Plant cells lack structures called centrosomes; in other higher organisms, the spindle that pulls chromosomes into each daughter cell during mitosis forms from the two centrosomes. The project asks, how do plant cells form mitotic spindles? In the past, many researchers viewed plant spindle assembly as a special case in which unique plant-specific mechanisms facilitate the construction of this evolutionarily conserved structure. Data from Dr. Cyr's laboratory challenge this notion and indicate there is more than one mechanistic pathway that leads to spindle assembly, and that even animal cells may possess the ancestral pathways used by plant cells. The hypothesis is that these pathways operate in redundant/synergistic manners, which adapt cells to divide quickly and with high fidelity under a variety of conditions. Higher plant cells, uncluttered by the presence of centrosomes, are ideal model systems in which to study these alternate paths. This project seeks further understanding of how the early plant mitotic spindle forms, and focuses on the role that one Kinesin-14 family member plays in the process of early spindle formation. ATK5 has been partially characterized and mitotic mutants in Arabidopsis are known; the mitotic spindle morphology in mutant plant cells is altered in a manner consistent with a defect in gathering microtubules that comprise the spindle and/or producing an inward force. Furthermore, cell biological studies have shown the protein accumulates in the spindle midzone just as the nuclear envelope breaks down, poising it to gather anti-parallel microtubules during the assemblage of the early spindle. This motor likely acts redundantly with a close relative, ATK1, because double homozygous mutant plants cannot be recovered. Different functions for the two forms may exist, however, because ATK1 appears to accumulate in the nucleus of interphase cells while ATK5 does not, and there is 17% divergence in amino acid sequence between these two kinesins. The outlined experiments focus on the behavior and characterization of ATK5 (although, where appropriate, ATK1 will also be tested) in two model systems, the BY-2 cultured tobacco cell line and Arabidopsis plants. In this manner the cultured cells will be used to examine how various domains of ATK5 contribute to its biochemical and cell biological properties, and then, using mutant plants of Arabidopsis, the biological significance of these findings will be ascertained (which, in turn, will likely stimulate new hypotheses for biochemical experiments). The outlined experiments will test whether ATK5 can bundle anti-parallel microtubules and transmit forces, and will probe the significance of the +tip tracking activity that is observed with ATK5. The intellectual merit of these experiments is that they will contribute to a better understanding of how cells divide, not just in plant cells, but perhaps also to eukaryotes in general. The broader impact of these studies will involve the training of graduate and undergraduate students in bench science and the resulting data will be used in the production of on-line course materials for introductory biology courses that are being disseminated nationally. Moreover, in the preparation of instructional materials, the evolutionary relationships in mitotic pathways will be emphasized.
