Phosphorylation of MCAK on Ser715 improves its MT-binding affinity.
(a) MT plus-end location of GFP-MCAKWT 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 MCAKWT 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-MCAKWT 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 MCAKS715E. ***P < 0.001, Student’s t-test. For full blots, see Supplementary Fig. S1e. (c) MT cosedimentation analysis of MCAKWT 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 MCAKWT, MCAKS715A and MCAKS715E. Data represent the average amounts from two independent experiments. (e) Imaging the binding affinity of MCAKWT 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 MCAKWT 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).