Examining PRC1 and EB1 localization in cross-sectional planes of dividing cells. (A and B) Near-simultaneous two-color LLSM was used to image Halo-PRC1 and GFP-EB1 during anaphase in hTERT-RPE1 cells. Cell volumes (58 images in each channel at 350-nm step size) were captured at 1-s intervals. A select time point from an early anaphase (A) and a late anaphase (B) spindle from two different cells is shown. Single-channel images (maximum-intensity projections) and overlays show Halo-PRC1 (green) and GFP-EB1 (magenta). Time-lapse recording is provided in Video 2. Scale bar, 3 µm. (C and D) Schematics of anaphase spindles show PRC1 and EB1 decoration in a plane incident with the pole-to-pole axis (C) or in the spindle midplane, the plane equidistant from the two spindle poles and perpendicular to the spindle long axis (D). (C) The position of the spindle midplane is indicated (dashed line). (D) Inset shows schematic of nearest neighbor distances measured between two PRC1 spots (dashed line), two EB1 spots (dotted line), or one PRC1 and one EB1 spot (solid line). (E) Midplane of the cell shown in B. T = 0 indicates the start of movie. Time-lapse recording is provided in Video 3. (F–H) Nearest-neighbor distances versus time show mean ± SD for each frame of the video for the cell shown in B and E. T = 0 indicates the start of movie. Measurements between pairs of PRC1 spots (F), pairs of EB1 spots (G), and PRC1 and EB1 spots (H) are shown. (I) Average nearest-neighbor distance measurements (pooled from the first 30 frames of each movie, n = 3 cells; PRC1–PRC1: 1.03 ± 0.12 µm; EB1–EB1: 1.04 ± 0.09 µm; PRC1–EB1: 0.71 ± 0.06 µm; **, P < 0.02; ***, P < 0.004). Error bars are SD. (J) Time series of a select region (single plane) in the midzone from the cell shown in B, indicating relative position of a single microtubule bundle and GFP-EB1 signal. Single-channel images and overlays show Halo-PRC1 (green) and GFP-EB1 (magenta) in consecutive frames. Scale bar, 3 µm.