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

LP Cheeseman, EF Harry, AD McAinsh… - Journal of cell …, 2013 - journals.biologists.com
Journal of cell science, 2013journals.biologists.com
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 …
Summary
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.
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