Hec1 is a critical structural component at the kinetochore outer layer, where it provides a major microtubule attachment site for establishing a stable bipolar spindle. Depletion of Hec1 results in defective spindle assembly checkpoint (SAC), unstable kinetochore-microtubule attachment, and multipolar mitotic spindles. Using siRNA technology and retrovirus infection, I am able to replace endogenous Hec1 with siRNA resistant Hec1 point mutants and deletion mutants. This allows me to precisely identify the amino acid residue or regions of Hec1 responsible for its role in spindle assembly checkpoint, bipolar spindle formation, and kinetochore targeting, and to elucidate the mechanism by which it is achieved.

First, I discovered that Nek2-dependent phosphorylation of Hec1 S165 was required to recruit SAC proteins Mad1 and Mad2 to the kinetochores. Dephosphorylation mediated by protein phosphatase 1 is most likely responsible for dephosphorylation Hec1 S165, thus allowing for anaphase onset. In addition, I demonstrated that Hec1 and Hice1 contributes to centrosome-dependent microtubule growth during mitosis and their interaction at the centrosome is required for bipolar spindle formation. Lastly, Hec1's coiled-coil domain, which accounts for two-thirds of the encoded protein, functions as a scaffold for kinetochore-associated proteins. To identify novel Hec1 functions mediated by the coiled-coil domain, we performed a structure-function analysis of Hec1's first coiled-coil domain by generating a series of GFP-tagged deletion mutants spanning the entire first coiled-coil domain of Hec1 in which two consecutive leucine heptad repeats were removed at a time. Surprisingly, we identified two consecutive leucine heptad repeats that were required for Hec1 localization to the kinetochore. Consistently, this Hec1 deletion mutant has a dramatic reduction in its association with Nuf2, Spc24, and Spc25 compared to wildtype Hec1.