The proposed studies seek to further understanding of phagocytosis, the process by which cells engulf and process particulates. Past studies indicate that phagocytosis is a multi-step process involving scores of proteins. Nonetheless, the identity and/or function of many of these proteins remain unknown or poorly understood. The proposed studies will utilize the ciliated protozoan Tetrahymena thermophila as a model for dissecting phagocytosis. T. thermophila has long been used as a model organism for the study of many eukaryotic processes, and a strong array of genetic and molecular genetic tools exist for the organism. In nature, Tetrahymena efficiently engulfs other microorganisms to meet its nutritional needs, but the process is conditionally non-essential, as growth without phagocytosis is possible on nutrient medium. The specific aims for the project are to: (1) Isolate and characterize a panel of phagocytosis mutants. An efficient antisense ribosome mutagenesis procedure will be used in combination with two screening/selection procedures to identify genes that affect a variety of steps in the process of phagocytosis. (2) Characterize mutants in phagocytosis. This will include identifying the affected gene in mutants, and generating gene knockout strains to determine the phenotype of null mutations. In addition, mutants will be analyzed using a number of cytological procedures to gain insight into which stage of phagocytosis is affected, to quantitate the level of the defect in phagocytosis, and to determine the subcellular location of identified gene products. (3) Initiate studies aimed at identifying and characterizing the major protein components of the Tetrahymena phagosome proteome. Two types of studies are planned. First, the role of phagosome proteins already identified in the mouse will be investigated by isolating gene homologs in Tetrahymena and then constructing and analyzing gene knockouts. Second, methods of phagosome purification have been adapted to Tetrahymena, and a preliminary analysis of the Tetrahymena phagosome proteome by mass spectrometry will be carried out. This will ultimately lead to the identification of the conserved set of phagosome proteins, and set the stage for functional analysis of many phagosome components. The project will serve as a vehicle for the scientific training of graduate students, as well as providing summer research experiences for students at earlier stages of their careers.

Agency
National Science Foundation (NSF)
Institute
Division of Molecular and Cellular Biosciences (MCB)
Application #
0343813
Program Officer
Richard Rodewald
Project Start
Project End
Budget Start
2004-05-15
Budget End
2009-04-30
Support Year
Fiscal Year
2003
Total Cost
$461,859
Indirect Cost
Name
University of Connecticut Health Center
Department
Type
DUNS #
City
Farmington
State
CT
Country
United States
Zip Code
06030