Video 6.
TPX2, Aurora A, WDR62, and katanin form an efficient module to sever GDP-MTs in vitro. MTs were first polymerized in the tubulin polymerization reaction mixture (20 µM unlabeled tubulin and 0.5 µM rhodamine-tubulin in MRB80 buffer) supplemented with 30 nM WDR62-GFP and 30 nM (top panels) or 60 nM (bottom panels) TPX2/TagBFP–Aurora A (AurA). After reaction for 10 min on the microscope stage (30°C), the sample in the unsealed flow chamber was imaged for ∼1 min. Subsequently, the second-round reaction mixture, which contained 10 µM tubulin together with 30 nM p60/SNAP-Alexa Fluor 647–p80, 30 nM WDR62-GFP, 30 nM (top panels) or 60 nM (bottom panels) TPX2/TagBFP–Aurora A, and 1 mM ATP was gently flowed into the chamber. Images were collected with a TIRF microscope at 10-s intervals. Video is sped up 60 times (6 frames/s). Time is shown in the format min:s.