Fig. 1: The MAD1:MAD2 complex can assume a folded conformation in vivo enabled by the hinge of MAD1.

A Representative models of the C-terminal region of the MAD1:MAD2 complex predicted by the ColabFold advanced algorithm²⁵ show either extended (left) or folded (right) conformations. These predicted structures agree with published crystal structures, from which labeled length measurements were taken (PDB IDs: 1GO4¹³ and 4DZO¹⁸). B A cartoon demonstrating how the folded conformation of MAD1 may help present the MIM of CDC20 to MAD2. The N-terminal region of CDC20 (including BOX1 and the MIM) —light-gray line; the WD40 fold— light-gray circle^16,21,40. C Left: exemplary images of the genome-edited HeLa-A12 cells expressing both MAD1-mNG and MAD2∨mScarlet-I acquired for FLIM (scale bar ~10 μm). The photon count heatmaps in the MAD1-mNG insets display regions of interest manually thresholded to isolate pixels corresponding to the nuclear envelope. Middle: The excited-state lifetime of MAD1-mNG in the MAD1-mNG or the MAD1-mNG/MAD2∨mScarlet-I genome-edited HeLa-A12 cell lines (N = 40 and 52 nuclei, respectively, pooled from four technical repeats). Right: calculated FRET efficiency for MAD1-mNG and MAD2∨mScarlet-I. Two-sided, unpaired t tests with Welch’s correction were performed. D Partial sequence of the hinge region of human wild-type MAD1, MAD1^ΔL, and MAD1^Lmut. E A representative model of the C-terminal region of MAD1^ΔL:MAD2 (top) and MAD1^Lmut:MAD2 (bottom) predicted by the ColabFold advanced algorithm. F Left: the lifetime of MAD1-mNG alone, MAD1-mNG in the presence of MAD2∨mScarlet-I, and MAD1^ΔL-mNG in the presence of MAD2∨mScarlet-I (N = 11, 20, and 16 nuclei respectively pooled from four independent experiments). Right: The average lifetime of MAD1-mNG alone, MAD1-mNG in the presence of MAD2∨mScarlet-I, and MAD1^Lmut-mNG in the presence of MAD2∨mScarlet-I (N = 28, 31, and 32 nuclei, respectively, pooled from three independent experiments). C, F Each dot represents a single cell. Mean values ± 95% confidence intervals are overlaid. F Observations are color-coded by trial number. Source data are provided as a Source Data file.