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

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.