Regulation of the Ipl1p kinase during chromosome bi-orientation in yeast

当细胞分裂时，染色体首先被复制或复制。这两个相同的拷贝保持关联，但会附着在微管上，微管是一种特殊的分子电缆，用于在分裂过程中将两个拷贝分开。至关重要的是，两个子细胞中的每一个都要收到每个染色体的一个拷贝，这样它们才能继承适当的遗传信息补充，这就要求这两个拷贝从细胞的相反两端附着在微管上，这样当细胞分裂成两个时，它们就会被分开。如果它们从同一端附着在微管上，染色体的两个副本将仅被拉入其中一个子细胞，导致一个子细胞获得额外的染色体副本，而另一个子细胞将失去其副本。染色体不会自动正确地附着在微管上，我们感兴趣的是在染色体附着错误有机会导致染色体丢失或获得事件之前，用于纠正染色体附着错误的分子机制。我们使用酵母作为这项研究的模式生物，因为所涉及的细胞成分在从酵母到人类的所有高等细胞中都能找到。然而，酵母菌构成了一个简单得多的实验系统，在其中研究这些基本问题。纠正酵母细胞中错误的微管附着涉及一种名为Ipl1p的酶，它催化着动粒中的蛋白质添加磷酸基团，动粒是在每条染色体上的一个特定点组装的蛋白质复合体，它构成了微管可以抓住的‘柄’。当染色体的两个副本上的动点从细胞的一端附着到微管上时，Ipl1p被激活，将磷酸基团添加到一个动粒上，导致它释放附着的微管，并允许它抓住来自细胞另一端的不同的微管。这纠正了附着错误，导致复制的染色体副本变得“双向”，这种状态确保它们在细胞分裂时被拉向相反的方向。我们工作的目标是了解是什么激活了Ipl1p来执行这一功能，更重要的是，当Ipl1p完成了工作后，它的活动是如何关闭的。Ipl1p最有可能的调节方式是通过张力/当复制的染色体复制是双向的时候施加在它们身上的拉力--因为然后连接它们的微管试图将它们分开。在它们是双向的之前，这种作用力会小得多，因为它们附着在微管上，试图将它们拉向相同(而不是相反)的方向。Ipl1p与另外两种蛋白质有关，这两种蛋白质和Ipl1p本身在人类细胞中也有对应的蛋白质，我们将研究紧张如何通过与这两种蛋白质的结合来调节Ipl1p。通过在分子水平上研究这一机制是如何工作的，我们将提高我们对一个基本过程的理解，该过程确保在细胞分裂过程中保持基因组的完整性，并与癌症和唐氏综合症等人类疾病相关，在这些疾病中，染色体丢失或获得事件起着重要作用。

When cells divide, chromosomes are first copied or replicated. The two identical copies remain associated but become attached to microtubules, special molecular cables that are used to pull the two copies apart during division. It is vital that each of the two daughter cells receive exactly one copy of each chromosome so that they inherit a proper complement of genetic information, and this requires that the two copies attach to microtubules from opposite ends of the cell so that they are pulled apart when the cell divides in two. Should they attach to microtubule from the same end, both copies of the chromosome will be pulled into just one of the daughter cells causing one daughter cell to gain an extra copy of the chromosome while the other daughter cell will lose its copy. Chromosomes do not automatically achieve correct attachment to microtubules and we are interested in the molecular mechanisms that are used to correct chromosome attachment errors before they have a chance to result in chromosome loss or gain events. We are using yeast as a model organism for this research because the cellular components involved are found in all higher cells from yeast to man. However, yeast constitutes a much simpler experimental system in which to investigate fundamental questions such as these. Correction of incorrect microtubule attachments in yeast cells involves an enzyme called Ipl1p that catalyses the addition of phosphate groups to proteins in the kinetochore, a complex of proteins assembled at a specific point on each chromosome and that constitutes the 'handle' onto which the microtubules can grab. When the kinetochores on both copies of a chromosome attach to microtubules from one end of the cell, Ipl1p is activated to add phosphate groups to one kinetochore, causing it to let go of its attached microtubule and allowing it to grab a different microtubule originating from the other end of the cell. This corrects the attachment error, causing the duplicated chromosomal copies to become 'bi-oriented', the state that ensures they are pulled in opposite directions when the cell divides. The objectives of our work are to understand what activates Ipl1p to perform this function, and more importantly, how its activity is then turned off when it has done its job. The most likely way that Ipl1p is regulated is through tension / the pulling force exerted on duplicated chromosomal copies when they are bi-oriented - because then the microtubules attached to them are trying to pull them apart. Before they are bi-oriented, this force would be much smaller because of their attachment to microtubules attempting to pull them both in the same (rather than opposing) directions. Ipl1p associates with two other proteins that, like Ipl1p itself also have counterparts in human cells and we will examine how tension might regulate Ipl1p through its association with these two proteins. By investigating how this mechanism works at the molecular level, we will improve our understanding of a fundamental process that ensures maintenance of genome integrity during cell division and that is relevant to human conditions such as cancer and Down's syndrome, where chromosome loss or gain events play an important role.