Regulation of cell division by mitotic kinases

有丝分裂激酶对细胞分裂的调节

基本信息

批准号：
9275657
负责人：
Ekaterina L Grishchuk
金额：
$ 13.06万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-05-01 至 2018-02-28
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）：准确的染色体分离取决于双向（即姐妹着丝粒从相反的两极附着到纺锤体微管（MT）上），这依赖于着丝粒 MT 的张力依赖性稳定性。多种证据表明，Aurora B 激酶（染色体乘客复合物 (CPC) 的酶促成分）是该机制的关键要素。引人注目的是，CPC 结合位点富集在内着丝粒处，而不是 Aurora B 底物结合 MT 末端的外着丝粒处。此外，随着着丝粒到着丝粒的距离随着张力的增加而增加，这些着丝粒底物的磷酸化会减少 MT 结合。这些观察结果建立了 Aurora B 信号传导与张力之间的相关联系，并产生了直观上有吸引力的“空间分离”模型，其中距离依赖性磷酸化在双向中起着至关重要的作用。然而，Aurora B 距离依赖性磷酸化的机制尚不清楚。此外，是否如体外实验所示，Aurora B 独立通路可以在体内感知张力尚未得到测试。已知 Aurora B 通过反式自磷酸化自动激活，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。该机制的基本特征是：1) 由于高密度的 CPC 结合位点（即聚类），Aurora B 在着丝粒处自动激活，2) 该活性通过活性激酶的解离、扩散和激酶传播到动粒溶液中的激活/失活反应，这取决于可溶性激酶/磷酸酶的比率。目标 1 将检验 Aurora B 通过体内反应扩散过程调节动粒-MT 相互作用的假设。我们将操纵着丝粒处的 CPC 聚类、着丝粒解绑动力学以及可溶性激酶/磷酸酶比率，并测量这些扰动对着丝粒功能的影响。目标 2 将在体外重建 Aurora B 磷酸化动力学和空间调节 MT 结合。体内情况很复杂，体外重构将使我们能够在输入和行为之间建立直接的定量关系。系统。这一目标建立在我们使用重组 Aurora B、磷酸酶和荧光底物开发的体外系统的基础上。目标 3 将定义张力和 Aurora B 对调节体内动粒 MT 的贡献。为了将张力与 Aurora B 分开，我们开发了一种化学诱导的二聚化策略，使我们能够直接操纵 Aurora B 在动粒处的活性，而不受张力的影响。我们将 (1) 确定着丝点 Aurora B 活性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 Aurora B 的间接影响。这些实验的结果将详细了解生化和着丝粒的机械变化控制着 MT 的相互作用。

项目成果

期刊论文数量（20）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Aurora B dynamics at centromeres create a diffusion-based phosphorylation gradient.

着丝粒的 Aurora B 动力学创建了基于扩散的磷酸化梯度。

DOI：
发表时间：
2011-08-22
期刊：
影响因子：
0
作者：
Wang, Enxiu;Ballister, Edward R;Lampson, Michael A
通讯作者：
Lampson, Michael A

Feedback control of chromosome separation by a midzone Aurora B gradient.

通过中区 Aurora B 梯度对染色体分离进行反馈控制。

DOI：
发表时间：
2014-07-18
期刊：
影响因子：
0
作者：
Afonso, Olga;Matos, Irina;Pereira, António J;Aguiar, Paulo;Lampson, Michael A;Maiato, Helder
通讯作者：
Maiato, Helder

Regulated targeting of protein phosphatase 1 to the outer kinetochore by KNL1 opposes Aurora B kinase.

KNL1 将蛋白磷酸酶 1 靶向外着丝粒，从而对抗 Aurora B 激酶。

DOI：
发表时间：
2010-03-22
期刊：
影响因子：
0
作者：
Liu, Dan;Vleugel, Mathijs;Backer, Chelsea B;Hori, Tetsuya;Fukagawa, Tatsuo;Cheeseman, Iain M;Lampson, Michael A
通讯作者：
Lampson, Michael A

Molecular control of kinetochore-microtubule dynamics and chromosome oscillations.

动粒微管动力学和染色体振荡的分子控制。

DOI：
发表时间：
2010-04
期刊：
影响因子：
21.3
作者：
Amaro, Ana C;Samora, Catarina P;Holtackers, René;Wang, Enxiu;Kingston, Isabel J;Alonso, Maria;Lampson, Michael;McAinsh, Andrew D;Meraldi, Patrick
通讯作者：
Meraldi, Patrick

Sensing chromosome bi-orientation by spatial separation of aurora B kinase from kinetochore substrates.

通过极光 B 激酶与着丝粒底物的空间分离来感知染色体双向。