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

细胞分裂是一个基本而复杂的过程，在这个过程中，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主轴成为可能。然后，染色体可以沿着纺锤体移动，并在细胞的赤道上对齐，然后才能发生染色体分离。在有丝分裂的早期阶段，许多对于维持染色体对齐和在后期染色体向相反两极移动非常重要的角色也发挥了作用。目前，灭活蛋白质的最好方法是使用RNAi、CRISPR、生长素降解或抑制来耗尽它们。这些事件在几分钟或几小时的时间尺度上发生，并且缺乏空间控制。因此，到目前为止，研究分子马达在有丝分裂后期的功能一直是困难的：它们的耗尽或抑制阻止了处于前中期的细胞。在这个项目中，学生将揭示运动在发动机CENP-E中的作用，这是染色体聚集、中期排列和后期分离所必需的。CENP-E也是一个主要的抗增殖药物靶点，其细胞水平与非整倍体和癌症有关。该项目使用创新和多学科方法来开发工具，以高空间和时间分辨率控制运动蛋白。这一方法也将适用于许多其他系统，因此将对其他生物学领域产生很大影响。该项目的第一部分将涉及设计和优化能够以光敏方式控制序列特异性蛋白酶的小分子化合物。学生将学习如何进行小分子的化学合成，提纯它们(高效液相色谱和超PLC)，并使用一系列技术(核磁共振，MS等)对它们进行分析。然后，学生将在体外测试这些抑制剂与我们的工程蛋白酶的作用，使用生物化学来纯化工程酶并测试其活性。学生还将确定与工程调控结构域结合的蛋白酶的结构，以优化配体抑制物的设计(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基因结构，这样它们就可以被蛋白酶(分子生物学)切割。然后，在光敏小分子抑制剂存在的情况下添加蛋白酶将允许学生探索CENP-E马达在中期和后期的功能，目前很难了解其在染色体运动中的功能(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，同时利用光诱导方法确定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成员在有丝分裂后期的功能，这些蛋白质也在使用这一策略的有丝分裂的早期阶段发挥关键作用。

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.