The centriole is a highly conserved intracellular structure composed of microtubule triplets arranged in a 9-fold symmetrical configuration. It is essential for cilia/flagella formation and the organization of centrosomes, which play a key role in a number of biological processes, including chromosome segregation during cell division. To perform proper functions, cells must produce the precise number of centrioles during the cell cycle and development. This project is aimed at development of a novel system of in vitro centriogenesis to facilitate the analysis of centriole formation and its regulation. Studies on centriole biogenesis in somatic cell lysates has been hampered by a low copy number of centrioles, which duplicate only once each relatively longer period of cell cycle time. To overcome this problem, the project uses DT40 chicken lymphocytes which continue to accumulate centriolar components, but fail to assemble centrioles by expressing genetically inactivated Cyclin-dependent kinases. The robust centriole assembly is triggered by supplementing exogenous Plk4 (Polo-like kinase 4) to cells. Using lysates prepared from mutant DT40 cells, the project will establish and optimize the in vitro condition of centriole biogenesis, and further assess the role of Plk4 in the control of centriole copy numbers. The scientific outcomes of the project will have great impact by opening a new avenue for the study of centrioles.
Broader Impact The project provides excellent opportunities for the scientific training of undergraduate students - future educators, scientists, engineers, and technicians. By participating in different parts of the project, students will receive intellectual as well as a wide range of technical trainings, including DNA cloning, protein preparation, cell culture/analysis, and light/electron microscopy. The PI has been involved in undergraduate lab education as an instructor of cell biology laboratory classes as well as a mentor of significant numbers of students performing research projects in the lab. Particular attention will be paid to women and underrepresented minorities for future recruitment. The PI will mentor student training by setting up several basic principles: 1) close mentoring at each critical stage; 2) selection of appropriate projects for individual students: 3) providing long-term opportunities of training. Students will be assigned to present data in formal/information meetings and submit summary statements/manuscripts to improve communication and writing skills. The scientific outcomes of the project will have broad impacts in other areas of cell and developmental biology, as the centriole is involved in many important biological functions. Information on experimental conditions will be accessible through open internet sites, such as mtocDB (microtubule-organizing center databases providing molecular and morphological data of centrioles, and other microtubule-organizing centers), which will facilitate to address important unanswered questions, such as morphological diversity of the centriole evolved among species, by forming variable structures under different assembly conditions.
