Fig. 3: The structural flexibility provided by the hinge of MAD1 is critical to the SAC signaling activity in vivo.

A The first two columns on the left used the MAD1-mNG genome-edited HeLa-A12 cell line which served as a reference for the endogenous level of MAD1 (see “Methods”). In situ tagging of MAD1 did not affect the 3’-UTR which siMAD1’s target. The two columns on the right used HeLa-A12 cells treated with siMAD1’s and induced to express exogenous MAD1-mNG or MAD1^ΔL-mNG. Each dot represents a cell (N = 86, 89, 55, and 50, respectively). B In the predicted structure of the core region of the MAD1:MAD1^ΔL heterodimer (in complex with MAD2, using the ColabFold advanced algorithm), the hinge of the wild-type copy introduces a bulge but the overall conformation is extended due to the stiffness of the now fused α-helix of MAD1^ΔL. C As in (A), the first two columns on the left used the MAD1-mNG genome-edited HeLa-A12 cell line which served as a reference for the endogenous level of MAD1. The two columns on the right used HeLa-A12 cell lines treated with siMAD1’s and induced to express exogenous MAD1^AL11-mNG or MAD1^Lmut-mNG. Each dot represents a cell (N = 265, 219, 80, and 78, respectively). A, C Results were pooled from at least two technical repeats. The mean value ± the 95% confidence interval of each group is overlaid. Unpaired two-sided t tests with Welch’s correction are performed in Prism. Source data are provided as a Source Data file.