Molecular Structure and Function of the Human Kinetochore Outer Plate

描述(由申请人提供)：在理解动粒外板的结构和分子组成方面的显著差距限制了对潜在功能机制的详细剖析。这项研究的长期目标是对脊椎动物动节外板的分子组织进行足够详细的剖析，以确定其主要作用机制。这里的目标是创建一个精确的外板结构模型，该模型指定选定分子组分的空间排列，并解释这些组分在形成外板及其与微管的连接中的作用。中心假设是动粒外板由纤维网络组成，AF15q14、CENP-F和脚跟复合体参与形成网络和端接的MT附着，Mis12复合体主要参与将网络附着到着丝粒内，Cep57主要参与MT附着，Zwint主要参与日冕的形成。这个项目的基本原理是，它产生的新模型将为理解动粒外板如何连接微管和末端并控制它们的动力学提供基础。将追求三个具体目标：1)构建描绘微管附着前后其固有结构的外板地图；2)筛选可能的外板组件，以确定其在外板形成和微管附着中的位置和作用；3)确定CENP-F和Heel在侧向和末端附着到微管中的作用。这种方法是创新的，因为它结合了电子断层扫描与分子鉴定和功能耗竭，并将提供第一批高分辨率的动粒外板天然水合形式的图像。这项研究意义重大，因为它提供的结构知识对于理解动粒外板中分子成分的位置、微管结合时发生的重排以及外板蛋白在形成纤维网络和结合微管+末端中的作用至关重要。这个项目与公共卫生有关，因为动粒功能障碍与许多主要的健康问题有关，包括癌症、出生缺陷和流产。对动粒结构和功能的了解的增加将指导开发药物策略来控制和调节癌症和其他细胞增殖性疾病治疗过程中的细胞分裂。

DESCRIPTION (provided by applicant): A significant gap in understanding the structure and molecular composition of the kinetochore outer plate limits a detailed dissection of the underlying functional mechanisms. The long-term goal of this research is to dissect the molecular organization of the outer plate in vertebrate kinetochores with sufficient detail in order to determine its major functional mechanisms. The objective here is to create an accurate structural model of the outer plate that specifies spatial arrangements of selected molecular components and explains the role of these components in forming the outer plate and its attachments to microtubules. The central hypothesis is that the kinetochore outer plate consists of a fibrous network with AF15q14, CENP-F, and the Heel complex involved in forming both the network and end-on MT attachments, the Mis12 complex primarily involved in attaching the network to inner centromere, Cep57 primarily involved in MT attachments, and Zwint primarily involved in corona formation. The rationale for this project is that the new model it produces will provide a foundation for understanding how the kinetochore outer plate attaches microtubule plus ends and controls their dynamics. Three specific aims will be pursued: 1) Construct a map of the outer plate that portrays its native structure before and after microtubule attachment; 2) Screen putative outer plate components for location and role in outer plate formation and microtubule attachment; 3) Determine the roles of CENP-F and Heel in lateral and end-on attachments to microtubules. The approach is innovative because it combines electron tomography with molecular identification and functional depletions, and will provide the first high-resolution images of the kinetochore outer plate in its native, hydrated form. The research is significant because the structural knowledge it provides will be critical for understanding the location of molecular components in the kinetochore outer plate, rearrangements that occur upon microtubule binding, and the role of outer plate proteins in forming a fibrous network and binding microtubule plus ends. This project is relevant to public health because kinetochore dysfunction has been linked to a number of major health problems including cancer, birth defects, and miscarriages. The increased understanding of kinetochore structure and function will guide efforts for developing pharmacological strategies to control and regulate cell division during the treatment of cancer and other cellular proliferative disorders.