Fig. 5: HURP preferentially binds the GDP-lattice of microtubules.

A Schematic of barcoded porcine-tubulin microtubule with the GMPCPP-, GTPγS- and GDP-tubulin sections (top), along with corresponding fluorescence channels of GDP- (HiLyte647), GTPγS- (HiLyte488) tubulin sections (unlabelled GMPCPP-tubulin, see “Methods”) and TagRFP-HURP (bottom); scale bars = 1.5 µm. B Boxplots of the TagRFP-HURP intensity/µm normalised to its median intensity on GMPCPP microtubules (N = 3 flow chambers; n = 171 (GMPCPP), 300 (GTPγS) and 238 (GDP); P = Kruskal–Wallis test). C Representative image of porcine-tubulin GMPCPP seeds extended with either human WT- (green) or E254A tubulin (magenta), labelled with HiLyte488 and HiLyte647 porcine brain tubulin (1:7 labelled:unlabelled ratio) respectively (right panel) and incubated with TagRFP-HURP (left panel); scale bars = 3 µm. D Boxplots of HURP intensity/µm normalised to its median intensity on GMPCPP seeds (N = 3 flow chambers; n = 121 (GMPCPP), 43 (WT) and 87 (E254A tubulin); P = Kruskal–Wallis test). E Immunofluorescence images of metaphase hTERT-RPE1 cells transducted either with RFP-wt-α-tubulin or RFP-E254A-α-tubulin and stained for EB1. Scale bars = 5 µm. F Live-cell imaging of hTERT-RPE1 EGFP-HURP/HaloTag-CENP-A cells transducted either with RFP-wt-α-tubulin or RFP-E254A-α-tubulin mutant (left), Z-projections of 5 × 0.5 µm, Scale bar = 5 µm; and boxplots of in vivo relative HURP intensities (right). N = 2; n = 38 and 42 cells. Boxplots indicate the 25th and 75th percentiles, the bars are medians, and the whiskers indicate values within 1.5 times the interquartile range in B, D, and minima and maxima in F; P = two-sided Mann–Whitney test. Source data for all graphs are provided as a Source Data file.