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存在重大差距，限制了对潜在功能机制的详细解剖。本研究的长期目标是充分详细地解剖脊椎动物动粒外板的分子组织，以确定其主要的功能机制。这里的目标是创建一个精确的结构模型的外板，指定选定的分子组件的空间排列，并解释这些组件在形成外板及其附件的微管的作用。中心假设是，动粒外板由纤维网络与AF 15 q14，CENP-F，和脚跟复杂的参与形成网络和端MT附件，Mis 12复杂的主要参与连接网络的内部着丝粒，Cep 57主要参与MT附件，和Zwint主要参与电晕形成。该项目的基本原理是，它产生的新模型将为理解动粒外板如何连接微管加端并控制其动力学提供基础。将追求三个具体的目标：1）构建一个地图的外板，描绘其天然结构之前和之后的微管附着; 2）筛选推定的外板组件的位置和作用，在外板形成和微管附着; 3）确定CENP-F和脚跟的作用，在横向和端对微管的附件。这种方法是创新的，因为它结合了电子断层扫描与分子识别和功能耗竭，并将提供第一个高分辨率的图像动粒外板在其原生的，水合的形式。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组分的位置，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加末端中的作用至关重要。该项目与公共卫生有关，因为动粒功能障碍与许多重大健康问题有关，包括癌症，出生缺陷和流产。对动粒结构和功能的理解的增加将指导在癌症和其他细胞增殖性疾病的治疗期间开发控制和调节细胞分裂的药理学策略的努力。

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.