Mps1 is a component of the M phase timer. (A) Mps1^wt and Mps1^as cells stably expressing mCherry-tagged histone H2B were treated with 10 µM 3MB-PP1 and filmed at 1-min intervals by spinning-disk confocal microscopy. Maximum intensity projections are shown (Videos 1–6). Bar, 10 µm. (B) Quantification of the mitotic timing defect in Mps1-inhibited cells. Cumulative frequency of anaphase onset is plotted as a function of time after NEB. (C) Strong aurora B inhibition does not block the autophosphorylation of Mps1. HEK293 cells were transfected with GFP-tagged Mps1 expression plasmids, arrested in M phase with nocodazole (noc), and treated with or without 2 µM ZM for 2 h. Cell extracts were resolved by SDS-PAGE and probed with antibodies to serine 10–phosphorylated histone H3 (to confirm suppression of aurora B kinase activity) and GFP (to assess Mps1’s electrophoretic mobility, which becomes retarded by its mitotic activation and autophosphorylation; Stucke et al., 2004).