FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1980779852> ?p ?o ?g. }

• W1980779852 endingPage "861" @default.
• W1980779852 startingPage "856" @default.
• W1980779852 abstract "Compromising the activity of the spindle checkpoint permits mitotic exit in the presence of unattached kinetochores and, consequently, greatly increases the rate of aneuploidy in the daughter cells [1Cleveland D.W. Mao Y. Sullivan K.F. Centromeres and kinetochores: From epigenetics to mitotic checkpoint signaling.Cell. 2003; 112: 407-421Abstract Full Text Full Text PDF PubMed Scopus (793) Google Scholar, 2Chan G.K. Yen T.J. The mitotic checkpoint: A signaling pathway that allows a single unattached kinetochore to inhibit mitotic exit.Prog. Cell Cycle Res. 2003; 5: 431-439PubMed Google Scholar, 3Musacchio A. Hardwick K.G. The spindle checkpoint: Structural insights into dynamic signalling.Nat. Rev. Mol. Cell Biol. 2002; 3: 731-741Crossref PubMed Scopus (459) Google Scholar]. The metazoan checkpoint mechanism is more complex than in yeast in that it requires additional proteins and activities besides the classical Mads and Bubs. Among these are Rod, Zw10, and Zwilch, components of a 700 Kdal complex (Rod/Zw10) [4Basto R. Gomes R. Karess R.E. Rough deal and Zw10 are required for the metaphase checkpoint in Drosophila.Nat. Cell Biol. 2000; 2: 939-943Crossref PubMed Scopus (110) Google Scholar, 5Chan G.K. Jablonski S.A. Starr D.A. Goldberg M.L. Yen T.J. Human Zw10 and ROD are mitotic checkpoint proteins that bind to kinetochores.Nat. Cell Biol. 2000; 2: 944-947Crossref PubMed Scopus (157) Google Scholar, 6Williams B.C. Li Z. Liu S. Williams E.V. Leung G. Yen T.J. Goldberg M.L. Zwilch, a new component of the ZW10/ROD complex required for kinetochore functions.Mol. Biol. Cell. 2003; 14: 1379-1391Crossref PubMed Scopus (77) Google Scholar] that is required for recruitment of dynein/dynactin to kinetochores [7Basto R. Scaerou F. Mische S. Wojcik E. Lefebvre C. Gomes R. Hays T. Karess R. In vivo dynamics of the rough deal checkpoint protein during Drosophila mitosis.Curr. Biol. 2004; 14: 56-61Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 8Starr D.A. Williams B.C. Hays T.S. Goldberg M.L. ZW10 helps recruit dynactin and dynein to the kinetochore.J. Cell Biol. 1998; 142: 763-774Crossref PubMed Scopus (211) Google Scholar] but whose role in the checkpoint is poorly understood. The dynamics of Rod and Mad2, examined in different organisms, show intriguing similarities as well as apparent differences [7Basto R. Scaerou F. Mische S. Wojcik E. Lefebvre C. Gomes R. Hays T. Karess R. In vivo dynamics of the rough deal checkpoint protein during Drosophila mitosis.Curr. Biol. 2004; 14: 56-61Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 9Howell B.J. Hoffman D.B. Fang G. Murray A.W. Salmon E.D. Visualization of Mad2 dynamics at kinetochores, along spindle fibers, and at spindle poles in living cells.J. Cell Biol. 2000; 150: 1233-1250Crossref PubMed Scopus (279) Google Scholar] . Here we simultaneously follow GFP-Mad2 and RFP-Rod and find they are in fact closely associated throughout early mitosis. They accumulate simultaneously on kinetochores and are shed together along microtubule fibers after attachment. Their behavior and position within attached kinetochores is distinct from that of BubR1; Mad2 and Rod colocalize to the outermost kinetochore region (the corona), whereas BubR1 is slightly more interior. Moreover, Mad2, but not BubR1, Bub1, Bub3, or Mps1, requires Rod/Zw10 for its accumulation on unattached kinetochores. Rod/Zw10 thus contributes to checkpoint activation by promoting Mad2 recruitment and to checkpoint inactivation by recruiting dynein/dynactin that subsequently removes Mad2 from attached kinetochores. Compromising the activity of the spindle checkpoint permits mitotic exit in the presence of unattached kinetochores and, consequently, greatly increases the rate of aneuploidy in the daughter cells [1Cleveland D.W. Mao Y. Sullivan K.F. Centromeres and kinetochores: From epigenetics to mitotic checkpoint signaling.Cell. 2003; 112: 407-421Abstract Full Text Full Text PDF PubMed Scopus (793) Google Scholar, 2Chan G.K. Yen T.J. The mitotic checkpoint: A signaling pathway that allows a single unattached kinetochore to inhibit mitotic exit.Prog. Cell Cycle Res. 2003; 5: 431-439PubMed Google Scholar, 3Musacchio A. Hardwick K.G. The spindle checkpoint: Structural insights into dynamic signalling.Nat. Rev. Mol. Cell Biol. 2002; 3: 731-741Crossref PubMed Scopus (459) Google Scholar]. The metazoan checkpoint mechanism is more complex than in yeast in that it requires additional proteins and activities besides the classical Mads and Bubs. Among these are Rod, Zw10, and Zwilch, components of a 700 Kdal complex (Rod/Zw10) [4Basto R. Gomes R. Karess R.E. Rough deal and Zw10 are required for the metaphase checkpoint in Drosophila.Nat. Cell Biol. 2000; 2: 939-943Crossref PubMed Scopus (110) Google Scholar, 5Chan G.K. Jablonski S.A. Starr D.A. Goldberg M.L. Yen T.J. Human Zw10 and ROD are mitotic checkpoint proteins that bind to kinetochores.Nat. Cell Biol. 2000; 2: 944-947Crossref PubMed Scopus (157) Google Scholar, 6Williams B.C. Li Z. Liu S. Williams E.V. Leung G. Yen T.J. Goldberg M.L. Zwilch, a new component of the ZW10/ROD complex required for kinetochore functions.Mol. Biol. Cell. 2003; 14: 1379-1391Crossref PubMed Scopus (77) Google Scholar] that is required for recruitment of dynein/dynactin to kinetochores [7Basto R. Scaerou F. Mische S. Wojcik E. Lefebvre C. Gomes R. Hays T. Karess R. In vivo dynamics of the rough deal checkpoint protein during Drosophila mitosis.Curr. Biol. 2004; 14: 56-61Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 8Starr D.A. Williams B.C. Hays T.S. Goldberg M.L. ZW10 helps recruit dynactin and dynein to the kinetochore.J. Cell Biol. 1998; 142: 763-774Crossref PubMed Scopus (211) Google Scholar] but whose role in the checkpoint is poorly understood. The dynamics of Rod and Mad2, examined in different organisms, show intriguing similarities as well as apparent differences [7Basto R. Scaerou F. Mische S. Wojcik E. Lefebvre C. Gomes R. Hays T. Karess R. In vivo dynamics of the rough deal checkpoint protein during Drosophila mitosis.Curr. Biol. 2004; 14: 56-61Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 9Howell B.J. Hoffman D.B. Fang G. Murray A.W. Salmon E.D. Visualization of Mad2 dynamics at kinetochores, along spindle fibers, and at spindle poles in living cells.J. Cell Biol. 2000; 150: 1233-1250Crossref PubMed Scopus (279) Google Scholar] . Here we simultaneously follow GFP-Mad2 and RFP-Rod and find they are in fact closely associated throughout early mitosis. They accumulate simultaneously on kinetochores and are shed together along microtubule fibers after attachment. Their behavior and position within attached kinetochores is distinct from that of BubR1; Mad2 and Rod colocalize to the outermost kinetochore region (the corona), whereas BubR1 is slightly more interior. Moreover, Mad2, but not BubR1, Bub1, Bub3, or Mps1, requires Rod/Zw10 for its accumulation on unattached kinetochores. Rod/Zw10 thus contributes to checkpoint activation by promoting Mad2 recruitment and to checkpoint inactivation by recruiting dynein/dynactin that subsequently removes Mad2 from attached kinetochores. To gain insight into the role of Rod/Zw10 relative to other checkpoint proteins, we undertook a study of fluorescently tagged (GFP and mRFP1 [10Campbell R.E. Tour O. Palmer A.E. Steinbach P.A. Baird G.S. Zacharias D.A. Tsien R.Y. A monomeric red fluorescent protein.Proc. Natl. Acad. Sci. USA. 2002; 99: 7877-7882Crossref PubMed Scopus (1917) Google Scholar]) Rod (CG1569), Mad2 (CG17498), and BubR1 (CG7838) in a single cell type, the Drosophila larval neuroblast. All three fusion proteins are controlled by their natural promotors, and all three retain their biological activity ([7Basto R. Scaerou F. Mische S. Wojcik E. Lefebvre C. Gomes R. Hays T. Karess R. In vivo dynamics of the rough deal checkpoint protein during Drosophila mitosis.Curr. Biol. 2004; 14: 56-61Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar]; Figure S1 and Table S1 in the Supplemental Data available with this article online; data not shown). Consistent with earlier reports, Rod and BubR1 are cytoplasmic in interphase [7Basto R. Scaerou F. Mische S. Wojcik E. Lefebvre C. Gomes R. Hays T. Karess R. In vivo dynamics of the rough deal checkpoint protein during Drosophila mitosis.Curr. Biol. 2004; 14: 56-61Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 11Howell B.J. Moree B. Farrar E.M. Stewart S. Fang G. Salmon E.D. Spindle checkpoint protein dynamics at kinetochores in living cells.Curr. Biol. 2004; 14: 953-964Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 12Taylor S.S. Hussein D. Wang Y. Elderkin S. Morrow C.J. Kinetochore localisation and phosphorylation of the mitotic checkpoint components Bub1 and BubR1 are differentially regulated by spindle events in human cells.J. Cell Sci. 2001; 114: 4385-4395Crossref PubMed Google Scholar], whereas Mad2 is associated with the nucleoplasm and nuclear envelope [11Howell B.J. Moree B. Farrar E.M. Stewart S. Fang G. Salmon E.D. Spindle checkpoint protein dynamics at kinetochores in living cells.Curr. Biol. 2004; 14: 953-964Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 13Campbell M.S. Chan G.K. Yen T.J. Mitotic checkpoint proteins HsMAD1 and HsMAD2 are associated with nuclear pore complexes in interphase.J. Cell Sci. 2001; 114: 953-963Crossref PubMed Google Scholar, 14Shah 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] (Figure 1A ). In fly neuroblasts, as in Hela cells [15Johnson V.L. Scott M.I. Holt S.V. Hussein D. Taylor S.S. Bub1 is required for kinetochore localization of BubR1, Cenp-E, Cenp-F and Mad2, and chromosome congression.J. Cell Sci. 2004; 117: 1577-1589Crossref PubMed Scopus (255) Google Scholar] but unlike in PtK cells [11Howell B.J. Moree B. Farrar E.M. Stewart S. Fang G. Salmon E.D. Spindle checkpoint protein dynamics at kinetochores in living cells.Curr. Biol. 2004; 14: 953-964Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar], BubR1 is the first to accumulate on kinetochores during prophase (at very low levels initially); it precedes Mad2 and Rod by 2–5 min (Movie S1). Mad2 and Rod begin to label kinetochores only during nuclear-envelope breakdown (NEB), easily recognized by the invasion of Rod into the nucleoplasm. The first kinetochore-associated Mad2 signals above the nucleoplasmic background are seen simultaneously with the first Rod signal (Figure 1A and Movie S2). In prometaphase, the kinetochores brightly label with all three proteins (Figure 1B). Because cytoplasmic Mad2 signal is consistently higher than either BubR1 or Rod, Mad2 kinetochore labeling appears relatively less prominent. As the kinetochores capture MTs, Mad2 and Rod both are transported poleward (Figures 1B–1D;Movies S3 and S4; and Figures S2 and S4), again consistent with previous reports [7Basto R. Scaerou F. Mische S. Wojcik E. Lefebvre C. Gomes R. Hays T. Karess R. In vivo dynamics of the rough deal checkpoint protein during Drosophila mitosis.Curr. Biol. 2004; 14: 56-61Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 9Howell B.J. Hoffman D.B. Fang G. Murray A.W. Salmon E.D. Visualization of Mad2 dynamics at kinetochores, along spindle fibers, and at spindle poles in living cells.J. Cell Biol. 2000; 150: 1233-1250Crossref PubMed Scopus (279) Google Scholar]. This process, called “shedding,” requires dynein/dynactin and may be important for shutting off the checkpoint once MTs are properly attached [7Basto R. Scaerou F. Mische S. Wojcik E. Lefebvre C. Gomes R. Hays T. Karess R. In vivo dynamics of the rough deal checkpoint protein during Drosophila mitosis.Curr. Biol. 2004; 14: 56-61Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 16Howell B.J. McEwen B.F. Canman J.C. Hoffman D.B. Farrar E.M. Rieder C.L. Salmon E.D. Cytoplasmic dynein/dynactin drives kinetochore protein transport to the spindle poles and has a role in mitotic spindle checkpoint inactivation.J. Cell Biol. 2001; 155: 1159-1172Crossref PubMed Scopus (400) Google Scholar, 17Wojcik E. Basto R. Serr M. Scaerou F. Karess R. Hays T. Kinetochore dynein: Its dynamics and role in the transport of the Rough deal checkpoint protein.Nat. Cell Biol. 2001; 3: 1001-1007Crossref PubMed Scopus (167) Google Scholar, 18Maiato H. DeLuca J. Salmon E.D. Earnshaw W.C. The dynamic kinetochore-microtubule interface.J. Cell Sci. 2004; 117: 5461-5477Crossref PubMed Scopus (313) Google Scholar]. These live images reveal a robustness that was not evident for Mad2 transport in earlier studies in PtK cells and Drosophila cells [9Howell B.J. Hoffman D.B. Fang G. Murray A.W. Salmon E.D. Visualization of Mad2 dynamics at kinetochores, along spindle fibers, and at spindle poles in living cells.J. Cell Biol. 2000; 150: 1233-1250Crossref PubMed Scopus (279) Google Scholar, 16Howell B.J. McEwen B.F. Canman J.C. Hoffman D.B. Farrar E.M. Rieder C.L. Salmon E.D. Cytoplasmic dynein/dynactin drives kinetochore protein transport to the spindle poles and has a role in mitotic spindle checkpoint inactivation.J. Cell Biol. 2001; 155: 1159-1172Crossref PubMed Scopus (400) Google Scholar, 19Logarinho E. Bousbaa H. Dias J.M. Lopes C. Amorim I. Antunes-Martins A. Sunkel C.E. Different spindle checkpoint proteins monitor microtubule attachment and tension at kinetochores in Drosophila cells.J. Cell Sci. 2004; 117: 1757-1771Crossref PubMed Scopus (89) Google Scholar], although it can be seen sometimes even by immunostaining (Figure S5). It is difficult to quantify these signals, but the films clearly show that new cytosolic Mad2 is continuously recruited to kinetochores even after MT capture and replaces that lost to shedding; the total Mad2 signal on KMTs over the duration of prometaphase and metaphase is far greater than the original kinetochore-associated signal. This is particularly evident in Movie S4, where metaphase is prolonged. Thus Mad2, like Rod, establishes a flux of recruitment to and shedding from attached kinetochores. GFP-Rod and RFP-Mad2 show a near-perfect coincidence of signal in prometaphase and early metaphase, not only on kinetochores but also along the KMTs (Figures 1C and 1D; Movies S3 and S4). The overall patterns of the two proteins are superimposable (Figures 1C and 1D, Figure S4). Where discrete particles of GFP-Mad2 could be followed, they always contained RFP-Rod (Figure 1D and Figure S4). These results suggest that Mad2 and Rod/Zw10 remain associated as they leave the kinetochore along the KMTs. By late metaphase, Mad2 signal has essentially disappeared from kinetochores and is only faintly visible on the spindle above the cytoplasmic Mad2 background, whereas Rod shedding continues robustly up to anaphase onset (Figure 1C and Movie S3). In larval neuroblasts, the timing of NEB to anaphase onset is typically 7–12 min, of which metaphase lasts 2–8 min (see also [20Savoian M.S. Rieder C.L. Mitosis in primary cultures of Drosophila melanogaster larval neuroblasts.J. Cell Sci. 2002; 115: 3061-3072Crossref PubMed Google Scholar]). There does not appear to be much delay between Mad2 disappearance from the spindle and anaphase onset. On average, Mad2 is gone less than 1 min prior to anaphase (average is 35 s, range 0–2 min, n = 16), and sometimes just seconds before (compare Movies S3 and S5). This contrasts with the situation in PtK cells [9Howell B.J. Hoffman D.B. Fang G. Murray A.W. Salmon E.D. Visualization of Mad2 dynamics at kinetochores, along spindle fibers, and at spindle poles in living cells.J. Cell Biol. 2000; 150: 1233-1250Crossref PubMed Scopus (279) Google Scholar], where anaphase occurs on average 10 min after the disappearance of the last detectable Mad2 signal. The significance of this difference is for now unclear. It may reflect simply an adaptation to the very rapid mitosis in flies (7–12 min NEB-anaphase, compared to 25 min after alignment of the last chromosome for Ptk cells). Alternatively, it may reflect a more fundamental difference in the way the spindle checkpoint is turned off. The behavior of Mad2 and Rod was distinguishable from that of BubR1 in several ways. BubR1 remained tightly associated with kinetochores and was not detectable along the spindle after MT capture (compare Mad2 and BubR1 in Figure 1B and Movies S1 and S2). Although in PtK cells BubR1 may be transported from kinetochores to poles after energy depletion [16Howell B.J. McEwen B.F. Canman J.C. Hoffman D.B. Farrar E.M. Rieder C.L. Salmon E.D. Cytoplasmic dynein/dynactin drives kinetochore protein transport to the spindle poles and has a role in mitotic spindle checkpoint inactivation.J. Cell Biol. 2001; 155: 1159-1172Crossref PubMed Scopus (400) Google Scholar], in normal fly neuroblasts shedding does not appear to be a major route by which BubR1 levels are reduced on attached kinetochores. Moreover, close inspection of in vivo double-labeled cells revealed that, as the metaphase plate develops, BubR1 becomes enriched in a kinetochore domain slightly internal to that of Rod and Mad2 (Figure 1E; Movie S1; see also Figure S3 and Table S2). Rod/Zw10, dynein/dynactin, Mad2 and BubR1, and all the transient kinetochore proteins are normally classified as outer-domain kinetochore components [12Taylor S.S. Hussein D. Wang Y. Elderkin S. Morrow C.J. Kinetochore localisation and phosphorylation of the mitotic checkpoint components Bub1 and BubR1 are differentially regulated by spindle events in human cells.J. Cell Sci. 2001; 114: 4385-4395Crossref PubMed Google Scholar, 18Maiato H. DeLuca J. Salmon E.D. Earnshaw W.C. The dynamic kinetochore-microtubule interface.J. Cell Sci. 2004; 117: 5461-5477Crossref PubMed Scopus (313) Google Scholar, 21Jablonski S. Chan G.K.T. Cooke C. Earnshaw W. Yen T.J. The hBUB1 and hBUBR1 kinases sequentially assemble onto kinetochores during prophase with hBUIBR1 concentrating at the kinetochore plates in mitosis.Chromosoma. 1998; 107: 386-396Crossref PubMed Scopus (151) Google Scholar], and indeed they all form enlarged crescents around the MT-free kinetochores [7Basto R. Scaerou F. Mische S. Wojcik E. Lefebvre C. Gomes R. Hays T. Karess R. In vivo dynamics of the rough deal checkpoint protein during Drosophila mitosis.Curr. Biol. 2004; 14: 56-61Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 18Maiato H. DeLuca J. Salmon E.D. Earnshaw W.C. The dynamic kinetochore-microtubule interface.J. Cell Sci. 2004; 117: 5461-5477Crossref PubMed Scopus (313) Google Scholar, 21Jablonski S. Chan G.K.T. Cooke C. Earnshaw W. Yen T.J. The hBUB1 and hBUBR1 kinases sequentially assemble onto kinetochores during prophase with hBUIBR1 concentrating at the kinetochore plates in mitosis.Chromosoma. 1998; 107: 386-396Crossref PubMed Scopus (151) Google Scholar, 22Hoffman D.B. Pearson C.G. Yen T.J. Howell B.J. Salmon E.D. Microtubule-dependent changes in assembly of microtubule motor proteins and mitotic spindle checkpoint proteins at PtK1 kinetochores.Mol. Biol. Cell. 2001; 12: 1995-2009Crossref PubMed Scopus (270) Google Scholar]. The outer domain can be further subdivided into a more interior “outer plate” which appears to be the MT attachment site as well as the location of BubR1 [21Jablonski S. Chan G.K.T. Cooke C. Earnshaw W. Yen T.J. The hBUB1 and hBUBR1 kinases sequentially assemble onto kinetochores during prophase with hBUIBR1 concentrating at the kinetochore plates in mitosis.Chromosoma. 1998; 107: 386-396Crossref PubMed Scopus (151) Google Scholar], and an outer fibrous corona that is believed to contain Rod/Zw10, dynein/dynactin, and CenpE [7Basto R. Scaerou F. Mische S. Wojcik E. Lefebvre C. Gomes R. Hays T. Karess R. In vivo dynamics of the rough deal checkpoint protein during Drosophila mitosis.Curr. Biol. 2004; 14: 56-61Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 23Cooke C. Scharr B. Yen T. Earnshaw W. Localization of CENP-E in the fibrous corona and outer plate of mammalian kinetochores from prometaphase through anaphase.Chromosoma. 1997; 106: 446-455Crossref PubMed Scopus (125) Google Scholar, 24Yao X. Anderson K. Cleveland D. The microtubule-dependent motor centromere-associated protein E (CENP-E) is an integral component of the kintochore corona fibers that link centromeres to spindle microtubules.J Cell Biol. 1997; 139: 435-447Crossref PubMed Scopus (178) Google Scholar]. The relative locations of the various checkpoint proteins have not been compared in attached kinetochores of living cells. Our observation that Mad2 colocalizes with Rod but not with BubR1 (Figure 1E; Figure S3 and Table S2) is to our knowledge the first demonstration that Mad2 is part of the corona. The different locations of Mad2 and BubR1 are consistent with certain distinct features of their behavior. For example, Mad2 accumulation is highly sensitive to MT attachment and is depleted from kinetochores by shedding along KMTs. BubR1 by contrast is not depleted significantly by shedding and responds more to changes in tension (for example, [19Logarinho E. Bousbaa H. Dias J.M. Lopes C. Amorim I. Antunes-Martins A. Sunkel C.E. Different spindle checkpoint proteins monitor microtubule attachment and tension at kinetochores in Drosophila cells.J. Cell Sci. 2004; 117: 1757-1771Crossref PubMed Scopus (89) Google Scholar]). If this correlation holds, perhaps other proteins with robust shedding (for example, CenpF [25Yang Z.Y. Guo J. Li N. Qian M. Wang S.N. Zhu X.L. Mitosin/CENP-F is a conserved kinetochore protein subjected to cytoplasmic dynein-mediated poleward transport.Cell Res. 2003; 13: 275-283Crossref PubMed Scopus (41) Google Scholar]) will prove to colocalize in the corona with Mad2, Rod/Zw10, and dynein. In summary, Mad2 and Rod/Zw10 behavior on kinetochores and spindles are qualitatively closely linked. They are simultaneously recruited and are shed together during prometaphase and early metaphase. BubR1, by contrast, is independently recruited to a different kinetochore domain and does not undergo detectable shedding. To further probe the relationship of Mad2 and Rod/Zw10, we examined the behavior of GFP-Mad2 in rod and zw10 null-mutant cells. Given the importance of dynein-dynactin for shedding [16Howell B.J. McEwen B.F. Canman J.C. Hoffman D.B. Farrar E.M. Rieder C.L. Salmon E.D. Cytoplasmic dynein/dynactin drives kinetochore protein transport to the spindle poles and has a role in mitotic spindle checkpoint inactivation.J. Cell Biol. 2001; 155: 1159-1172Crossref PubMed Scopus (400) Google Scholar, 17Wojcik E. Basto R. Serr M. Scaerou F. Karess R. Hays T. Kinetochore dynein: Its dynamics and role in the transport of the Rough deal checkpoint protein.Nat. Cell Biol. 2001; 3: 1001-1007Crossref PubMed Scopus (167) Google Scholar] and the role of Rod/Zw10 in dynein recruitment [8Starr D.A. Williams B.C. Hays T.S. Goldberg M.L. ZW10 helps recruit dynactin and dynein to the kinetochore.J. Cell Biol. 1998; 142: 763-774Crossref PubMed Scopus (211) Google Scholar], we had anticipated that rod or zw10 mutants would show abnormal retention of Mad2 on kinetochores. In fact, however, in these cells kinetochore-associated GFP-Mad2 was significantly reduced (Figure 2A , frames 3–5; Movie S6), although Mad2 was still prominent on interphase rod nuclei (Figure 2A, frame 9). The reduction of kinetochore-associated Mad2 was evident in every rod or zw10 mutant cell examined, although the extent of reduction was somewhat variable. In three of 15 rod cells (20%) filmed from NEB to anaphase onset, no kinetochore-associated Mad2 was detectable above the cytoplasmic background at any stage. In the rest, a weak signal was briefly detectable on some kinetochores during prometaphase. Quantitation of these signals revealed that the kinetochore intensity in rod cells was only about 20% above the cytoplasmic level, (range 0%–50%, n = 15) at their maximum, whereas in wild-type cells kinetochore Mad2 signals averaged 4.4-fold higher than cytoplasmic signals (range 2.5–8, n = 19). Depolymerizing microtubules with colchicine, which normally elevates kinetochore levels of checkpoint proteins, including Mad2 [22Hoffman D.B. Pearson C.G. Yen T.J. Howell B.J. Salmon E.D. Microtubule-dependent changes in assembly of microtubule motor proteins and mitotic spindle checkpoint proteins at PtK1 kinetochores.Mol. Biol. Cell. 2001; 12: 1995-2009Crossref PubMed Scopus (270) Google Scholar], did not increase Mad2 kinetochore signals in rod cells, (Figure 2A, frame 6). These observations indicated that Mad2 requires the Rod/Zw10 complex to achieve its normal levels on kinetochores. An earlier report did not find that inactivating Rod by antibody injection of Hela cells had any effect on Mad2 recruitment [5Chan G.K. Jablonski S.A. Starr D.A. Goldberg M.L. Yen T.J. Human Zw10 and ROD are mitotic checkpoint proteins that bind to kinetochores.Nat. Cell Biol. 2000; 2: 944-947Crossref PubMed Scopus (157) Google Scholar], although the antibody did block Rod recruitment at the kinetochore and did lead to premature mitotic exit. The discrepancy with our results may be due to the different methodologies employed. We also examined several other checkpoint proteins in rod and zw10 mutants (Figure 2B). BubR1 and Bub3 were still present, as had been reported before [26Basu J. Logarinho E. Herrmann S. Bousbaa H. Li Z. Chan G.K. Yen T.J. Sunkel C.E. Goldberg M.L. Localization of the Drosophila checkpoint control protein Bub3 to the kinetochore requires Bub1 but not Zw10 or Rod.Chromosoma. 1998; 107: 376-385Crossref PubMed Scopus (74) Google Scholar]. Mps1 and Bub1 were also unaffected by rod mutants (Figure 2B). Thus, the requirement for Rod/Zw10 seems to be specific to Mad2. By contrast, treatments that remove Mad2 from kinetochores in vertebrate cells have no effect on Rod/Zw10 [27Liu S.T. Chan G.K. Hittle J.C. Fujii G. Lees E. Yen T.J. Human MPS1 kinase is required for mitotic arrest induced by the loss of CENP-E from kinetochores.Mol. Biol. Cell. 2003; 14: 1638-1651Crossref PubMed Scopus (144) Google Scholar]. It was possible that the failure of rod and zw10 mutant cells to recruit Mad2 was caused by the premature degradation of cyclin B in these checkpoint-defective cells [4Basto R. Gomes R. Karess R.E. Rough deal and Zw10 are required for the metaphase checkpoint in Drosophila.Nat. Cell Biol. 2000; 2: 939-943Crossref PubMed Scopus (110) Google Scholar]; perhaps Mad2 cannot bind kinetochores when cyclinB/cdc2 kinase activity is low. To test this possibility, we examined Mad2 behavior in cells doubly mutant for rod and ida, the gene encoding APC5, a component of the APC/C. ida cells arrest in M phase with consistently elevated cyclin B [28Bentley A.M. Williams B.C. Goldberg M.L. Andres A.J. Phenotypic characterization of Drosophila ida mutants: Defining the role of APC5 in cell cycle progression.J. Cell Sci. 2002; 115: 949-961PubMed Google Scholar]. The ida phenotype is epistatic to rod: i.e., ida rod double mutants do not exit mitosis, and they retain elevated cyclin B (Figure S6). In ida cells, chromosomes are frequently found unattached to spindles [28Bentley A.M. Williams B.C. Goldberg M.L. Andres A.J. Phenotypic characterization of Drosophila ida mutants: Defining the role of APC5 in cell cycle progression.J. Cell Sci. 2002; 115: 949-961PubMed Google Scholar], and Mad2 accumulation on kinetochores is therefore prominent even without colchicine (Figure 2A, frame 7). Significantly, in ida rod or ida zw10 double mutants, Mad2 signal on kinetochores was greatly reduced, just as in rod or zw10 mutants alone (Figure 2A, frame 8). This result argues that the Rod/Zw10 complex is physically required, directly or indirectly, for normal Mad2 accumulation on kinetochores. We have shown that many aspects of Mad2 behavior are intimately associated with the Rod/Zw10 complex. Rod/Zw10 accompanies Mad2 as it accumulates on unattached kinetochores and as it leaves kinetochores after MT attachment, and in the absence of Rod/Zw10, little or no Mad2 accumulates on kinetochores. Given that Rod/Zw10 is also required for dynein/dynactin recruitment, which removes Mad2 from attached kinetochores, one can say that the entire kinetochore cycle of Mad2 depends, directly or indirectly, on Rod/Zw10. The checkpoint defect of rod and zw10 mutants is now presumably explained by this failure to recruit Mad2. These results suggest that Rod/Zw10 is physically interacting with a complex containing Mad2 (or Mad1, see below) throughout mitosis. However, two-hybrid screening, immunoaffinity columns [6Williams B.C. Li Z. Liu S. Williams E.V. Leung G. Yen T.J. Goldberg M.L. Zwilch, a new component of the ZW10/ROD complex required for kinetochore functions.Mol. Biol. Cell. 2003; 14: 1379-1391Crossref PubMed Scopus (77) Google Scholar, 8Starr D.A. Williams B.C. Hays T.S. Goldberg M.L. ZW10 helps recruit dynactin and dynein to the kinetochore.J. Cell Biol. 1998; 142: 763-774Crossref PubMed Scopus (211) Google Scholar], and coimmunoprecipitation experiments (our data not shown) have not revealed any interaction between Rod/Zw10 and Mad1 or Mad2. Thus, unlike dynein/dynactin, Mad1/Mad2 may be binding only indirectly to Rod/Zw10, perhaps via an unknown protein. Alternatively, there may be direct interactions between Rod/Zw10 and Mad1/Mad2, but only under native conditions on intact kinetochores. We have summarized our findings and some speculations in a model shown in Figure 2C; Mad1/Mad2 binding sites are depicted as comprising multiple components whose affinity for Mad1/Mad2 can be enhanced by the Rod/Zw10 complex and reduced by MT capture during spindle assembly. MT capture also leads to depletion of Mad1/Mad2 by another route, as it is dragged off the kinetochores (along with Rod/Zw10) by dynein-mediated transport. Kinetochore recruitment of Mad2 initially occurs as part of a complex with Mad1, to which it is tightly bound even in interphase [29Chen R.H. Shevchenko A. Mann M. Murray A.W. Spindle checkpoint protein Xmad1 recruits Xmad2 to unattached kinetochores.J. Cell Biol. 1998; 143: 283-295Crossref PubMed Scopus (250) Google Scholar, 30Chung 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]. The Mad1/Mad2 complex is relatively stable at unattached kinetochores [11Howell B.J. Moree B. Farrar E.M. Stewart S. Fang G. Salmon E.D. Spindle checkpoint protein dynamics at kinetochores in living cells.Curr. Biol. 2004; 14: 953-964Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 14Shah 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], but a second Mad2 population, which depends on the first, turns over rapidly and presumably becomes an activated form, the “wait anaphase” signal [14Shah 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, 31Sironi 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]. Once MTs have attached, however, the Mad1/Mad2 complex is rapidly depleted, at least partially by dynein-mediated shedding along KMTs [11Howell B.J. Moree B. Farrar E.M. Stewart S. Fang G. Salmon E.D. Spindle checkpoint protein dynamics at kinetochores in living cells.Curr. Biol. 2004; 14: 953-964Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 14Shah 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], and this is believed to be part of the mechanism that extinguishes the checkpoint signal [16Howell B.J. McEwen B.F. Canman J.C. Hoffman D.B. Farrar E.M. Rieder C.L. Salmon E.D. Cytoplasmic dynein/dynactin drives kinetochore protein transport to the spindle poles and has a role in mitotic spindle checkpoint inactivation.J. Cell Biol. 2001; 155: 1159-1172Crossref PubMed Scopus (400) Google Scholar, 17Wojcik E. Basto R. Serr M. Scaerou F. Karess R. Hays T. Kinetochore dynein: Its dynamics and role in the transport of the Rough deal checkpoint protein.Nat. Cell Biol. 2001; 3: 1001-1007Crossref PubMed Scopus (167) Google Scholar]. It is therefore likely that the Rod/Zw10 complex is exerting its effect on the Mad1/Mad2 complex and not on Mad2 alone. Recent work in Hela cells supports this contention by showing that depletion of Zw10 by RNAi reduces both Mad1 and Mad2 recruitment to unattached kinetochores [32Kops G.J. Kim Y. Weaver B.A. Mao Y. McLeod I. Yates III, J.R. Tagaya M. Cleveland D.W. ZW10 links mitotic checkpoint signaling to the structural kinetochore.J. Cell Biol. 2005; 169: 49-60Crossref PubMed Scopus (184) Google Scholar]. It is unclear what kinetochore components constitute the Mad1/Mad2 “binding site.” The hierarchy of kinetochore assembly has been studied in several model systems, not always with consistent results. However, it appears that the Ndc80 complex [33Hori T. Haraguchi T. Hiraoka Y. Kimura H. Fukagawa T. Dynamic behavior of Nuf2-Hec1 complex that localizes to the centrosome and centromere and is essential for mitotic progression in vertebrate cells.J. Cell Sci. 2003; 116: 3347-3362Crossref PubMed Scopus (121) Google Scholar, 34DeLuca J.G. Howell B.J. Canman J.C. Hickey J.M. Fang G. Salmon E.D. Nuf2 and Hec1 are required for retention of the checkpoint proteins Mad1 and Mad2 to kinetochores.Curr. Biol. 2003; 13: 2103-2109Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 35Bharadwaj R. Qi W. Yu H. Identification of two novel components of the human NDC80 kinetochore complex.J. Biol. Chem. 2004; 279: 13076-13085Crossref PubMed Scopus (90) Google Scholar, 36Martin-Lluesma S. Stucke V.M. Nigg E.A. Role of Hec1 in spindle checkpoint signaling and kinetochore recruitment of Mad1/Mad2.Science. 2002; 297: 2267-2270Crossref PubMed Scopus (365) Google Scholar, 37Meraldi P. Draviam V.M. Sorger P.K. Timing and checkpoints in the regulation of mitotic progression.Dev. Cell. 2004; 7: 45-60Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar, 38Deluca J.G. Dong Y. Hergert P. Strauss J. Hickey J.M. Salmon E.D. McEwen B.F. Hec1 and Nuf2 are core components of the kinetochore outer plate essential for organizing microtubule attachment sites.Mol Biol Cell. 2004; 16 (Published online November 17, 2004): 519-531https://doi.org/10.1091/mbc.E04-09-0852Crossref PubMed Scopus (192) Google Scholar], Bub1 [39Brady D.M. Hardwick K.G. Complex formation between Mad1p, Bub1p and Bub3p is crucial for spindle checkpoint function.Curr. Biol. 2000; 10: 675-678Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 40Sharp-Baker H. Chen R.H. Spindle checkpoint protein Bub1 is required for kinetochore localization of Mad1, Mad2, Bub3, and CENP-E, independently of its kinase activity.J. Cell Biol. 2001; 153: 1239-1250Crossref PubMed Scopus (183) Google Scholar], and Mps1 kinase activity [27Liu S.T. Chan G.K. Hittle J.C. Fujii G. Lees E. Yen T.J. Human MPS1 kinase is required for mitotic arrest induced by the loss of CENP-E from kinetochores.Mol. Biol. Cell. 2003; 14: 1638-1651Crossref PubMed Scopus (144) Google Scholar, 41Abrieu A. Magnaghi-Jaulin L. Kahana J.A. Peter M. Castro A. Vigneron S. Lorca T. Cleveland D.W. Labbe J.C. Mps1 is a kinetochore-associated kinase essential for the vertebrate mitotic checkpoint.Cell. 2001; 106: 83-93Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar] are required for the subsequent assembly of Mad1/Mad2 on kinetochores. Conversely, interfering with Mps1 [27Liu S.T. Chan G.K. Hittle J.C. Fujii G. Lees E. Yen T.J. Human MPS1 kinase is required for mitotic arrest induced by the loss of CENP-E from kinetochores.Mol. Biol. Cell. 2003; 14: 1638-1651Crossref PubMed Scopus (144) Google Scholar] or the Ndc80 complex in Hela cells has no effect on Rod or dynein recruitment [33Hori T. Haraguchi T. Hiraoka Y. Kimura H. Fukagawa T. Dynamic behavior of Nuf2-Hec1 complex that localizes to the centrosome and centromere and is essential for mitotic progression in vertebrate cells.J. Cell Sci. 2003; 116: 3347-3362Crossref PubMed Scopus (121) Google Scholar, 34DeLuca J.G. Howell B.J. Canman J.C. Hickey J.M. Fang G. Salmon E.D. Nuf2 and Hec1 are required for retention of the checkpoint proteins Mad1 and Mad2 to kinetochores.Curr. Biol. 2003; 13: 2103-2109Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 36Martin-Lluesma S. Stucke V.M. Nigg E.A. Role of Hec1 in spindle checkpoint signaling and kinetochore recruitment of Mad1/Mad2.Science. 2002; 297: 2267-2270Crossref PubMed Scopus (365) Google Scholar, 37Meraldi P. Draviam V.M. Sorger P.K. Timing and checkpoints in the regulation of mitotic progression.Dev. Cell. 2004; 7: 45-60Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar], and rod and zw10 mutants have no effect on BubR1, Bub3 [26Basu J. Logarinho E. Herrmann S. Bousbaa H. Li Z. Chan G.K. Yen T.J. Sunkel C.E. Goldberg M.L. Localization of the Drosophila checkpoint control protein Bub3 to the kinetochore requires Bub1 but not Zw10 or Rod.Chromosoma. 1998; 107: 376-385Crossref PubMed Scopus (74) Google Scholar], CenpMeta (the fly homolog of CenpE) [6Williams B.C. Li Z. Liu S. Williams E.V. Leung G. Yen T.J. Goldberg M.L. Zwilch, a new component of the ZW10/ROD complex required for kinetochore functions.Mol. Biol. Cell. 2003; 14: 1379-1391Crossref PubMed Scopus (77) Google Scholar], Bub1, or Mps1 (this study), nor in all likelihood on the Ndc80 complex (in rod mutants, chromosomes are efficiently captured by MTs and congress). Thus Rod/Zw10, with Ndc80 complex, Bub1, and Mps1, all contribute to Mad2 kinetochore recruitment. The role of Rod/Zw10 may be to enhance the affinity of Mad1/Mad2 for its binding site (because some Mad2 binds even in rod mutants), increasing its stability on kinetochores prior to MT capture, perhaps by interacting with Ndc80 complex. Our results also demonstrate that, just like Rod/Zw10 [7Basto R. Scaerou F. Mische S. Wojcik E. Lefebvre C. Gomes R. Hays T. Karess R. In vivo dynamics of the rough deal checkpoint protein during Drosophila mitosis.Curr. Biol. 2004; 14: 56-61Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar], Mad1/Mad2 is continuously recruited to and then released from kinetochores, even following MT capture (Figure 1C and Movies S3 and S4), and only disappears from spindles just prior to anaphase onset. This differs significantly from the behavior reported in vertebrate cells, in which MT capture appears to shut off new Mad2 recruitment [16Howell B.J. McEwen B.F. Canman J.C. Hoffman D.B. Farrar E.M. Rieder C.L. Salmon E.D. Cytoplasmic dynein/dynactin drives kinetochore protein transport to the spindle poles and has a role in mitotic spindle checkpoint inactivation.J. Cell Biol. 2001; 155: 1159-1172Crossref PubMed Scopus (400) Google Scholar]. The difference need not conflict with the basic model in which kinetochore Mad2 generates the anaphase inhibitor. In both cases, there is a rapid decline, perhaps below a critical threshold, in the net steady-state abundance of Mad2 on attached kinetochores. Alternatively, MT capture may render the remaining kinetochore-associated Mad2 inactive. Perhaps the difference is in the rate of Mad2 recruitment in the two cell types. Even in PtK cells there is some evidence that Mad2 is capable of recruitment to attached kinetochores: If dynein activity (and therefore shedding) is blocked after chromosome alignment, Mad2 eventually reaccumulates at attached kinetochores [16Howell B.J. McEwen B.F. Canman J.C. Hoffman D.B. Farrar E.M. Rieder C.L. Salmon E.D. Cytoplasmic dynein/dynactin drives kinetochore protein transport to the spindle poles and has a role in mitotic spindle checkpoint inactivation.J. Cell Biol. 2001; 155: 1159-1172Crossref PubMed Scopus (400) Google Scholar], suggesting that prior to dynein inhibition, Mad2 was being recruited and immediately shed from these kinetochores. This continuous recruitment of Mad1/Mad2 to attached kinetochores may ensure that it will always be available to begin generating anaphase inhibitor should one or more MTs inadvertently detach. At the same time, the continued presence of Rod/Zw10 ensures the dynein levels required both to remove unneeded Mad1/Mad2 and, later, to power anaphase movement. We thank Claudio Sunkel, Greg Rogers, Christian Lehner, and Alvaro Tavares for antibodies and strains. This work was supported in part by grants to R.K. from the French Centre National de la Recherche Scientifique (CNRS), the Association pour la Recherche sur le Cancer (ARC), and La Ligue Nationale Contre le Cancer. E.B. was supported by Le Ministère de l’Education Nationale de la Recherche et de la Technologie, C.L by ARC, J-Y. H. by the Wellcome Trust, and M.E.G. by contract HPRN-CT-2002-00260 (MADCD) (European Community Human Potential Program). Download .pdf (.3 MB) Help with pdf files Document S1. Supplemental Experimental Procedures, Six Figures, and Tables Download .mov (6.35 MB) Help with mov files Movie S1. BubR1 and Rod Behavior Compared: Separate Kinetochore Recruitment and Localization to Distinct DomainsBubR1 is recruited to kinetochores prior to Rod. In this field of GFP-Rod and mRFP1-BubR1-labeled cells, two neuroblasts in division are seen. In the left, a cell has just entered prophase. In this cell, BubR1 accumulates on kinetochores first, and Rod (yellow arrow) follows 2 min later. In this and all movies, frames were taken every 10 s. The cell to the right, already in prometaphase as the film begins, shows the RFP-BubR1 clearly localized internally to the GFP-Rod. As this cell enters anaphase, Rod persists on kinetochores as BubR1 rapidly declines. Download .mov (1.31 MB) Help with mov files Movie S2. Mad2 and Rod Behavior Compared: Simultaneous kinetochore RecruitmentMad2 and Rod are recruited to kinetochores within seconds of each other. GFP-Mad2- and mRFP1-Rod-labeled neuroblast. The first kinetochores visibly labeled with Rod are simultaneously labeled with Mad2 (arrows). Download .mov (1.87 MB) Help with mov files Movie S3. GFP-Mad2 and RFP-Rod Behavior Compared: Shedding and FluxThe migration pattern along KMTs is strikingly similar for the two proteins until near anaphase onset. Note how Mad2 signal disappears just prior to anaphase. Download .mov (1.85 MB) Help with mov files Movie S4. GFP-Mad2 and RFP-Rod Behavior Compared: Shedding and Flux in a Neuroblast with a Prolonged MetaphaseMad2 is continuously recruited to aligned kinetochores and immediately shed again over a period of several minutes, establishing a robust flux of GFP-Mad2 very similar to that of RFP-Rod. Download .mov (1.47 MB) Help with mov files Movie S5. A GFP-Mad2 Neuroblast in which There Is Almost No Delay between Metaphase Alignment and Anaphase OnsetMad2 signal disappears immediately prior to anaphase. Download .mov (.77 MB) Help with mov files Movie S6. GFP-Mad2 Fails to Accumulate on Kinetochores in rod Mutant NeuroblastsDuring interphase, Mad2 associates normally with the nuclear membrane, but afterg NEB, little or no Mad2 can be seen on kinetochores. Two central neuroblasts enter mitosis (for the lower cell, NEB approx 13:30, and anaphase onset 19:50; for the upper cell, NEB 16:00 and anaphase onset 24:20). A weak Mad2 signal is briefly visible in the upper cell from 22:30–22:50" @default.
• W1980779852 created "2016-06-24" @default.
• W1980779852 creator A5024316523 @default.
• W1980779852 creator A5054995174 @default.
• W1980779852 creator A5059337168 @default.
• W1980779852 creator A5059664711 @default.
• W1980779852 creator A5075821680 @default.
• W1980779852 date "2005-05-01" @default.
• W1980779852 modified "2023-10-15" @default.
• W1980779852 title "Recruitment of Mad2 to the Kinetochore Requires the Rod/Zw10 Complex" @default.
• W1980779852 cites W1508230049 @default.
• W1980779852 cites W1535467971 @default.
• W1980779852 cites W1536109612 @default.
• W1980779852 cites W1540858422 @default.
• W1980779852 cites W1927775801 @default.
• W1980779852 cites W1961562730 @default.
• W1980779852 cites W1963713396 @default.
• W1980779852 cites W1965475092 @default.
• W1980779852 cites W2005383283 @default.
• W1980779852 cites W2007074379 @default.
• W1980779852 cites W2007388359 @default.
• W1980779852 cites W2015187344 @default.
• W1980779852 cites W2032422144 @default.
• W1980779852 cites W2034700471 @default.
• W1980779852 cites W2036068405 @default.
• W1980779852 cites W2037607661 @default.
• W1980779852 cites W2045369188 @default.
• W1980779852 cites W2050911321 @default.
• W1980779852 cites W2062117947 @default.
• W1980779852 cites W2067897696 @default.
• W1980779852 cites W2068176108 @default.
• W1980779852 cites W2079741946 @default.
• W1980779852 cites W2084221077 @default.
• W1980779852 cites W2084567406 @default.
• W1980779852 cites W2088006036 @default.
• W1980779852 cites W2089844705 @default.
• W1980779852 cites W2094533427 @default.
• W1980779852 cites W2101712721 @default.
• W1980779852 cites W2107913200 @default.
• W1980779852 cites W2111644689 @default.
• W1980779852 cites W2129543447 @default.
• W1980779852 cites W2131863700 @default.
• W1980779852 cites W2143471239 @default.
• W1980779852 cites W2145009672 @default.
• W1980779852 cites W2157471438 @default.
• W1980779852 cites W2161522127 @default.
• W1980779852 cites W2161791847 @default.
• W1980779852 cites W2161965142 @default.
• W1980779852 cites W2162768809 @default.
• W1980779852 cites W2170535602 @default.
• W1980779852 doi "https://doi.org/10.1016/j.cub.2005.03.052" @default.
• W1980779852 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15886105" @default.
• W1980779852 hasPublicationYear "2005" @default.
• W1980779852 type Work @default.
• W1980779852 sameAs 1980779852 @default.
• W1980779852 citedByCount "181" @default.
• W1980779852 countsByYear W19807798522012 @default.
• W1980779852 countsByYear W19807798522013 @default.
• W1980779852 countsByYear W19807798522014 @default.
• W1980779852 countsByYear W19807798522015 @default.
• W1980779852 countsByYear W19807798522016 @default.
• W1980779852 countsByYear W19807798522017 @default.
• W1980779852 countsByYear W19807798522018 @default.
• W1980779852 countsByYear W19807798522019 @default.
• W1980779852 countsByYear W19807798522020 @default.
• W1980779852 countsByYear W19807798522021 @default.
• W1980779852 countsByYear W19807798522022 @default.
• W1980779852 countsByYear W19807798522023 @default.
• W1980779852 crossrefType "journal-article" @default.
• W1980779852 hasAuthorship W1980779852A5024316523 @default.
• W1980779852 hasAuthorship W1980779852A5054995174 @default.
• W1980779852 hasAuthorship W1980779852A5059337168 @default.
• W1980779852 hasAuthorship W1980779852A5059664711 @default.
• W1980779852 hasAuthorship W1980779852A5075821680 @default.
• W1980779852 hasBestOaLocation W19807798521 @default.
• W1980779852 hasConcept C104317684 @default.
• W1980779852 hasConcept C192838845 @default.
• W1980779852 hasConcept C2777563682 @default.
• W1980779852 hasConcept C30481170 @default.
• W1980779852 hasConcept C54355233 @default.
• W1980779852 hasConcept C86803240 @default.
• W1980779852 hasConcept C95444343 @default.
• W1980779852 hasConceptScore W1980779852C104317684 @default.
• W1980779852 hasConceptScore W1980779852C192838845 @default.
• W1980779852 hasConceptScore W1980779852C2777563682 @default.
• W1980779852 hasConceptScore W1980779852C30481170 @default.
• W1980779852 hasConceptScore W1980779852C54355233 @default.
• W1980779852 hasConceptScore W1980779852C86803240 @default.
• W1980779852 hasConceptScore W1980779852C95444343 @default.
• W1980779852 hasIssue "9" @default.
• W1980779852 hasLocation W19807798521 @default.
• W1980779852 hasLocation W19807798522 @default.
• W1980779852 hasOpenAccess W1980779852 @default.
• W1980779852 hasPrimaryLocation W19807798521 @default.
• W1980779852 hasRelatedWork W1965475092 @default.
• W1980779852 hasRelatedWork W1976238904 @default.
• W1980779852 hasRelatedWork W2007969738 @default.
• W1980779852 hasRelatedWork W2010145895 @default.

• next