Mad1 contribution to spindle assembly checkpoint signalling goes beyond presenting Mad2 at kinetochores

The spindle assembly checkpoint inhibits anaphase until all chromosomes have become attached to the mitotic spindle. A complex between the checkpoint proteins Mad1 and Mad2 provides a platform for Mad2:Mad2 dimerization at unattached kinetochores, which enables Mad2 to delay anaphase. Here, we show that mutations in Bub1 and within the Mad1 C‐terminal domain impair the kinetochore localization of Mad1:Mad2 and abrogate checkpoint activity. Artificial kinetochore recruitment of Mad1 in these mutants co‐recruits Mad2; however, the checkpoint remains non‐functional. We identify specific mutations within the C‐terminal head of Mad1 that impair checkpoint activity without affecting the kinetochore localization of Bub1, Mad1 or Mad2. Hence, Mad1 potentially in conjunction with Bub1 has a crucial role in checkpoint signalling in addition to presenting Mad2.     

Spindle checkpoint protein dynamics at kinetochores in living cells

BACKGROUND: To test current models for how unattached and untense kinetochores prevent Cdc20 activation of the anaphase-promoting complex/cyclosome (APC/C) throughout the spindle and the cytoplasm, we used GFP fusions and live-cell imaging to quantify the abundance and dynamics of spindle checkpoint proteins Mad1, Mad2, Bub1, BubR1, Mps1, and Cdc20 at kinetochores during mitosis in living PtK2 cells. RESULTS: Unattached kinetochores in prometaphase bound on average only a small fraction (estimated at 500-5000 molecules) of the total cellular pool of each spindle checkpoint protein. Measurements of fluorescence recovery after photobleaching (FRAP) showed that GFP-Cdc20 and GFP-BubR1 exhibit biphasic exponential kinetics at unattached kinetochores, with approximately 50% displaying very fast kinetics (t1/2 of approximately 1-3 s) and approximately 50% displaying slower kinetics similar to the single exponential kinetics of GFP-Mad2 and GFP-Bub3 (t1/2 of 21-23 s). The slower phase of GFP-Cdc20 likely represents complex formation with Mad2 since it was tension insensitive and, unlike the fast phase, it was absent at metaphase kinetochores that lack Mad2 but retain Cdc20 and was absent at unattached prometaphase kinetochores for the Cdc20 derivative GFP-Cdc20delta1-167, which lacks the major Mad2 binding domain but retains kinetochore localization. GFP-Mps1 exhibited single exponential kinetics at unattached kinetochores with a t1/2 of approximately 10 s, whereas most GFP-Mad1 and GFP-Bub1 were much more stable components. CONCLUSIONS: Our data support catalytic models of checkpoint activation where Mad1 and Bub1 are mainly resident, Mad2 free of Mad1, BubR1 and Bub3 free of Bub1, Cdc20, and Mps1 dynamically exchange as part of the diffuse wait-anaphase signal; and Mad2 interacts with Cdc20 at unattached kinetochores.     

Dynein-dependent transport of spindle assembly checkpoint proteins off kinetochores toward spindle poles

A predominant mechanism of spindle assembly checkpoint (SAC) silencing is dynein-mediated transport of certain kinetochore proteins along microtubules. There are still conflicting data as to which SAC proteins are dynein cargoes. Using two ATP reduction assays, we found that the core SAC proteins Mad1, Mad2, Bub1, BubR1, and Bub3 redistributed from attached kinetochores to spindle poles, in a dynein-dependent manner. This redistribution still occurred in metaphase-arrested cells, at a time when the SAC should be satisfied and silenced. Unexpectedly, we found that a pool of Hec1 and Mis12 also relocalizes to spindle poles, suggesting KMN components as additional dynein cargoes. The potential significance of these results for SAC silencing is discussed.     

The closed form of Mad2 is bound to Mad1 and Cdc20 at unattached kinetochores

The spindle assembly checkpoint (SAC) ensures accurate chromosome segregation by delaying anaphase onset in response to unattached kinetochores. Anaphase is delayed by the generation of the mitotic checkpoint complex (MCC) composed of the checkpoint proteins Mad2 and BubR1/Bub3 bound to the protein Cdc20. Current models assume that MCC production is catalyzed at unattached kinetochores and that the Mad1/Mad2 complex is instrumental in the conversion of Mad2 from an open form (O-Mad2) to a closed form (C-Mad2) that can bind to Cdc20. Importantly the levels of Mad2 at kinetochores correlate with SAC activity but whether C-Mad2 at kinetochores exclusively represents its complex with Mad1 is not fully established. Here we use a recently established C-Mad2 specific monoclonal antibody to show that Cdc20 and C-Mad2 levels correlate at kinetochores and that depletion of Cdc20 reduces Mad2 but not Mad1 kinetochore levels. Importantly reintroducing wild type Cdc20 but not Cdc20 R132A, a mutant form that cannot bind Mad2, restores Mad2 levels. In agreement with this live cell imaging of fluorescent tagged Mad2 reveals that Cdc20 depletion strongly reduces Mad2 localization to kinetochores. These results support the presence of Mad2-Cdc20 complexes at kinetochores in agreement with current models of the SAC but also argue that Mad2 levels at kinetochores cannot be used as a direct readout of Mad1 levels.     

Yeast kinetochores do not stabilize Stu2p-dependent spindle microtubule dynamics

The interaction of kinetochores with dynamic microtubules during mitosis is essential for proper centromere motility, congression to the metaphase plate, and subsequent anaphase chromosome segregation. Budding yeast has been critical in the discovery of proteins necessary for this interaction. However, the molecular mechanism for microtubule-kinetochore interactions remains poorly understood. Using live cell imaging and mutations affecting microtubule binding proteins and kinetochore function, we identify a regulatory mechanism for spindle microtubule dynamics involving Stu2p and the core kinetochore component, Ndc10p. Depleting cells of the microtubule binding protein Stu2p reduces kinetochore microtubule dynamics. Centromeres remain under tension but lack motility. Thus, normal microtubule dynamics are not required to maintain tension at the centromere. Loss of the kinetochore (ndc10-1, ndc10-2, and ctf13-30) does not drastically affect spindle microtubule turnover, indicating that Stu2p, not the kinetochore, is the foremost governor of microtubule dynamics. Disruption of kinetochore function with ndc10-1 does not affect the decrease in microtubule turnover in stu2 mutants, suggesting that the kinetochore is not required for microtubule stabilization. Remarkably, a partial kinetochore defect (ndc10-2) suppresses the decreased spindle microtubule turnover in the absence of Stu2p. These results indicate that Stu2p and Ndc10p differentially function in controlling kinetochore microtubule dynamics necessary for centromere movements.     

Tankyrase function at telomeres, spindle poles, and beyond

Telomeres have special needs; they require distinct mechanisms for their protection, replication, and separation at mitosis. A dedicated six-subunit protein complex termed shelterin attends to these needs. But shelterin cannot do it alone and often relies on recruits from other cellular locales. One such recruit is tankyrase 1, a poly(ADP-ribose) polymerase that is brought to telomeres by the shelterin DNA binding subunit TRF1, where it functions in telomere length regulation and sister chromatid separation. An understanding of how tankyrase 1 functions at telomeres has been confounded by its complexity; it localizes to multiple subcellular sites, it has many diverse binding partners, and it has a closely related homolog (tankyrase 2) with which it may functionally overlap. This review summarizes our current knowledge of tankyrases focusing on their localization, binding partners, and function.     

Visualization of PtdIns3P dynamics in living plant cells

To investigate PtdIns3P localization and function in plants, a fluorescent PtdIns3P-specific biosensor (YFP-2xFYVE) was created. On lipid dot blots it bound specifically and with high affinity to PtdIns3P. Transient expression in cowpea protoplasts labelled vacuolar membranes and highly motile structures undergoing fusion and fission. Stable expression in tobacco BY-2 cells labelled similar motile structures, but labelled vacuolar membranes hardly at all. YFP-2xFYVE fluorescence strongly co-localized with the pre-vacuolar marker AtRABF2b, partially co-localized with the endosomal tracer FM4-64, but showed no overlap with the Golgi marker STtmd-CFP. Treatment of cells with wortmannin, a PI3 kinase inhibitor, caused the YFP-2xFYVE fluorescence to redistribute into the cytosol and nucleus within 15 min. BY-2 cells expressing YFP-2xFYVE contained twice as much PtdIns3P as YFP-transformed cells, but this had no effect on cell-growth or stress-induced phospholipid signalling responses. Upon treatment with wortmannin, PtdIns3P levels were reduced by approximately 40% within 15 min in both cell lines. Stable expression of YFP-2xFYVE in Arabidopsis plants labelled different subcellular structures in root compared with shoot tissues. In addition labelling the motile structures common to all cells, YFP-2xFYVE strongly labelled the vacuolar membrane in leaf epidermal and guard cells, suggesting that cell differentiation alters the distribution of PtdIns3P. In dividing BY-2 cells, YFP-2xFYVE-labelled vesicles surrounded the newly formed cell plate, suggesting a role for PtdIns3P in cytokinesis. Together, these data show that YFP-2xFYVE may be used as a biosensor to specifically visualize PtdIns3P in living plant cells.     

Dynein/Dynactin-mediated transport of kinetochore components off kinetochores and onto spindle poles induced by nordihydroguaiaretic acid

The mitotic checkpoint functions to ensure accurate chromosome segregation by regulating the progression from metaphase to anaphase. Once the checkpoint has been satisfied, it is inactivated in order to allow the cell to proceed into anaphase and complete the cell cycle. The minus end-directed microtubule motor dynein/dynactin has been implicated in the silencing of the mitotic checkpoint by "stripping" checkpoint proteins off kinetochores. A recent study suggested that Nordihydroguaiaretic acid (NDGA) stimulates dynein/dynactin-mediated transport of its cargo including ZW10 (Zeste White 10). We analyzed the effects of NDGA on dynein/dynactin dependent transport of the RZZ (Zeste White 10, Roughdeal, Zwilch) complex as well as other kinetochore components from kinetochores to spindle poles. Through this approach we have catalogued several kinetochore and centromere components as dynein/dynactin cargo. These include hZW10, hZwilch, hROD, hSpindly, hMad1, hMad2, hCENP-E, hCdc27, cyclin-B and hMps1. Furthermore, we found that treatment with NDGA induced a robust accumulation and complete stabilization of hZW10 at spindle poles. This finding suggests that NDGA may not induce dynein/dynactin transport but rather interfere with cargo release. Lastly, we determined that NDGA induced accumulation of checkpoint proteins at the poles requires dynein/dynactin-mediated transport, hZW10 kinetochore localization and kinetochore-microtubule attachments but not tension or Aurora B kinase activity.     

Dual regulation of Mad2 localization on kinetochores by Bub1 and Dam1/DASH that ensure proper spindle interaction

The spindle assembly checkpoint monitors the state of spindle–kinetochore interaction to prevent premature onset of anaphase. Although checkpoint proteins, such as Mad2, are localized on kinetochores that do not interact properly with the spindle, it remains unknown how the checkpoint proteins recognize abnormalities in spindle–kinetochore interaction. Here, we report that Mad2 localization on kinetochores in fission yeast is regulated by two partially overlapping but distinct pathways: the Dam1/DASH and the Bub1 pathways. We show that Mad2 is localized on “unattached” as well as “tensionless” kinetochores. Our observations suggest that Bub1 is required for Mad2 to detect tensionless kinetochores, whereas Dam1/DASH is crucial for Mad2 to detect unattached kinetochores. In cells lacking both Bub1 and Dam1/DASH, Mad2 localization on kinetochores is diminished, and mitotic progression appears to be accelerated despite the frequent occurrence of abnormal chromosome segregation. Furthermore, we found that Dam1/DASH is required for promotion of spindle association with unattached kinetochores. In contrast, there is accumulating evidence that Bub1 is involved in resolution of erroneous spindle attachment on tensionless kinetochores. These pathways may act as molecular sensors determining the state of spindle association on each kinetochore, enabling proper regulation of the checkpoint activation as well as promotion/resolution of spindle attachment.     

DDA3 recruits microtubule depolymerase Kif2a to spindle poles and controls spindle dynamics and mitotic chromosome movement

Dynamic turnover of the spindle is a driving force for chromosome congression and segregation in mitosis. Through a functional genomic analysis, we identify DDA3 as a previously unknown regulator of spindle dynamics that is essential for mitotic progression. DDA3 depletion results in a high frequency of unaligned chromosomes, a substantial reduction in tension across sister kinetochores at metaphase, and a decrease in the velocity of chromosome segregation at anaphase. DDA3 associates with the mitotic spindle and controls microtubule (MT) dynamics. Mechanistically, DDA3 interacts with the MT depolymerase Kif2a in an MT-dependent manner and recruits Kif2a to the mitotic spindle and spindle poles. Depletion of DDA3 increases the steady-state levels of spindle MTs by reducing the turnover rate of the mitotic spindle and by increasing the rate of MT polymerization, which phenocopies the effects of partial knockdown of Kif2a. Thus, DDA3 represents a new class of MT-destabilizing protein that controls spindle dynamics and mitotic progression by regulating MT depolymerases.     

Rapid microtubule-independent dynamics of Cdc20 at kinetochores and centrosomes in mammalian cells

Cdc20 is a substrate adaptor and activator of the anaphase-promoting complex/cyclosome (APC/C), the E3 ubiquitin ligase whose activity is required for anaphase onset and exit from mitosis. A green fluorescent protein derivative, Cdc20-GFP, bound to centrosomes throughout the cell cycle and to kinetochores from late prophase to late telophase. We mapped distinct domains of Cdc20 that are required for association with kinetochores and centrosomes. FRAP measurements revealed extremely rapid dynamics at the kinetochores (t1/2 = 5.1 s) and spindle poles (t1/2 = 4.7 s). This rapid turnover is independent of microtubules. Rapid transit of Cdc20 through kinetochores may ensure that spindle checkpoint signaling at unattached/relaxed kinetochores can continuously inhibit APC/CCdc20 targeting of anaphase inhibitors (securins) throughout the cell until all the chromosomes are properly attached to the mitotic spindle.     

Nsk1 ensures accurate chromosome segregation by promoting association of kinetochores to spindle poles during anaphase B

Type 1 phosphatase (PP1) antagonizes Aurora B kinase to stabilize kinetochore-microtubule attachments and to silence the spindle checkpoint. We screened for factors that exacerbate the growth defect of Δdis2 cells, which lack one of two catalytic subunits of PP1 in fission yeast, and identified Nsk1, a novel protein required for accurate chromosome segregation. During interphase, Nsk1 resides in the nucleolus but spreads throughout the nucleoplasm as cells enter mitosis. Following dephosphorylation by Clp1 (Cdc14-like) phosphatase and at least one other phosphatase, Nsk1 localizes to the interface between kinetochores and the inner face of the spindle pole body during anaphase. In the absence of Nsk1, some kinetochores become detached from spindle poles during anaphase B. If this occurs late in anaphase B, then the sister chromatids of unclustered kinetochores segregate to the correct daughter cell. These unclustered kinetochores are efficiently captured, retrieved, bioriented, and segregated during the following mitosis, as long as Dis2 is present. However, if kinetochores are detached from a spindle pole early in anaphase B, then these sister chromatids become missegregated. These data suggest Nsk1 ensures accurate chromosome segregation by promoting the tethering of kinetochores to spindle poles during anaphase B.     

The Mad1/Mad2 complex as a template for Mad2 activation in the spindle assembly checkpoint

Background: The spindle assembly checkpoint (SAC) imparts fidelity to chromosome segregation by delaying anaphase until all sister chromatid pairs have become bipolarly attached. Mad2 is a component of the SAC effector complex that sequesters Cdc20 to halt anaphase. In prometaphase, Mad2 is recruited to kinetochores with the help of Mad1, and it is activated to bind Cdc20. These events are linked to the existence of two distinct conformers of Mad2: a closed conformer bound to its kinetochore receptor Mad1 or its target in the checkpoint Cdc20 and an open conformer unbound to these ligands. Results: We investigated the mechanism of Mad2 recruitment to the kinetochore during checkpoint activation and subsequent transfer to Cdc20. We report that a closed conformer of Mad2 constitutively bound to Mad1, rather than Mad1 itself, is the kinetochore receptor for cytosolic open Mad2 and show that the interaction of open and closed Mad2 conformers is essential to sustain the SAC. Conclusions: We propose that closed Mad2 bound to Mad1 represents a template for the conversion of open Mad2 into closed Mad2 bound to Cdc20. This simple model, which we have named the "Mad2 template" model, predicts a mechanism for cytosolic propagation of the spindle checkpoint signal away from kinetochores.     

Mad2-independent spindle assembly checkpoint activation and controlled metaphase-anaphase transition in Drosophila S2 cells

The spindle assembly checkpoint is essential to maintain genomic stability during cell division. We analyzed the role of the putative Drosophila Mad2 homologue in the spindle assembly checkpoint and mitotic progression. Depletion of Mad2 by RNAi from S2 cells shows that it is essential to prevent mitotic exit after spindle damage, demonstrating its conserved role. Mad2-depleted cells also show accelerated transit through prometaphase and premature sister chromatid separation, fail to form metaphases, and exit mitosis soon after nuclear envelope breakdown with extensive chromatin bridges that result in severe aneuploidy. Interestingly, preventing Mad2-depleted cells from exiting mitosis by a checkpoint-independent arrest allows congression of normally condensed chromosomes. More importantly, a transient mitotic arrest is sufficient for Mad2-depleted cells to exit mitosis with normal patterns of chromosome segregation, suggesting that all the associated phenotypes result from a highly accelerated exit from mitosis. Surprisingly, if Mad2-depleted cells are blocked transiently in mitosis and then released into a media containing a microtubule poison, they arrest with high levels of kinetochore-associated BubR1, properly localized cohesin complex and fail to exit mitosis revealing normal spindle assembly checkpoint activity. This behavior is specific for Mad2 because BubR1-depleted cells fail to arrest in mitosis under these experimental conditions. Taken together our results strongly suggest that Mad2 is exclusively required to delay progression through early stages of prometaphase so that cells have time to fully engage the spindle assembly checkpoint, allowing a controlled metaphase-anaphase transition and normal patterns of chromosome segregation.     

Nuf2 and Hec1 are required for retention of the checkpoint proteins Mad1 and Mad2 to kinetochores

Members of the Ndc80/Nuf2 complex have been shown in several systems to be important in formation of stable kinetochore-microtubule attachments and chromosome alignment in mitosis. In HeLa cells, we have shown that depletion of Nuf2 by RNA interference (RNAi) results in a strong prometaphase block with an active spindle checkpoint, which correlates with low but detectable Mad2 at kinetochores that have no or few stable kinetochore microtubules. Another RNAi study in HeLa cells reported that Hec1 (the human Ndc80 homolog) is required for Mad1 and Mad2 binding to kinetochores and that kinetochore bound Mad2 does not play a role in generating and maintaining the spindle assembly checkpoint. Here, we show that depletion of either Nuf2 or Hec1 by RNAi in HeLa cells results in reduction of both proteins at kinetochores and in the cytoplasm. Mad1 and Mad2 concentrate at kinetochores in late prophase/early prometaphase but become depleted by 5-fold or more over the course of the prometaphase block, which is Mad2 dependent. The reduction of Mad1 and Mad2 is reversible upon spindle depolymerization. Our observations support a model in which Nuf2 and Hec1 function to prevent microtubule-dependent stripping of Mad1 and Mad2 from kinetochores that have not yet formed stable kinetochore-microtubule attachments.     

Structural activation of Mad2 in the mitotic spindle checkpoint: the two-state Mad2 model versus the Mad2 template model

The inheritance of a normal assortment of chromosomes during each cell division relies on a cell-cycle surveillance system called the mitotic spindle checkpoint. The existence of sister chromatids that do not achieve proper bipolar attachment to the mitotic spindle in a cell activates this checkpoint, which inhibits the ubiquitin ligase activity of the anaphase-promoting complex or cyclosome (APC/C) and delays the onset of anaphase. The mitotic arrest deficiency 2 (Mad2) spindle checkpoint protein inhibits APC/C through binding to its mitotic-specific activator, Cdc20. Binding of Mad2 to Cdc20 involves a large conformational change of Mad2 and requires the Mad1-Mad2 interaction in vivo. Two related but distinct models of Mad1-assisted activation of Mad2, the "two-state Mad2" and the "Mad2 template" models, have been proposed. I review the recent structural, biochemical, and cell biological data on Mad2, discuss the differences between the two models, and propose experiments that test their key principles.