title: Abnormal kinetochore-generated pulling forces from expressing a N-terminally modified Hec1.
abstract: BACKGROUND
  Highly Expressed in Cancer protein 1 (Hec1) is a constituent of the Ndc80 complex, a kinetochore component that has been shown to have a fundamental role in stable kinetochore-microtubule attachment, chromosome alignment and spindle checkpoint activation at mitosis. HEC1 RNA is found up-regulated in several cancer cells, suggesting a role for HEC1 deregulation in cancer. In light of this, we have investigated the consequences of experimentally-driven Hec1 expression on mitosis and chromosome segregation in an inducible expression system from human cells.
  
  
  RESULTS
  Overexpression of Hec1 could never be obtained in HeLa clones inducibly expressing C-terminally tagged Hec1 or untagged Hec1, suggesting that Hec1 cellular levels are tightly controlled. On the contrary, a chimeric protein with an EGFP tag fused to the Hec1 N-terminus accumulated in cells and disrupted mitotic division. EGFP- Hec1 cells underwent altered chromosome segregation within multipolar spindles that originated from centriole splitting. We found that EGFP-Hec1 assembled a mutant Ndc80 complex that was unable to rescue the mitotic phenotypes of Hec1 depletion. Kinetochores harboring EGFP-Hec1 formed persisting lateral microtubule-kinetochore interactions that recruited the plus-end depolymerase MCAK and the microtubule stabilizing protein HURP on K-fibers. In these conditions the plus-end kinesin CENP-E was preferentially retained at kinetochores. RNAi-mediated CENP-E depletion further demonstrated that CENP-E function was required for multipolar spindle formation in EGFP-Hec1 expressing cells.
  
  
  CONCLUSIONS
  Our study suggests that modifications on Hec1 N-terminal tail can alter kinetochore-microtubule attachment stability and influence Ndc80 complex function independently from the intracellular levels of the protein. N-terminally modified Hec1 promotes spindle pole fragmentation by CENP-E-mediated plus-end directed kinetochore pulling forces that disrupt the fine balance of kinetochore- and centrosome-associated forces regulating spindle bipolarity. Overall, our findings support a model in which centrosome integrity is influenced by the pathways regulating kinetochore-microtubule attachment stability.
authors.affiliation: Institute of Molecular Biology and Pathology, National Research Council of Italy, Rome, Italy.
authors.forename: Marta
authors.initials: M
authors.lastname: Mattiuzzo
authors.forename: Giulia
authors.initials: G
authors.lastname: Vargiu
authors.forename: Pierangela
authors.initials: P
authors.lastname: Totta
authors.forename: Mario
authors.initials: M
authors.lastname: Fiore
authors.forename: Claudio
authors.initials: C
authors.lastname: Ciferri
authors.forename: Andrea
authors.initials: A
authors.lastname: Musacchio
authors.forename: Francesca
authors.initials: F
authors.lastname: Degrassi
mesh_terms: D001696:Biomechanical Phenomena; D002460:Cell Line; D002868:Chromosomal Proteins, Non-Histone / Q000502:physiology; D003598:Cytoskeletal Proteins; D015972:Gene Expression Regulation, Neoplastic; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D018386:Kinetochores / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism; D008938:Mitosis; D050505:Mutant Proteins; D009687:Nuclear Proteins / Q000235:genetics* / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis; D011993:Recombinant Fusion Proteins; D008941:Spindle Apparatus / Q000378:metabolism
chemical_list: D002868:Chromosomal Proteins, Non-Histone; D003598:Cytoskeletal Proteins; D050505:Mutant Proteins; C120529:NDC80 protein, human; D009687:Nuclear Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C068688:centromere protein E; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins