During development of diverse organisms, many cells become polarized as part of the differentiation process. One example of this event occurs in the one-cell C. elegans embryo, a process that establishes the anterior-posterior (AP) body axis. In many cell types, this polarization event is cued by the centrosome, the microtubule organizing center of the cell. Despite much work implicating the centrosome, little is known about how it is positioned in the cell to ensure polarity establishment. PAM-1 is a puromycin-sensitive aminopeptidase required for centrosome positioning during polarity establishment in C. elegans. Puromycin-sensitive aminopeptidases (PSA) are widely conserved and have important developmental roles in meiosis, cell cycle progression, and reproduction in organisms from plants to mice. Additionally, PSAs have been suggested to play a protective role in the development of neurodegenerative diseases such as Alzheimer's and Huntington's. The proposed work seeks to isolate, characterize and identify suppressors of pam-1 in C. elegans. Genetic analysis will be used to characterize the nature of each suppressor. Time-lapse microscopy will be used to track centrosome movements in each suppressed strain. Single-nucleotide polymorphism mapping and Whole genome sequencing will be employed to identify each suppressor mutation. It is hypothesized that cloning and characterization of these suppressor mutations will reveal novel proteins that work in conjunction with PAM-1 and as targets of the aminopeptidase during polarity establishment. Given the implication of PSAs in neurodegeneration, identification of targets in C. elegans may also advance the understanding of disease mechanisms. Additionally, new players in centrosome positioning will be identified which may be applicable to other systems. The proposed experiments will be largely carried out by undergraduates who will be mentored to provide them the guidance and expertise needed for success in science careers and graduate work.
) Puromycin-sensitive aminopeptidases have been implicated in the degradation of neurotoxic peptides in diseases such as Alzheimer's and Huntington's. Additionally, they are involved in cell polarization, a process necessary for normal cell function. This work will lead to the discovery of protein targets and regulators of enzyme activity that will not only provide a better understanding of the role of these proteins during development, but may also impact our understanding of neurodegenerative disease.