Plant Mitosis; + Tips and Minus Motors

植物细胞缺少中心体，而在其他高等生物中，有丝分裂时将染色体拉入子细胞的纺锤体是由两个中心体形成的。 该项目的问题是，植物细胞如何形成有丝分裂纺锤体？ 在过去，许多研究人员认为植物纺锤体组装是一个特殊的情况下，独特的植物特异性机制促进了这种进化上保守的结构的构建。 Cyr博士实验室的数据挑战了这一观点，并表明导致纺锤体组装的机制途径不止一种，甚至动物细胞也可能拥有植物细胞使用的祖先途径。 假设这些途径以冗余/协同的方式运作，使细胞在各种条件下快速分裂并高保真。 高等植物细胞，整齐的中心体的存在下，是理想的模型系统，在其中研究这些交替的路径。 该项目旨在进一步了解早期植物有丝分裂纺锤体的形成，并关注驱动蛋白-14家族成员在早期纺锤体形成过程中的作用。 ATK 5已经部分表征，并且已知拟南芥中的有丝分裂突变体;突变植物细胞中的有丝分裂纺锤体形态以与聚集微管（其包括纺锤体和/或产生向内的力）中的缺陷一致的方式改变。 此外，细胞生物学研究表明，这种蛋白质在纺锤体中间区积累，就像核膜分解一样，使其在早期纺锤体组装过程中聚集反平行微管。 这种马达可能与近亲ATK 1冗余地起作用，因为双纯合突变体植物不能恢复。 然而，这两种形式可能存在不同的功能，因为ATK 1似乎在间期细胞的细胞核中积累，而ATK 5则没有，并且这两种驱动蛋白之间的氨基酸序列存在17%的差异。 概述的实验集中在两个模型系统，BY-2培养的烟草细胞系和拟南芥植物中ATK 5的行为和表征（尽管在适当的情况下，ATK 1也将被测试）。 以这种方式，培养的细胞将被用于检查ATK 5的各个结构域如何有助于其生物化学和细胞生物学特性，然后，使用拟南芥的突变体植物，这些发现的生物学意义将被确定（这反过来可能会激发生化实验的新假设）。 概述的实验将测试ATK 5是否可以捆绑反平行微管和传递力，并将探测ATK 5观察到的+tip跟踪活动的意义。 这些实验的智力价值在于，它们将有助于更好地理解细胞是如何分裂的，不仅是在植物细胞中，而且可能也是在一般的真核生物中。 这些研究的更广泛影响将涉及对研究生和本科生进行实验台科学培训，所产生的数据将用于制作正在全国传播的生物学入门课程的在线课程材料。 此外，在编写教学材料时，将强调有丝分裂途径中的进化关系。

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.