Figure 1.

The majority of MT plus ends that reach metaphase chromosomes do not originate from spindle poles. (A–G) Live-cell confocal microscopy of HeLa cells expressing EB3-EGFP (green) and mCherry-CENPA (magenta), stained with SiR-Hoechst (blue). (A) Metaphase cell imaged for 1 min at 2 s/frame (see Video 1). (B and C) Quantification of MT plus end distribution from the spindle poles (see Materials and methods for details). (B) All time frames were registered to correct for spindle rotation (see Video 3) and projected to obtain mean-intensity images. EB3-EGFP fluorescence was measured in interpolar spindle regions, along a series of circumferential lines of increasing radius centered on the spindle poles. The quantification region extends from the pole to the spindle center (full white line; arrow indicates direction of the pole–pole axis). (C) Mean EB3-EGFP fluorescence measured as in B (n = 25 cells; individual measurements normalized to the centrosome rim). The black line indicates predicted signal dilution by radial geometry. (D and E) Quantification of EB3-EGFP and SiR-Hoechst fluorescence across the chromosome–cytoplasm boundary. (D) Mean intensities were profiled along lines placed either inside the spindle (full white line) or in its immediate periphery (dotted white line); results are plotted in E (n = 25 cells). (F) Quantification of fluorescence of EB3-EGFP and mCherry-CENPA along curved lines connecting pairs of sister KTs (dashed white line) to one of the spindle poles in maximum-intensity projections of three sequential video frames. (G) Mean intensity profiles, aligned to the midpoint between sister KTs (n = 42 profiles in 7 cells). (H–K) HeLa cells imaged during metaphase by 3D lattice light-sheet microscopy (n = 11). (H) Maximum-intensity projections of 5 (left) and 60 (right) consecutive slices of a deconvolved z-stack. (I) EB3-EGFP fluorescence was measured in nondeconvolved stacks inside conical ROIs defined around the interpolar axis (yellow). Shown is the same cell as in H; bounding box is 23 × 24 × 11 µm. The slice highlighted in red follows the plane defined by the spindle poles and a random KT. (J) Distribution of MT plus ends in interpolar spindle regions, estimated from the EB3-EGFP fluorescence measured as in I (green) or from the EB3-EGFP particles detected as in Fig. S1 F (black). Fluorescence intensities were normalized to the centrosome rim; the count profiles shown in Fig. S1 G were normalized to 2 µm from the spindle poles. (K) Fractions of MT plus ends attributable (light green) and not attributable (dark green) to nucleation at the spindle poles as a function of distance from the pole. Computed from the fluorescence measurements shown in J as detailed in Materials and methods. Lines and shaded areas denote mean ± SD, respectively. Scale bars, 10 µm. Yellow dotted lines indicate cell boundaries. AU, arbitrary units.