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 min) 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 of kinetochore dynamics. Our results indicate both that TACC3/ch-TOG/clathrin complexes are important for the maintenance of spindle structure and function, in addition to initial spindle assembly.