Through analyzing the spindle matrix proteome, which we defined in our previous funding periods, we have made a number of exciting observations. Most importantly we have demonstrated that the spindle matrix protein, BuGZ, undergoes oligomerization via phase transition or coacervation to form liquid droplets in vitro mediated in part by the hydrophobic residues found in the intrinsically disorder region of BuGZ. The coacervation activity of BuGZ is essential for assembly of the spindle and its matrix. BuGZ coacervation in vitro as pure protein or in the spindle matrix concentrates tubulin and promotes MT assembly and bundling. Our published and unpublished findings suggest that the interaction and/or coacervation of BuGZ and another spindle assembly factor, TPX2, promotes spindle assembly. Here we propose to use biophysical, biochemical, and cell biological approaches to dissect the molecular mechanism by which these spindle assembly factors synergize to regulate Aurora A activation, microtubule assembly, and kinetochore-microtubule interactions in mitosis.
Most studies of spindle assembly have focused on how spindle microtubules interact with the kinetochores to ensure proper chromosome segregation. Many observations, however, have shown that an amorphous assemblage besides the spindle microtubules also contribute toward spindle morphogenesis and chromosome segregation. Historically, this assemblage has been called the spindle matrix. In the last over 10 years, my lab has developed biochemical assays to define and characterize the proteins in the spindle matrix. We have shown that a number of spindle matrix proteins are required for proper spindle assembly. Yet the major challenge has been to understand the structural principle of spindle matrix assembly. By studying one of the spindle matrix proteins, we have recently demonstrated that protein oligomerization via phase separation or coacervation is required for spindle matrix assembly. With our newly identified molecular handles and biochemical assays, we propose to further dissect how spindle assembly factors synergize via coacervation in spindle matrix to promote spindle functions. This proposal addresses an area of cell division that has received much less attention than the other areas of mitosis studies. We aim to uncover novel insights about spindle assembly mechanisms that had not been considered previously. Since the spindle matrix proteome contains signaling molecules and cell fate determinants, we believe understanding how the spindle matrix interacts with microtubules during cell division will provide new avenues to explore therapeutic targets for halting uncontrolled cell divisions.
