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© The Rockefeller University Press, 0021-9525/2000//1233 $5.00
The Journal of Cell Biology, Volume 150, Number 6, , 2000 1233-1250

Visualization of Mad2 Dynamics at Kinetochores, along Spindle Fibers, and at Spindle Poles in Living Cells

^a Department of Biology, CB#3280, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
^b Department of Biological Sciences, Stanford University, Stanford, California 94305-5020
^c Department of Physiology, University of California at San Francisco, San Francisco, California 94143-0444
Department of Biology, CB#3280, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.(919) 962-1625(919) 962-2354

The spindle checkpoint prevents errors in chromosome segregation by inhibiting anaphase onset until all chromosomes have aligned at the spindle equator through attachment of their sister kinetochores to microtubules from opposite spindle poles. A key checkpoint component is the mitotic arrest–deficient protein 2 (Mad2), which localizes to unattached kinetochores and inhibits activation of the anaphase-promoting complex (APC) through an interaction with Cdc20. Recent studies have suggested a catalytic model for kinetochore function where unattached kinetochores provide sites for assembling and releasing Mad2–Cdc20 complexes, which sequester Cdc20 and prevent it from activating the APC. To test this model, we examined Mad2 dynamics in living PtK1 cells that were either injected with fluorescently labeled Alexa 488-XMad2 or transfected with GFP-hMAD2. Real-time, digital imaging revealed fluorescent Mad2 localized to unattached kinetochores, spindle poles, and spindle 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 24–28 s. Cells entered anaphase 10 min after Mad2 was no longer detectable on the kinetochores of the last chromosome to congress to the metaphase plate. Several observations indicate that Mad2 binding sites are translocated from kinetochores 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 disappearance at the kinetochores. Third, ATP depletion resulted in microtubule-dependent depletion of Mad2 fluorescence at kinetochores and increased fluorescence at spindle poles. Finally, in normal cells, the half-life of Mad2 turnover at poles, 23 s, was similar to kinetochores. Thus, kinetochore-derived sites along spindle fibers and at spindle poles may also catalyze Mad2 inhibitory complex formation.

Key Words: cell cycle • mitosis • spindle checkpoint • chromosome • microtubule

© 2000 The Rockefeller University Press

Abbreviations used in this paper: APC, anaphase-promoting complex; Bub, budding uninhibited by benzimidazole; GFP, green fluorescent protein; hMad2, human Mad2; Mad2, mitotic arrest–deficient protein 2; XMad2, Xenopus Mad2.

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