title: Specific removal of TACC3-ch-TOG-clathrin at metaphase deregulates kinetochore fiber tension.
abstract: Microtubule-associated proteins of the mitotic spindle are thought to be important for the initial assembly and the maintenance of spindle structure and function. However, distinguishing assembly and maintenance roles for a given protein is difficult. Most experimental methods for protein inactivation are slow and therefore affect both assembly and maintenance. Here, we have used 'knocksideways' to rapidly (∼5 minutes) and specifically remove TACC3-ch-TOG-clathrin non-motor complexes from kinetochore fibers (K-fibers). This method allows the complex to be inactivated at defined stages of mitosis. Removal of TACC3-ch-TOG-clathrin after nuclear envelope breakdown caused severe delays in chromosome alignment. Inactivation at metaphase, following a normal prometaphase, significantly delayed progression to anaphase. In these cells, K-fiber tension was reduced and the spindle checkpoint was not satisfied. Surprisingly, there was no significant loss of K-fiber microtubules, even after prolonged removal. TACC3-ch-TOG-clathrin removal during metaphase also resulted in a decrease in spindle length and significant alteration in kinetochore dynamics. Our results indicate that TACC3-ch-TOG-clathrin complexes are important for the maintenance of spindle structure and function as well as for initial spindle assembly.
authors.affiliation: Department of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Crown Street, Liverpool L69 3BX, UK.
authors.forename: Liam P
authors.initials: LP
authors.lastname: Cheeseman
authors.forename: Edward F
authors.initials: EF
authors.lastname: Harry
authors.forename: Andrew D
authors.initials: AD
authors.lastname: McAinsh
authors.forename: Ian A
authors.initials: IA
authors.lastname: Prior
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Royle
mesh_terms: D002877:Chromosomes, Human / Q000235:genetics / Q000378:metabolism*; D002966:Clathrin / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D018386:Kinetochores / Q000378:metabolism*; D008677:Metaphase / Q000502:physiology*; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D008870:Microtubules / Q000235:genetics / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D008941:Spindle Apparatus / Q000235:genetics / Q000378:metabolism*
chemical_list: D002966:Clathrin; D008869:Microtubule-Associated Proteins; D046912:Multiprotein Complexes; C120551:TACC3 protein, human
keywords: Checkpoint; Knocksideways; Microtubule; Mitotic spindle; Rapid inactivation