(A) Zoom-in view of the atomic model of the TPX2 ridge (left) and wedge (right). (B) Schematic of TPX2micro indicating the ‘ridge’ and the ‘wedge’ regions as well as the residues that were mutated to test for MT interaction. (C) TIRFM images depicting mGFP-TPX2micro (green) binding to growing Atto565-labeled MTs (magenta). Yellow arrowheads indicate the GMPCPP-‘seed’ region. (D–E) TIRFM images depicting mutant mGFP-TPX2micro (green) binding to MTs (magenta). Tubulin and mGFP-TPX2micro concentrations were 15 μM and 1 µM, respectively. Note that in all cases background subtracted 25-frame averages are shown to allow visualization of the differences between the faint signals of the mutants on the MT lattice. (F) Box-and-whiskers plot depicting average fluorescence intensity measurements for mGFP-TPX2micro GMPCPP MT ‘seed’ binding comparing wild-type and mutant proteins. The boxes extend from 25th to 75th percentiles, the whiskers extend from minimum to maximum values, and the mean value is plotted as a line in the middle of the box. 500 timeframes were averaged for each MT ‘seed’. Number of ‘seeds’ analyzed: WT – 18, F307A – 30, F307E – 25, F334A H335A – 25, F334E H334 – 29, F307A F334A H335E – 29, F307E F334E H335E – 24.