Phosphorylation of MCAK on Ser715 stimulates its MT depolymerization activity.
(a) Pi release displayed over time in reactions containing 1 mM ATP and 320 nM MCAKWT or its mutants. The data of three independent experiments was fitted to two-phase exponential curves. (b) Representative images of MTs incubated with 4 nM MCAKWT or its mutants. Rhodamine-labelled GMPCPP-MTs (1 μM) were incubated with FLAG-MCAKWT or its mutants for 10 min at 25 °C. MTs were then examined by TIRF microscope at the indicated time. Scale bar, 10 μm. (c) Plot of MT depolymerization against MCAK concentration. Ratio of MT length at 10 min (post-reaction) over that at 0 min (pre-reaction) was used to reflect MT depolymerization. (d) Real-time images of MT depolymerization in the presence of MCAKWT or its mutants. Rhodamine-labelled GMPCPP-stabilized MTs were immobilized as described in Fig. 1e. FLAG-MCAK (2 nM) was then added to initialize MT depolymerization. Scale bar, 1 μm. (e) Statistical analysis of the average rate of MT depolymerization during a 5 minutes interval (d). Data are presented as means ± SD. At least 40 MTs were measured for each condition. *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t-test. (f) Negative stained transmission electron micrographs showing the microtubules in the presence of MCAKWT or its mutants. GMPCPP-MTs were incubated with 2 nM MCAK for 5 min at 25 °C. Insets show details of MT ends. Scale bars, 100 nm and 20 nm (enlarged images). (g) Statistical analyses of different structures of MT ends seen in (f). The blunt ends were considered to be stabilized, while the pared ends were seen as shrinking ends (see also Supplementary Table S2). Data are derived from approximately 60 MT ends for each condition. (h) Images of MT density in HeLa cells overexpressing GFP-MCAKWT or its mutants. At 48 hr post-transfection, cells were fixed with methanol and stained with anti-α-tubulin antibody (red) and DAPI (blue). Scale bar, 20 μm. (i) Statistical analysis of relative MT intensity in (h). Data are presented as means ± SE and derived from at least 60 cells in three independent experiments. **P < 0.01, Student’s t-test.