Localization of Mad2 to Kinetochores Depends on Microtubule Attachment, Not Tension

Abstract

A single unattached kinetochore can delay anaphase onset in mitotic tissue culture cells (Rieder, C.L., A. Schultz, R. Cole, G. Sluder. 1994. J. Cell Biol. 127:1301–1310). Kinetochores in vertebrate cells contain multiple binding sites, and tension is generated at kinetochores after attachment to the plus ends of spindle microtubules. Checkpoint component Mad2 localizes selectively to unattached kinetochores (Chen, R.-H., J.C. Waters, E.D. Salmon, and A.W. Murray. 1996. Science. 274:242–246; Li, Y., and R. Benezra. Science. 274: 246–248) and disappears from kinetochores by late metaphase, when chromosomes are properly attached to the spindle. Here we show that Mad2 is lost from PtK1 cell kinetochores as they accumulate microtubules and re-binds previously attached kinetochores after microtubules are depolymerized with nocodazole. We also show that when kinetochore microtubules in metaphase cells are stabilized with taxol, tension at kinetochores is lost. The phosphoepitope 3f3/2, which has been shown to become dephosphorylated in response to tension at the kinetochore (Nicklas, R.B., S.C. Ward, and G.J. Gorbsky. 1995. J. Cell Biol. 130:929–939), is phosphorylated on all 22 kinetochores after tension is reduced with taxol. In contrast, Mad2 only localized to an average of 2.6 out of the 22 kinetochores in taxol-treated PtK1 cells. Therefore, loss of tension at kinetochores occupied by microtubules is insufficient to induce Mad2 to accumulate on kinetochores, whereas unattached kinetochores consistently bind Mad2. We also found that microinjecting antibodies against Mad2 caused cells arrested with taxol to exit mitosis after ∼12 min, while uninjected cells remained in mitosis for at least 6 h, demonstrating that Mad2 is necessary for maintenance of the taxol-induced mitotic arrest. We conclude that kinetochore microtubule attachment stops the Mad2 interactions at kinetochores which are important for inhibiting anaphase onset.

Inducing precocious anaphase in cultured mammalian cells

“…Our studies show that either Mad2⌬C or GST-Mad1F10 can overcome the checkpoint in nocodazole-treated PtK1 cells where kinetochores lack both tension as well as attached microtubules. This result indicates that BubR1 is not able to maintain the spindle checkpoint independently of Mad2 function, a conclusion supported by other studies where Mad2 function was inhibited with antibodies or deleted by genetic manipulations [Canman et al, 2000;Dobles et al, 2000;Gorbsky et al, 1998;Hoyt, 2001;Kitagawa and Rose, 1999;Li and Murray, 1991;Li and Benezra, 1996;Michel et al, 2001;Waters et al, 1998]. How the kinetochore mediates inhibition of APC/C by Cdc20 via Mad2, BubR1, or a direct effect on APC/C remains an important unresolved issue [Gillett and Sorger, 2001;Hoyt, 2001;Sudakin et al, 2001].…”

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

“…Spindle checkpoint proteins including Mps1p, Mad1p, Mad2p, Mad3p/BubR1p, Bub1p, and Bub3p are recruited specifically to unattached kinetochores in animal cells [reviewed in Cleveland et al, 2003], and Mad2p recruitment is independent of tension [Gillett et al, 2004;Waters et al, 1998]. However, experiments in insect spermatocytes have suggested that the lack of tension signals checkpoint activation because applying artificial tension to the kinetochore of a misaligned chromosome is able to override the checkpoint arrest in this system [Li and Nicklas, 1995].…”

“…However, experiments in insect spermatocytes have suggested that the lack of tension signals checkpoint activation because applying artificial tension to the kinetochore of a misaligned chromosome is able to override the checkpoint arrest in this system [Li and Nicklas, 1995]. Although stabilization of microtubules in mammalian cells with drugs such as taxol decreases tension across sister kinetochores and activates the SAC, these treatments do not visibly alter microtubule attachment to kinetochores [Skoufias et al, 2001;Waters et al, 1998]. The SAC also appears to monitor tension across sister chromatids in budding yeast, and Ipl1p is required for this response but not for SAC activation in response to a loss of kinetochoremicrotubule attachment [Biggins and Murray, 2001].…”

CENP-meta, an Essential Kinetochore Kinesin Required for the Maintenance of Metaphase Chromosome Alignment in Drosophila

“…These include rod (Karess and Glover 1989; Starr et al, 1998), the MAP kinases ppERK (Shapiro et al 1998; Zecevic et al 1998) and ppMEK (Shapiro et al 1998), and an APC activator protein fizzy (called cdc20/cdh1 in other organisms; Kallio et al 1998). Likewise, the protein kinase polo (Logarinho and Sunkel 1998) and a number of components of the highly conserved spindle assembly checkpoint, including Bub1 (Taylor and McKeon 1997; Taylor et al 1998; Basu et al 1999), Bub3 (Taylor et al 1998; Basu et al 1998; Martinez-Exposito et al 1999), Mad1 (Jin et al 1998; Chen et al 1998), and Mad2 (Waters et al 1998; Chen et al 1996, Chen et al 1998), are transiently kinetochore-bound.…”