Kinetochore-microtubule attachment relies on the disordered N-terminal tail domain of Hec1

Curr Biol. 2008 Nov 25;18(22):1778-84. doi: 10.1016/j.cub.2008.08.012.

Abstract

Accurate chromosome segregation is dependent upon stable attachment of kinetochores to spindle microtubules during mitosis. A long-standing question is how kinetochores maintain stable attachment to the plus ends of dynamic microtubules that are continually growing and shortening. The 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. We show that the 80 amino acid N-terminal unstructured "tail" of Hec1 is required for generating stable kinetochore-microtubule attachments. PtK1 cells depleted of endogenous Hec1 and rescued with Hec1-GFP fusion proteins deleted of the entire N terminus or the disordered N-terminal 80 amino acid tail domain fail to generate stable kinetochore-microtubule attachments. Mutation of nine amino acids within the Hec1 tail to reduce its positive charge also abolishes stable attachment. Furthermore, the mitotic checkpoint remains functional after deletion of the N-terminal 80 amino acid tail, but not after deletion of the N-terminal 207 amino acid region containing both the tail domain and a calponin homology (CH) domain. These results demonstrate that 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Binding Sites
  • Cell Line
  • Green Fluorescent Proteins / analysis
  • Kinetochores / metabolism*
  • Kinetochores / ultrastructure
  • Microtubules / metabolism*
  • Microtubules / ultrastructure
  • Mutation
  • Nuclear Proteins / chemistry*
  • Nuclear Proteins / metabolism
  • Nuclear Proteins / physiology
  • Potoroidae
  • Protein Structure, Tertiary
  • RNA Interference
  • Recombinant Fusion Proteins / analysis

Substances

  • Nuclear Proteins
  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins