Mutations in PRC1 that reduce microtubule binding affinity can form microtubule bundles but cannot rescue chromosome hypersegregation defects. (A–C) Immunofluorescence analysis of HeLa cells in anaphase. Single-channel images (maximum-intensity projections) and overlays show DNA (blue), PRC1 (red), and tubulin (green). GFP fluorescence (maximum-intensity projection) is shown as reference (gray). HeLa cells coexpressing shRNA to endogenous PRC1 (shPRC1) and shRNA-resistant GFP-PRC1^FL (A), GFP-PRC1^AA (B), or GFP-PRC1^ΔC (C). Scale bar, 3 µm. (D) Analysis of the number of PRC1-tagged microtubule bundles in cells with interchromosome distances between 12 and 16 µm. Error bars are SD. (E–G) Analysis of PRC1 signal intensity in fixed cells. (E) PRC1 channel image from A showing position of linescan (yellow) used to measure PRC1 signal intensity along the pole-to-pole axis. (F and G) Plots show example traces from cells expressing shRNA to PRC1 (shPRC1) and shRNA-resistant GFP-PRC1^FL (F) or GFP-PRC1^AA (G). Signal intensity data were fit to a Gaussian (red traces) to determine the FWHM. Gaussian fits with R² values >0.90 were retained for analysis. (H) Bar chart of average FWHM, binned by interchromosome distances. Error bars are SD. (I) Histogram of the number of anaphase cells, binned by interchromosome distance. n = 53 (shPRC1+GFP-PRC1^FL) and 28 (shPRC1+GFP-PRC1^AA) cells.