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

背景：异常的纺锤体样小头相关蛋白(ASPM)是正常有丝分裂所必需的。ASPM基因突变影响细胞分裂平面，导致以小脑和认知障碍为特征的原发性常染色体隐性小头症(MCPH)。它预测的N端微管结合结构域和串联的Calponin同源结构域可能将其定位于有丝分裂的纺锤体极。它的81个‘IQ’(异亮氨酸-谷氨酰胺)基序的大中心块的结构尚不清楚，可能与钙调蛋白结合，可能是长的和线性的，也可能是紧凑的，这取决于钙调蛋白与IQ基序的相互作用。C-末端区域包含进一步相互作用的蛋白质的结合部位，其特性才刚刚被发现(Bond Lab)。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，使我们能够确定它在主轴极内可能是如何组织的。我们的第二个目标是在体外表征它与其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在体内对其功能做出贡献。新颖性和及时性：这一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，并为研究ASPM在细胞分裂中的结构和功能提供了一个新的机会，细胞分裂是关键生物学过程之一。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，使用从蛋白质表达和纯化、生化分析、高含量成像、电子显微镜、结晶和超分辨率显微镜方法等一系列技术，结合邦德实验室用于研究ASPM细胞的强大背景、工具、技术和新型试剂来研究其结构和功能。

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.