Sensor localization during S phase and CDK-1-dependent phosphorylation of the sensor.
(A) Quantification of DNA content by propidium iodide staining in L1 larvae at 2, 3, 4 and 5 hours after hatching. Muscle cells were used as the reference for a 2N DNA content. Error bars indicate SEM. (B) Boxplots of ratios calculated from confocal fluorescence microscopy images of larvae staged at 2, 3 and 4 hours after hatching (n = 9 for single seam cells, n = 27 for average of cells). Avg. refers to the average ratio of V2, V3 and V4 together. The borders of the boxes are the 25th and 75th percentile, • indicates the mean, error bars correspond to 1.5× the interquartile range, outliers are shown. (C) Boxplots containing the quantification of cytoplasmic-to-nuclear fluorescence ratio in control and HU-treated larvae, 5 hours after hatching (n = 9). The borders of the boxes are the 25th and 75th percentile, the mean is indicated by +, error bars correlate to 1.5× the interquartile range. (D) Live-cell imaging of WT (top) and cdk-1(he5) mutant (bottom) larvae expressing the CDK sensor, at 300 minutes (5 hours) after hatching. (E) Spinning disk confocal fluorescence microscopy time-lapse movie analysis of sensor localization in control animals (n = 3 cells) and cdk-1(he5) mutants in L1 (n = 3). Control cells before anaphase in grey, control anterior cells in orange, control posterior cells in blue, cdk-1(he5) mutant cells in green. Average ratio is indicated by a bold line, individual cells are shown with thin lines. Dotted black lines indicate the time between anaphase and nuclear envelope reformation, where ratios could not be determined because of absence of the nucleus. (F) Comparison between the maximal calculated ratio’s in control (n = 10) and cdk-1(he5) (n = 14) animals during L1 development. The borders of the boxes are the 25th and 75th percentile, the mean is indicated by–, error bars correlate to 1.5× the interquartile range.