Fin1 is associated with kinetochores from metaphase to anaphase. (a) Dsn1-Flag was immunoprecipitated to purify kinetochore complexes from G1-arrested cultures and cdc15-1 anaphase-arrested cultures. Purifications were performed in triplicate for each condition. MudPIT analysis of purified kinetochores detected Fin1 in the anaphase samples but not the G1 samples. (b) Live-cell imaging of Fin1-GFP shows that Fin1 localizes in a kinetochore-like focus from metaphase to anaphase that dissolves in G1. Eight cells were analyzed; a representative time course is shown. Bar, 5 µm. (c) Quantification of Fin1-GFP from b shows increased normalized intensity as the kinetochore distance increases in anaphase, and a decrease in G1. (d) Snapshots of FRAP on Fin1-GFP at the indicated times show recovery during anaphase following bleach in early anaphase. Heat map (lower panel) of the FRAP shows the intensity change before and after bleaching. Eight cells were analyzed; a representative time course is shown. Bar, 5 µm. (e) Quantification of Fin1-GFP from d shows recovery in anaphase. Intensity of mother and daughter kinetochore cluster was normalized to 1 at 0 min. The second time point represents the photobleach step. (f) Calibrated imaging was used to determine copy number of Fin1-GFP from metaphase to anaphase, plotted as a function of spindle length calculated from the inter-kinetochore distance. Fin1-GFP copy number increases as kinetochore clusters separate during anaphase. For the box plot, 200–250 kinetochore clusters were quantified, and for statistical analysis, a two-tailed t test was performed (*, P < 0.0001). In the box plot, the middle line represents the mean, the box represents SD, and whiskers represent SEM.