The maize kinetochore: Composition, structure and roles in meiotic chromosome segregation

Kinetochores are key organelles that ensure chromosome transmission with high fidelity in both mitosis and meiosis. However, little is known about the molecular composition of the plant kinetochore. Functional analysis of the plant kinetochore has therefore been severely hampered. Here I describe detailed cytological and molecular experiments to explore the maize plant kinetochore.; First, a novel three-dimensional living culture system was developed to observe neocentromere-mediated chromosome movement. This was the first time it was shown in living cells that maize neocentromeres can be activated and lead chromosome movement during meiotic anaphase II. Experiments with combined fluorescence immunocytochemistry and in situ hybridization further showed that neocentromeres in maize mobilize on the spindle differently from the kinetochores and that maize neocentromeres do not bind to kinetochore components CENPC and MAD2. High resolution three-dimensional immunofluorescence microscopy studies on CENPC and MAD2, as well as the 3F3/2 antigen, an unknown and yet conserved kinetochore component, revealed that the maize kinetochore is structurally divided into functional sub-domains.; Comparative functional analysis of the maize kinetochore focused on the roles of MAD2 and the 3F3/2 antigen in the meiotic spindle checkpoint. In contrast to the previous observation in mitosis that the dissociation of MAD2 protein from the kinetochore is sensitive to microtubule attachment, my observations suggest that tension at the kinetochore, rather than microtubule attachment, is responsible for MAD2 dislocation from the meiotic kinetochore. The contrasting localization patterns of MAD2 imply that two fundamentally different spindle assembly pathways are employed in mitosis and meiosis. Using the maize meiotic mutant afd1 (a&barbelow;bsence of f&barbelow;irst d&barbelow;ivision 1&barbelow;), I extended my analysis into cells possessing only single-kinetochore chromosomes. I show here that maize single kinetochores align at the spindle equator and are able to interact with both spindle poles in a tension-sensitive manner. These observations indicate that the meiotic maize kinetochores have an inherent plasticity and functional redundancy.; The structural and functional studies of the maize kinetochore presented in this dissertation support the idea that eukaryotic kinetochores are evolutionarily conserved in their molecular composition, structure and function.