The establishment of future body axes (e.g., head to tail or top to bottom) is a crucial early step in animal development. In the soil nematode Caenorhabditis elegans, the anterior-posterior (head to tail) axis is established in the one-cell stage embryo when the centrosome, the microtubule organizing center of the cell, contacts the cell cortex. Although the contact appears to be essential for axis establishment, the timing and mechanism governing this centrosome-cortical association is unknown. The PI has identified the PAM-1 protein (puromycin-sensitive aminopeptidase) as a crucial regulator of these events. This protein is found in diverse animals from C. elegans to humans. In this project, the timing and mechanism of centrosome movements will be elucidated using genetic and molecular approaches with study of PAM-1 as a central component of the work. The roles of the microtubule motor protein dynein and microtubule growth will be examined as well as new proteins identified through genetic screens. This project will reveal the proteins that control centrosome movement and cortical association in C. elegans as well as how these events are coordinated with the cell cycle. The outcomes are likely to have far reaching impacts on the fields of microtubule dynamics, axis establishment and puromycin-sensitive aminopeptidase function. This work will be performed at an undergraduate college; hence, a major goal of this project is to immerse undergraduates in meaningful research projects. All the work will be performed in conjunction with undergraduates and portions of the research will be incorporated into an open-ended laboratory course in Molecular Genetics. Students will present their work at national conferences and will be prepared for entry into research-based graduate programs.
Throughout development of all multi-cellular organisms, cells that are at first similar in potential, accept different fates. This process ensures the proper development of a complex organism with specialized cells to perform all the necessary functions. Many of the cells become polarized as part of this process, resulting in the localization of proteins to different cellular domains. This polarization process is triggered by the centrosome in numerous cells, a structure necessary for cell division. To better understand this process, we studied the role of the centrosome in polarization of the one-cell embryo of the small soil nematode C. elegans. Through this study, we uncovered a role for the PAM-1 aminopeptidase in proper positioning of the centrosome to enable cell polarization. PAM-1 is the first protein known to be critical in regulating centrosome position. In addition, we were able to track the centrosomes position over time to reveal the dynamics of the positioning process and its role in cuing cell polarity. Because little is known about how PAM-1 acts to position the centrosome, we conducted a suppressor screen to identify additional proteins involved in the process. We have been able to characterize six new mutations that suppress the centrosome positioning defect of pam-1 mutants. All of this work was completed at a small liberal arts school and performed by undergraduate researchers. This grant allowed for the training of over 30 undergraduate students in the design and implementation of experiments and the interpretation and dissemination of the results through publications and conference presentations. Their scientific work has prepared them well. Three of these students were awarded the prestigious Goldwater Scholarship, and one a Rhodes Scholarship. Three of these students are pursuing advanced graduate degrees in research, six are attending medical school, and one is enrolled in an MD-PhD student. Thus, this grant has resulted in successful scientific careers for these students. In addition, three high school students took part in research for six weeks as a part of a career study.
