Alterations in the BUB-1 kinase domain perturb MAD-1 localization to unattached kinetochores independently of kinase activity. Video shows GFP::MAD-1 and mCh::H2b in two-cell C. elegans embryos expressing untagged BUB-1 variants and depleted of endogenous BUB-1, which are undergoing monopolar division caused by ZYG-1 depletion. Images were acquired using a spinning-disk confocal system (Andor Revolution XD; Andor Technology). Two different sequences are shown for each condition. The images in the video from left to right show BUB-1 (WT), BUB-1 (L777K, N781K), BUB-1 (K718R, D847N), and BUB-1 (D814N). Top row images are GFP::MAD-1; bottom row images are a merge of GFP::MAD-1 (green) and mCh::H2b (red). Frame seven is when pseudometaphase plate formation occurs. Each frame in the video is 15 s apart and is a maximum intensity projection of 6 × 2–µm z sections. Playback rate is six frames per second (90× real time). Images in the related figure (Fig. 5 D) are taken from the first sequence shown for each condition.
