Many human tumors that are anueploid and CIN and overexpress the conserved checkpoint protein Mad2 [7
]. Moreover, mice engineered to overexpress Mad2 develop tumors that are CIN [14
]. Interestingly, cells derived from mouse models overexpressing Mad2 display elevated frequencies of lagging chromosomes in anaphase [14
] raising the possibility that Mad2 plays a role as a component of the machinery that regulates k-MT attachment stability to promote the correction of attachment errors needed for faithful chromosome segregation. To determine if Mad2 influences the stability of k-MT attachments during mitosis we measured the dynamics of k-MTs in cells expressing photoactivatable GFP-tubulin (PA-GFP-tubulin). We expressed PA-GFP-tubulin in non-transformed, diploid, chromosomally stable human RPE-1 cells and used fluorescence dissipation after photoactivation of spindle microtubules to determine the stability of k-MT attachments () [3
]. Fluorescence decay of the activated region fit a double exponential curve (r2
>0.99) where the rapidly decaying fluorescence corresponds to non-k-MTs and the slowly decaying fluorescence corresponds to k-MTs (). Non-k-MTs turn over with a half-life (t1/2
) of approximately 14.4 ± 0.1 sec in prometaphase and 15.6 ± 0.1 sec in metaphase and there was no detectable change in these rates in any of our experimental conditions. K-MTs are more stable during mitosis and display t1/2
of 1.8 ± 0.6 min and 3.8 ± 1.1 min in prometaphase and metaphase, respectively, in untreated RPE-1 cells ().
Figure 1 Measurement of kinetochore-microtubule dynamics in cells overexpressing Mad2. (A) DIC and time-lapse fluorescent images of prometaphase and metaphase spindles in untreated (control) and Mad2 overexpression (Mad2OX) in PA-GFP tubulin expressing RPE1 cells (more ...)
Figure 2 Kinetochore-microtubule half-life. kMT half-life calculated from the exponential decay curve of photoactivated fluorescence (r2 > 0.99) under different conditions for (A) prometaphase and (B) metaphase. Error bars represent SD, *p<0.05, (more ...)
Next, we examined k-MT attachment stability in cells depleted of the conserved checkpoint protein Mad2. Mad2 depletion was confirmed by immunoblot and by reductions in the mitotic index of cells in the presence of the microtubule perturbing drug nocodazole indicating functional loss of checkpoint activity (Figure S1
). To prevent premature anaphase onset caused by the absence of a functional mitotic checkpoint in the absence of Mad2, we measured k-MT turnover in Mad2-deficient mitotic cells treated with the proteasome inhibitor MG-132 (5 µM). Delaying anaphase onset using MG-132 does not significantly change k-MT stability relative to control cells in either prometaphase or metaphase () regardless of the length of time that cells were blocked in metaphase (we tested up to 4 hours). The half-life of k-MT attachments in Mad2-deficient cells was significantly reduced relative to control cells in both prometaphase and metaphase (). To eliminate potential off-target effects of the siRNA, we depleted Mad2 using an siRNA sequence derived from the 3’ UTR and rescued the loss of Mad2 by exogenous expression of mCherry-Mad2 (Figure S1
). Mad2 depletion using this siRNA sequence was confirmed by immunoblot and loss of checkpoint function in the presence of nocodazole (Figure S1
). The half-life of k-MT attachments in cells depleted of Mad2 using this siRNA sequence was reduced compared to control cells to an extent that was equivalent to cells depleted with the other siRNA sequence (Figure S1
). Overexpression of mCherry-Mad2 in these Mad2-deficient cells restored both the checkpoint function and k-MT stabililty indicating a specific requirement for Mad2 to regulate k-MT attachment stability.
To determine if the change in k-MT attachment stability was accounted for by loss of checkpoint activity we measured k-MT turnover in cells depleted of another conserved checkpoint protein Mad1. Mad1 depletion was confirmed by immunoblot and by reductions in the mitotic index of cells in the presence of the microtubule perturbing drug nocodazole indicating functional loss of checkpoint activity (Figure S1
). In contrast to Mad2-deficient cells, there was no significant change in the half-life of k-MT attachments in Mad1-deficient cells in either prometaphase or metaphase relative to control cells (). This demonstrates that Mad2 stabilizes k-MT attachments during both prometaphase and metaphase independently of Mad1.
We next tested the contribution of other proteins involved in the metaphase-anaphase transition to k-MT attachment stability by depleting cells of either BubR1 or Cdc20. We confirmed efficient depletion of BubR1 by immunoblot and by reductions in the mitotic index of cells in the presence of the microtubule perturbing drug nocodazole indicating functional loss of checkpoint activity (Figure S1
). BubR1 has a known role in stabilizing k-MT attachments [18
]. Accordingly, we observed significant reductions in the half-life of k-MT attachments in BubR1-deficient cells in both prometaphase and metaphase (). Kinetochore-MTs in cells simultaneously depleted of both Mad2 and BubR1 are significantly less stable than cells depleted of BubR1 alone in both prometaphase and metaphase (Student’s t-test; p=0.04 in prometaphase, p=0.02 in metaphase) demonstrating that BubR1 and Mad2 contribute to the stabilization of k-MTs through independent pathways (). The depletion of Cdc20 was confirmed by mmunoblot and by increases in mitotic index indicating a failure to efficiently activate the APC/C to induce anaphase onset (Figure S1
). The half-life of k-MT attachments in Cdc20-deficient mitotic cells was significantly reduced relative to control cells in both prometaphase and metaphase (), yet indistinguishable from cells lacking Mad2 alone. The half-life of k-MT attachments in cells depleted of both Cdc20 and Mad2 in both prometaphase and metaphase was not significantly different from cells depleted of either Cdc20 or Mad2 alone ( and S1
) demonstrating that Mad2 and Cdc20 act in the same pathway to stabilize k-MT attachments during mitosis.
To test if excess levels of Mad2 hyperstabilize k-MT attachments we overexpressed Mad2 and measured k-MT attachment stability. Transient expression of mCherry-Mad2 was detected by immunoblot and fluorescence imaging in most cells (Figure S2
). As expected, it localized to kinetochores in prometaphase cells (Figure S2
). The expression level of mCherry-Mad2 under these conditions was estimated to be approximately 5- to 10-fold higher than endogenous protein levels. There was little change in the mitotic index of cells overexpressing mCherry-Mad2 consistent with only a 2-fold increase in time spent between prometaphase and anaphase measured previously in mouse cells overexpressing Mad2 [14
]. The attachment stability of k-MTs in cells overexpressing mCherry-Mad2 was significantly increased compared to control cells in both prometaphase and metaphase ( and ). We next tested the effects of overexpression of mutant versions of Mad2 including mCherry-Mad2ΔC, -Mad2V193N, and -Mad2R133A [21
]. The level of overexpression of each protein was equivalent to mCherry-Mad2 (Figure S2
). Neither Mad2ΔC nor Mad2V193N altered k-MT attachment stability indicating that Mad2 must be capable of converting to a closed conformation to influence k-MT attachments (). In contrast, Mad2R133A stabilized k-MT attachments indicating that Mad2 does not need to dimerize to influence k-MT attachments (). Finally, to test if k-MT stabilization by excess Mad2 requires a functional mitotic checkpoint we overexpressed mCherry-Mad2 in Mad1-deficient cells. Immunoblot analyses verify the overexpression of mCherry-Mad2 and the efficient depletion of Mad1 (Figure S1
). Overexpression of mCherry-Mad2 increased the half-life of k-MT attachments to similar extents in mitotic cells with or without Mad1 (). Thus, overexpression of Mad2 hyperstabilizes k-MT attachments through a mechanism that is independent of the Mad1-dependent mitotic checkpoint.
The consequence of increasing k-MT attachment stability is reduced correction of k-MT attachment errors such as merotely that manifest as lagging chromosomes in anaphase [3
]. Overexpression of mCherry-Mad2 significantly increases the frequency of anaphase cells with lagging chromosomes in human RPE-1 cells (). Overexpression of mCherry-Mad2ΔC does not alter the frequency of lagging chromosomes in anaphase () consistent with the lack of change in k-MT attachment stability when this mutant version of Mad2 is overexpressed. To verify that the hyperstabilization of k-MT attachments induced by Mad2 overexpression is the root cause of lagging chromosomes in anaphase we overexpressed the kinesin-13 protein MCAK which has been demonstrated to destabilize k-MT attachments [3
]. The frequency of lagging chromosomes in anaphase in cells overexpressing both Mad2 and MCAK was much lower than cells expressing Mad2 alone and not significantly different from untreated cells (). Similar results were obtained using U2OS cells that are aneuploid and CIN and have an inherently high basal rate of lagging chromosomes in anaphase owing to CIN (Figure S3
). Thus, Mad2 overexpression undermines the correction of k-MT attachment errors by hyperstabilizing k-MT attachments. This elevates chromosome mis-segregation rates through the most common mechanism known to cause CIN in human tumor cells.
Figure 3 MCAK overexpression suppresses lagging chromosomes in anaphase in Mad2 overexpressing cells. (A) Anaphase in RPE-1 cells expressing mCherry-Mad2 (red) alone or both mCherry-Mad2 and GFP-MCAK (green) and stained for DNA (blue). The lagging chromosomes (more ...)
To explore the mechanism through which Mad2 stabilizes k-MT attachments we examined the localization of Aurora B and BubR1, two known regulators of k-MT attachment stability [18
]. The quantity of Aurora B localized to centromeres was significantly increased by Mad2 depletion and significantly decreased by Mad2 overexpression in both prometaphase and metaphase (). The functional activity of Aurora B was similarly influenced by Mad2 levels as judged by alterations in the quantities of Aurora B substrates CenpA () [26
] and histone H3 (data not shown). Mad2 levels also affect the quantity and activity of Aurora B kinase at centromeres in nocodazole-treated mitotic cells (Figure S4
) indicating that these events are independent of microtubule attachment at kinetochores. Moreover, sister kinetochore spacing was unchanged in both prometaphase and metaphase by either overexpression or depletion of Mad2 indicating that Aurora B activity is regulated independently of the spatial positioning of sister kinetochores under these conditions. In contrast, changing Mad2 levels did not significantly affect the quantity of BubR1 localized at kinetochores indicating a specific response of Aurora B to Mad2 levels (). It has been shown that k-MT attachment stability is acutely sensitive to Aurora B function [23
], and these data demonstrate that Mad2 attenuates the degree to which Aurora B kinase destabilizes k-MT attachment stability.
Figure 4 Mad2 affects Aurora B localization and activity at the centromeres. (A) Metaphase of untreated RPE-1 cells (control) and RPE-1 cells overexpressing Mad2 (Mad2OX) and depleted of Mad2 (Mad2KD). Scale bar 5µm. Fluorescence intensities of centromeres (more ...)