Regulation of cell division by mitotic kinases

描述(申请人提供)：准确的染色体分离依赖于双向(即姐妹动点从相反的两极附着到纺锤体微管)，这依赖于动点微管的张力依赖的稳定。多条证据表明，染色体乘客复合体(CPC)的酶成分极光B激酶是这一机制的关键元件。值得注意的是，CPC结合位点集中在着丝粒内侧，而不是在Aurora B底物与MT末端结合的外部着丝粒。此外，这些动粒底物的磷酸化减少了MT的结合，随着着丝粒到动粒的距离随着张力的增加而减少。这些观察结果建立了Aurora B信号和张力之间的关联，并导致了一个直观的吸引人的“空间分离”模型，在该模型中，距离依赖的磷酸化在双向过程中起着关键作用。然而，Aurora B的距离依赖性磷酸化的机制尚不清楚。此外，体外实验表明，在体内是否可以通过Aurora B非依赖的途径感受到张力，这一点还没有得到测试。已知Aurora B通过反式作用中的自动磷酸化而自动激活，因此我们开发了一个定量模型来描述由此产生的非直观的空间非线性动力学。我们的模型预测，Aurora B的距离依赖性磷酸化是通过反应扩散机制建立的。这种机制的基本特征是：1)Aurora B由于高密度的CPC结合位点(即聚集)而在着丝粒上自动激活，以及2)这种活性通过解离活性激酶、扩散和溶液中的激酶激活/失活反应而传播到动点，这取决于可溶激酶/磷酸酶的比率。目的1验证Aurora B在体内通过反应-扩散过程调节动粒-MT相互作用的假说。我们将操纵着丝粒上的CPC聚集，着丝粒解离的动力学，以及可溶性激酶/磷酸酶的比率，并测量这些扰动对着丝粒功能的影响。目的2将在体外重建Aurora B的磷酸化动力学和空间调控的MT结合。体内情况是复杂的，体外重建将使我们能够在输入和行为之间建立直接的、定量的关系 系统。这一目标建立在我们开发的使用重组Aurora B、磷酸酶和荧光底物的体外系统上。目标3将确定张力和极光B在体内调节动粒MTS中的作用。为了将张力从Aurora B中分离出来，我们开发了一种化学诱导的二聚化策略，允许我们在运动中心直接操纵Aurora B的活动，而不依赖于张力。我们将(1)确定运动中枢Aurora B活动的受控变化如何影响MT动力学，以及(2)剖析张力的直接影响和通过Aurora B的间接影响。这些实验的结果将详细了解运动中枢的生化和机械变化如何控制与MT的相互作用。

DESCRIPTION (provided by applicant): Accurate chromosome segregation depends on bi-orientation (i.e., sister kinetochores attaching to spindle microtubules (MTs) from opposite poles), which relies on tension-dependent stabilization of kinetochore MTs. Multiple lines of evidence suggest that Aurora B kinase, the enzymatic component of the chromosome passenger complex (CPC), is a key element of this mechanism. Strikingly, CPC binding sites are enriched at the inner centromere, not at the outer kinetochore where Aurora B substrates bind MT ends. Moreover, phosphorylation of these kinetochore substrates, which reduces MT binding, decreases as the distance from the centromere to the kinetochore increases with tension. These observations established a correlative link between Aurora B signaling and tension and led to an intuitively attractive "spatial separation" model, in which distance-dependent phosphorylation plays a crucial role in bi-orientation. However, the mechanisms underlying distance-dependent phosphorylation by Aurora B are unknown. Furthermore, whether tension can be sensed by Aurora B-independent pathways in vivo, as suggested by in vitro experiments has not been tested. Aurora B is known to auto-activate by auto-phosphorylation in trans, so we developed a quantitative model to describe the resulting non-intuitive spatial nonlinear dynamics. Our model predicts that distance- dependent phosphorylation by Aurora B is established by a reaction-diffusion mechanism. The essential features of this mechanism are: 1) Aurora B auto-activates at centromeres due to a high density of CPC binding sites (i.e., clustering), and 2) this activity propagates to kinetochores by unbinding of active kinase, diffusion, and kinase activation/inactivation reactions in solution, which depend on the soluble kinase/phosphatase ratio. Aim 1 will test the hypothesis that Aurora B regulates kinetochore-MT interactions through a reaction-diffusion process in vivo. We will manipulate CPC clustering at centromeres, the kinetics of centromere unbinding, and the soluble kinase/phosphatase ratio, and measure the effects of these perturbations on kinetochore function. Aim 2 will reconstitute Aurora B phosphorylation dynamics and spatially-regulated MT binding in vitro. The in vivo situation is complex, and in vitro reconstitution will allow us to establish direct, quantitative relationships between the inputs and the behavior of the system. This aim builds on an in vitro system that we developed using recombinant Aurora B and phosphatase and fluorescent substrates. Aim 3 will define the contributions of tension and Aurora B to regulating kinetochore MTs in vivo. To uncouple tension from Aurora B, we developed a chemically-induced dimerization strategy that allows us to directly manipulate Aurora B activity at kinetochores and independent of tension. We will (1) determine how controlled changes in Aurora B activity at kinetochores affect MT dynamics, and (2) dissect the direct effects of tension and indirect effects via Aurora B. The result of these experiments will b detailed understanding of how both biochemical and mechanical changes at kinetochores control interactions with MTs.