Structural and functional studies on mitotic spindle orientation in <em>Saccharomyces cerevisiae</em>. &nbsp;

Accurate positioning of the mitotic spindle relative to the plane of cytokinesis ensures proper distribution of the genetic material and of cell fate determinants. Failure in spindle orientation and migration can cause severe defects such as aberrant chromosome numbers, which is often correlated with the development of cancer. In all eukaryotes, spindle positioning is accomplished through interactions between cytoplasmic microtubules, actin filaments and the cell cortex. Many of the relevant proteins are conserved from yeast to human and the in vivo functions of the proteins are well understood in yeast. Therefore, budding yeast is a powerful model system to investigate the molecular details and control mechanisms required for accurate spindle positioning. In yeast, the spindle is aligned along the axis of division by a complex consisting of the type-V myosin Myo2p, the adapter Kar9p and the microtubule-binding protein Bim1p. The goal of this study was to elucidate the molecular principles underlying mitotic spindle positioning in budding yeast. In order to reach this goal, different structural techniques such as X-ray crystallography and small angle X-ray scattering (SAXS) were combined with various in vitro and in vivo assays to study the regulation of the Kar9p-Bim1p interaction. In this study, crystals of full-length Kar9p and Kar9p variants were obtained, but the X-ray diffraction properties were not sufficient to solve a three-dimensional structure. Yet, the crystal structure of the C-terminal cargo-binding domain of Bim1p was determined. The crystal structure revealed that the C-terminal domain of Bim1p is formed by a dimer. SAXS analysis confirmed that Bim1p is dimeric in solution. Structure-guided mutational analysis of Bim1p resulted in the identification of residues that either reduced or increased the interaction with Kar9p. Remarkably, the residues involved in cargo-binding in Bim1p are within a highly conserved region on the surface of the protein. Other studies have shown that this highly conserved region mediates cargo binding in the Bim1p homolog EB1, suggesting that both proteins interact with their cargo in a similar manner. To gain further insight into the regulation of complex assembly, the role of posttranslational modifications of both proteins was analysed. Sumoylation of Kar9p is required for binding to Bim1p, whereas phosphorylation of Kar9p is not. Furthermore, a yet unknown binding region for Kar9p in Bim1p was identified in the unstructured middle region. The middle region of Bim1p was only capable of Kar9p binding when the protein was dimerised and it was shown that the interaction via this region is regulated by phosphorylation by Aurora B. These results in combination with findings from other studies lead to a model that explains the cell-cycle dependent regulation of the assembly and disassembly of the Myo2p-Kar9p- Bim1p complex. Complex assembly during metaphase is regulated by sumoylation of Kar9p, whereas the complex disassembly in late anaphase is regulated by phosphorylation of Bim1p by Aurora B