Dynamics of microtubule bundles and FRAP analysis of PRC1. (A) Schematic indicating position of PRC1-tagged microtubules (green bars) and position of the spindle midplane, the plane formed by passing through yellow and blue vectors, orthogonal to the red vector, equidistant to the two spindle poles (green spheres). (B) Schematic of an anaphase spindle shows GFP-PRC1 (green) localization in a plane incident with the pole-to-pole axis and in the spindle midplane. (C–E) LLSM was used to image GFP-PRC1 during anaphase in hTERT-RPE1 cells. T = 0 s was assigned to the frame immediately before detectable pole separation. Cells were imaged at 4.4-s intervals. (C) Single-channel images (single cross-sectional plane) at select time points. Scale bar, 3 µm. (D) Inset from yellow boxes in C, magnified 5.3×. Two spots of GFP-PRC1 intensity in consecutive frames are indicated (yellow circles). (E) Single-channel images (single cross-sectional plane) after watershed processing. Images from two consecutive frames are shown. Scale bar, 3 µm. (F) Plot of the number of spots versus time detected in watershed-processed images (mean ± SD). (G) Schematic of FRAP experiment. Single microtubule bundles were targeted for photobleaching and the fluorescence recovery monitored over time. (H) Analysis of GFP-PRC1 FRAP in hTERT-RPE1 cells imaged using spinning disk confocal microscopy. Time = 0 is time of photobleach laser pulse. Overlay indicates region targeted for photobleaching (red box) and an eqivalent unphotobleached control area (gray box). Scale bar, 3 µm. (I) Plot of normalized recovery for photobleached GFP-PRC1 signal (red dots) and unbleached GFP-PRC1 control signal (gray dots) for the cell shown in H. Data were fitted to the following equation: f(x) = A[1 − exp(−kx)]; k = 0.035 (95% confidence bounds 0.023 to 0.047).