Visualization of Mad2 dynamics at kinetochores, along spindle fibers, and at spindle poles in living cells

The spindle checkpoint prevents errors in chromosome segregation by inhibit ing anaphase onset until all chromosomes have aligned at the spindle equato r through attachment of their sister kinetochores to microtubules from oppo site spindle poles. A key checkpoint component is the mitotic arrest-defici ent protein 2 (Mad2), which localizes to unattached kinetochores and inhibi ts activation of the anaphase-promoting complex (APC) through an interactio n with Cdc20. Recent studies have suggested a catalytic model for kinetocho re function where unattached kinetochores provide sites for assembling and releasing Mad2-Cdc20 complexes, which sequester Cdc20 and prevent it from a ctivating the APC. To test this model, we examined Mad2 dynamics in living PtK1 cells that were either injected with fluorescently labeled Alexa 488-X Mad2 or transfected with GFP-hMAD2. Real-time, digital imaging revealed flu orescent Mad2 localized to unattached kinetochores, spindle poles, and spin dle fibers depending on the stage of mitosis, FRAP measurements showed that Mad2 is a transient component of unattached kinetochores, as predicted by the catalytic model, with a t(1/2) of similar to 24-28 s. Cells entered ana phase similar to 10 min after Mad2 was no longer detectable on the kinetoch ores of the last chromosome to congress to the metaphase plate. Several obs ervations indicate that Mad2 binding sites are translocated from kinetochor es to spindle poles along microtubules. First, Mad2 that bound to sites on a kinetochore was dynamically stretched in both directions upon microtubule interactions, and Mad2 particles moved from kinetochores toward the poles. Second, spindle fiber and pole fluorescence disappeared upon Mad2 disappea rance at the kinetochores. Third, ATP depletion resulted in microtubule-dep endent depletion of Mad2 fluorescence at kinetochores and increased fluores cence at spindle poles. Finally, in normal cells, the half-life of Mad2 tur nover at poles, 23 s. was similar to kinetochores. Thus, kinetochore-derive d sites along spindle fibers and at spindle poles may also catalyze Mad2 in hibitory complex formation.