Plant Mitosis; + Tips and Minus Motors

植物细胞缺乏被称为中心体的结构；在其他高等生物中，在有丝分裂期间将染色体拉入每个子细胞的纺锤体由两个中心体形成。该项目提出的问题是，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种特殊情况，在这种情况下，独特的植物特有的机制有助于构建这种进化上保守的结构。来自西尔博士实验室的数据挑战了这一概念，并表明，导致纺锤体组装的机械途径不止一种，甚至动物细胞也可能拥有植物细胞使用的祖先途径。假说是这些通路以冗余/协同的方式运行，使细胞在各种条件下快速、高保真地分裂。高等植物细胞不会因中心体的存在而杂乱无章，是研究这些替代途径的理想模型系统。这个项目试图进一步了解早期植物有丝分裂纺锤体的形成，并重点研究一个Kinesin-14家族成员在早期纺锤体形成过程中所起的作用。ATK5已被部分鉴定，拟南芥中已知有丝分裂突变体；突变植物细胞中有丝分裂纺锤体的形态发生改变，其方式与收集组成纺锤体的微管和/或产生内向力的缺陷一致。此外，细胞生物学研究表明，就在核膜破裂时，蛋白质在纺锤体中部积累，使其在早期纺锤体的组装过程中聚集反平行的微管。这个马达可能与近亲ATK1冗余地发挥作用，因为双重纯合突变植物无法恢复。然而，这两种形式可能存在不同的功能，因为ATK1似乎积聚在间期细胞的细胞核中，而ATK5不积聚，并且两者的氨基酸序列有17%的差异。概述的实验重点是ATK5的行为和特征(尽管在适当的情况下，也将测试ATK1)在两个模型系统中，即BY-2培养烟草细胞系和拟南芥植物。通过这种方式，培养的细胞将被用来研究ATK5的不同结构域如何影响其生化和细胞生物学特性，然后，使用拟南芥的突变植株，这些发现的生物学意义将被确定(这反过来可能会激发新的生化实验假设)。概述的实验将测试ATK5是否可以捆绑反平行微管和传递力，并将探索ATK5观察到的+TIP跟踪活动的意义。这些实验的学术价值在于，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，也许对整个真核生物也是如此。这些研究的更广泛影响将涉及对研究生和本科生进行板凳科学方面的培训，由此产生的数据将用于编制生物入门课程的在线课程材料，这些材料正在全国范围内传播。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.