Abstract
The mitotic checkpoint protein MAD1 is overexpressed in cancers, weakening the mitotic checkpoint and promoting mitotic slippage. Overexpressed MAD1 forms ectopic foci in mitotic cells, but the biophysical nature of these foci and their roles in mitotic slippage remain unclear. Here, we report that MAD1 ectopic foci are phase-separated condensates that shorten mitosis by sequestering checkpoint proteins. We show that MAD1 ectopic foci display dynamic condensate behaviors, and Mad1 phase separation is driven by interactions between its coiled-coil and disordered domains at the N-terminus. By inducing MAD1 ectopic condensates in mitotic cells with low MAD1 levels, we decouple the effects of condensation from concentration and demonstrate the causal role of condensation in mitotic slippage. Mechanistically, MAD1 ectopic condensates trap the diffusive pool of MAD2, weakening the MAD2 conversion cycle needed for a robust checkpoint. Our work highlights a loss of function caused by ectopic condensates in cancer cells.