权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Defining the spatial and temporal function of the CENP-E motor during mitosis using rapid light-induced regulation

使用快速光诱导调节定义有丝分裂期间 CENP-E 电机的空间和时间功能

基本信息

批准号：
1868240
负责人：
金额：
--
依托单位：
University of Edinburgh
依托单位国家：
英国
项目类别：
Studentship
财政年份：
2017
资助国家：
英国
起止时间：
2017 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=studentship-1868240
关键词：
Defining spatial temporal function CENP

项目摘要

Cell division is an essential and complex process during which the cell distributes its genetic information equally to daughter cells. Microtubules and molecular motors enable the self-assembly spindle. The chromosomes can then move along the spindle and align in the equator of the cell before chromosome segregation can occur. Many of the players important for the maintenance of chromosome alignment and for chromosome movement to opposite poles during anaphase also play roles at earlier stages during mitosis. Currently, the best ways to inactivate proteins is to deplete them using RNAi, CRISPR, Auxin-degradation or inhibition. These events occur on a timescale of minutes or hours and lack spatial control. Thus it has been difficult until now to study the function of molecular motors during the late stages of mitosis: their depletion or inhibition arrests the cell in prometaphase. In this project, the student will uncover the role of the kinesin motor CENP-E, which is essential for chromosome congression, alignment at metaphase and segregation during anaphase. CENP-E is also a major anti-proliferative drug target and its cellular levels are associated with aneuploidy and cancer. The project uses an innovative and multidisciplinary approach to develop tools to control kinesins with high spatial and temporal resolution. This methodology will also be applicable to many other systems and therefore will have a high impact on other areas of biology.The first part of the project will involve designing and optimizing small molecule compounds that can control a sequence specific protease in a light-sensitive manner. The student will learn to perform the chemical synthesis of small molecules, to purify them (HPLC and UPLC) and analyse them using a range of techniques (NMR, MS etc.). Then the student will test the inhibitors in vitro against our engineered protease, using biochemistry to purify the engineered protease and test its activity. The student will also determine the structure of the protease bound to the engineered regulatory domain to optimize the design of the ligand inhibitor (protein purification, general biochemistry and structural biology). The student will design CENP-E gene constructs using gene assembly, such that they can be cleaved by the protease (molecular biology). Then addition of the protease in the presence of the light-sensitive small molecule inhibitor will allow the student to probe the function of CENP-E motor during metaphase and anaphase for which it is currently difficult to understand its function during chromosome movement (live cell imaging, super-resolution microscopy). From these studies, the student will gain expertise in multiple complementary disciplines while defining the role of mitotic molecular motors using light-induced approaches. They will also be able to use this approach to examine the function of other mitotic players during anaphase for proteins that also play key roles during the earlier stages of mitosis using this strategy.

细胞分裂是一个重要而复杂的过程，在此过程中，细胞将其遗传信息平等地分配给子细胞。微管和分子马达使自组装纺锤体成为可能。然后，染色体可以沿着纺锤体移动，并在染色体分离发生之前在细胞的赤道上排列。在有丝分裂后期，许多对维持染色体排列和染色体向相反两极运动很重要的参与者也在有丝分裂的早期阶段发挥作用。目前，抑制蛋白质的最佳方法是使用RNAi、CRISPR、生长素降解或抑制来耗尽它们。这些事件发生在几分钟或几小时的时间尺度上，缺乏空间控制。因此，直到现在，研究分子马达在有丝分裂后期的功能仍然很困难：它们的消耗或抑制使细胞停滞在前中期。在这个项目中，学生将揭示驱动蛋白马达CENP-E的作用，它对染色体的聚集，中期的对齐和后期的分离至关重要。CENP-E也是一种主要的抗增殖药物靶标，其细胞水平与非整倍性和癌症相关。该项目采用创新和多学科的方法来开发工具，以控制具有高空间和时间分辨率的驱动蛋白。该方法也将适用于许多其他系统，因此将对生物学的其他领域产生重大影响。该项目的第一部分将涉及设计和优化小分子化合物，这些化合物可以以光敏方式控制序列特异性蛋白酶。学生将学习进行小分子的化学合成，纯化它们（HPLC和UPLC），并使用一系列技术（NMR，MS等）进行分析。然后，学生将在体外测试抑制剂对我们的工程蛋白酶，使用生物化学纯化工程蛋白酶，并测试其活性。学生还将确定与工程调控结构域结合的蛋白酶的结构，以优化配体抑制剂的设计（蛋白质纯化，一般生物化学和结构生物学）。学生将使用基因组装设计CENP-E基因构建体，以便它们可以被蛋白酶切割（分子生物学）。然后，在光敏小分子抑制剂存在下添加蛋白酶将允许学生在中期和后期期间探测CENP-E马达的功能，目前难以理解其在染色体运动期间的功能（活细胞成像，超分辨率显微镜）。从这些研究中，学生将获得多个互补学科的专业知识，同时使用光诱导方法定义有丝分裂分子马达的作用。他们还将能够使用这种方法来检查其他有丝分裂参与者在后期的功能，这些蛋白质也在使用这种策略的有丝分裂早期阶段发挥关键作用。