Anaphase

Proliferation index, mitotic duration and chromosome missegregation were scored by visual inspection of time-lapse movies. Proliferation was calculated as the ratio of all live cells in the last movie frame (24 h) divided by all live cells from the first movie frame. Mitotic duration was defined as the interval from prometaphase onset until anaphase onset for all cells entering mitosis within the first 12 h of the experiment, and determined based on the chromatin morphology. The incidence of chromosome missegregation was calculated by dividing the number of anaphase cells with lagging or bridged anaphase chromosomes by the total number of anaphase cells.
DNA labelling specificity was quantified 2 h after adding the different dyes by automated image analysis using the open-source CellCognition software20 (link). Cell nuclei were automatically segmented in five consecutive image frames per experimental condition by local adaptive thresholding in the H2B-mCherry channel. Mitotic cells and dead cells were excluded from analysis based on automated classification by supervised machine learning. The mean fluorescence was then measured in the far-red channel. To calculate the nucleo/cytoplasmic fluorescence ratio, a cytoplasmic region was defined for each cell as a 5 pixel wide rim around the nuclear segmentation mask, spaced at a distance of 1 pixel. Extracellular background fluorescence was manually measured and subtracted from intracellular mean fluorescence measurements. The nucleo/cytoplasmic fluorescence ratio was first calculated for individual cells to derive the mean nucleo/cytoplasmic fluorescence ratio of the cell population. The mean and s.e.m. was then calculated for each dye condition based on three independent biological replicates. Fluorescence cross-talk from the H2B-mCherry channel was very low (<2% of the signal detected in 500 nM SiR–Hoechst-treated cells), as assessed by measuring fluorescence intensity in dimethylsulfoxide-treated control cells.

To determine the number of ovarioles in wild type and mutant ovaries, ovaries were stained with antibodies against Vasa and Engrailed, a transcription factor that is expressed only in niche cells of the germaria [52 (link)]. The number of niches was determined by counting Engrailed positive cells adjacent to Vasa positive cells.
Several nuclear parameters were assessed. To determine intensity of GFP-BAF, emerin, and lamin at the nuclear envelope or within the GSC nucleus, a line segment was drawn across each nucleus. The rim intensity was defined as the average grey value of the two intersection points between the line and the nuclear envelope. The interior intensity was defined as the mean grey value along the line that is inside of the nucleus. For these experiments, three replicates were performed, corresponding to three different slides made for each genotype. In each case, imaging parameters were kept the same among experiments and samples. The middle section of each GSC was chosen for quantification. Background was subtracted for these quantifications. To measure nuclear roundness, each nucleus was traced based on lamin staining in ImageJ and roundness was determined as 4*area/(π*major_axis^2).
Several mitotic parameters were assessed. Mitotic stages were determined based on staining patterns of α-tubulin, Cnn, and H3S10p using the following criteria (Fig. S2): 1) Prophase was defined as H3S10p staining that was restricted to the periphery and evidence of microtubules nucleation at the periphery of the nuclear envelope, 2) Prometaphase was defined as chromosomes marked by H3S10p that were partially condensed and microtubules that were beginning to connect chromosomes to the spindle poles, 3) Metaphase was defined as chromosomes marked with H3S10p that were fully condensed and aligned at the metaphase plates, and microtubules formed the characteristic fusiform metaphase spindle, 4) Anaphase was defined as chromosomes marked with H3S10p that were separated into two clusters that each had individual chromosome arms that were visible, and 5) Telophase was defined as H3S10p staining that was weaker and the presence of a central spindle at the mid-plane of the cell. To determine alignment of microtubules (MTs), images were visualized in ImageJ and misaligned MTs were defined as MTs that cross each other’s path at the metaphase plate. The percentage of MT coverage of chromosomes was quantified in maximum projection images of metaphase GSCs that were stained with antibodies against H3S10p and α-tubulin. The coverage was defined as a ratio of the chromosome length that was covered by MTs divided by the entire length of the chromosome mass. The intensity of CID and CENP-C in images of metaphase GSCs were quantified in Image J using the sum slices projection method. Only well-separated CID and CENP-C foci were included. For each centromere, background was subtracted with 50 pixels rolling ball radius. Total grey values were reported for both CID and CENP-C staining. Different genotypes were imaged using the same parameters and at the same time.
To quantify DCP-1 intensity in wing discs, discs were hand dissected from third instar larvae of the indicated genotypes and stained with antibodies against cleaved DCP-1 and DAPI. Samples were imaged using a confocal microscopy under the same setting at the same time. DCP-1 intensity was quantified from a summed z-projection with background subtracted using image J (50 pixel rolling ball radius). The total intensity was divided by the area of the wing disc to control for size differences. Resulting DCP-1 mean intensity was graphed. At least three replicates were performed for each genotype.