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。这两个相同的拷贝仍然相互联系，但会附着在微管上，微管是一种特殊的分子电缆，在分裂过程中用来将两个拷贝分开。至关重要的是，两个子细胞中的每一个都得到每条染色体的一个拷贝，这样它们就能继承适当的遗传信息，这就需要两个拷贝从细胞的两端附着在微管上，这样当细胞分裂成两半时，它们就会被分开。如果它们从同一端附着在微管上，两条染色体的拷贝就会被拉入一个子细胞中，导致一个子细胞获得额外的染色体拷贝，而另一个子细胞将失去它的拷贝。染色体不会自动实现对微管的正确附着，我们对用于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纠正它们的分子机制感兴趣。我们在这项研究中使用酵母作为模式生物，因为涉及的细胞成分在从酵母到人类的所有高等细胞中都有发现。然而，酵母构成了一个更简单的实验系统，在其中研究诸如此类的基本问题。在酵母细胞中，纠正不正确的微管附着需要一种名为Ipl1p的酶，这种酶可以催化向着丝点中的蛋白质添加磷酸基团，着丝点是在每条染色体上特定点上组装的蛋白质复合体，它构成了微管可以抓住的“把手”。当染色体的两个拷贝上的着丝点附着在细胞一端的微管上时，Ipl1p被激活，将磷酸基团添加到一个着丝点上，使它脱离附着的微管，并允许它抓住来自细胞另一端的不同微管。这纠正了附着错误，导致复制的染色体副本变成“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来执行这个功能，更重要的是，当它完成它的工作时，它的活动是如何被关闭的。Ipl1p调控的最可能的方式是通过张力/拉力施加在复制的染色体副本上，当它们是双向的，因为附着在它们上的微管试图将它们分开。在它们是双向的之前，这个力会小得多，因为它们附着在微管上，试图把它们拉向相同（而不是相反）的方向。Ipl1p与另外两种蛋白质相关，就像Ipl1p本身一样，在人类细胞中也有对应的蛋白质，我们将研究张力如何通过Ipl1p与这两种蛋白质的关联来调节Ipl1p。通过研究这种机制在分子水平上是如何工作的，我们将提高我们对细胞分裂过程中确保基因组完整性维持的基本过程的理解，这与癌症和唐氏综合症等人类疾病有关，其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.