To analyze cell division, the beginning of division was determined by morphological rounding of cells using phase-contrast imaging before DNA condensation determined by the H2BK-GFP or mCherry signal. The number of frames until the end of DNA separation or complete cell division (cell spreading of daughter cells) was quantified and used to determine the overall time of cell division. Cells were considered separated if less than a third of the cell boundaries of the daughter cells were attached to each other; this was analyzed up to 90 min after nuclear division. To analyze CAR localization at junctions, videos were opened in ImageJ and line scans (4.2 μm) were drawn perpendicular to junctions. The intensity profile of CAR-GFP was then calculated; 30 junctions were analyzed per condition, which were then averaged and normalized. To analyze CAR-GFP and EGFR-mCherry dynamics in live cells, identically sized region-of-interest boxes were placed over central (5 μm wide) junctional regions at time 0, and the resulting intensity measurements over time were calculated for both GFP and mCherry channels. These values were then exported into GraphPad Prism and normalized to the starting intensity for each junction analyzed to provide relative intensity changes over time. For analysis of single microtubules at the cell periphery (microtubule tracking assay), acquired movies were subjected to a band-pass filter (20:2 pixels) in ImageJ (background-subtracted using a rolling ball radius of 15 pixels), and a three-dimensional Gaussian blur filter was applied. Resulting movies were overlaid with the originals to avoid image processing–derived artifacts, and single microtubule growth was measured over time from a defined starting point proximal to the cell periphery. Frequency of catastrophe, growth rate, and time spent in growth phase were quantified as in (56). Example representative images of time-dependent changes to microtubule dynamics were generated using the Temporal Color Code plug-in in FIJI (Centre for Molecular and Cellular Imaging, European Molecular Biology Laboratory, Germany).

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