Slipping past the spindle assembly checkpoint Radhika Subramanian and Tarun M. Kapoor Error-free genome segregation depends on the spindle assembly checkpoint (SAC), a signalling network that delays anaphase onset until chromosomes have established proper spindle attachments. Three reports now quantitatively examine the sensitivity and robustness of the SAC response. A proofreading network comprised of proteins, known as the spindle assembly checkpoint (SAC), is required to ensure the accurate partitioning of genomic material during cell division. The SAC inhibits anaphase onset until all the chromosomes in the dividing cell are properly attached to the spindle microtubules1. As even a single mis-segregation event can result in altered chromosome numbers or aneuploidy 2 (a condition frequently observed in cancer cells), understanding the checkpoint response has been of long-standing interest in the field. A protein-based network that is designed to prevent the loss of even 1 of 46 chromatids must be very sensitive to the error signal. Rather than responding to a ‘proceed to anaphase’ signal and somehow measuring a signal generated when all 46 chromosome–microtubule attachments are established, the SAC responds to a ‘stop anaphase’ signal that is generated by unattached kinetochores. In current models, anaphase is blocked by only one unattached sister chromatid, suggesting that the minimal signal to obtain a full response of the SAC is equal to that generated by a single failure in spindle attachment 3. In mammalian cells, approximately 25 microtubules attach to each kinetochore, the site of chromosome–microtubule attachment4. Does a kinetochore with only 15 microtubules attached generate 60% of the error signal and, if so, can two partially attached kinetochores (that together generate a greater than 100% signal) block anaphase? In addition to sensitivity, the checkpoint must also be robust and function Radhika Subramanian and Tarun M. Kapoor are in the Laboratory of Chemistry and Cell Biology, The Rockefeller University, 1230 York Avenue, New York, New York 10065, USA. e-mail: [email protected]

in the face of intracellular or extracellular fluctuations. How the spindle checkpoint generates a sensitive yet robust response remains poorly understood. Three new studies using complementary approaches analyse the SAC response and begin to answer these questions5–7. In general, the response generated by a biochemical network to an input signal may be graded or binary. When the response is graded, the output correlates with the input signal. This type of a response has been described for tissues during development, wherein morphogen gradients can determine different cell fates in a concentration-dependent manner 8. When the network’s response is binary, the system reacts to a threshold in an ultrasensitive switch-like fashion. One of the best understood examples of this type of response is the MAP kinase signalling module, which can generate an all-ornone response to a stimulus9. Is the response of the SAC to unattached chromosomes graded or binary? The prevailing view in the field, which is based on the observation that even single unattached chromosomes can delay mitosis by many hours3, has been that the SAC generates a switch-like ‘all-or-none’ response. The main elements of the SAC signalling network have been elucidated1,10. In current models, inhibition of the anaphase-promoting complex/cyclosome (APC/C)-dependent ubiquitylation and subsequent proteolysis of substrates, including securin and cyclin  B (cell-cycle-progression regulatory proteins), depends on the formation of the MCC (mitotic checkpoint complex). Each unattached kinetochore catalyses a conformational change in the spindle checkpoint protein Mad2 so as to bind cell-division cycle protein 20 (Cdc20), and, together with the mitotic checkpoint proteins Bub3 and BubR1, forms the MCC (Fig.  1a,

NATURE CELL BIOLOGY VOLUME 15 | NUMBER 11 | NOVEMBER 2013 © 2013 Macmillan Publishers Limited. All rights reserved

inset). Despite these advances, a systematic analysis of the SAC response to the number of unattached chromosomes has been lacking, and it also remains unclear how a single kinetochore can generate enough MCC to inhibit all of the APC/C in a cell. In one of three studies of the SAC in this issue, Dick and Gerlich acutely sever kinetochore fibres using a laser-based method to detach one chromosome from the metaphase spindle and monitor the checkpoint response in a single living cell6. They first demonstrate that a laser-detached chromosome rapidly (in less than 2 minutes) accumulates Mad2 at the kinetochore, an indicator of the SAC response. Surprisingly, they find that around 30% of cells enter anaphase even in the presence of chromosomes that fail to align at the metaphase plate, most probably due to the lack of proper microtubule attachments. This is in striking contrast to the persistent mitotic arrest that is induced when all chromosomes are unattached in the presence of the microtubule destabilizing agent nocodazole, suggesting that the strength of the SAC response may be graded. To gain insight into the SAC response, the authors analyse securin degradation as a measurement of APC/C activity. They find that the rate of securin degradation varies in response to the checkpoint, ranging from being very slow when many chromosomes are detached, to an approximately 60-fold faster rate when all chromosomes are properly connected to the spindle. Furthermore, they show that after most chromosomes have attached to the spindle and the APC/C has been activated, a laser-detached chromosome takes more than 5 min to generate enough signal to inhibit the APC/C. This suggests that there is a critical time-window during mitosis for mounting a SAC response, 1261


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Figure 1 Sensitivity and robustness of the spindle assembly checkpoint. (a) Schematic showing that the SAC response scales with increasing numbers of unattached chromosomes. Orange lines represent microtubules, chromosomes are coloured grey and black circles are kinetochores. Inset: unattached chromosomes accumulate Mad1 and C-Mad2 (closed Mad2), and catalyse the conversion of O-Mad2 (open Mad2) to cytoplasmic C-Mad2, which binds BubR1, Bub3 and Cdc20 to assemble into the mitotic checkpoint complex (MCC). The MCC inhibits the activity of the APC/C to block anaphase. (b) The SAC response changes with increasing the number of laser-detached chromosomes (dashed line)6 and in the presence of different spindle poisons (taxol; di-methyl anastron, DMA; and nocodazole), indicated by the grey bands5. (c) The SAC response also changes with increasing Mad2 concentration7. Highlighted are situations when Mad2 efficiently sequesters Cdc20 to mount a strong checkpoint response (red), and when Cdc20 is in excess (green), which results in a weak checkpoint response.

and chromosome attachment errors occurring just before anaphase have a higher probability of slipping past the checkpoint. Importantly, their analyses also shows that the extent of APC/C inhibition correlates with the number of unattached chromosomes, consistent with a graded SAC response (Fig. 1a,b). In another report published in this issue, Collin et al. use a different approach to measure the strength of the spindle assembly checkpoint response5. Building on their earlier work, the authors demonstrate that degradation of cyclin A2 by APC/C before anaphase can be used to quantitatively measure the SAC response11. Using this assay and different spindle poisons, they test the hypothesis that variable duration of mitotic arrest may be governed by the strength of the SAC signal. Interestingly, these studies reveal a correlation between the strength of the SAC signal and the number of 1262

Mad2 foci at kinetochores (Fig. 1b). Together with the findings of Dick and Gerlich, these results indicate that the SAC response is likely to be graded. Measurements by Collin et al. also indicate that, under their experimental conditions, the amount of Mad2 per kinetochore correlates with the SAC response. They further show that partial reduction of Mad2 levels in single cells leads to shorter mitotic arrest, consistent with the amount of Mad2 controlling the strength of the SAC response. This raises the question of how robust the checkpoint is to fluctuations in the cellular abundance of the core SAC components. The third paper, by Heinrich et al., addresses this by quantitative analysis of the SAC response in fission yeast 7. First, the authors determine the absolute and relative concentrations of core SAC proteins. The GFP-fusions of the SAC

genes are expressed from their endogenous promoters, and protein abundance is quantified using fluorescence correlation spectroscopy and quantitative immunoblotting. Next, they systematically analyse the mitotic delay to assess the SAC response after altering Mad1, Mad2 and Mad3 concentrations by promoter modification. These studies reveal that of these three proteins, the checkpoint is most sensitive to the abundance of Mad2, as well as to the relative amounts of the SAC proteins (Fig. 1c). Strikingly, altering protein concentrations can not only inactivate the checkpoint, but can under some conditions also result in a bimodal population split, with only one population of cells retaining proper checkpoint function. Given the observed sensitivity of the checkpoint to protein concentrations, how does the checkpoint function reliably in the face of inherent fluctuations due to transcriptional noise? To address this question, Heinrich et al. examine the cell-to-cell variation in SAC protein expression levels. Remarkably, they find that the variability in the expression of SAC components is quite low (around 10%), providing an explanation for why the checkpoint functions reliably under normal conditions. They also find that at least some fluctuations can be ‘buffered’ by compensatory changes in protein concentrations. For example, they report that a reduction in Mad2 levels by 40–65% can be rescued by a concomitant reduction in Cdc20 (Slp1 in fission yeast) concentration. This suggests that the checkpoint can function reliably as long as a compensatory mechanism ensures that cytoplasmic Cdc20 is efficiently sequestered in the MCC. The findings from these papers, along with earlier studies, indicate that there are several ways to slip past the spindle assembly checkpoint. First, it has been shown that merotelic attachments (when one kinetochore is attached to both spindle poles) do not generate a checkpoint response and can lead to ‘lagging’ chromosomes that get left behind during anaphase, typically near the middle of the dividing cell12. Second, if a chromosome detaches from the spindle too close to anaphase, there is insufficient time to block the cell cycle and prevent mis-segregation6. Third, a cell that cannot properly regulate the total and relative levels of the SAC proteins may fail to ensure error-free chromosome segregation7. An open question is whether the required precise balance in the levels of the SAC components is maintained at the level of transcription, translation or protein

NATURE CELL BIOLOGY VOLUME 15 | NUMBER 11 | NOVEMBER 2013 © 2013 Macmillan Publishers Limited. All rights reserved

NEWS AND VIEWS degradation. It is likely that combining the use of quantitative proteomics with next-generation sequencing-based methods will answer this question. Contrary to the previously held view, these studies reveal that the checkpoint response is graded rather than binary 5,6. Why didn’t a toggle-switch-like and more robust checkpoint evolve? It is possible that cells need to balance the time needed to biochemically amplify the APC/C inhibitory signal from a single (rather than a few) unattached chromosome, against the cost of prolonging cell division, a stage of the cell cycle during which several other basic

cellular processes (for example, transcription, translation and protein trafficking) are turned off. Kinetochore–microtubule interactions can be reconstituted with purified components13 and it seems likely that a biochemically defined system to study the SAC response will be devised in the near future. These minimal systems should allow further testing of why a graded response that allows some errors is the mechanism that is safeguarding our genomes. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. 1. Musacchio, A. & Salmon, E. D. Nat. Rev. Mol. Cell Biol. 8, 379–393 (2007).

2. Kops, G. J., Weaver, B. A. & Cleveland, D. W. Nat. Rev. Cancer 5, 773–785 (2005). 3. Rieder, C. L., Cole, R. W., Khodjakov, A. & Sluder, G. J. Cell Biol. 130, 941–948 (1995). 4. Cheeseman, I. M. & Desai, A. Nat. Rev. Mol. Cell Biol. 9, 33–46 (2008). 5. Collin, P., Nashchekina, O., Walker, R. & Pines, J. Nat. Cell Biol. 15, 1378–1385 (2013). 6. Dick, A. E. & Gerlich, D. W. Nat. Cell Biol. 15, 1370– 1377 (2013). 7. Heinrich, S. et  al. Nat. Cell Biol. 15, 1328–1339 (2013). 8. Grimm, O., Coppey, M. & Wieschaus, E. Development 137, 2253–2264 (2010). 9. Ferrell, J. E., Jr. Trends Biochem. Sci. 21, 460–466 (1996). 10. Yu, H. J. Cell Biol. 173, 153–157 (2006). 11. Di Fiore, B. & Pines, J. J. Cell Biol. 190, 501–509 (2010). 12. Cimini, D. et al. J. Cell Biol. 153, 517–527 (2001). 13. Akiyoshi, B. et al. Nature 468, 576–579 (2010).

Crosstalk between mTOR complexes Jianling Xie and Christopher G. Proud The mTOR protein kinase controls anabolic processes as part of mTOR complexes 1 and 2 (mTORC1 and mTORC2). The two complexes are now shown to be involved in a negative feedback regulatory mechanism, in which mTORC1 stimulation inactivates mTORC2 through the inhibitory phosphorylation of the mTORC2 component Sin1. The mammalian target of rapamycin (mTOR) protein kinase plays a central role in regulating cellular processes including, among others, cell growth, proliferation, cell survival and autophagy 1. mTOR phosphorylates distinct sets of substrates as a component of two different protein complexes, mTORC1 and mTORC2. Signalling through the mTORC1 pathway is stimulated by growth factors and hormones such as insulin, which coordinate the activation of mTORC1 by the G  protein Rheb, and the inhibition of Rheb by tuberous sclerosis 2 (TSC2)2. Specifically, insulin triggers a signalling cascade involving phosphatidylinositide-3-kinase (PI(3)K) and protein kinase B, also known as Akt. In turn, Akt phosphorylates and inactivates TSC2, allowing Rheb to activate mTORC1 (ref.  3). Phosphorylation of some mTORC1 substrates is inhibited by rapamycin, a macrolide antibiotic. Among other effects, rapamycin can inhibit cell proliferation. As a consequence, it has a number of clinical applications1. Rapamycin-sensitive Jianling Xie and Christopher G. Proud are at the Centre for Biological Sciences, Life Sciences Building (B85), University of Southampton, Southampton SO17 1BJ, UK. e-mail: [email protected]

mTORC1 substrates include the ribosomal protein S6 kinases (S6K1 and S6K2), which play an important role in controlling cell and animal size and modulating insulin signalling 4. mTORC1 substrates are thought to be recruited to this complex by its component Raptor 1. mTORC2 contains mTOR, Rictor, mLst8 (also found in mTORC1) and Sin1, plus additional components5. Sin1 seems crucial for the integrity of mTORC2 (ref. 6) and for recruiting substrates such as Akt and protein kinase C (PKC) to mTORC2 to facilitate their phosphorylation7. Insulin and insulin-like growth factors (IGFs) activate Akt by stimulating its phosphorylation at two sites. Akt Thr  308, which is phosphorylated by PDK1 (ref. 8), is crucial for activation. Akt Ser 473 is phosphorylated by mTORC2, and this is important for Akt to phosphorylate some of its substrates (but not TSC2, for example)9. Much less is known about the regulation of mTORC2 than about mTORC1. Interestingly, mTORC1 can impair the activation of mTORC2 by phosphorylating insulin receptor substrate-1 (IRS-1, an S6K substrate10) and Grb10 (refs 11,12), both of which are involved in signalling events upstream of PI(3)K and therefore of both mTOR complexes (Fig. 1a). Previously, neither a specific

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feedback regulatory link between mTORC1 and mTORC2, nor a negative feedback mechanism towards mTOR complexes in response to other growth stimuli, had been identified. In this issue, Liu et al.13 show that negative regulation of mTORC2 by mTORC1 is still observed in cells deleted for IRS-1 and Grb10, through the mTORC1-dependent phosphorylation of Sin1. The authors showed that Sin1 undergoes phosphorylation in response to various growth stimuli in HeLa epithelial cells (Fig.  1b). Moreover, this phosphorylation was eliminated by compounds that inhibit mTORC1 or S6K1. These findings point to a potential regulatory link between mTORC1 and mTORC2 through S6K1. S6Ks and Akt both phosphorylate serines or threonines that have adjacent N-terminal arginyl residues. Liu et al.13 show that Sin1 contains two such phosphorylation sites, Thr 86 and Thr 398, both of which can be phosphorylated by S6K1. Consistent with this, all stimuli that activate S6K1 also induce phosphorylation of these sites, and this is impaired by interfering with mTORC1 or S6K1. In a separate large-scale phosphoproteomic study in mouse 3T3-L1 adipocytes, Humphrey et al.14 also found that insulin rapidly enhances phosphorylation of Sin1 at Thr 86. Judging by its timing and sensitivity to 1263

Slipping past the spindle assembly checkpoint.

Error-free genome segregation depends on the spindle assembly checkpoint (SAC), a signalling network that delays anaphase onset until chromosomes have...
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