Molecular Structure and Function of the Human Kinetochore Outer Plate

描述（由申请人提供）：在理解着丝点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解剖脊椎动物动着点外板的分子组织，以确定其主要的功能机制。这里的目标是创建一个精确的外板结构模型，该模型指定选定的分子成分的空间排列，并解释这些成分在形成外板及其附着于微管中的作用。中心假设是着丝点外板由纤维网络组成，其中AF15q14、CENP-F和Heel复合体参与形成网络和端端MT附着物，Mis12复合体主要参与将网络连接到内部着丝粒，Cep57主要参与MT附着物，Zwint主要参与日冕的形成。该项目的基本原理是，它产生的新模型将为理解着丝点外板如何附着微管+端并控制其动力学提供基础。将追求三个具体目标：1)构建外板的地图，描绘其在微管附着之前和之后的天然结构；2)筛选假定的外板组件，以确定外板形成和微管附着的位置和作用；3)确定CENP-F和Heel在微管外侧和末端附着中的作用。该方法是创新的，因为它结合了电子断层扫描与分子识别和功能消耗，并将提供第一个高分辨率图像的着丝点外板在其天然的，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着丝点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正端的作用至关重要。该项目与公共卫生有关，因为着丝点功能障碍与许多主要健康问题有关，包括癌症、出生缺陷和流产。对着丝点结构和功能的进一步了解将指导在癌症和其他细胞增生疾病的治疗过程中开发控制和调节细胞分裂的药理学策略。

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.