Mechanisms of kinetochore-microtubule attachment and regulation

着丝粒-微管附着和调节机制

• 批准号: 10116423
• 负责人: Jennifer G DeLuca
• 金额: $ 36.63万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-01 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10116423
• 关键词: Aneuploidy; Architecture; Area; Biochemical; Biological; Biological Assay; Biological Models; Cell Cycle Progression; Cell division; Cells; Centromere; Characteristics; Chromosome Segregation; Chromosomes; Congenital Abnormality; Defect; Ensure; Event; Genetic Materials; Goals; Health; Human; Image; Kinetochores; Lead; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Microtubule Stabilization; Microtubules; Mitosis; Mitotic; Mitotic Chromosome; Modification; Molecular; Mutagenesis; Oncogenic; Phosphorylation; Positioning Attribute; Process; Proteins; Regulation; Research; Resolution; Signal Transduction; Structure; Sum; Techniques; Time; aurora B kinase; cancer cell; cancer therapy; chromosome missegregation; daughter cell; insight; recruit; segregation; temporal measurement; tool; tumor progression

Project Summary The goal of cell division is to reliably produce two identical daughter cells, each with an exact copy of the original genetic material. When this process goes awry, a common result is aneuploidy, which is a leading cause of birth defects and a key characteristic of cancer. Mitotic cell division critically depends on kinetochores, structures that orchestrate chromosome segregation and integrate all aspects of the mitotic machinery to ensure mitosis is executed with high fidelity. Kinetochores physically connect chromosomes to spindle microtubules (MTs), and they regulate the strength of these connections so that erroneously-attached MTs are released, and correctly-attached MTs are stabilized. Importantly, kinetochores ensure that cells do not exit mitosis if chromosomes fail to attach, or are incorrectly attached, to MTs. Much progress has been made in identifying the kinetochore proteins that participate in these processes; however, how these molecules function in concert to ensure the accuracy of chromosome attachment and segregation, and to ensure timely mitotic progression remains poorly understood. Our lab has been instrumental in defining how kinetochores regulate MT attachment, and how this fundamental aspect of mitosis is integrated into controlling cell cycle progression. Additionally, our lab has begun to make significant inroads to understanding how oncogenic transformation leads to deregulated kinetochore function, and how these defects lead to cancer cell-specific vulnerabilities that can potentially be exploited for cancer therapies. Our expertise in studying kinetochore function in combination with our newly developed experimental approaches – especially those that provide increased spatial and temporal resolution of kinetochore proteins – puts us in a strategic position to resolve how kinetochores ensure accurate chromosome segregation and drive mitotic progression. In the next five years, our research will focus on four areas. We will: (1) Examine the mechanisms of kinetochore-MT attachment regulation using biochemical and cell biological tools, as well as new assays to track the dynamics of specific phosphorylation events in cells with high temporal resolution; (2) Investigate how Aurora B kinase, the “master” regulator of attachment, is recruited to discrete centromere and kinetochore regions with precise temporal control using in-cell mutagenesis approaches, proximity-dependent interaction/mass spectrometry analysis, and phospho-modification tracking techniques; (3) Probe how kinetochore-MT attachment status is communicated to the spindle assembly checkpoint, in part by using super-resolution imaging to map the changes in kinetochore architecture that occur upon stable MT attachment; (4) Determine how oncogenic signaling leads to kinetochore-MT attachment deregulation using a tumor progression model system built from primary cells. In sum, our studies will provide critical insight into the fundamental mechanisms that regulate kinetochore-MT attachment, and that integrate this critical mitotic function with other mitotic processes including chromosome architecture, spindle MT dynamics, and checkpoint signaling. !

项目摘要 细胞分裂的目标是可靠地产生两个相同的子细胞，每个子细胞都有一个完全相同的复制品。 原始遗传物质当这一过程出错时，一个常见的结果是非整倍体，这是一种主要的染色体变异。 出生缺陷的原因和癌症的关键特征。有丝分裂细胞的分裂主要依赖于动粒， 这些结构协调染色体分离并整合有丝分裂机制的所有方面， 确保有丝分裂以高保真度执行。动粒将染色体与纺锤体连接起来 微管（MT），它们调节这些连接的强度，使错误连接的MT 释放，并稳定正确连接的MT。重要的是，动粒确保细胞不会退出 有丝分裂，如果染色体未能附着或错误地附着到MT。取得了很大进展 确定参与这些过程的动粒蛋白;然而，这些分子如何发挥作用 以确保染色体附着和分离的准确性，并确保及时的有丝分裂 进展仍然知之甚少。我们的实验室在确定动粒如何调节 MT附着，以及有丝分裂的这一基本方面如何整合到控制细胞周期进程中。 此外，我们的实验室已经开始在理解致癌转化如何 导致动粒功能失调，以及这些缺陷如何导致癌细胞特异性脆弱性 有可能被用于癌症治疗。我们在研究动粒功能方面的专业知识， 与我们新开发的实验方法相结合-特别是那些提供增加的实验方法。 动粒蛋白质的空间和时间分辨率-使我们处于一个战略地位，以解决如何 着丝粒确保染色体精确分离并驱动有丝分裂进程。在接下来的五年里， 我们的研究会集中在四个范畴。我们将：（1）研究运动舞蹈-MT附着的机制 使用生物化学和细胞生物学工具进行调控，以及新的检测方法来跟踪特定的 （2）研究细胞中的极光B激酶（“主”激酶）是如何在细胞中以高时间分辨率检测磷酸化事件的; 附着调节子，以精确的时间间隔被募集到离散的着丝粒和动粒区域。 使用细胞内诱变方法的控制，邻近依赖性相互作用/质谱分析， 和磷酸化修饰跟踪技术;（3）探讨着丝粒-MT的附着状态如何影响 传送到主轴组件检查点，部分地通过使用超分辨率成像来映射主轴组件检查点。 在稳定的MT附着后发生的动粒结构的变化;（4）确定致癌性如何 使用由以下构建的肿瘤进展模型系统， 主要细胞总之，我们的研究将提供关键的洞察力的基本机制，调节 动丝素-MT附着，并将这一关键的有丝分裂功能与其他有丝分裂过程整合 包括染色体结构、纺锤体MT动力学和检查点信号传导。 !