Molecular analysis of Hec1 kinetochore protein for accurate chromosome segregation during mitosis
My dissertation focuses on the kinetochore protein Hec1, which is essential for cell survival and is required for chromosome segregation. Hec1 plays an important role in proper kinetochore architecture, spindle checkpoint signaling and microtubule recruitment at kinetochores through its interaction with other proteins. Here, I demonstrate that Hec1 directly interacts with Zwint-1 and a novel protein 15A2. Zwint-1 is a kinetochore protein and interacts with ZW10, which is required for chromosome mobility and spindle checkpoint. My study shows that Hec1 is required for the kinetochore localization of both Zwint-1 and ZW10 to perform their functions. Disrupting this recruitment by inhibiting the expression of Hec1 or Zwint-1 causes chromosome missegregation, spindle checkpoint failure, and eventual cell death upon cytokinesis. Therefore, Hec1 is a critical component to assemble fully functional kinetochores, making it a key player in assuring accurate chromosome segregation and spindle checkpoint control.
In addition, kinetochores generate a microtubule-attaching interface that links chromosomes to the mitotic spindle. Hec1 is indispensable for stable microtubule attachment at kinetochores, although the precise molecular mechanism is unknown. Here, I show that Hec1 interacts with 15A2, which is associated with microtubules and centrosomes throughout the cell cycle. Depletion of 15A2 by RNA interference leads to a prometaphase-like stage with mis-aligned chromosomes and to an increased mitotic index. These phenotypes associated with by 15A2 depletion suggest that 15A2 may be involved in kinetochore-microtubule attachment during mitosis. In Hec1-depleted cells, similar phenotypes are also observed. Importantly, Hec1 interacts with 15A2 specifically at M phase in vivo, suggesting that they have an overlapping role in the interface of kinetochores and microtubules. Thus, these results, at least in part, demonstrate how Hec1 and 15A2 play a critical role in mediating stable kinetochore-microtubule attachment. (Abstract shortened by UMI.)
0307: Molecular biology