GTSE1 interacts directly with MCAK and inhibits its MT depolymerase activity in vitro. (A) Immunoblot (IB) showing MCAK pulled down in an in vitro GST pulldown using GST alone, GST-GTSE1 1–460, and GST-GTSE1 463–739 fragments. Input represents 2.5% of total MCAK protein used for the GST pulldown assay. (B) Kymograph depicting 50 nM MCAK depolymerizing a GMPCPP-stabilized MT (Video 5). The black dashed line represents the start of the experiment when MCAK was added. The second kymograph depicts a GMPCPP-stabilized MT maintaining constant length in the presence of 50 nM MCAK plus 250 nM GTSE1 (Video 6). The black dashed line represents the start of the experiment when MCAK and GTSE1 were added. Depolymerization rates of GMPCPP-stabilized MTs in the presence of 50 nM MCAK alone, 50 nM MCAK with a fivefold excess of GTSE1, and 50 nM MCAK with an equimolar amount of GTSE1 are shown in the box plot. n > 100 MTs; 4 experiments. (C) Kymographs of MT growth and catastrophe in the presence of 20 µM tubulin (Tb) alone or 20 µM tubulin and 250 nM GTSE1 from GMPCPP-MT seeds. Box plot shows the shrinkage rate after catastrophe of 20 µM tubulin alone and 20 µM tubulin with 250 nM GTSE1. n = 34 and 90 MTs, respectively.