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• W2119784671 abstract "The Mad2 protein is required to delay sister chromatid separation until all chromosomes have been aligned on the mitotic spindle. Two recent studies provide new insights into how Mad2 contributes to this remarkable task. The Mad2 protein is required to delay sister chromatid separation until all chromosomes have been aligned on the mitotic spindle. Two recent studies provide new insights into how Mad2 contributes to this remarkable task. The attachment of microtubules to kinetochores on chromosomes is key to mitotic spindle assembly. Microtubules ‘search and capture’ kinetochores in a largely stochastic process, making it impossible to predict when exactly a cell will have all of its chromosomes attached to both spindle poles [1Kirschner M. Mitchison T. Beyond self-assembly: from microtubules to morphogenesis.Cell. 1986; 45: 329-342Abstract Full Text PDF PubMed Scopus (925) Google Scholar]. Presumably for this reason, most cells do not simply rely on a ‘timer’ mechanism that would initiate sister chromatid separation after a defined period of time; instead, cells directly monitor the state of kinetochores and delay anaphase until all kinetochores are properly attached to the mitotic spindle. This sophisticated surveillance mechanism is called the spindle assembly checkpoint (SAC) [2Musacchio A. Hardwick K.G. The spindle checkpoint: structural insights into dynamic signalling.Nat. Rev. Mol. Cell Biol. 2002; 3: 731-741Crossref PubMed Scopus (460) Google Scholar, 3Nasmyth K. How do so few control so many?.Cell. 2005; 120: 739-746Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar]. When the SAC is active, Mad2 and other checkpoint proteins associate with Cdc20, a co-activator of the anaphase-promoting complex/cyclosome (APC/C) [4Yu H. Regulation of APC-Cdc20 by the spindle checkpoint.Curr. Opin. Cell Biol. 2002; 14: 706-714Crossref PubMed Scopus (284) Google Scholar]. Cdc20 bound by Mad2 is no longer capable of activating the APC/C, and therefore cells are not able to initiate anaphase and exit from mitosis, because both of these processes depend strictly on APC/C activity [4Yu H. Regulation of APC-Cdc20 by the spindle checkpoint.Curr. Opin. Cell Biol. 2002; 14: 706-714Crossref PubMed Scopus (284) Google Scholar]. How is Mad2 activated at unattached kinetochores? The ‘template model’, proposed last year by Andrea Musacchio and colleagues [5De Antoni A. Pearson C.G. Cimini D. Canman J.C. Sala V. Nezi L. Mapelli M. Sironi L. Faretta M. Salmon E.D. et al.The Mad1/Mad2 complex as a template for Mad2 activation in the spindle assembly checkpoint.Curr. Biol. 2005; 15: 214-225Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar], suggests that inactive Mad2 may be activated by interaction with an active Mad2 ‘template’ at unattached kinetochores. This dimerization would activate Mad2 by inducing a conformational change, similar to how prion proteins propagate conformational changes from one molecule to the next. Recent work by Vink et al.[6Vink M. Simonetta M. Transidico P. Ferrari K. Mapelli M. De Antoni A. Massimiliano L. Ciliberto A. Faretta M. Salmon E.D. Musacchio A. In vitro FRAP identifies the minimal requirements for Mad2 kinetochore dynamics.Curr. Biol. 2006; 16: 755-766Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar] and Mapelli et al.[7Mapelli M. Filipp F.V. Rancati G. Massimiliano L. Nezi L. Stier G. Hagan R.S. Confalonieri S. Piatti S. Sattler M. et al.Determinants of conformational dimerization of Mad2 and its inhibition by p31(comet).EMBO J. 2006; 25: 1273-1284Crossref PubMed Scopus (110) Google Scholar] has now provided important additional support for this template model. Before the template model was proposed, it was already known that Mad2 is recruited to unattached kinetochores by Mad1, a protein that forms a tight 2:2 complex with Mad2 [8Sironi L. Mapelli M. Knapp S. De Antoni A. Jeang K.T. Musacchio A. Crystal structure of the tetrameric Mad1-Mad2 core complex: implications of a ‘safety belt’ binding mechanism for the spindle checkpoint.EMBO J. 2002; 21: 2496-2506Crossref PubMed Scopus (230) Google Scholar, 9Chung E. Chen R.H. Spindle checkpoint requires Mad1-bound and Mad1-free Mad2.Mol. Biol. Cell. 2002; 13: 1501-1511Crossref PubMed Scopus (112) Google Scholar]. Surprisingly, structural studies showed that Mad1 competes with Cdc20 for the exact same binding site on Mad2 [10Luo X. Tang Z. Rizo J. Yu H. The Mad2 spindle checkpoint protein undergoes similar major conformational changes upon binding to either Mad1 or Cdc20.Mol. Cell. 2002; 9: 59-71Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar]. These studies revealed further that Mad2 exists in two distinct conformations, called ‘open’ and ‘closed’ [8Sironi L. Mapelli M. Knapp S. De Antoni A. Jeang K.T. Musacchio A. Crystal structure of the tetrameric Mad1-Mad2 core complex: implications of a ‘safety belt’ binding mechanism for the spindle checkpoint.EMBO J. 2002; 21: 2496-2506Crossref PubMed Scopus (230) Google Scholar, 11Luo X. Tang Z. Xia G. Wassmann K. Matsumoto T. Rizo J. Yu H. The Mad2 spindle checkpoint protein has two distinct natively folded states.Nat. Struct. Mol. Biol. 2004; 11: 338-345Crossref PubMed Scopus (214) Google Scholar]. When bound to Mad1 or Cdc20, Mad2 is found in its closed conformation (C-Mad2), in which two β-strands of Mad2 topologically trap Mad1 or Cdc20, like a passenger is embraced by a safety belt in a car seat [8Sironi L. Mapelli M. Knapp S. De Antoni A. Jeang K.T. Musacchio A. Crystal structure of the tetrameric Mad1-Mad2 core complex: implications of a ‘safety belt’ binding mechanism for the spindle checkpoint.EMBO J. 2002; 21: 2496-2506Crossref PubMed Scopus (230) Google Scholar]. In contrast, Mad2 that lacks these binding partners is present in the open conformation (O-Mad2). An obvious conclusion from these observations was that, during SAC activation, O-Mad2 has somehow to be converted to C-Mad2 which is bound to Cdc20. Surprisingly, however, both Mad2 confomers are rather stable and do not seem to be easily converted from one into the other spontaneously [5De Antoni A. Pearson C.G. Cimini D. Canman J.C. Sala V. Nezi L. Mapelli M. Sironi L. Faretta M. Salmon E.D. et al.The Mad1/Mad2 complex as a template for Mad2 activation in the spindle assembly checkpoint.Curr. Biol. 2005; 15: 214-225Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar]. If the two forms are stable, how is the transition from O-Mad2 to C-Mad2 catalyzed to efficiently sequester all cellular Cdc20? Even further, how is it possible that Mad1 is essential for Cdc20 sequestration in vivo, when Mad1 competes with Cdc20 for Mad2 binding? The Mad2 template model proposes that Mad1 and C-Mad2 form a stable core complex at unattached kinetochores. This core then binds additional molecules of O-Mad2 through formation of conformational heterodimers between the C-Mad2 subunits of the Mad1–Mad2 complex and O-Mad2 (Figure 1). This interaction would then somehow lead to conversion of O-Mad2 to C-Mad2 and its simultaneous association with Cdc20, resulting in the formation of Cdc20–C-Mad2 heterodimers and keeping APC/C inactive [5De Antoni A. Pearson C.G. Cimini D. Canman J.C. Sala V. Nezi L. Mapelli M. Sironi L. Faretta M. Salmon E.D. et al.The Mad1/Mad2 complex as a template for Mad2 activation in the spindle assembly checkpoint.Curr. Biol. 2005; 15: 214-225Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar]. The template model provided an elegant explanation for a number of previously enigmatic observations. Nevertheless, parts of the model remained speculative. Vink et al.[6Vink M. Simonetta M. Transidico P. Ferrari K. Mapelli M. De Antoni A. Massimiliano L. Ciliberto A. Faretta M. Salmon E.D. Musacchio A. In vitro FRAP identifies the minimal requirements for Mad2 kinetochore dynamics.Curr. Biol. 2006; 16: 755-766Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar] therefore challenged the template model by testing several of its predictions. For this purpose, they developed an in vitro fluorescence-recovery-after-photobleaching (FRAP) assay using Mad1–C-Mad2 complexes that were immobilized on beads and assayed the dynamics of O-Mad2 and Cdc20 on this ‘minimal kinetochore’. They found that beads coated with Mad1–C-Mad2-CFP were stably fluorescent, but when photobleached, fluorescence did not recover, indicating that Mad1–C-Mad2 forms a tight complex that does not exchange with the environment. On the other hand, when soluble O-Mad2-CFP was added to non-fluorescent Mad1–C-Mad2 on beads, FRAP showed almost full recovery, confirming that O-Mad2 dynamically associates with Mad1–C-Mad2 complexes. Finally, when Mad1–C-Mad2-CFP and O-Mad2-CFP were used simultaneously, FRAP revealed two pools of Mad2, one that exchanges on the beads and one that does not. All of these properties reflect exactly what would be predicted from the template model, and importantly they are in perfect agreement with previous in vivo FRAP data [12Shah J.V. Botvinick E. Bonday Z. Furnari F. Berns M. Cleveland D.W. Dynamics of centromere and kinetochore proteins; implications for checkpoint signaling and silencing.Curr. Biol. 2004; 14: 942-952Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar]. The reconstitution of Mad2 dynamics in vitro is an important achievement, because in such a system the reactants and their possible reactions are clearly defined. This reaction network can be written in the form of a quantitative model that in turn can be fitted to the FRAP curves and used to determine biochemical parameters, such as off-rate of a binding reaction [13Sprague B.L. McNally J.G. FRAP analysis of binding: proper and fitting.Trends Cell Biol. 2005; 15: 84-91Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar]. Recently, more technologies have become available that allow measurement of biochemical parameters in live cells, such as FRAP, fluorescence resonance energy transfer (FRET) and fluorescence cross-correlation spectroscopy (FCS) [13Sprague B.L. McNally J.G. FRAP analysis of binding: proper and fitting.Trends Cell Biol. 2005; 15: 84-91Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar, 14Bacia K. Schwille P. A dynamic view of cellular processes by in vivo fluorescence auto- and cross-correlation spectroscopy.Methods. 2003; 29: 74-85Crossref PubMed Scopus (196) Google Scholar]. In all cases, full understanding of the underlying molecules and their reactions would be essential to properly interpret and quantitatively model the system [13Sprague B.L. McNally J.G. FRAP analysis of binding: proper and fitting.Trends Cell Biol. 2005; 15: 84-91Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar]. Not the easiest, but one of the most elegant solutions to this problem is to reconstitute the system in vitro — as beautifully exemplified by the work of Vink et al.[6Vink M. Simonetta M. Transidico P. Ferrari K. Mapelli M. De Antoni A. Massimiliano L. Ciliberto A. Faretta M. Salmon E.D. Musacchio A. In vitro FRAP identifies the minimal requirements for Mad2 kinetochore dynamics.Curr. Biol. 2006; 16: 755-766Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar]. Vink et al.[6Vink M. Simonetta M. Transidico P. Ferrari K. Mapelli M. De Antoni A. Massimiliano L. Ciliberto A. Faretta M. Salmon E.D. Musacchio A. In vitro FRAP identifies the minimal requirements for Mad2 kinetochore dynamics.Curr. Biol. 2006; 16: 755-766Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar] also used their in vitro FRAP system to test the effects of p31/Comet, a cellular inhibitor of the SAC [15Habu T. Kim S.H. Weinstein J. Matsumoto T. Identification of a MAD2-binding protein, CMT2, and its role in mitosis.EMBO J. 2002; 21: 6419-6428Crossref PubMed Scopus (127) Google Scholar, 16Xia G. Luo X. Habu T. Rizo J. Matsumoto T. Yu H. Conformation-specific binding of p31(comet) antagonizes the function of Mad2 in the spindle checkpoint.EMBO J. 2004; 23: 3133-3143Crossref PubMed Scopus (156) Google Scholar]. It had remained mysterious how p31/Comet functions, because it does not prevent Mad2's interaction with either Mad1 or Cdc20 [15Habu T. Kim S.H. Weinstein J. Matsumoto T. Identification of a MAD2-binding protein, CMT2, and its role in mitosis.EMBO J. 2002; 21: 6419-6428Crossref PubMed Scopus (127) Google Scholar, 16Xia G. Luo X. Habu T. Rizo J. Matsumoto T. Yu H. Conformation-specific binding of p31(comet) antagonizes the function of Mad2 in the spindle checkpoint.EMBO J. 2004; 23: 3133-3143Crossref PubMed Scopus (156) Google Scholar]. Vink et al.[6Vink M. Simonetta M. Transidico P. Ferrari K. Mapelli M. De Antoni A. Massimiliano L. Ciliberto A. Faretta M. Salmon E.D. Musacchio A. In vitro FRAP identifies the minimal requirements for Mad2 kinetochore dynamics.Curr. Biol. 2006; 16: 755-766Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar] could now show that p31/Comet, like a Mad2 mutant that lacks its carboxyl terminus (Mad2-ΔC) competes with O-Mad2 for binding to Mad1–C-Mad2 complexes. Thus p31/Comet inhibits SAC without affecting Mad2 binding to Cdc20 or Mad1, but rather by blocking the O-Mad2–C-Mad2 interaction, a mechanism that is well explained by the template model. Mapelli et al.[7Mapelli M. Filipp F.V. Rancati G. Massimiliano L. Nezi L. Stier G. Hagan R.S. Confalonieri S. Piatti S. Sattler M. et al.Determinants of conformational dimerization of Mad2 and its inhibition by p31(comet).EMBO J. 2006; 25: 1273-1284Crossref PubMed Scopus (110) Google Scholar] set out to test another key prediction of the template model: does binding of O-Mad2 to the Mad1–C-Mad2 core complex change the conformation of O-Mad2 to help its binding to Cdc20? NMR spectroscopy revealed major structural rearrangements in O-Mad2 bound to C-Mad2 as compared to its free form. Upon binding to C-Mad2, O-Mad2 adopts a previously unknown intermediate conformation with a ‘loosened safety-belt’ (O-Mad2∗, Figure 1) that might allow ‘entry’ of Cdc20 into the open structure of Mad2. The data presented in the two new papers [6Vink M. Simonetta M. Transidico P. Ferrari K. Mapelli M. De Antoni A. Massimiliano L. Ciliberto A. Faretta M. Salmon E.D. Musacchio A. In vitro FRAP identifies the minimal requirements for Mad2 kinetochore dynamics.Curr. Biol. 2006; 16: 755-766Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 7Mapelli M. Filipp F.V. Rancati G. Massimiliano L. Nezi L. Stier G. Hagan R.S. Confalonieri S. Piatti S. Sattler M. et al.Determinants of conformational dimerization of Mad2 and its inhibition by p31(comet).EMBO J. 2006; 25: 1273-1284Crossref PubMed Scopus (110) Google Scholar] provide strong additional support for the template model of Mad2 activation. But many questions about the spindle assembly checkpoint remain open. For example, what is the rate of Cdc20–C-Mad2 formation in vivo? Would this rate be sufficient to sequester all cellular Cdc20 on time? Are Cdc20–C-Mad2 complexes only formed at kinetochores, or — as suggested by DeAntoni et al.[5De Antoni A. Pearson C.G. Cimini D. Canman J.C. Sala V. Nezi L. Mapelli M. Sironi L. Faretta M. Salmon E.D. et al.The Mad1/Mad2 complex as a template for Mad2 activation in the spindle assembly checkpoint.Curr. Biol. 2005; 15: 214-225Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar] — can Cdc20–C-Mad2 complexes function as cytosolic templates that can themselves catalyze formation of new Cdc20–C-Mad2? Is the interaction between Mad2 and Cdc20 alone sufficient to explain how the SAC keeps APC/C inactive? This is an important question because numerous other mechanisms besides Mad2 activation have also been proposed to control Cdc20 stability and activity [17Peters J.M. The anaphase-promoting complex: proteolysis in mitosis and beyond.Mol. Cell. 2002; 9: 931-943Abstract Full Text Full Text PDF PubMed Scopus (746) Google Scholar, 18Vanoosthuyse V. Hardwick K.G. Bub1 and the multilayered inhibition of Cdc20-APC/C in mitosis.Trends Cell Biol. 2005; 15: 231-233Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar]. How do other components of the spindle assembly checkpoint, such as BubR1 or Bub3, fit into this model? These proteins are also known to interact with Cdc20 and might even assemble into mitotic checkpoint complexes that contain Mad2, but how these complexes are activated is unknown [19Sudakin V. Chan G.K. Yen T.J. Checkpoint inhibition of the APC/C in HeLa cells is mediated by a complex of BUBR1, BUB3, CDC20, and MAD2.J. Cell Biol. 2001; 154: 925-936Crossref PubMed Scopus (629) Google Scholar]. Finally, how do Mad2 and other checkpoint proteins actually inhibit Cdc20 function? Understanding these mysteries of the SAC will keep many of us busy for some time to come." @default.
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• W2119784671 title "Checkpoint Activation: Don't Get Mad Too Much" @default.
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