Figure 6.

CLIP-170 H1 phosphomimetics promote MT rescues in vitro. Recombinant His-sfGFP^T187A-H1 in its WT or in its T25E-T45E-S147E (EEE) versions was added or not to purified tubulin in in vitro MT dynamics assays monitored by TIRF microscopy. (A) The sketch at the top indicates that MT (yellow, tubulin-TAMRA and His-sfGFP^T187A-H1) elongation and dynamics were initiated at GMPCPP seeds (cyan, tubulin-HiLyte 647) and took place on kinesin-1 heavy chain (KHC) kept in nonmotile conformation in the presence of AMPPNP. In the assay, His-sfGFP^T187-H1 phosphomimetic (EEE) binding was weaker than that of the WT form to GMPCPP seeds. Intensity profile of the images shown are plotted below, and the normalized values of GFP fluorescence/micrometer ± SD are shown on the right panel. (B) Sample kymographs derived from recordings of the assay in A show the occurrence of rescues at MT plus ends (magenta arrows). The cyan dashed lines correspond to the end of the GMPCPP seeds. The diamond graph shows the mean values of each dynamic instability parameter after normalization relative to WT. The values of overall rescue frequencies are reported in box plots showing representative percentiles and outliers. The numerical mean values ± SD of each parameter are shown, but also the numbers of MTs and rescues are detailed in Table S7. The statistical comparisons were performed using one-factor ANOVA followed by Fisher's protected t tests for pairwise comparisons (Table S8). n indicates the number of independent experiments. ****, P < 0.0001. Scale bars are indicated for images and kymographs. ns, not significant.