Figure 1

Phosphorylation of MCAK on Ser715 improves its MT-binding affinity.

(a) MT plus-end location of GFP-MCAK^WT or its phosphorylation site mutants in HeLa cells depleted of endogenous MCAK. Time-lapse imaging was performed at 3 sec intervals. Left column is the control for MCAK shRNA transfection as indicated by mCherry-H2B. The second column shows the single frame from the time-lapse images as grayscale. The third column displays projection of 6 consecutive frames, respectively shown as the sequence of red, green and blue. Fluorescence profile of MCAK^WT or its mutants along MT plus-end is shown on the right. Data are presented as means ± SD derived from at least 60 MT ends for each condition. (b) EB1-binding activity of GFP-MCAK^WT and its mutants. GST-EB1 was used as an affinity matrix to isolate the indicated GFP fusions from 293T cell extracts. Quantitative analysis of the relative binding affinity by anti-GFP blotting and Coomassie Brilliant Blue (CBB) staining indicated that EB1 bound less to MCAK^S715E. ***P < 0.001, Student’s t-test. For full blots, see Supplementary Fig. S1e. (c) MT cosedimentation analysis of MCAK^WT or its mutants (1 μM) with increasing concentrations of Taxol-stabilized MTs. Equal volumes of supernatant (S) and pellet (P) were run on SDS-PAGE gels and stained by CBB. (d) Graphs showing the MT-binding curves of MCAK^WT, MCAK^S715A and MCAK^S715E. Data represent the average amounts from two independent experiments. (e) Imaging the binding affinity of MCAK^WT or its mutants for MTs in vitro. Rhodamine-labelled GMPCPP-MTs were immobilized to the surface of coverslips and GTP-MTs were then polymerized by addition of tubulins and GTP. Equal concentration of GFP-His-tagged MCAK^WT or its mutants (4 nM) were separately introduced into the chambers and their binding activities for MTs were then observed by total internal reflection fluorescence (TIRF) microscope. Scale bars, 5 μm (all image panels).