title: Kinetochore-microtubule attachment relies on the disordered N-terminal tail domain of Hec1.
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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523-1870, USA.
authors.forename: Geoffrey J
authors.initials: GJ
authors.lastname: Guimaraes
authors.forename: Yimin
authors.initials: Y
authors.lastname: Dong
authors.forename: Bruce F
authors.initials: BF
authors.lastname: McEwen
authors.forename: Jennifer G
authors.initials: JG
authors.lastname: Deluca
mesh_terms: D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D049452:Green Fluorescent Proteins / Q000032:analysis; D018386:Kinetochores / Q000378:metabolism* / Q000648:ultrastructure; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D048328:Potoroidae; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000032:analysis
chemical_list: D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins