Figure 5.
Figure 5. Partial cohesin cleavage impairs mitotic progression. (A) Stills of embryos undergoing mitosis with full complement of cohesin complexes (100% Rad21WT, control) and upon removal of a large percentage of cohesin from chromosomes before mitotic entry (strain D after TEV injection). The first column depicts time of injection. Times are relative to NEBD. (B) Mitotic timings (NEBD to anaphase [top] and NEBD to nuclear envelope formation [NEF; bottom]) in different conditions. Five nuclei per embryo were followed across time. n = 5 (control, genetic WT Rad21), 6 (100% Rad21WT), 4 (strain D + TEV buffer), and 10 (strain D + TEV protease) embryos. ***, P < 0.001, Kruskal-Wallis test relative to control. (C) Mitotic errors observed during chromosome segregation in strain D upon Rad21TEV cleavage. Mitotic errors were measured in 5 (control), 5 (strain D + buffer), and 7 (strain D + TEV protease) embryos corresponding with at least 111 mitotic figures analyzed per condition. Mean ± SD. (D) Representative images of common errors observed in strain D highlighting lagging Cid-EGFP signals (arrowheads). Normal division comes from a control condition. (E) Native chromosome squashes from brains in no HS control and 20/45 min after HS-induced TEV expression in strains carrying solely Rad21TEV-EGFP (green). Percentages depict average levels of chromatin-bound Rad21TEV-EGFP (see also Fig. S3 A). Mean ± SEM. Cells also express His-H2Av-mRFP1 (red). (F) Stills from neuroblasts in control and mild cohesin cleavage conditions (20 min HS). Partial cleavage of cohesin results in mild to severe metaphase centromere misalignment and mitotic errors. Times are relative to NEBD. Bars, 5 µm. (G) Frequency of partial/full PSCS in the indicated conditions. (H) Mitotic timings across different experimental conditions: control strains surviving on Rad21TEV without HS, cells containing WT Rad21 (TEV resistant) after HS, and cells surviving solely on Rad21TEV after 20 or 45 min induction of TEV protease. n ≥ 4 independent brains corresponding with ≥40 cells analyzed per experimental condition. (I) Frequency of segregation errors observed upon partial cohesin cleavage (20 min HS). Cells undergoing full PSCS were excluded from analysis. n = 5 brains; n = 27 cells.

Partial cohesin cleavage impairs mitotic progression. (A) Stills of embryos undergoing mitosis with full complement of cohesin complexes (100% Rad21WT, control) and upon removal of a large percentage of cohesin from chromosomes before mitotic entry (strain D after TEV injection). The first column depicts time of injection. Times are relative to NEBD. (B) Mitotic timings (NEBD to anaphase [top] and NEBD to nuclear envelope formation [NEF; bottom]) in different conditions. Five nuclei per embryo were followed across time. n = 5 (control, genetic WT Rad21), 6 (100% Rad21WT), 4 (strain D + TEV buffer), and 10 (strain D + TEV protease) embryos. ***, P < 0.001, Kruskal-Wallis test relative to control. (C) Mitotic errors observed during chromosome segregation in strain D upon Rad21TEV cleavage. Mitotic errors were measured in 5 (control), 5 (strain D + buffer), and 7 (strain D + TEV protease) embryos corresponding with at least 111 mitotic figures analyzed per condition. Mean ± SD. (D) Representative images of common errors observed in strain D highlighting lagging Cid-EGFP signals (arrowheads). Normal division comes from a control condition. (E) Native chromosome squashes from brains in no HS control and 20/45 min after HS-induced TEV expression in strains carrying solely Rad21TEV-EGFP (green). Percentages depict average levels of chromatin-bound Rad21TEV-EGFP (see also Fig. S3 A). Mean ± SEM. Cells also express His-H2Av-mRFP1 (red). (F) Stills from neuroblasts in control and mild cohesin cleavage conditions (20 min HS). Partial cleavage of cohesin results in mild to severe metaphase centromere misalignment and mitotic errors. Times are relative to NEBD. Bars, 5 µm. (G) Frequency of partial/full PSCS in the indicated conditions. (H) Mitotic timings across different experimental conditions: control strains surviving on Rad21TEV without HS, cells containing WT Rad21 (TEV resistant) after HS, and cells surviving solely on Rad21TEV after 20 or 45 min induction of TEV protease. n ≥ 4 independent brains corresponding with ≥40 cells analyzed per experimental condition. (I) Frequency of segregation errors observed upon partial cohesin cleavage (20 min HS). Cells undergoing full PSCS were excluded from analysis. n = 5 brains; n = 27 cells.

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