Understanding the structure and function of ASPM, a protein essential for normal mitosis.

背景：异常的纺锤体样小头畸形相关蛋白（ASPM）是正常有丝分裂所必需的。ASPM的突变影响细胞分裂平面，导致原发性常染色体隐性小头畸形（MCPH），其特征是小脑和认知障碍。预测其n端微管结合域和串联对钙钙蛋白同源域可能定位于有丝分裂纺锤极。它的81个“IQ”（异亮氨酸-谷氨酰胺）基序的大中心块的结构是未知的，可能与钙调素结合，可能是长而线性的，也可能是紧凑的，这取决于钙调素如何与智商基序相互作用。c端区域包含进一步相互作用的蛋白质的结合位点，其身份只是刚刚被发现（Bond实验室）。目的：我们的主要目的是了解ASPM在有丝分裂中的作用机制。我们的第一个目标是确定ASPM的结构，使我们能够确定它如何在主轴杆内组织。我们的第二个目标是表征其与体外结合伙伴的相互作用，并利用这些信息来询问这些相互作用如何有助于其在体内的功能。新颖性和及时性：这次新的合作汇集了两位主管在细胞和分子/结构生物学方面的互补专业知识，并提供了一个新的机会来研究ASPM在细胞分裂中的结构和功能，这是关键的生物过程之一。实验方法：该项目将利用Peckham实验室表达和纯化ASPM结构域的能力，利用蛋白质表达和纯化、生化分析、高含量成像、电子显微镜、结晶和超分辨率显微镜等一系列技术，结合Bond实验室用于研究细胞中ASPM的强大背景、工具、技术和新型试剂，研究它们的结构和功能。

Background: Abnormal spindle-like, microcephaly-associated (ASPM) protein is essential for normal mitosis. Mutations in ASPM affect the plane of cell division resulting in the disorder primary autosomal recessive microcephaly (MCPH), which is characterized by a small brain and cognitive impairment. Its predicted the N-terminal microtubule binding domain and tandem pair of calponin homology domains probably localize it to mitotic spindle poles. The structure of its large central block of 81 'IQ' (isoleucine-glutamine) motifs, which probably bind calmodulin, is unknown, and could be long and linear, or compact depending on how calmodulin interacts with the IQ motifs. The C-terminal region contains binding sites for further interacting proteins, the identity of which are only just being discovered (Bond Lab). Objectives: Our main objective is to understand the mechanism by which ASPM functions in mitosis. Our first aim is to determine the structure of ASPM, to enable us to determine how it might be organized within the spindle pole. Our second aim is to characterize its interactions with its binding partners in vitro, and use this information to interrogate how these interactions contribute to its function in vivo. Novelty & Timeliness: This new collaboration brings together complimentary expertise from the two supervisors in cell and molecular/structural biology, and provides a novel opportunity to investigate the structure and function of ASPM in cell division, one of the key biological processes.Experimental approach: The project will exploit the ability of the Peckham lab to express and purify domains from ASPM to study their structure and function using a range of techniques from protein expression and purification, biochemical assays, high content imaging, electron microscopy, crystallization, and super-resolution microscopy approaches combined with the strong background, tools, techniques and novel reagents utilised to study ASPM in cells from the Bond Lab.