proteomics and bioinformatics by GFP-tagging, scanning-laser confocal micros-copy, and immuno-gold electron tomography. 4) Undergraduate research training. Develop an ultrastructure workstation for undergraduate analysis of protein localization through both scanning laser confocal fluorescence microscopy and electron tomography. Participate in creating a Ciliate Molecular Biology "Bootcamp" for undergraduate researchers.
PUBLIC HEALTH RELEVANCE: The Tetrahymena nuclear exchange junction represents a novel system for exploring the involvement of the "recycling endosome pathway", a pathway implicated in numerous medical disorders and in the proper functioning of the immune system. The "conjusome" is a non-membrane-bound electron-dense organelle resembling germ-plasm or nuage from metazoan embryos, and as such, it represents a potentially tractable system for exploring mechanisms of small-RNA-mediated remodeling of an organism's genome in service of genome defense. The use of small RNA molecules (the RNAi phenomenon) has become a leading area for therapeutic research.