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

Abstract

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.

SAC‐ing mitotic errors: How the spindle assembly checkpoint (SAC) plays defense against chromosome mis‐segregation

“…2) [reviewed in . For example, Bub1p binds to Bub3p throughout the cell cycle [reviewed in [Howell et al, 2000[Howell et al, , 2004Shah et al, 2004]. The association of SAC proteins with kinetochores is important for SAC function [Kadura et al, 2005;Taylor et al, 1998;Vanoosthuyse et al, 2004] perhaps because it alters the binding interactions between SAC proteins and influences the formation of the mitotic checkpoint complex (MCC) [Sudakin et al, 2001], which imposes a cell cycle arrest by binding to the Anaphase Promoting Complex (APC).…”

Microtubule-dependent Changes in Assembly of Microtubule Motor Proteins and Mitotic Spindle Checkpoint Proteins at PtK1 Kinetochores

“…There is qualitative evidence that unattached prometaphase kinetochores are larger in width (Rieder, 1982;Salmon, 1989;Cassimeris et al, 1990) and have greater amounts of microtubule motor proteins and mitotic spindle checkpoint proteins (Gorbsky and Ricketts, 1993;Chen et al, 1996;Echeverri et al, 1996;Li and Benezra, 1996;Taylor and McKeon, 1997;Yao et al, 1997Yao et al, , 2000Chan et al, 1998;Chen et al, 1998;Dujardin et al, 1998;Jablonksi et al, 1998;Waters et al, 1998;Martinez-Exposito et al, 1999; than metaphase kinetochores with their full complement of kinetochore microtubules. The enhanced assembly may be important for the recruiting kinetochore microtubules, increasing force production, and generating a strong "wait-anaphase" signal (Rieder and Salmon, 1998;Howell et al, 2000). There is also evidence that kinetochore assembly depends on interactions with nonkinetochore microtubules.…”

The Spindle Assembly Checkpoint: Clock or Domino?

“…The spindle assembly checkpoint proteins Mad1, Mad2, and BubR1, are removed from kinetochores upon microtubule attachment and transported along microtubules to the spindle pole (Howell et al 2000, a transition that is supposed to silence the signal emanating from kinetochores. This spindle checkpoint protein depletion system uses the dynein/dynactin motor protein and specialized dynein binding partners on kinetochores, composed of the RZZ complex (Rod, ZW10 and Zwilch) and Spindly (Barisic et al 2010;Chan et al 2009;Clute and Pines 1999;Gassmann et al 2008Gassmann et al , 2010Griffis et al 2007;Howell et al 2001;Karess 2005;Yamamoto et al 2008).…”