Figure 1.
Figure 1. 3D analysis of microtubule bundles and chromosomes in dividing cells. (A and C) Near-simultaneous two-color LLSM was used to image GFP-PRC1 and chromosomes during anaphase in hTERT-RPE1 cells. Cell volumes (101 images in each channel at 300-nm step size) were captured at 3-s intervals. T = 0 was assigned to the frame immediately before that with detectable chromatid separation. (A) Single-channel images (maximum-intensity projections) and overlays from select time points show GFP-PRC1 (green) and chromosomes (magenta). Time-lapse recording is provided in Video 1. Scale bar, 3 µm. (B) Schematic of an anaphase spindle shows PRC1 (green) and chromosomes (magenta) at a plane incident with the pole-to-pole axis and at the spindle midplane, a cross-sectional plane orthogonal to the pole-to-pole axis and equidistant between the two poles. (C) Single-channel images (single image planes) and overlays show the spindle midplane of the cell shown in A. Scale bar, 3 µm. (D) Schematic of an anaphase spindle shows region of microtubule overlap marked by PRC1 (green) and chromosomes (magenta). The length of microtubule overlap and interchromosome distance is indicated. (E) Plots of average microtubule overlap length (green dots) and interchromosome distance (magenta dots) versus time in hTERT-RPE1 cells. Average microtubule overlap length was determined for each cell at each frame (n = 5 cells). (F) Average chromosome segregation rates following anaphase onset. Data were binned in 50-s time intervals. Error bars are SD. (G) LLSM was used to image Halo-PRC1 during anaphase in HeLa cells. Cell volumes (101 images at 300-nm step size) were captured at 9.5–20.5-s intervals. Single-channel images (maximum-intensity projections) show select time points from an example cell imaged at 20.5-s intervals. T = 0 was assigned to the frame immediately before that with detectable spindle elongation. Scale bar, 3 µm. (H) Plot of average microtubule overlap length versus time in HeLa cells. Average microtubule overlap length was determined for each cell at each frame (n = 3 cells).

3D analysis of microtubule bundles and chromosomes in dividing cells. (A and C) Near-simultaneous two-color LLSM was used to image GFP-PRC1 and chromosomes during anaphase in hTERT-RPE1 cells. Cell volumes (101 images in each channel at 300-nm step size) were captured at 3-s intervals. T = 0 was assigned to the frame immediately before that with detectable chromatid separation. (A) Single-channel images (maximum-intensity projections) and overlays from select time points show GFP-PRC1 (green) and chromosomes (magenta). Time-lapse recording is provided in Video 1. Scale bar, 3 µm. (B) Schematic of an anaphase spindle shows PRC1 (green) and chromosomes (magenta) at a plane incident with the pole-to-pole axis and at the spindle midplane, a cross-sectional plane orthogonal to the pole-to-pole axis and equidistant between the two poles. (C) Single-channel images (single image planes) and overlays show the spindle midplane of the cell shown in A. Scale bar, 3 µm. (D) Schematic of an anaphase spindle shows region of microtubule overlap marked by PRC1 (green) and chromosomes (magenta). The length of microtubule overlap and interchromosome distance is indicated. (E) Plots of average microtubule overlap length (green dots) and interchromosome distance (magenta dots) versus time in hTERT-RPE1 cells. Average microtubule overlap length was determined for each cell at each frame (n = 5 cells). (F) Average chromosome segregation rates following anaphase onset. Data were binned in 50-s time intervals. Error bars are SD. (G) LLSM was used to image Halo-PRC1 during anaphase in HeLa cells. Cell volumes (101 images at 300-nm step size) were captured at 9.5–20.5-s intervals. Single-channel images (maximum-intensity projections) show select time points from an example cell imaged at 20.5-s intervals. T = 0 was assigned to the frame immediately before that with detectable spindle elongation. Scale bar, 3 µm. (H) Plot of average microtubule overlap length versus time in HeLa cells. Average microtubule overlap length was determined for each cell at each frame (n = 3 cells).

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