The Experts below are selected from a list of 72963 Experts worldwide ranked by ideXlab platform
Andrea Musacchio - One of the best experts on this subject based on the ideXlab platform.
-
role of the MAD2 dimerization interface in the spindle assembly checkpoint independent of kinetochores
Current Biology, 2012Co-Authors: Luca Mariani, Luigi Nezi, Andrea Musacchio, Simonetta Piatti, Elena Chiroli, Heiko Muller, Andrea CilibertoAbstract:Summary Background The spindle assembly checkpoint (SAC) arrests cells when kinetochores are unattached to spindle microtubules. The signaling pathway is initiated at the kinetochores by one SAC component, MAD2, which catalyzes the initial steps of the cascade via the conformational dimerization of its open and closed conformers. Away from kinetochores, the dimerization surface of MAD2 has been proposed, based on data in vitro, to either interact with SAC activators or inactivators and thus to contribute to SAC activation or silencing. Here, we analyze its role in vivo. Results To analyze the putative pathway downstream of the kinetochores, we used two complementary approaches: we activated the SAC ectopically and independently from kinetochores, and we separated genetically the kinetochore-dependent and independent pools of MAD2. We found that the dimerization surface is required also downstream of kinetochores to mount a checkpoint response. Conclusion Our results show that away from kinetochores the dimerization surface is required for stabilizing the end-product of the pathway, the mitotic checkpoint complex. Surprisingly, downstream of kinetochores the surface does not mediate MAD2 dimerization. Instead, our results are consistent with a role of Mad3 as the main interactor of MAD2 via the dimerization surface.
-
sustained mps1 activity is required in mitosis to recruit o MAD2 to the mad1 c MAD2 core complex
Journal of Cell Biology, 2010Co-Authors: Laura Hewi, Anthony Tighe, Stefano Santaguida, Anne White, Clifford David Jones, Andrea Musacchio, Stephe Gree, Stephe S TayloAbstract:Mps1 is an essential component of the spindle assembly checkpoint. In this study, we describe a novel Mps1 inhibitor, AZ3146, and use it to probe the role of Mps1’s catalytic activity during mitosis. When Mps1 is inhibited before mitotic entry, subsequent recruitment of Mad1 and MAD2 to kinetochores is abolished. However, if Mps1 is inhibited after mitotic entry, the Mad1–C-MAD2 core complex remains kinetochore bound, but O-MAD2 is not recruited to the core. Although inhibiting Mps1 also interferes with chromosome alignment, we see no obvious effect on aurora B activity. In contrast, kinetochore recruitment of centromere protein E (CENP-E), a kinesin-related motor protein, is severely impaired. Strikingly, inhibition of Mps1 significantly increases its own abundance at kinetochores. Furthermore, we show that Mps1 can dimerize and transphosphorylate in cells. We propose a model whereby Mps1 transphosphorylation results in its release from kinetochores, thus facilitating recruitment of O-MAD2 and CENP-E and thereby simultaneously promoting checkpoint signaling and chromosome congression.
-
The MAD2 Conformational Dimer: Structure and Implications for the Spindle Assembly Checkpoint
Cell, 2007Co-Authors: Marina Mapelli, Stefano Santaguida, Lucia Massimiliano, Andrea MusacchioAbstract:The 25 kDa MAD2 protein is a key player in the spindle assembly checkpoint, a safeguard against chromosome segregation errors in mitosis. MAD2 combines three unusual properties. First, MAD2 adopts two conformations with distinct topologies, open (O) and closed (C) MAD2. Second, C-MAD2 forms topological links with its two best-characterized protein ligands, Mad1 and Cdc20. Third, O-MAD2 and C-MAD2 engage in a "conformational" dimer that is essential for spindle checkpoint function in different organisms. The crystal structure of the O-MAD2-C-MAD2 conformational dimer, reported here, reveals an asymmetric interface that explains the selective dimerization of the O-MAD2 and C-MAD2 conformers. The structure also identifies several buried hydrophobic residues whose rearrangement correlates with the MAD2 topological change. The structure of the O-MAD2-C-MAD2 conformational dimer is consistent with a catalytic model in which a C-MAD2 template facilitates the binding of O-MAD2 to Cdc20, the target of MAD2 in the spindle checkpoint.
-
Accumulation of MAD2–Cdc20 complex during spindle checkpoint activation requires binding of open and closed conformers of MAD2 in Saccharomyces cerevisiae
Journal of Cell Biology, 2006Co-Authors: Luigi Nezi, Anna De Antoni, Simonetta Piatti, Giulia Rancati, Sebastiano Pasqualato, Andrea MusacchioAbstract:The spindle assembly checkpoint (SAC) coordinates mitotic progression with sister chromatid alignment. In mitosis, the checkpoint machinery accumulates at kinetochores, which are scaffolds devoted to microtubule capture. The checkpoint protein MAD2 (mitotic arrest deficient 2) adopts two conformations: open (O-MAD2) and closed (C-MAD2). C-MAD2 forms when MAD2 binds its checkpoint target Cdc20 or its kinetochore receptor Mad1. When unbound to these ligands, MAD2 folds as O-MAD2. In HeLa cells, an essential interaction between C- and O-MAD2 conformers allows Mad1-bound C-MAD2 to recruit cytosolic O-MAD2 to kinetochores. In this study, we show that the interaction of the O and C conformers of MAD2 is conserved in Saccharomyces cerevisiae. MAD2 mutant alleles impaired in this interaction fail to restore the SAC in a MAD2 deletion strain. The corresponding mutant proteins bind Mad1 normally, but their ability to bind Cdc20 is dramatically impaired in vivo. Our biochemical and genetic evidence shows that the interaction of O- and C-MAD2 is essential for the SAC and is conserved in evolution.
-
accumulation of MAD2 cdc20 complex during spindle checkpoint activation requires binding of open and closed conformers of MAD2 in saccharomyces cerevisiae
Journal of Cell Biology, 2006Co-Authors: Luigi Nezi, Anna De Antoni, Simonetta Piatti, Giulia Rancati, Sebastiano Pasqualato, Andrea MusacchioAbstract:The spindle assembly checkpoint (SAC) coordinates mitotic progression with sister chromatid alignment. In mitosis, the checkpoint machinery accumulates at kinetochores, which are scaffolds devoted to microtubule capture. The checkpoint protein MAD2 (mitotic arrest deficient 2) adopts two conformations: open (O-MAD2) and closed (C-MAD2). C-MAD2 forms when MAD2 binds its checkpoint target Cdc20 or its kinetochore receptor Mad1. When unbound to these ligands, MAD2 folds as O-MAD2. In HeLa cells, an essential interaction between C- and O-MAD2 conformers allows Mad1-bound C-MAD2 to recruit cytosolic O-MAD2 to kinetochores. In this study, we show that the interaction of the O and C conformers of MAD2 is conserved in Saccharomyces cerevisiae. MAD2 mutant alleles impaired in this interaction fail to restore the SAC in a MAD2 deletion strain. The corresponding mutant proteins bind Mad1 normally, but their ability to bind Cdc20 is dramatically impaired in vivo. Our biochemical and genetic evidence shows that the interaction of O- and C-MAD2 is essential for the SAC and is conserved in evolution.
Helder Maiato - One of the best experts on this subject based on the ideXlab platform.
-
spindle assembly checkpoint robustness requires tpr mediated regulation of mad1 MAD2 proteostasis
Journal of Cell Biology, 2013Co-Authors: Nina Schweizer, Cristina Ferrás, David M Kern, Elsa Logarinho, Iain M. Cheeseman, Helder MaiatoAbstract:Tpr is a conserved nuclear pore complex (NPC) protein implicated in the spindle assembly checkpoint (SAC) by an unknown mechanism. Here, we show that Tpr is required for normal SAC response by stabilizing Mad1 and MAD2 before mitosis. Tpr coimmunoprecipitated with Mad1 and MAD2 (hereafter designated as Tpr/Mad1/MAD2 or TM2 complex) during interphase and mitosis, and is required for Mad1–c-MAD2 recruitment to NPCs. Interestingly, Tpr was normally undetectable at kinetochores and dispensable for Mad1, but not for MAD2, kinetochore localization, which suggests that SAC robustness depends on MAD2 levels at kinetochores. Protein half-life measurements demonstrate that Tpr stabilizes Mad1 and MAD2, ensuring normal Mad1–c-MAD2 production in an mRNA- and kinetochore-independent manner. Overexpression of GFP-MAD2 restored normal SAC response and MAD2 kinetochore levels in Tpr-depleted cells. Mechanistically, we provide evidence that Tpr might spatially regulate SAC proteostasis through the SUMO-isopeptidases SENP1 and SENP2 at NPCs. Thus, Tpr is a kinetochore-independent, rate-limiting factor required to mount and sustain a robust SAC response.
-
Spindle assembly checkpoint robustness requires Tpr-mediated regulation of Mad1/MAD2 proteostasis
Journal of Cell Biology, 2013Co-Authors: Nina Schweizer, Cristina Ferrás, David M Kern, Elsa Logarinho, Iain M. Cheeseman, Helder MaiatoAbstract:Tpr is a conserved nuclear pore complex (NPC) protein implicated in the spindle assembly checkpoint (SAC) by an unknown mechanism. Here, we show that Tpr is required for normal SAC response by stabilizing Mad1 and MAD2 before mitosis. Tpr coimmunoprecipitated with Mad1 and MAD2 (hereafter designated as Tpr/Mad1/MAD2 or TM2 complex) during interphase and mitosis, and is required for Mad1–c-MAD2 recruitment to NPCs. Interestingly, Tpr was normally undetectable at kinetochores and dispensable for Mad1, but not for MAD2, kinetochore localization, which suggests that SAC robustness depends on MAD2 levels at kinetochores. Protein half-life measurements demonstrate that Tpr stabilizes Mad1 and MAD2, ensuring normal Mad1–c-MAD2 production in an mRNA- and kinetochore-independent manner. Overexpression of GFP-MAD2 restored normal SAC response and MAD2 kinetochore levels in Tpr-depleted cells. Mechanistically, we provide evidence that Tpr might spatially regulate SAC proteostasis through the SUMO-isopeptidases SENP1 and SENP2 at NPCs. Thus, Tpr is a kinetochore-independent, rate-limiting factor required to mount and sustain a robust SAC response.
-
Spatiotemporal control of mitosis by the conserved spindle matrix protein Megator
The Journal of cell biology, 2009Co-Authors: Mariana Lince-faria, Stefano Maffini, Bernardo Orr, Yun Ding, Cláudia Florindo, Claudio E. Sunkel, Álvaro A. Tavares, Jørgen Johansen, Kristen M. Johansen, Helder MaiatoAbstract:A putative spindle matrix has been hypothesized to mediate chromosome motion, but its existence and functionality remain controversial. In this report, we show that Megator (Mtor), the Drosophila melanogaster counterpart of the human nuclear pore complex protein translocated promoter region (Tpr), and the spindle assembly checkpoint (SAC) protein MAD2 form a conserved complex that localizes to a nuclear derived spindle matrix in living cells. Fluorescence recovery after photobleaching experiments supports that Mtor is retained around spindle microtubules, where it shows distinct dynamic properties. Mtor/Tpr promotes the recruitment of MAD2 and Mps1 but not Mad1 to unattached kinetochores (KTs), mediating normal mitotic duration and SAC response. At anaphase, Mtor plays a role in spindle elongation, thereby affecting normal chromosome movement. We propose that Mtor/Tpr functions as a spatial regulator of the SAC, which ensures the efficient recruitment of MAD2 to unattached KTs at the onset of mitosis and proper spindle maturation, whereas enrichment of MAD2 in a spindle matrix helps confine the action of a diffusible “wait anaphase” signal to the vicinity of the spindle.
Kevin G. Hardwick - One of the best experts on this subject based on the ideXlab platform.
-
The Bub1-TPR Domain Interacts Directly with Mad3 to Generate Robust Spindle Checkpoint Arrest.
Current biology : CB, 2019Co-Authors: Ioanna Leontiou, Karen M May, Nitobe London, Lucile Grzesiak, Bethan Medina-pritchard, Priya Amin, A. Arockia Jeyaprakash, Sue Biggins, Kevin G. HardwickAbstract:Summary The spindle checkpoint monitors kinetochore-microtubule interactions and generates a “wait anaphase” delay when any defects are apparent [ 1 , 2 , 3 ]. This provides time for cells to correct chromosome attachment errors and ensure high-fidelity chromosome segregation. Checkpoint signals are generated at unattached chromosomes during mitosis. To activate the checkpoint, Mps1 Mph1 kinase phosphorylates the kinetochore component KNL1 Spc105/Spc7 on conserved MELT motifs to recruit Bub3-Bub1 complexes [ 4 , 5 , 6 ] via a direct Bub3 interaction with phospho-MELT motifs [ 7 , 8 ]. Mps1 Mph1 then phosphorylates Bub1, which strengthens its interaction with Mad1-MAD2 complexes to produce a signaling platform [ 9 , 10 ]. The Bub1-Mad1 platform is thought to recruit Mad3, Cdc20, and MAD2 to produce the mitotic checkpoint complex (MCC), which is the diffusible wait anaphase signal [ 9 , 11 , 12 ]. The MCC binds and inhibits the mitotic E3 ubiquitin ligase, known as Cdc20-anaphase promoting complex/cyclosome (APC/C), and stabilizes securin and cyclin to delay anaphase onset [ 13 , 14 , 15 , 16 , 17 ]. Here we demonstrate, in both budding and fission yeast, that kinetochores and KNL1 Spc105/Spc7 can be bypassed; simply inducing heterodimers of Mps1 Mph1 kinase and Bub1 is sufficient to trigger metaphase arrest that is dependent on Mad1, MAD2, and Mad3. We use this to dissect the domains of Bub1 necessary for arrest, highlighting the need for Bub1-CD1, which binds Mad1 [ 9 ], and Bub1’s highly conserved N-terminal tetratricopeptide repeat (TPR) domain [ 18 , 19 ]. We demonstrate that the Bub1 TPR domain is both necessary and sufficient to bind and recruit Mad3. We propose that this brings Mad3 into close proximity to Mad1-MAD2 and Mps1 Mph1 kinase, enabling efficient generation of MCC complexes.
-
identification of a sgo2 dependent but MAD2 independent pathway controlling anaphase onset in fission yeast
Cell Reports, 2017Co-Authors: John C Meadows, Kevin G. Hardwick, Maria Morasantos, Theresa C Lancaster, Alicja M Sochaj, Graham J Buttrick, Liam J Messin, Jonathan B A MillarAbstract:Summary The onset of anaphase is triggered by activation of the anaphase-promoting complex/cyclosome (APC/C) following silencing of the spindle assembly checkpoint (SAC). APC/C triggers ubiquitination of Securin and Cyclin B, which leads to loss of sister chromatid cohesion and inactivation of Cyclin B/Cdk1, respectively. This promotes relocalization of Aurora B kinase and other components of the chromosome passenger complex (CPC) from centromeres to the spindle midzone. In fission yeast, this is mediated by Clp1 phosphatase-dependent interaction of CPC with Klp9/MKLP2 (kinesin-6). When this interaction is disrupted, kinetochores bi-orient normally, but APC/C activation is delayed via a mechanism that requires Sgo2 and some (Bub1, Mph1/Mps1, and Mad3), but not all (Mad1 and MAD2), components of the SAC and the first, but not second, lysine, glutamic acid, asparagine (KEN) box in Mad3. These data indicate that interaction of CPC with Klp9 terminates a Sgo2-dependent, but MAD2-independent, APC/C-inhibitory pathway that is distinct from the canonical SAC.
-
kinase activity of fission yeast mph1 is required for MAD2 and mad3 to stably bind the anaphase promoting complex
Current Biology, 2012Co-Authors: Judith Zich, Alicja M Sochaj, Heather M Syred, Laura Milne, Atlanta G Cook, Hiro Ohkura, Juri Rappsilber, Kevin G. HardwickAbstract:Summary Defects in chromosome segregation result in aneuploidy, which can lead to disease or cell death [1, 2]. The spindle checkpoint delays anaphase onset until all chromosomes are attached to spindle microtubules in a bipolar fashion [3, 4]. MAD2 is a key checkpoint component that undergoes conformational activation, catalyzed by a Mad1-MAD2 template enriched at unattached kinetochores [5]. MAD2 and Mad3 (BubR1) then bind and inhibit Cdc20 to form the mitotic checkpoint complex (MCC), which binds and inhibits the anaphase promoting complex (APC/C). Checkpoint kinases (Aurora, Bub1, and Mps1) are critical for checkpoint signaling, yet they have poorly defined roles and few substrates have been identified [6–8]. Here we demonstrate that a kinase-dead allele of the fission yeast MPS1 homolog (Mph1) is checkpoint defective and that levels of APC/C-associated MAD2 and Mad3 are dramatically reduced in this mutant. Thus, MCC binding to fission yeast APC/C is dependent on Mph1 kinase activity. We map and mutate several phosphorylation sites in MAD2, producing mutants that display reduced Cdc20-APC/C binding and an inability to maintain checkpoint arrest. We conclude that Mph1 kinase regulates the association of MAD2 with its binding partners and thereby mitotic arrest.
-
the spindle checkpoint functions of mad3 and MAD2 depend on a mad3 ken box mediated interaction with cdc20 anaphase promoting complex apc c
Journal of Biological Chemistry, 2008Co-Authors: Matylda Sczaniecka, Julie Blyth, Jun Song Chen, Anna Feoktistova, Kathleen L Gould, Karen M May, Kevin G. HardwickAbstract:Mitotic progression is driven by proteolytic destruction of securin and cyclins. These proteins are labeled for destruction by an ubiquitin-protein isopeptide ligase (E3) known as the anaphase-promoting complex or cyclosome (APC/C). The APC/C requires activators (Cdc20 or Cdh1) to efficiently recognize its substrates, which are specified by destruction (D box) and/or KEN box signals. The spindle assembly checkpoint responds to unattached kinetochores and to kinetochores lacking tension, both of which reflect incomplete biorientation of chromosomes, by delaying the onset of anaphase. It does this by inhibiting Cdc20-APC/C. Certain checkpoint proteins interact directly with Cdc20, but it remains unclear how the checkpoint acts to efficiently inhibit Cdc20-APC/C activity. In the fission yeast, Schizosaccharomyces pombe, we find that the Mad3 and MAD2 spindle checkpoint proteins interact stably with the APC/C in mitosis. Mad3 contains two KEN boxes, conserved from yeast Mad3 to human BubR1, and mutation of either of these abrogates the spindle checkpoint. Strikingly, mutation of the N-terminal KEN box abolishes incorporation of Mad3 into the mitotic checkpoint complex (Mad3-MAD2-Slp1 in S. pombe, where Slp1 is the Cdc20 homolog that we will refer to as Cdc20 hereafter) and stable association of both Mad3 and MAD2 with the APC/C. Our findings demonstrate that this Mad3 KEN box is a critical mediator of Cdc20-APC/C inhibition, without which neither Mad3 nor MAD2 can associate with the APC/C or inhibit anaphase onset.
-
The spindle checkpoint: structural insights into dynamic signalling
Nature Reviews Molecular Cell Biology, 2002Co-Authors: Andrea Musacchio, Kevin G. HardwickAbstract:The spindle checkpoint monitors the interactions between chromosomes and microtubules. The Mad and Bub proteins are core components of this cell-cycle control. The checkpoint can monitor kinetochore–microtubule attachment and/or the tension exerted at kinetochores upon bipolar attachment. Kinetochores are a key site for monitoring defects and signalling, and for effecting the cell-cycle delay. They can act as the catalytic site for the production of the checkpoint signal ('wait anaphase'), which must then be transmitted globally. Microtubule motors (CENP-E and dynein) and protein kinases (Aurora B) have roles in kinetochore binding and bipolar attachment, and could be involved in sensing tension. MAD2 and other checkpoint proteins have a very dynamic association with kinetochores (half-life of ∼20 s). These dynamics might also be involved in checkpoint silencing. Several checkpoint components (MAD2, BubR1, CENP-E and Rod) are continuously removed from kinetochores by a dynein-dependent mechanism. Two related interactions of MAD2 (with Mad1 and Cdc20) are central to the checkpoint, and crystal structures of a Mad1–MAD2 complex have revealed a 'safety-belt' bind-and-release mechanism. MAD2 and/or BubR1 complexes might stoichiometrically inhibit Cdc20 and thereby the activity of the anaphase-promoting complex. Chromosome segregation is a complex and astonishingly accurate process whose inner working is beginning to be understood at the molecular level. The spindle checkpoint plays a key role in ensuring the fidelity of this process. It monitors the interactions between chromosomes and microtubules, and delays mitotic progression to allow extra time to correct defects. Here, we review and integrate findings on the dynamics of checkpoint proteins at kinetochores with structural information about signalling complexes.
Xuelian Luo - One of the best experts on this subject based on the ideXlab platform.
-
Structure of an intermediate conformer of the spindle checkpoint protein MAD2
Proceedings of the National Academy of Sciences, 2015Co-Authors: Mayuko Hara, Hongbin Sun, Engin Özkan, Xuelian LuoAbstract:The spindle checkpoint senses unattached kinetochores during prometaphase and inhibits the anaphase-promoting complex or cyclosome (APC/C), thus ensuring accurate chromosome segregation. The checkpoint protein mitotic arrest deficient 2 (MAD2) is an unusual protein with multiple folded states. MAD2 adopts the closed conformation (C-MAD2) in a Mad1–MAD2 core complex. In mitosis, kinetochore-bound Mad1–C-MAD2 recruits latent, open MAD2 (O-MAD2) from the cytosol and converts it to an intermediate conformer (I-MAD2), which can then bind and inhibit the APC/C activator cell division cycle 20 (Cdc20) as C-MAD2. Here, we report the crystal structure and NMR analysis of I-MAD2 bound to C-MAD2. Although I-MAD2 retains the O-MAD2 fold in crystal and in solution, its core structural elements undergo discernible rigid-body movements and more closely resemble C-MAD2. Residues exhibiting methyl chemical shift changes in I-MAD2 form a contiguous, interior network that connects its C-MAD2–binding site to the conformationally malleable C-terminal region. Mutations of residues at the I-MAD2–C-MAD2 interface hinder I-MAD2 formation and impede the structural transition of MAD2. Our study provides insight into the conformational activation of MAD2 and establishes the basis of allosteric communication between two distal sites in MAD2.
-
Structure of human Mad1 C-terminal domain reveals its involvement in kinetochore targeting
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Soonjoung Kim, Diana R. Tomchick, Hongbin Sun, Xuelian LuoAbstract:The spindle checkpoint prevents aneuploidy by delaying anaphase onset until all sister chromatids achieve proper microtubule attachment. The kinetochore-bound checkpoint protein complex Mad1-MAD2 promotes the conformational activation of MAD2 and serves as a catalytic engine of checkpoint signaling. How Mad1 is targeted to kinetochores is not understood. Here, we report the crystal structure of the conserved C-terminal domain (CTD) of human Mad1. Mad1 CTD forms a homodimer and, unexpectedly, has a fold similar to those of the kinetochore-binding domains of Spc25 and Csm1. Nonoverlapping Mad1 fragments retain detectable kinetochore targeting. Deletion of the CTD diminishes, does not abolish, Mad1 kinetochore localization. Mutagenesis studies further map the functional interface of Mad1 CTD in kinetochore targeting and implicate Bub1 as its receptor. Our results indicate that CTD is a part of an extensive kinetochore-binding interface of Mad1, and rationalize graded kinetochore targeting of Mad1 during checkpoint signaling.
-
Phosphorylation of the spindle checkpoint protein MAD2 regulates its conformational transition
Proceedings of the National Academy of Sciences, 2010Co-Authors: Soonjoung Kim, Katja Wassmann, Hongbin Sun, Haydn L. Ball, Xuelian LuoAbstract:Regulated conformational changes of proteins are critical for cellular signal transduction. The spindle checkpoint protein MAD2 is an unusual protein with two native folds: the latent open conformer (O-MAD2) and the activated closed conformer (C-MAD2). During mitosis, cytosolic O-MAD2 binds to the Mad1-MAD2 core complex at unattached kinetochores and undergoes conformational activation to become C-MAD2. C-MAD2 binds to and inhibits Cdc20, an activator of APC/C, to prevent precocious anaphase onset. Here, we show that the conformational transition of MAD2 is regulated by phosphorylation of S195 in its C-terminal region. The phospho-mimicking MAD2(S195D) mutant and the phospho-S195 MAD2 protein obtained using intein-mediated semisynthesis do not form C-MAD2 on their own. MAD2(S195D) fails to bind to Cdc20, a low-affinity ligand, but still binds to high-affinity ligands, such as Mad1 and MBP1, forming ligand-bound C-MAD2. Overexpression of MAD2(S195D) in human cells causes checkpoint defects. Our results indicate that MAD2 phosphorylation inhibits its function through differentially regulating its binding to Mad1 and Cdc20 and establish that the conformational change of MAD2 is regulated by posttranslational mechanisms.
-
Insights Into MAD2 Regulation in the Spindle Checkpoint Revealed by the Crystal Structure of the Symmetric MAD2 Dimer.
PLoS Biology, 2008Co-Authors: Maojun Yang, Josep Rizo, Diana R. Tomchick, Mischa Machius, Chyong Jy Liu, Xuelian LuoAbstract:In response to misaligned sister chromatids during mitosis, the spindle checkpoint protein MAD2 inhibits the anaphase-promoting complex or cyclosome (APC/C) through binding to its mitotic activator Cdc20, thus delaying anaphase onset. Mad1, an upstream regulator of MAD2, forms a tight core complex with MAD2 and facilitates MAD2 binding to Cdc20. In the absence of its binding proteins, free MAD2 has two natively folded conformers, termed N1-MAD2/open-MAD2 (O-MAD2) and N2-MAD2/closed MAD2 (C-MAD2), with C-MAD2 being more active in APC/CCdc20 inhibition. Here, we show that whereas O-MAD2 is monomeric, C-MAD2 forms either symmetric C-MAD2–C-MAD2 (C–C) or asymmetric O-MAD2–C-MAD2 (O–C) dimers. We also report the crystal structure of the symmetric C–C MAD2 dimer, revealing the basis for the ability of unliganded C-MAD2, but not O-MAD2 or liganded C-MAD2, to form symmetric dimers. A MAD2 mutant that predominantly forms the C–C dimer is functional in vitro and in living cells. Finally, the Mad1–MAD2 core complex facilitates the conversion of O-MAD2 to C-MAD2 in vitro. Collectively, our results establish the existence of a symmetric MAD2 dimer and provide insights into Mad1-assisted conformational activation of MAD2 in the spindle checkpoint.
-
p31comet blocks MAD2 activation through structural mimicry.
Cell, 2007Co-Authors: Maojun Yang, Josep Rizo, Diana R. Tomchick, Mischa Machius, Xuelian LuoAbstract:Summary The status of spindle checkpoint signaling depends on the balance of two opposing dynamic processes that regulate the highly unusual two-state behavior of MAD2. In mitosis, a Mad1-MAD2 core complex recruits cytosolic MAD2 to kinetochores through MAD2 dimerization and converts MAD2 to a conformer amenable to Cdc20 binding, thereby facilitating checkpoint activation. p31 comet inactivates the checkpoint through binding to Mad1- or Cdc20-bound MAD2, thereby preventing MAD2 activation and promoting the dissociation of the MAD2-Cdc20 complex. Here, we report the crystal structure of the MAD2-p31 comet complex. The C-terminal region of MAD2 that undergoes rearrangement in different MAD2 conformers is a major structural determinant for p31 comet binding, explaining the specificity of p31 comet toward Mad1- or Cdc20-bound MAD2. p31 comet adopts a fold strikingly similar to that of MAD2 and binds at the dimerization interface of MAD2. Thus, p31 comet exploits the two-state behavior of MAD2 to block its activation by acting as an "anti-MAD2."
E D Salmon - One of the best experts on this subject based on the ideXlab platform.
-
In Vitro FRAP Identifies the Minimal Requirements for MAD2 Kinetochore Dynamics
Current Biology, 2006Co-Authors: Martin Vink, Anna De Antoni, Marina Mapelli, Mario Faretta, Lucia Massimiliano, Marco Simonetta, Pietro Transidico, Karin Johanna Ferrari, Andrea Ciliberto, E D SalmonAbstract:Summary Background Mad1 and MAD2 are constituents of the spindle-assembly checkpoint, a device coupling the loss of sister-chromatid cohesion at anaphase to the completion of microtubule attachment of the sister chromatids at metaphase. Fluorescence recovery after photobleaching (FRAP) revealed that the interaction of cytosolic MAD2 with kinetochores is highly dynamic, suggesting a mechanism of catalytic activation of MAD2 at kinetochores followed by its release in a complex with Cdc20. The recruitment of cytosolic MAD2 to kinetochores has been attributed to a stable receptor composed of a distinct pool of MAD2 tightly bound to Mad1. Whether specifically this interaction accounts for the kinetochore dynamics of MAD2 is currently unknown. Results To gain a precise molecular understanding of the interaction of MAD2 with kinetochores, we reconstituted the putative MAD2 kinetochore receptor and developed a kinetochore recruitment assay with purified components. When analyzed by FRAP in vitro, this system faithfully reproduced the previously described in vivo dynamics of MAD2, providing an unequivocal molecular account of the interaction of MAD2 with kinetochores. Using the same approach, we dissected the mechanism of action of p31 comet , a spindle-assembly checkpoint inhibitor. Conclusions In vitro FRAP is a widely applicable approach to dissecting the molecular bases of the interaction of a macromolecule with an insoluble cellular scaffold. The combination of in vitro fluorescence recovery after photobleaching with additional fluorescence-based assays in vitro can be used to unveil mechanism, stoichiometry, and kinetic parameters of a macromolecular interaction, all of which are important for modeling protein interaction networks.
-
the mad1 MAD2 complex as a template for MAD2 activation in the spindle assembly checkpoint
Current Biology, 2005Co-Authors: Anna De Antoni, Chad G Pearson, Daniela Cimini, Julie C Canman, Valeria Sala, Luigi Nezi, Marina Mapelli, Lucia Sironi, Mario Faretta, E D SalmonAbstract:Abstract 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.
-
The Mad1/MAD2 Complex as a Template for MAD2 Activation in the Spindle Assembly Checkpoint
Current Biology, 2005Co-Authors: Anna De Antoni, Chad G Pearson, Daniela Cimini, Julie C Canman, Valeria Sala, Luigi Nezi, Marina Mapelli, Lucia Sironi, Mario Faretta, E D SalmonAbstract:Abstract 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.
-
spindle checkpoint protein dynamics at kinetochores in living cells
Current Biology, 2004Co-Authors: Bonnie Howell, Guowei Fang, Ben Moree, Emily M Farrar, Scott Stewart, E D SalmonAbstract:Abstract 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 PtK 2 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 ∼50% displaying very fast kinetics ( t 1/2 of ∼1–3 s) and ∼50% displaying slower kinetics similar to the single exponential kinetics of GFP-MAD2 and GFP-Bub3 ( t 1/2 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-Cdc20 Δ1–167 , which lacks the major MAD2 binding domain but retains kinetochore localization. GFP-Mps1 exhibited single exponential kinetics at unattached kinetochores with a t 1/2 of ∼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.
-
Nuf2 and Hec1 are required for retention of the checkpoint proteins Mad1 and MAD2 to kinetochores.
Current Biology, 2003Co-Authors: Jennifer G. Deluca, Julie C Canman, Bonnie Howell, Jennifer M. Hickey, Guowei Fang, E D SalmonAbstract:Abstract 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 [1–9]. 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 [5]. 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 [6]. 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.
