Figure 4.
Figure 4. PRC1 knockdown results in increased chromosome segregation rate and distance. (A) Western blot analysis of cell lysates of HeLa control cells and HeLa cells containing shRNA to PRC1 before (− tet) and after (+ tet) tetracycline induction of shRNA construct. Antibodies against α-tubulin (α-tub) and PRC1 are indicated. Expected position of PRC1 protein is indicated (red arrow). Full blots are provided in Figs. S3 and S5. (B) Analysis of mitotic index from control cells (ctr) and those expressing shRNA to PRC1 (+sh) cells. (C and D) Immunofluorescence of HeLa cells. Single-channel images (maximum-intensity projections) and overlays show DNA (blue), tubulin (green), and PRC1 (red) in HeLa control cells (C) and cells expressing shRNA to PRC1 (D). Scale bar, 3 µm. (E–G) Live-cell imaging of HeLa cells. Single-channel (single z slice) and overlay images show differential interference contrast (DIC) images (gray), chromosomes (magenta), and GFP-PRC1 (green) in HeLa control cells (E), HeLa cells expressing shRNA to PRC1 (shPRC1; F), and HeLa cells expressing shRNA to PRC1 and shRNA-resistant GFP-tagged full-length PRC1 (shPRC1+GFP-PRC1FL; G). T = 0 was assigned to the frame immediately before that with detectable chromatid separation. Time-lapse recordings are provided in Videos 4 and 5. Scale bar, 3 µm. (H) Segmented binary image showing outline of cortex and chromosome mask. The position of chromosome centroid (yellow squares), the axis of chromosome segregation (blue line), the position of the midplane (dotted line), the distance from the midplane to the cortex (gray line), and the distance from chromosome to cortex (pink line) are indicated. (I–N) Analysis of chromosome and cortical position in HeLa control (black; n = 15), shRNA (blue; n = 19), and shPRC1+GFP-PRC1 (gray; n = 14) cells. Mean ± SD. (I and J) Interchromosome distance over time (I) and at T = 5 min (J; control: 11.9 ± 1.5 µm; shPRC1: 14.5 ± 1.4 µm; shPRC1+GFP-PRC1FL: 12.1 ± 1.5 µm; P < 0.003). (K and L) Chromosome-to-cortex distance over time (K) and at T = 5 min (L; control: 6.0 ± 0.9 µm; shPRC1: 4.2 ± 0.5 µm; shPRC1+GFP-PRC1FL: 5.9 ± 0.7 µm; P << 0.0001). (M and N) Relative chromosome distance to cortex over time (M) and at T = 5 min (N; control: 0.50 ± 0.06; shPRC1: 0.63 ± 0.04; shPRC1+GFP-PRC1FL: 0.50 ± 0.06; P < 0.0001). n.s., not significant.

PRC1 knockdown results in increased chromosome segregation rate and distance. (A) Western blot analysis of cell lysates of HeLa control cells and HeLa cells containing shRNA to PRC1 before (− tet) and after (+ tet) tetracycline induction of shRNA construct. Antibodies against α-tubulin (α-tub) and PRC1 are indicated. Expected position of PRC1 protein is indicated (red arrow). Full blots are provided in Figs. S3 and S5. (B) Analysis of mitotic index from control cells (ctr) and those expressing shRNA to PRC1 (+sh) cells. (C and D) Immunofluorescence of HeLa cells. Single-channel images (maximum-intensity projections) and overlays show DNA (blue), tubulin (green), and PRC1 (red) in HeLa control cells (C) and cells expressing shRNA to PRC1 (D). Scale bar, 3 µm. (E–G) Live-cell imaging of HeLa cells. Single-channel (single z slice) and overlay images show differential interference contrast (DIC) images (gray), chromosomes (magenta), and GFP-PRC1 (green) in HeLa control cells (E), HeLa cells expressing shRNA to PRC1 (shPRC1; F), and HeLa cells expressing shRNA to PRC1 and shRNA-resistant GFP-tagged full-length PRC1 (shPRC1+GFP-PRC1FL; G). T = 0 was assigned to the frame immediately before that with detectable chromatid separation. Time-lapse recordings are provided in Videos 4 and 5. Scale bar, 3 µm. (H) Segmented binary image showing outline of cortex and chromosome mask. The position of chromosome centroid (yellow squares), the axis of chromosome segregation (blue line), the position of the midplane (dotted line), the distance from the midplane to the cortex (gray line), and the distance from chromosome to cortex (pink line) are indicated. (I–N) Analysis of chromosome and cortical position in HeLa control (black; n = 15), shRNA (blue; n = 19), and shPRC1+GFP-PRC1 (gray; n = 14) cells. Mean ± SD. (I and J) Interchromosome distance over time (I) and at T = 5 min (J; control: 11.9 ± 1.5 µm; shPRC1: 14.5 ± 1.4 µm; shPRC1+GFP-PRC1FL: 12.1 ± 1.5 µm; P < 0.003). (K and L) Chromosome-to-cortex distance over time (K) and at T = 5 min (L; control: 6.0 ± 0.9 µm; shPRC1: 4.2 ± 0.5 µm; shPRC1+GFP-PRC1FL: 5.9 ± 0.7 µm; P << 0.0001). (M and N) Relative chromosome distance to cortex over time (M) and at T = 5 min (N; control: 0.50 ± 0.06; shPRC1: 0.63 ± 0.04; shPRC1+GFP-PRC1FL: 0.50 ± 0.06; P < 0.0001). n.s., not significant.

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