Cells reproduce by duplicating their genomic content and then dividing the genome into two identical daughter cells. The final step of division is cytokinesis, where the two daughters are physically separated from each other; its success is absolutely essential for normal growth and development, as well as reproduction in all organisms. Eukaryotic organisms from different kingdoms have developed cytokinesis mechanisms that share certain features but differ in others. In advanced green algae and plants, cytokinesis is brought about by a sophisticated apparatus called the phragmoplast, which contains a core framework of structural microtubules. Phragmoplast microtubules serve as tracks that transport vesicles used to assemble the cell plate, the structure that physically separates the two daughter cells. Microtubules undergo rapid remodeling while the developing cell plate expands outward toward the cell cortex. The phragmoplast is considered to be an evolutionary landmark that enabled the emergence of land plants on earth, but little is known about how plant cells assemble this structure. In order to understand plant cytokinesis, this project will dissect mechanisms that regulate microtubule reorganization in the phragmoplast. This work will not only advance knowledge of plant cell division but also shed light on how eukaryotic cells harness protein-based machineries to accomplish sophisticated tasks. Knowledge obtained here will be applicable to all plants, and aid understanding of how marine green algae transitioned into land plants during evolution. Undergraduate and high school students from the UC Davis campus area will join the discovery, and receive hands-on training in modern cell biology. The PI & Co-PI emphasize training undergraduate students whose curiosity and talent in research are shadowed by their relatively low GPAs. The impacts of the proposed project will broaden participation of underrepresented groups, and aim for their advanced training upon graduation or prepare them to be technically competitive when seeking jobs in academia and the biotechnology industry.
The PI's group uses the mustard plant Arabidopsis thaliana as a model system to dissect cytokinesis because of its advanced genetics/genomics and well developed cell biology tools. As preliminary work, the PI has prepared necessary reagents, insightful mutants, and informative fluorescent marker lines to make the system ready for the proposed experiments. Prior accomplishments have resulted in a model hypothesizing that the phragmoplast is assembled in modular form, where a core of interdigitating microtubules are surrounded by non-interdigitating ones. The current project will test this modular model to learn how this apparatus is assembled to execute cytokinesis and taken apart upon the completion of cell division. Specifically, the project will examine proteins that act at microtubule plus ends in the phragmoplast and integrate their functions in order to generate the dynamic array. The proposed work has two objectives: 1) Towards establishing a quantitative model of microtubule organization in the phragmoplast. Efforts will be devoted to analyzing the kinetics of the phragmoplast microtubule array by the state-of-the-art live-cell imaging technologies. Research will use a photoswitchable microtubule marker to test whether microtubule translocation takes place in the phragmoplast. Available mutants will be employed to test specific functions of the microtubule-bundling protein MAP65-3, the motor Kinesin-12, and the kinase MPK4 in the assembly and disassembly of the array. Results from these studies will be integrated into a quantitative model recapitulating microtubule dynamics and reorganization during cytokinesis. 2) To uncover the novel function of MAP65-4 in the phragmoplast. The project will examine MAP65-4 localization by immunofluorescence and live-cell imaging in transgenic lines. Genetic experiments will be followed to test for any redundant functions of MAP65-3 and MAP65-4 in the phragmoplast. The outcome of these investigations will advance our knowledge on how the anti-parallel microtubule array is established. Overall, this project will provide training opportunities for undergraduate and graduate students
