Kinetochore-Microtubule Attachment Relies on the Disordered N-Terminal Tail Domain of Hec1

Abstract

SUMMARY Accurate chromosome segregation is dependent upon kinetochores stably attaching chromosomes to spindle microtubules during mitosis. A long-standing question is how kinetochores are able to maintain stable attachment to the plus-ends of dynamic microtubules that are continually growing and shrinking. The hetero-tetrameric Ndc80 complex is essential for persistent, end-on kinetochore-microtubule attachment in cells [1,2], but how the Ndc80 complex forms functional microtubule binding sites remains unknown. Here we show the 80 amino acid N-terminal “tail” of Hec1 is a key domain required for generating stable kinetochore-microtubule attachments in cells. PtK1 cells depleted of endogenous Hec1 and rescued with Hec1-GFP fusion proteins deleted of the entire N-terminal tail domain or the disordered N-terminal 80 amino acid tail domain fail to generate stable kinetochore-microtubule attachments. Mutation of 9 amino acids within the Hec1 tail domain to reduce its positive charge also abolishes stable kinetochore-microtubule attachment. Furthermore, the mitotic checkpoint remains functional after deletion of the N-terminal 80 amino acids, but not after deletion of the N-terminal 207 amino acid calponin homology (CH) domain. These results demonstrate kinetochore-microtubule binding is dependent on electrostatic interactions mediated through the disordered N-terminal 80 amino acid tail domain and mitotic checkpoint function is dependent on the CH domain of Hec1.