FIGURE 1.

Endogenous HURP dynamics after photobleaching half of the mitotic spindle. (A) Left: Representative image of HeLa Kyoto cell endogenously expressing eGFP-HURP, arrested in metaphase. Immunofluorescence was performed against α-tubulin and DNA was counterstained with Hoechst. Right: Endogenous eGFP-HURP and α-tubulin intensity plot profiles along HeLa Kyoto metaphase spindle long axis. Bold-lines indicate the average of n = 16 cells, and the green dashed-lines indicate the 95% CI. (B) Schematic representation of a FRAP experiment performed on HeLa Kyoto cells endogenously expressing eGFP-HURP, counterstained with SiR-tubulin and arrested in metaphase. Tubulin signal was used as a spatial reference for drawing a polygon (bleaching geometry) to photobleach HURP molecules. Time-point 0 s represents the first acquired frame post photobleaching. (C) Representative images of control (upper panel) and Nocodazole (lower panel) treated HeLa Kyoto cells at different timepoints post photobleaching. (D) Left: Fluorescence recovery curves of eGFP-HURP in control and Nocodazole treated cells. Bold-lines indicate the mean and dashed-lines represent the ± S.D. Right: One-phase exponential fitting resulted in half-lives of $t_{1/2}=25\pm 5\mathit{sec}$ for control cells, (n = 18 cells) and $t_{1/2}=22\pm 5\mathit{sec}$ for Nocodazole treated cells (n = 20 cells) (mean ± S.D.; *p = 0.032). (E) Left: eGFP-HURP fluorescence decay curves of the unbleached spindle half in control and Nocodazole treated cells. Bold-lines indicate the mean and dashed-lines represent the ±S.D. Right: One-phase decay fitting resulted in half-lives of $t_{1/2}=27\pm 5\mathit{sec}$ for control, (n = 18 cells) and $t_{1/2}=23\pm 5\mathit{sec}$ for Nocodazole treated cells (n = 20 cells). (mean ± S.D.; *p = 0.016). (F) Spindle-bound endogenous eGFP-HURP quantification (relative to SiR-tubulin signal), derived from the pre-photobleached frames. (control, n = 23 cells; Nocodazole, n = 30 cells). Scale bar denotes 5 μm.