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. 2022 Jul 1;221(8):e202108156. doi: 10.1083/jcb.202108156

Figure 4.

Figure 4.

Multivalent interactions between Sgo1 and different CPC components are essential for high-affinity CPC–Sgo1 binding and efficient CPC recruitment. (A–C) Isotherms for the analyses of Survivin interaction with Sgo11–130 (40 μl of 312 μM Survivin was injected into 200 μl of 20 μM Sgo11–130; A), CPCISB10–280 interaction with Sgo11–130 (40 μl of 100 μM CPCISB10–280 was injected into 200 μl of 10 μM Sgo11–130; B), and CPCISB10–221 interaction with Sgo11–130 (40 μl of 120 μM CPCISB10–221 was injected into 200 μl of 12 μM Sgo11–130; C). The ITC experiments were performed with 16 × 2.5-μl injections (0.5 μl first injection), 180 s apart, at 20°C. Top panels show raw ITC data; bottom panels show integrated heat data corrected for heat of dilution and fitted to a standard 1:1 binding model (Malvern Instruments MicroCal Origin software, v1.3). (D and E) Representative micrographs (left) and quantifications (right) for the analysis of endogenous Aurora B and Borealin recruitment to the LacO array in U-2 OS-LacO Haspin CM cells expressing different Sgo1-LacI-GFP constructs: LacI-GFP (n = 22 for Aurora B; n = 24 for Borealin), Sgo1130–280-LacI-GFP (n = 20 for Aurora B; n = 22 for Borealin), Sgo1130–240-LacI-GFP (n = 24 for Aurora B; n = 20 for Borealin), Sgo1150–280-LacI-GFP (n = 16 for Aurora B; n = 21 for Borealin), Sgo1159–240-LacI-GFP (n = 10 for Aurora B; n = 22 for Borealin), Sgo1130–280 4A-LacI-GFP (n = 22 for Aurora B; n = 21 for Borealin) or Sgo1159–240 4A-LacI-GFP (n = 23 for Aurora B; n = 21 for Borealin; D); LacI-GFP (n = 20 for Aurora B; n = 20 for Borealin), Sgo11–527-LacI-GFP (n = 17 for Aurora B; n = 22 for Borealin), Sgo11–527 Nmut-LacI-GFP (n = 14 for Aurora B; n = 23 for Borealin), and Sgo11–527 4A-LacI-GFP (n = 19 for Aurora B; n = 21 for Borealin) or Sgo11–527 Nmut/4A-LacI-GFP (n = 22 for Aurora B; n = 22 for Borealin; E). The graphs show the intensities of Aurora B and Borealin over GFP (dots) and the means (red bar). Data are representative of four biological replicates. Scale bar, 5 μm. One-way ANOVA with Dunnett’s multiple comparison test (****, P < 0.0001; ***, P < 0.001). DP, differential power.