Roles of chromosomal factors in chromosome segregation

描述(申请人提供)：在细胞分裂过程中，染色体必须均匀分离。真核生物使用微管来控制染色体的定位和移动。除了动点和中心体，构成染色体臂的染色质在组织纺锤体微管方面也起着重要作用。这项建议的长期目标是了解染色体因素如何影响当地环境并协调染色体分离。为了达到这一目标，我们使用表达筛选来鉴定新的中期染色体结合蛋白，并发现了脊椎动物染色体乘客复合体的两个新成分Dasra A和Dasra B，该复合体包含Aurora B激酶。该复合体在纺锤体检查点和细胞质分裂中发挥作用，并被认为调节微管动力学。Dasra B在脊椎动物中是保守的，而Dasra A的同源物可以在鱼、青蛙和鸡中找到，但在哺乳动物中不存在。含有Dasra A的脊椎动物区别于哺乳动物的特征之一是早期胚胎细胞的快速分裂，在此过程中检查点控制被抑制，这表明Dasra A可能专门调节这些快速细胞分裂周期中的纺锤体组装。利用非洲爪哇卵提取物，我们建议：1)确定染色体乘客复合体的全部成分。2)区分Dasra A和Dasra B在染色质诱导的纺锤体组装、纺锤体组装检查点的激活/维持以及Aurora B的激酶活性调节中的功能。3)研究染色体乘客复合体是如何在染色体上调节的。是染色体生化激活了染色体乘客复合体，还是染色体只是用来增加复合体的局部浓度？这项提议的发现将帮助我们了解正常细胞如何准确分离它们的染色体，以及这一过程如何在癌细胞和出生缺陷中失败。

DESCRIPTION (provided by applicant): Chromosomes must be equally segregated during cell division. Eukaryotes use microtubules to control the positioning and movement of chromosomes. In addition to kinetochores and centrosomes, the chromatin that makes up the chromosome arms plays important roles in organizing spindle microtubules. The long-term goal of this proposal is to understand how chromosomal factors influence the local environment and coordinate chromosome segregation. To approach this goal, we used expression screening to identify new metaphase chromosome-binding proteins, and discovered Dasra A and Dasra B, two new components of the vertebrate chromosomal passenger complex, which contains the kinase Aurora B. This complex plays roles in the spindle checkpoint and cytokinesis, and it has been proposed to regulate microtubule dynamics. Whereas Dasra B is conserved among vertebrates, Dasra A homologs can be found in fishes, frogs and chicken, but not in mammals. One of the characteristics distinguishing Dasra A-containing vertebrates from mammals is the rapid early embryonic cell divisions during which the checkpoint controls are suppressed, suggesting that Dasra A may be specialized to regulate spindle assembly in these rapid cell division cycles. Using Xenopus egg extracts, we propose to: 1) Determine the full constituents of the chromosomal passenger complex. 2) Distinguish the functions of Dasra A and Dasra B in chromatin-induced spindle assembly, in activation/maintenance of the spindle assembly checkpoint, and in the regulation of the kinase activity of Aurora B. 3) Examine how the chromosomal passenger complex is regulated on chromosomes. Do chromosomes biochemically activate the chromosomal passenger complex, or are chromosomes simply used for increasing local concentration of the complex? The findings of this proposal will help us understand how normal cells segregate their chromosomes accurately and how this process fails in cancer cells and in birth defects.