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.

SUBMITTER: Cheeseman LP 

PROVIDER: S-EPMC3666260 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature