Structural studies on phospho-regulation of the TACC3 / ChTOG protein complex in mitotic spindle assembly

Viewed through a microscope, cells undergo a spectacular transformation as they enter mitosis, the phase of their existence just before they divide. The formation of the mitotic spindle, where the cell?s network of microtubule fibres is completely rearranged to span from either end to the chromosomes at the centre, is particularly striking. This spindle is a molecular machine that ensures the cell?s chromosomes are accurately distributed between its two daughter cells. Errors in the workings of the spindle are a known driving force of cancer and are also responsible for a congenital brain disease. Several control mechanisms ensure the mitotic spindle is normally assembled correctly. At an early stage in assembly, two proteins called TACC3 and ChTOG promote microtubule stability and hence promote assembly. These proteins are more effective when TACC3 is modified by a phosphate group: one phosphorous atom and three oxygen atoms that is commonly used by cells to alter the activity of their proteins. In the case of TACC3, the protein that adds the phosphate is called Aurora-A. Spindle assembly is thus controlled by the activity of Aurora-A, which is itself controlled by many other proteins under the influence of events within and outside the cell. We propose to investigate how the phosphate group influences the effectiveness of the TACC3/ChTOG partnership at the level of atoms. How this works is currently a mystery as the phosphate is only four atoms big, and yet it changes the activity of TACC3/ChTOG which total tens of thousands of atoms. We will use electron microscopy, a technique that allows us to see directly the shapes of proteins, to study the changes in TACC3 upon phosphorylation, and the effect on ChTOG. We will also use X-ray crystallography to determine the location of every atom within the proteins and to map the atoms by which TACC3 and ChTOG cooperate. This information will allow us to make a hypothesis for the details of how the TACC3/ChTOG partnership works and how phosphorylation enhances their effectiveness. We will use our protein structure models to design subtle modifications to TACC3 and ChTOG to test this hypothesis in human cells grown in culture. An overabundance of TACC3, ChTOG or Aurora-A have been linked with cancer, and TACC3 and Aurora-A are also important in brain development. These studies will provide the impetus for future investigations to understand the role of these proteins in human disease.

At the entry to mitosis microtubules assemble into a bipolar mitotic spindle, a molecular machine for the accurate segregation of chromosomes in cell division. Improper mitotic spindle function is a driver of carcinogenesis and is implicated in autosomal recessive primary microcephaly, a congenital brain disease. Mitotic spindle assembly is mediated by the interactions of microtubules with many scaffolding and motor proteins. These interactions are regulated by protein kinases. For example, the conserved assembly-promoting TACC3 / ChTOG protein complex is regulated by Aurora-A kinase. Phosphorylation of TACC3 by Aurora-A enhances the centrosomal localisation of the protein and increases the assembly-promoting activity of ChTOG. We propose to determine the structural mechanism of TACC3 / ChTOG phospho-regulation by Aurora-A and verify the mechanism in human tissue culture cells. The first aim of our proposal is to address whether Aurora-A induces a conformational change in TACC3, and deduce the nature of the change. We will apply biochemical and biophysical techniques such as circular dichroism spectroscopy, analytical ultracentrifugation and multi-angle light scattering to compare the structure and oligomerisation state of phosphorylated and unphosphorylated TACC3. We plan to solve the TACC3 structure using X-ray crystallography. Our second aim is to determine the structural basis of the TACC3/ChTOG interaction and devise a model for the mechanism of ChTOG regulation by TACC3. We will use X-ray crystallography to determine a high resolution structure of the minimal TACC3 / ChTOG complex. We will use electron microscopy to visualise longer fragments of the proteins, and place the high resolution structure in the context of the longer proteins. Electron microscopy data will be analysed to define structural changes upon complex formation and/or phosphorylation. Putting this information together, we will devise a structural model for the mechanisms of TACC3 / ChTOG regulation and function. Our third aim is to validate the structural mechanisms and models in human tissue culture cells. Our structural studies will inform the design of fragments or point mutants of TACC3 and ChTOG to interfere with their function and regulation. We will use these mutants to test our structural models in human tissue culture cells, a model system in which TACC3 / ChTOG function and regulation is poorly understood. Notably, overexpression of all three proteins has been linked with cancer. The structural mechanisms, models and reagents we produce will be invaluable in understanding the role of these proteins in human disease.