Uncovering the Mechanisms that Control Unattached Kinetochore Clustering

Abstract

The equal partitioning of the genome during cell division is essential for the survival of an organism. Errors in this process are hallmarks of many human cancers. Central to ensuring faithful chromosome segregation are kinetochores, megadalton protein complexes that form on duplicated sister chromatids during cell division. Kinetochores bridge the connection between chromosomes and the microtubules of the mitotic spindle which generate the forces necessary to pull chromosomes to opposite sides of the cell. A challenge in this process is that sister chromatids must attach to microtubules from opposite spindle poles, a process called biorientation. Kinases play an essential role in this process. Mps1 is a kinase that halts the cell cycle until all kinetochores are bioriented through signaling the spindle assembly checkpoint (SAC). Mps1 phosphorylates Spc105 at its MELT motifs which recruits the Bub3:Bub1 and Mad1:Mad2 complexes to kinetochores to catalyze the formation of a cell cycle inhibitor. Kinetochores signaling the SAC also recruit Stu1 and Slk19, two spindle proteins involved in microtubule stability and crosslinking. Studies suggest Stu1 and Slk19 are involved in promoting the capture of kinetochores to microtubules, but how they are recruited and regulated remains unknown. Here, I investigated the role of Stu1 and Slk19 at unattached kinetochores and mechanisms that promote their recruitment. I found that Stu1 and Slk19 act to cluster unattached kinetochores and this depends on Mps1 activity. Unexpectedly, I found that Stu1 contains conserved MELT motifs like Spc105 that are phosphorylated by Mps1 to promote Slk19 binding. Preventing phosphorylation of the MELTs prevents Stu1 and Slk19 localization to kinetochores and disrupts kinetochore clustering. My work underscores the importance of Mps1 as the master regulator of unattached kinetochores. In addition to controlling kinetochore clustering and the SAC, phosphorylation plays important roles in kinetochore assembly and error correction, of which only a handful have been studied in detail. To further explore the importance of phosphorylation in kinetochore regulation, I have compiled a list of > 500 phosphorylation events that my colleagues and I have detected in vivo from the purification of kinetochore and kinetochore adjacent complexes by mass spectrometry. Using computational methods, I predicted the kinases that are likely responsible for each phosphorylation event. Together, these findings will further accelerate the discoveries of other key phosphorylation events at the kinetochore.