Fig. 1: HURP recruits tubulin and regulates microtubule dynamics.
From: HURP binding to the vinca domain of β-tubulin accounts for cancer drug resistance
a Domain structures of HURP. The microtubule-binding domain is colored black. Residue numbers indicate domain boundaries of HURP. b Schematic representation of a kymograph illustrating microtubule growth events from a microtubule seed. Microtubule seed is colored by red and dynamic microtubule is shown by blue. Microtubule growth events, including polymerization, depolymerization, catastrophe and rescue, are indicated. Plus and minus ends of microtubules are indicated. c–e Representative kymographs show microtubules grown from X-rhodamine-labeled GMPCPP-stabilized seeds with HiLyte FluorTM647 tubulin in the (c) absence and presence of 500 nM (d) GFP-HURP (1-280) and (e) GFP-HURP FL. Vertical scale bar = 90 sec, and horizontal scale bar = 10 μm. f–j Quantification of microtubule dynamicity, including (f) polyermization rate, (g) depolymerization rate, (h) catastrophe frequency, (i) rescue frequency and (j) microtubule length, as a function of the indicated GFP-HURP (1-280) and GFP-HURP FL concentrations. Data are mean ± SD (Standard deviation) from three independent experiments (n = 25 (Control), 17 (GFP-HURP (1-280) and 26 (GFP-HURP FL) microtubules). Two-tailed Student’s t test; Statistical differences: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., not significant. k A summary of recruitment of tubulin to the microtubule in the presence of HURP. X-rhodamine-labeled microtubules were immobilized on the glass surface. GFP-HURP (indicated as HURP; GFP is shown as a black dot) and HiLyte FluorTM 647 labeled tubulin (indicated as α and β; HiLyte FluorTM647 is shown as an open dot) were imaged using TIRF microscopy. l, m GMPCPP-stabilized microtubules (X-rhodamine-labeled) were incubated with HiLyte FluorTM647 tubulin in the (l) absence and (m) presence of GFP-HURP FL. Scale bars, 10 μm. n HiLyte FluorTM 647 intensity for each condition (arbitrary units (arb. units)) was analyzed and plotted. Data are mean ± SD from three independent experiments (n = 99 microtubules for each condition). Two-tailed Student’s t test; Statistical differences: ****, P < 0.0001.
