NOD-mCherry expression stabilizes syntelic attachments. (A, B, E, and F) Two-color confocal imaging of Ndc80-GFP (green)– and NOD-mCherry (red)–expressing S2 cells. (A) Selected frames from a confocal time lapse of a cell with both bioriented (B) and syntelic (S) attachments (approximate pole positions are marked with asterisks). Note that the syntelic attachments persist for the duration of the time lapse. See Video 4. (B) Selected confocal Z-sections showing a combination of syntelic and bioriented kinetochore pairs in the same cell. The mCherry fluorescence intensity for each cell was quantified from the maximum intensity projection of the Z-sections (rightmost panel). (C) Plotting the percentage of syntelic attachments versus NOD-mCherry fluorescence reveals that NOD-mCherry stabilizes syntelic attachments in a dose-dependent fashion (n = 60 cells). Inset shows the mean percentage of syntelic attachments found in monopolar spindles assembled in the absence of Klp61F. (D) Syntelic stabilization by NOD-mCherry requires the motor domain of NOD (NOD, n = 57 cells; motorless NOD, n = 70 cells). (E and F) Maximum intensity projections of representative NOD-mCherry– and motorless NOD-mCherry–expressing cells with comparable expression levels (highlighted in D). Error bar represents the SEM. Bars, 10 µm.