Figure 2
Naa50/San is required for sister chromatid cohesion in Drosophila S2 cells.
Drosophila S2 cells depleted for Naa50/San (san RNAi-treated cells) showed sister chromatid cohesion defects (Supplementary Fig. 2A,B and D), with a metaphase arrest, detectable single chromatids during metaphase, and chromosome segregation defects during anaphase. All cells in this figure were analyzed 96 hours after RNAi-treatment. (A–E) Selected frames from time-lapse videos (see Movies S1, S2, S5 and S6) of control RNAi and san RNAi-treated S2 cells. (A,C) Control RNAi-treated cells showed no significant arrest in metaphase or chromosome segregation defects during anaphase (see Movies S1 and S5). (B,D,E) san RNAi-treated cells show an arrest in metaphase (see Movies S2,S3,S4 and S6) with a significant increase in their mitotic index (F), and chromosome segregation defects during anaphase (G). (F) Mitotic index (% of phospho-H3 (pSer10) positive cells) for control RNAi and san RNAi-treated cells (96 hours after RNAi-treatment) was, respectively, 4.3% ± 0.2 (n = 1139) and 12.0% ± 0.7 (n = 983) (p < 0.001 Student’s t-test). san RNAi but not control RNAi-treated cells showed single chromatids with their typical oscillatory behavior during metaphase (see arrowheads in B,D and E; see Movies S1,S2,S3,S4,S5,S6), which is suggestive of a loss of cohesion. (G) Frequency of cells with single chromatids after san RNAi-treatment was 5.6% ± 0.1 (n = 17) (72 hours after RNAi-treatment) (see Fig. 3) and 61% ± 11.7 (n = 77) (96 hours after RNAi-treatment). Single chromatids were never detected during mitosis and before anaphase in control RNAi-treated cells. Drosophila S2 cells stably expressed GFP-Histone H2B (green) and α-Tubulin-mCherry (red) (A,B)70 or GFP-α-Tubulin (green) and CID-mCherry (red) (C–E)71. All images were obtained using maximum intensity projections of Z-stacks (0.8 μm stacks of 5 sections each). For movies, Z-stacks were collected every 30 seconds. Scale bars equal 10 μm. San protein levels after control RNAi and san RNAi-treatment are shown in Supplementary Fig. 3A.
