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J Cell Sci. 2013 May 1;126(Pt 9):2102-13. doi: 10.1242/jcs.124834. Epub 2013 Mar 26.

Specific removal of TACC3-ch-TOG-clathrin at metaphase deregulates kinetochore fiber tension.

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Department of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Crown Street, Liverpool L69 3BX, UK.


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.


Checkpoint; Knocksideways; Microtubule; Mitotic spindle; Rapid inactivation

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