@article{mills_wang_bhatt_grimsrud_matson_lahiri_burke_cook_hirschey_emanuele_2021, title={Sirtuin 5 Is Regulated by the SCFCyclin (F) Ubiquitin Ligase and Is Involved in Cell Cycle Control}, volume={41}, ISSN={["1098-5549"]}, DOI={10.1128/MCB.00269-20}, abstractNote={The ubiquitin-proteasome system is essential for cell cycle progression. Cyclin F is a cell cycle-regulated substrate adapter F-box protein for the Skp1, CUL1, and F-box protein (SCF) family of E3 ubiquitin ligases. ABSTRACT The ubiquitin-proteasome system is essential for cell cycle progression. Cyclin F is a cell cycle-regulated substrate adapter F-box protein for the Skp1, CUL1, and F-box protein (SCF) family of E3 ubiquitin ligases. Despite its importance in cell cycle progression, identifying cyclin F-bound SCF complex (SCFCyclin F) substrates has remained challenging. Since cyclin F overexpression rescues a yeast mutant in the cdc4 gene, we considered the possibility that other genes that genetically modify cdc4 mutant lethality could also encode cyclin F substrates. We identified the mitochondrial and cytosolic deacylating enzyme sirtuin 5 (SIRT5) as a novel cyclin F substrate. SIRT5 has been implicated in metabolic processes, but its connection to the cell cycle is not known. We show that cyclin F interacts with and controls the ubiquitination, abundance, and stability of SIRT5. We show SIRT5 knockout results in a diminished G1 population and a subsequent increase in both S and G2/M. Global proteomic analyses reveal cyclin-dependent kinase (CDK) signaling changes congruent with the cell cycle changes in SIRT5 knockout cells. Together, these data demonstrate that SIRT5 is regulated by cyclin F and suggest a connection between SIRT5, cell cycle regulation, and metabolism.}, number={2}, journal={MOLECULAR AND CELLULAR BIOLOGY}, author={Mills, Christine A. and Wang, Xianxi and Bhatt, Dhaval P. and Grimsrud, Paul A. and Matson, Jacob Peter and Lahiri, Debojyoti and Burke, Daniel J. and Cook, Jeanette Gowen and Hirschey, Matthew D. and Emanuele, Michael J.}, year={2021}, month={Feb} }
 @article{choudhury_bonacci_arceci_lahiri_mills_kernan_branigan_decaprio_burke_emanuele_2016, title={APC/C and SCFcyclin F Constitute a Reciprocal Feedback Circuit Controlling S-Phase Entry}, volume={16}, ISSN={["2211-1247"]}, DOI={10.1016/j.celrep.2016.08.058}, abstractNote={The anaphase promoting complex/cyclosome (APC/C) is an ubiquitin ligase and core component of the cell-cycle oscillator. During G1 phase, APC/C binds to its substrate receptor Cdh1 and APC/C(Cdh1) plays an important role in restricting S-phase entry and maintaining genome integrity. We describe a reciprocal feedback circuit between APC/C and a second ubiquitin ligase, the SCF (Skp1-Cul1-F box). We show that cyclin F, a cell-cycle-regulated substrate receptor (F-box protein) for the SCF, is targeted for degradation by APC/C. Furthermore, we establish that Cdh1 is itself a substrate of SCF(cyclin F). Cyclin F loss impairs Cdh1 degradation and delays S-phase entry, and this delay is reversed by simultaneous removal of Cdh1. These data indicate that the coordinated, temporal ordering of cyclin F and Cdh1 degradation, organized in a double-negative feedback loop, represents a fundamental aspect of cell-cycle control. This mutual antagonism could be a feature of other oscillating systems.}, number={12}, journal={CELL REPORTS}, author={Choudhury, Rajarshi and Bonacci, Thomas and Arceci, Anthony and Lahiri, Debojyoti and Mills, Christine A. and Kernan, Jennifer L. and Branigan, Timothy B. and DeCaprio, James A. and Burke, Daniel J. and Emanuele, Michael J.}, year={2016}, month={Sep}, pages={3359–3372} }
 @article{stukenberg_burke_2015, title={Connecting the microtubule attachment status of each kinetochore to cell cycle arrest through the spindle assembly checkpoint}, volume={124}, ISSN={0009-5915 1432-0886}, url={http://dx.doi.org/10.1007/S00412-015-0515-Z}, DOI={10.1007/S00412-015-0515-Z}, abstractNote={Kinetochores generate a signal that inhibits anaphase progression until every kinetochore makes proper attachments to spindle microtubules. This spindle assembly checkpoint (SAC) increases the fidelity of chromosome segregation. We will review the molecular mechanisms by which kinetochores generate the SAC and extinguish the signal after making proper attachments, with the goal of identifying unanswered questions and new research directions. We will emphasize recent breakthroughs in how phosphorylation changes drive the activation and inhibition of the signal. We will also emphasize the dramatic changes in kinetochore structure that occur after attaching to microtubules and how these coordinate SAC function with microtubule attachment status. Finally, we will review the emerging cross talk between the DNA damage response and the SAC.}, number={4}, journal={Chromosoma}, publisher={Springer Science and Business Media LLC}, author={Stukenberg, P. Todd and Burke, Daniel J.}, year={2015}, month={Apr}, pages={463–480} }
 @article{burke_platanias_fish_2013, title={31}, volume={63}, ISSN={1043-4666}, url={http://dx.doi.org/10.1016/J.CYTO.2013.06.034}, DOI={10.1016/J.CYTO.2013.06.034}, abstractNote={Type I IFNs induce an antiviral immune response within hours of virus infection, mediated by activation of signaling pathways downstream of the type I IFN receptor. An IFN-induced antiviral response requires both transcriptional activation of genes and the rapid translation of mRNAs into proteins that effect an antiviral response. Both these processes are energy taxing. Cognizant that IFNs activate both the PI3K-Akt/mTOR pathway that is associated with nutrient sensing, and AMPK, associated with stress-induced metabolic activation, we undertook studies to investigate whether IFN-alpha/beta treatment influences metabolic events to enable an antiviral response. Using a panel of mouse embryonic fibroblasts null for Akt1/2, p85s, AMPKa, TSC2 or 4E-BP1, i.e. with defects in various signaling intermediates along the PI3K/mTOR pathway, we provide evidence for IFN-induced regulation of glucose uptake, mediated by activation of the PI3K/mTOR pathway. Notably, we demonstrate that IFN regulation of glucose uptake is required for an antiviral response to infection with the cardiotropic virus, coxsackievirus B3 (CVB3). In addition, we provide evidence for IFN regulated increases in ATP production. In vivo, we provide evidence that activation of AMPK by the pharmacological agent metformin enhances the antiviral effects of IFN-s treatment in mice infected with CVB3, lending further support that metabolic activation enhances an IFN-induced antiviral response.}, number={3}, journal={Cytokine}, publisher={Elsevier BV}, author={Burke, Daniel J. and Platanias, Leonidas C. and Fish, Eleanor N.}, year={2013}, month={Sep}, pages={250} }
 @article{burke_2013, title={Unbiased segregation of yeast chromatids in Saccharomyces cerevisiae}, volume={21}, ISSN={0967-3849 1573-6849}, url={http://dx.doi.org/10.1007/S10577-013-9348-X}, DOI={10.1007/S10577-013-9348-X}, abstractNote={The budding yeast Saccharomyces cerevisiae is characterized by asymmetric cell division and the asymmetric inheritance of spindle components during normal vegetative growth and during certain specialized cell divisions. There has been a longstanding interest in the possibility that yeast chromosomes segregate non-randomly during mitosis and that some of the differences between mother and daughter cells could be explained by selective chromatid segregation. This review traces the history of the experiments to determine if there is biased chromatid segregation in yeast. The special aspects of spindle morphogenesis and behavior in yeast that could accommodate a mechanism for biased segregation are discussed. Finally, a recent experiment demonstrated that yeast chromatids segregate randomly without mother-daughter bias in a common laboratory strain grown under routine laboratory conditions.}, number={3}, journal={Chromosome Research}, publisher={Springer Science and Business Media LLC}, author={Burke, Daniel J.}, year={2013}, month={May}, pages={193–202} }
 @article{keyes_burke_2009, title={Irc15 Is a Microtubule-Associated Protein that Regulates Microtubule Dynamics in Saccharomyces cerevisiae}, volume={19}, ISSN={0960-9822}, url={http://dx.doi.org/10.1016/j.cub.2009.01.068}, DOI={10.1016/j.cub.2009.01.068}, abstractNote={Microtubules are polymers composed of α-β tubulin heterodimers that assemble into microtubules [1Nogales E. Wolf S.G. Downing K.H. Structure of the alpha beta tubulin dimer by electron crystallography.Nature. 1998; 391: 199-203Crossref PubMed Scopus (1715) Google Scholar]. Microtubules are dynamic structures that have periods of both growth and shrinkage by addition and removal of subunits from the polymer [2Desai A. Mitchison T.J. Microtubule polymerization dynamics.Annu. Rev. Cell Dev. Biol. 1997; 13: 83-117Crossref PubMed Scopus (1822) Google Scholar]. Microtubules stochastically switch between periods of growth and shrinkage, termed dynamic instability [3Mitchison T. Kirschner M. Dynamic instability of microtubule growth.Nature. 1984; 312: 237-242Crossref PubMed Scopus (2160) Google Scholar]. Dynamic instability is coupled to the GTPase activity of the β-tubulin subunit of the tubulin heterodimer [4Davis A. Sage C.R. Dougherty C.A. Farrell K.W. Microtubule dynamics modulated by guanosine triphosphate hydrolysis activity of beta-tubulin.Science. 1994; 264: 839-842Crossref PubMed Scopus (51) Google Scholar]. Microtubule dynamics are regulated by microtubule-associated proteins (MAPs) that interact with microtubules to regulate dynamic instability [5Drewes G. Ebneth A. Mandelkow E.M. MAPs, MARKs and microtubule dynamics.Trends Biochem. Sci. 1998; 23: 307-311Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar]. MAPs in budding yeast have been identified that bind microtubule ends (Bim1), that stabilize microtubule structures (Stu2), that bundle microtubules by forming cross-bridges (Ase1), and that interact with microtubules at the kinetochore (Cin8, Kar3, Kip3) [6Tirnauer J.S. O'Toole E. Berrueta L. Bierer B.E. Pellman D. Yeast Bim1p promotes the G1-specific dynamics of microtubules.J. Cell Biol. 1999; 145: 993-1007Crossref PubMed Scopus (200) Google Scholar, 7Usui T. Maekawa H. Pereira G. Schiebel E. The XMAP215 homologue Stu2 at yeast spindle pole bodies regulates microtubule dynamics and anchorage.EMBO J. 2003; 22: 4779-4793Crossref PubMed Scopus (62) Google Scholar, 8Schuyler S.C. Liu J.Y. Pellman D. The molecular function of Ase1p: evidence for a MAP-dependent midzone-specific spindle matrix. Microtubule-associated proteins.J. Cell Biol. 2003; 160: 517-528Crossref PubMed Scopus (162) Google Scholar, 9de Gramont A. Barbour L. Ross K.E. Cohen-Fix O. The spindle midzone microtubule-associated proteins Ase1p and Cin8p affect the number and orientation of astral microtubules in Saccharomyces cerevisiae.Cell Cycle. 2007; 6: 1231-1241Crossref PubMed Scopus (11) Google Scholar, 10Endow S.A. Kang S.J. Satterwhite L.L. Rose M.D. Skeen V.P. Salmon E.D. Yeast Kar3 is a minus-end microtubule motor protein that destabilizes microtubules preferentially at the minus ends.EMBO J. 1994; 13: 2708-2713Crossref PubMed Scopus (232) Google Scholar]. IRC15 was previously identified in four different genetic screens for mutants affecting chromosome transmission or repair [11Daniel J.A. Keyes B.E. Ng Y.P.Y. Freeman C.O. Burke D.J. Diverse functions of spindle assembly checkpoint genes in Saccharomyces cerevisiae.Genetics. 2006; 172: 53-65Crossref PubMed Scopus (45) Google Scholar, 12Alvaro D. Lisby M. Rothstein R. Genome-wide analysis of Rad52 foci reveals diverse mechanisms impacting recombination.PLoS Genet. 2007; 3: e228Crossref PubMed Scopus (135) Google Scholar, 13Measday V. Baetz K. Guzzo J. Yuen K. Kwok T. Sheikh B. Ding H. Ueta R. Hoac T. Cheng B. et al.Systematic yeast synthetic lethal and synthetic dosage lethal screens identify genes required for chromosome segregation.Proc. Natl. Acad. Sci. USA. 2005; 102: 13956-13961Crossref PubMed Scopus (108) Google Scholar, 14Jordan P.W. Klein F. Leach D.R. Novel roles for selected genes in meiotic DNA processing.PLoS Genet. 2007; 3: e222Crossref PubMed Scopus (26) Google Scholar]. Here we present evidence that Irc15 is a microtubule-associated protein, localizing to microtubules in vivo and binding to purified microtubules in vitro. Irc15 regulates microtubule dynamics in vivo and loss of IRC15 function leads to delayed mitotic progression, resulting from failure to establish tension between sister kinetochores.}, number={6}, journal={Current Biology}, publisher={Elsevier BV}, author={Keyes, Brice E. and Burke, Daniel J.}, year={2009}, month={Mar}, pages={472–478} }
 @article{burke_stukenberg_2008, title={Linking Kinetochore-Microtubule Binding to the Spindle Checkpoint}, volume={14}, ISSN={1534-5807}, url={http://dx.doi.org/10.1016/j.devcel.2008.03.015}, DOI={10.1016/j.devcel.2008.03.015}, abstractNote={The spindle checkpoint blocks cell-cycle progression until chromosomes are properly attached to the mitotic spindle. Popular models propose that checkpoint proteins associate with kinetochores to produce a "wait anaphase" signal that inhibits anaphase. Recent data suggest that a two-state switch results from using the same kinetochore proteins to bind microtubules and checkpoint proteins. At least eight protein kinases are implicated in spindle checkpoint signaling, arguing that a traditional signal transduction cascade is integral to spindle checkpoint signaling. The spindle checkpoint blocks cell-cycle progression until chromosomes are properly attached to the mitotic spindle. Popular models propose that checkpoint proteins associate with kinetochores to produce a "wait anaphase" signal that inhibits anaphase. Recent data suggest that a two-state switch results from using the same kinetochore proteins to bind microtubules and checkpoint proteins. At least eight protein kinases are implicated in spindle checkpoint signaling, arguing that a traditional signal transduction cascade is integral to spindle checkpoint signaling. The spindle checkpoint is an evolutionarily conserved mechanism that regulates genome stability from yeast to humans (reviewed in Lew and Burke, 2003Lew D.J. Burke D.J. Annu. Rev. Genet. 2003; 37: 251-282Crossref PubMed Scopus (222) Google Scholar, Cleveland et al., 2003Cleveland D.W. Mao Y. Sullivan K.F. Cell. 2003; 112: 407-421Abstract Full Text Full Text PDF PubMed Scopus (793) Google Scholar, and Musacchio and Salmon, 2007Musacchio A. Salmon E.D. Nat. Rev. Mol. Cell Biol. 2007; 8: 379-393Crossref PubMed Scopus (1616) Google Scholar). A single chromosome detached from the mitotic spindle can activate the spindle checkpoint and inhibit the onset of anaphase. The chromosomal domain responsible for mitotic inhibition via the checkpoint is the kinetochore, and popular models suggest that the kinetochore is a platform that produces a diffusible "wait anaphase" signal that inhibits mitosis. Ultimately, the checkpoint inhibits Cdc20, a specificity factor for the Anaphase Promoting Complex (APC), an E3 ubiquitin ligase that regulates the metaphase-to-anaphase transition (Lew and Burke, 2003Lew D.J. Burke D.J. Annu. Rev. Genet. 2003; 37: 251-282Crossref PubMed Scopus (222) Google Scholar, Cleveland et al., 2003Cleveland D.W. Mao Y. Sullivan K.F. Cell. 2003; 112: 407-421Abstract Full Text Full Text PDF PubMed Scopus (793) Google Scholar). Conserved checkpoint proteins, originally identified in yeast, consist of Bub3, Mad1–3, and the two kinases Bub1 and Mps1. The Ipl1 protein kinase (Aurora B in higher cells) was later identified as a component of the checkpoint but may function in a more limited way (Biggins and Murray, 2001Biggins S. Murray A.W. Genes Dev. 2001; 15: 3118-3129Crossref PubMed Scopus (320) Google Scholar). The spindle checkpoint is more complex in higher cells. Mad3 is associated with a Bub1-related kinase domain on the C terminus and was named BubR1 (Taylor et al., 1998Taylor S.S. Ha E. McKeon F. J. Cell Biol. 1998; 142: 1-11Crossref PubMed Scopus (354) Google Scholar). BubR1 kinase activity is stimulated by the CENP-E plus end directed kinesin when it is not attached to microtubules (Mao et al., 2003Mao Y. Abrieu A. Cleveland D.W. Cell. 2003; 114: 87-98Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). In addition, a complex of Rough Deal, Zeste-White 10 (ZW10), and Zwilch, abbreviated as RZZ, identified in Drosophila and absent in yeast, functions in the checkpoint as described below (Basto et al., 2000Basto R. Gomes R. Karess R.E. Nat. Cell Biol. 2000; 2: 939-943Crossref PubMed Scopus (110) Google Scholar, Chan et al., 2000Chan G.K. Jablonski S.A. Starr D.A. Goldberg M.L. Yen T.J. Nat. Cell Biol. 2000; 2: 944-947Crossref PubMed Scopus (157) Google Scholar). There are homologs of all three proteins in higher cells, and their checkpoint functions are conserved. There are four protein kinases (p38 MAP kinase, Nek2A, Tao1, and Prp4) required for the checkpoint in higher cells that are not present in yeast (Minshull et al., 1994Minshull J. Sun H. Tonks N.K. Murray A.W. Cell. 1994; 79: 475-486Abstract Full Text PDF PubMed Scopus (349) Google Scholar, Lou et al., 2004Lou Y. Yao J. Zereshki A. Dou Z. Ahmed K. Wang H. Hu J. Wang Y. Yao X. J. Biol. Chem. 2004; 279: 20049-20057Crossref PubMed Scopus (107) Google Scholar, Draviam et al., 2007Draviam V.M. Stegmeier F. Nalepa G. Sowa M.E. Chen J. Liang A. Hannon G.J. Sorger P.K. Harper J.W. Elledge S.J. Nat. Cell Biol. 2007; 9: 556-564Crossref PubMed Scopus (83) Google Scholar, Montembault et al., 2007Montembault E. Duterte S. Prigent C. Giet R. J. Cell Biol. 2007; 179: 601-609Crossref PubMed Scopus (49) Google Scholar). Despite the obvious potential for protein kinases as mediators of an intracellular signal transduction pathway, the roles of the protein kinases in the spindle checkpoint have been de-emphasized. Models for the role of the kinetochore in the spindle checkpoint are derived from diverse experimental systems (yeast to human) and incorporate two important observations. The first is that checkpoint proteins (in systems where they can be measured) dynamically associate with unattached kinetochores (Shah et al., 2004Shah J.V. Botvinick E. Bonday Z. Furnari F. Berns M. Cleveland D.W. Curr. Biol. 2004; 14: 942-952Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, Howell et al., 2004Howell B.J. Moree B. Farrar E.M. Stewart S. Fang G. Salmon E.D. Curr. Biol. 2004; 14: 953-964Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). The second derives from in vitro assays for APC regulation showing that a complex of checkpoint proteins called the mitotic checkpoint complex (MCC), consisting of Mad3/BubR1, Mad2, Bub3, and Cdc20, is a potent inhibitor of the ubiquitin ligase activity of the APC. A popular model (Figure 1) that is meant to be "universal" is that the dynamic association of checkpoint proteins with kinetochores of unattached chromosomes reflects the catalytic assembly and then release of MCC, which then diffuses from the unoccupied kinetochore to inhibit the APC (Cleveland et al., 2003Cleveland D.W. Mao Y. Sullivan K.F. Cell. 2003; 112: 407-421Abstract Full Text Full Text PDF PubMed Scopus (793) Google Scholar, Musacchio and Salmon, 2007Musacchio A. Salmon E.D. Nat. Rev. Mol. Cell Biol. 2007; 8: 379-393Crossref PubMed Scopus (1616) Google Scholar). The key step is believed to be the formation of Mad2-Cdc20 complexes, and an elegant model for how this is catalyzed by Mad1 has been proposed (Musacchio and Salmon, 2007Musacchio A. Salmon E.D. Nat. Rev. Mol. Cell Biol. 2007; 8: 379-393Crossref PubMed Scopus (1616) Google Scholar). The current model for the role of the kinetochore in the spindle checkpoint is incomplete for several reasons. First, MCC formation does not require a kinetochore in yeast and perhaps human cells (Fraschini et al., 2001Fraschini R. Beretta A. Sironi L. Musacchio A. Lucchini G. Piatti S. EMBO J. 2001; 20: 6648-6659Crossref PubMed Scopus (154) Google Scholar, Rancati et al., 2005Rancati G. Crispo V. Lucchini G. Piatti S. Cell Cycle. 2005; 4: 972-980Crossref PubMed Scopus (21) Google Scholar). Second, the model does not explain why metazoans have increased the complexity of the signal by employing proteins such as the kinesin motor CENP-E and RZZ (Cleveland et al., 2003Cleveland D.W. Mao Y. Sullivan K.F. Cell. 2003; 112: 407-421Abstract Full Text Full Text PDF PubMed Scopus (793) Google Scholar, Musacchio and Salmon, 2007Musacchio A. Salmon E.D. Nat. Rev. Mol. Cell Biol. 2007; 8: 379-393Crossref PubMed Scopus (1616) Google Scholar). Third, the model does not describe how checkpoint proteins associate with the kinetochore. Finally, the model downplays the role that the protein kinases play in checkpoint signaling. Recent data has lead to a refined model where checkpoint proteins associate with the same proteins that bind microtubules, producing a competition between signaling and microtubule binding. There is a "super complex" of proteins called KMN (a complex of KNL-1/AF15Q14/Spc105/Blinkin, Mis12 complex, and Ndc80 complex) which is a critical microtubule-binding interface in the kinetochore required for microtubule attachments (Emanuele et al., 2007Emanuele M. Burke D.J. Stukenberg P.T. Nat. Struct. Mol. Biol. 2007; 14: 11-13Crossref PubMed Scopus (4) Google Scholar). KMN has at least two direct microtubule interacting proteins: the Ndc80 (Hec1 in human cells) subunit of the Ndc80 complex and KNL-1 (homologous to human Blinkin, for Bub-linking) (Cheeseman et al., 2006Cheeseman I.M. Chappie J.S. Wilson-Kubalek E.M. Desai A. Cell. 2006; 127: 983-997Abstract Full Text Full Text PDF PubMed Scopus (676) Google Scholar). Each of these proteins has been implicated in checkpoint signaling and may directly associate with checkpoint proteins. These associations suggest that there is a relationship in higher cells, as there is in yeast, between microtubule attachment and checkpoint signaling. Moreover, these microtubule attachment complexes are not strongly associated in solution but only come together at kinetochores (Emanuele et al., 2005Emanuele M.J. McCleland M.L. Satinover D.L. Stukenberg P.T. Mol. Biol. Cell. 2005; 16: 4882-4892Crossref PubMed Scopus (61) Google Scholar). This assembly mechanism could assure that checkpoint signals are produced only at kinetochores. An important suggestion of a molecular link between spindle attachment and checkpoint activity in the kinetochore came from yeast mutants that were defective for the four proteins of the evolutionarily conserved Ndc80 kinetochore complex (Ndc80, Nuf2, Spc24, and Spc25). These cells are unable to attach chromosomes to the spindle and are also checkpoint defective (He et al., 2001He X. Rines D.R. Espelin C.W. Sorger P.K. Cell. 2001; 106: 195-206Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, Janke et al., 2001Janke C. Ortiz J. Lechner J. Shevchenko A. Shevchenko A. Magiera M.M. Schramm C. Schiebel E. EMBO J. 2001; 20: 777-791Crossref PubMed Scopus (152) Google Scholar, Wigge and Kilmartin, 2001Wigge P.A. Kilmartin J.V. J. Cell Biol. 2001; 152: 349-360Crossref PubMed Scopus (263) Google Scholar, McCleland et al., 2003McCleland M.L. Gardner R.D. Kallio M.J. Daum J.R. Gorbsky G.J. Burke D.J. Stukenberg P.T. Genes Dev. 2003; 17: 101-114Crossref PubMed Scopus (200) Google Scholar). Mutations that eliminate proteins from other kinetochore complexes, Okp1 (COMA complex), Mtw1 (MIND complex), Duo1 (Dam1 complex), and Stu1, are checkpoint proficient (Gardner et al., 2001Gardner R.D. Poddar A. Yellman C. Tavormina P.A. Monteagudo M.C. Burke D.J. Genetics. 2001; 157: 1493-1502PubMed Google Scholar, Cheeseman et al., 2001Cheeseman I.M. Enquist-Newman M. Muller-Reichert T. Drubin D.G. Barnes G. J. Cell Biol. 2001; 152: 197-212Crossref PubMed Scopus (125) Google Scholar, Kosco et al., 2001Kosco K.A. Pearson C.G. Maddox P.S. Wang P.J. Adams I.R. Salmon E.D. Bloom K. Huffaker T.C. Mol. Biol. Cell. 2001; 12: 2870-2880Crossref PubMed Scopus (118) Google Scholar). The relationship between kinetochore-microtubule binding and checkpoint signaling is strengthened by the observation that Mps1, a protein kinase and checkpoint protein, is kinetochore associated and implicated in regulating microtubule attachment (Jones et al., 2005Jones M.H. Huneycutt B.J. Pearson C.G. Zhang C. Morgan G. Shokat K. Bloom K. Winey M. Curr. Biol. 2005; 15: 160-165Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). In addition, the C. elegans homolog of the yeast kinetochore protein Spc105 (KNL-1) binds microtubules in vitro, and the phenotype of temperature-sensitive spc105 mutants in yeast could be interpreted as lacking the spindle checkpoint, which further supports a molecular link between kinetochore-microtubule attachments and checkpoint signaling (Nekrasov et al., 2003Nekrasov V.S. Smith M.A. Peak-Chew S. Kilmartin J.V. Mol. Biol. Cell. 2003; 14: 4931-4946Crossref PubMed Scopus (73) Google Scholar, Cheeseman et al., 2006Cheeseman I.M. Chappie J.S. Wilson-Kubalek E.M. Desai A. Cell. 2006; 127: 983-997Abstract Full Text Full Text PDF PubMed Scopus (676) Google Scholar). In metazoans, all four members of the Ndc80 complex are required for congression of chromosomes to the metaphase plate and anaphase segregation of sister chromatids (Martin-Lluesma et al., 2002Martin-Lluesma S. Stucke V.M. Nigg E.A. Science. 2002; 297: 2267-2270Crossref PubMed Scopus (365) Google Scholar, DeLuca et al., 2002DeLuca J.G. Moree B. Hickey J.M. Kilmartin J.V. Salmon E.D. J. Cell Biol. 2002; 159: 549-555Crossref PubMed Scopus (217) Google Scholar, McCleland et al., 2003McCleland M.L. Gardner R.D. Kallio M.J. Daum J.R. Gorbsky G.J. Burke D.J. Stukenberg P.T. Genes Dev. 2003; 17: 101-114Crossref PubMed Scopus (200) Google Scholar, McCleland et al., 2004McCleland M.L. Kallio M.J. Barrett-Wilt G.A. Kestner C.A. Shabanowitz J. Hunt D.F. Gorbsky G.J. Stukenberg P.T. Curr. Biol. 2004; 14: 131-137Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). The N terminus of Ndc80 has an unstructured tail followed by a globular head that binds microtubules and has limited homology to the microtubule plus end binding protein EB1 (Wei et al., 2007Wei R.R. Al-Bassam J. Harrison S.C. Nat. Struct. Mol. Biol. 2007; 14: 54-59Crossref PubMed Scopus (240) Google Scholar). Together these data suggest that the Ndc80 complex may be the key microtubule interface in the kinetochore. The Ndc80 complex is also required to generate a spindle checkpoint signal in vertebrates (Martin-Lluesma et al., 2002Martin-Lluesma S. Stucke V.M. Nigg E.A. Science. 2002; 297: 2267-2270Crossref PubMed Scopus (365) Google Scholar, McCleland et al., 2003McCleland M.L. Gardner R.D. Kallio M.J. Daum J.R. Gorbsky G.J. Burke D.J. Stukenberg P.T. Genes Dev. 2003; 17: 101-114Crossref PubMed Scopus (200) Google Scholar, McCleland et al., 2004McCleland M.L. Kallio M.J. Barrett-Wilt G.A. Kestner C.A. Shabanowitz J. Hunt D.F. Gorbsky G.J. Stukenberg P.T. Curr. Biol. 2004; 14: 131-137Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, Meraldi et al., 2004Meraldi P. Draviam V.M. Sorger P.K. Dev. Cell. 2004; 7: 45-60Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar). KMN may function in the checkpoint as a scaffold that brings checkpoint proteins together. A two-hybrid interaction between Ndc80 and Mad1 has been reported, and the Ndc80 complex is required for kinetochore assembly of Mad1, Mad2, Mps1, and RZZ (Martin-Lluesma et al., 2002Martin-Lluesma S. Stucke V.M. Nigg E.A. Science. 2002; 297: 2267-2270Crossref PubMed Scopus (365) Google Scholar, McCleland et al., 2003McCleland M.L. Gardner R.D. Kallio M.J. Daum J.R. Gorbsky G.J. Burke D.J. Stukenberg P.T. Genes Dev. 2003; 17: 101-114Crossref PubMed Scopus (200) Google Scholar, McCleland et al., 2004McCleland M.L. Kallio M.J. Barrett-Wilt G.A. Kestner C.A. Shabanowitz J. Hunt D.F. Gorbsky G.J. Stukenberg P.T. Curr. Biol. 2004; 14: 131-137Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, Stucke et al., 2004Stucke V.M. Baumann C. Nigg E.A. Chromosoma. 2004; 113: 1-15Crossref PubMed Scopus (63) Google Scholar). This suggests that Ndc80 is a platform for Mad1 and perhaps Mps1 binding, although direct physical interactions between the proteins have not been shown. The role of Ndc80 in both kinetochore-microtubule attachment and checkpoint signaling suggests that recognition of microtubule binding by the checkpoint may be as simple as a mutually exclusive binding of the Ndc80 complex for microtubules and Mad1. However, Mad1 association with kinetochores is not dynamic (Shah et al., 2004Shah J.V. Botvinick E. Bonday Z. Furnari F. Berns M. Cleveland D.W. Curr. Biol. 2004; 14: 942-952Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, Howell et al., 2004Howell B.J. Moree B. Farrar E.M. Stewart S. Fang G. Salmon E.D. Curr. Biol. 2004; 14: 953-964Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar), suggesting that this is unlikely to be a shared binding site. Rather, models suggesting either a conformational change promoted by microtubule binding that displaces Mad1 or activation of attachment-sensitive kinases are more consistent with these data. A recent publication has documented interactions of other checkpoint proteins with a different member of KMN. The human homolog of KNL-1, Blinkin, is the platform that directs Bub1 and BubR1 to kinetochores (Kiyomitsu et al., 2007Kiyomitsu T. Obuse C. Yanagida M. Dev. Cell. 2007; 13: 663-676Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). Bub1 and BubR1 have TPR domains that interact with the Blinkin N terminus. Most importantly, siRNA of Blinkin abolishes the spindle checkpoint while maintaining kinetochore assembly. Point mutations that eliminate the interaction between the TPR domain of the Bubs and Blinkin cannot localize Bub proteins to the kinetochore or generate checkpoint signals. Similarly, loss of the N and middle domains of Blinkin prevents checkpoint signaling and mislocalizes the Bub proteins. Given that the Blinkin homolog in C. elegans, KNL-1, binds to microtubules, these data strongly implicate Blinkin in both microtubule binding and checkpoint signaling through the Bub proteins. The third member of KMN, Mis12, is required for checkpoint signaling (McAinsh et al., 2006McAinsh A.D. Meraldi P. Draviam V.M. Toso A. Sorger P.K. EMBO J. 2006; 25: 4033-4049Crossref PubMed Scopus (60) Google Scholar). Moreover, Mis12 recruits a third checkpoint complex RZZ to kinetochores through an interaction with Zwint-1, a protein originally identified because of its interaction with ZW10 (Emanuele et al., 2005Emanuele M.J. McCleland M.L. Satinover D.L. Stukenberg P.T. Mol. Biol. Cell. 2005; 16: 4882-4892Crossref PubMed Scopus (61) Google Scholar). RZZ regulates binding of the microtubule-based motor dynein to kinetochores and is also required to generate the checkpoint signal. Bub1 and BubR1 properly localize in human cells after RZZ knockdown, but Mad1 and Mad2 do not (Chan et al., 2000Chan G.K. Jablonski S.A. Starr D.A. Goldberg M.L. Yen T.J. Nat. Cell Biol. 2000; 2: 944-947Crossref PubMed Scopus (157) Google Scholar, Wang et al., 2004Wang H. Hu X. Ding X. Dou Z. Yang Z. Shaw A.W. Teng M. Cleveland D.W. Goldberg M.L. Niu L. et al.J. Biol. Chem. 2004; 279: 54590-54598Crossref PubMed Scopus (88) Google Scholar, Kops et al., 2005Kops G.J. Kim Y. Weaver B.A. Mao Y. McLeod I. Yates 3rd, J.R. Tagaya M. Cleveland D.W. J. Cell Biol. 2005; 169: 49-60Crossref PubMed Scopus (184) Google Scholar). Chromosomes align and undergo anaphase movements, suggesting that the KMN complexes are intact. These data suggest that KMN is not sufficient for Mad1 binding in higher cells and that there is an additional requirement for RZZ. Overall, KMN is implicated as a central coordinator of microtubule attachment and checkpoint signaling in the kinetochore. The event that is both evolutionarily conserved and most tightly correlated with spindle checkpoint signaling is Mad1 recruitment to the kinetochore. Mad1 levels are dramatically higher on unaligned kinetochores than those at the metaphase plate (Murray et al., 1999Murray D. Mirzayans R. Chen R.H. Br. J. Cancer. 1999; 81: 959-965Crossref PubMed Scopus (22) Google Scholar). How Mad1 is specifically recruited to unattached kinetochores is an important question for the field, but a number of indirect experiments have implicated KMN as the binding site. Mad1 has at least three independent interactions with kinetochore proteins: KNL-1 (via Bub1), Ndc80, and RZZ (Figure 2A). Kinetochores bind either Mad1 or microtubules, suggesting that Mad1 association is a microtubule-regulated step. There is a complex of Bub1, Bub3, and Mad1 in budding yeast, suggesting that there is an indirect association between Mad1 and KNL-1 through Bub1 (Brady and Hardwick, 2000Brady D.M. Hardwick K.G. Curr. Biol. 2000; 10: 675-678Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Thus, a simple model for the spindle checkpoint is that KMN binds either microtubules or Mad1, but cannot bind both simultaneously (Figure 2A). Microtubule attachment would block the generation of the signal at various points. First we propose that when Ndc80 and KNL-1 bind microtubules they lose the capacity to bind Mad1. Microtubule binding would also block the CENP-E/BubR1 interaction, shutting off BubR1 kinase activity (not shown in the model), and allow dynein to carry RZZ, Mad1, and Mad2 away from KMN. A simple model of mutually exclusive binding of Mad1 or a microtubule makes sense for yeast kinetochores that bind a single microtubule (Winey et al., 1995Winey M. Mamay C.L. O'Toole E.T. Mastronarde D.N. Giddings Jr., T.H. McDonald K.L. McIntosh J.R. J. Cell Biol. 1995; 129: 1601-1615Crossref PubMed Scopus (339) Google Scholar). This simple model, when applied to mammalian kinetochores, seems at odds with the observation that there is Mad2 localized to kinetochores with bound microtubules when dynein activity is inhibited (Howell et al., 2001Howell B.J. McEwen B.F. Canman J.C. Hoffman D.B. Farrar E.M. Rieder C.L. Salmon E.D. J. Cell Biol. 2001; 155: 1159-1172Crossref PubMed Scopus (400) Google Scholar). Yeast have approximately eight copies of the Ndc80 complex and six or seven copies of the Mis12 complex per kinetochore, suggesting that a microtubule binding site has approximately eight copies of KMN (Joglekar et al., 2006Joglekar A.P. Bouck D.C. Molk J.N. Bloom K.S. Salmon E.D. Nat. Cell Biol. 2006; 8: 581-585Crossref PubMed Scopus (210) Google Scholar). Vertebrate kinetochores are more complicated than yeast and bind approximately 30 microtubules (McEwen et al., 1998McEwen B.F. Hsieh C.E. Mattheyses A.L. Rieder C.L. Chromosoma. 1998; 107: 366-375Crossref PubMed Scopus (89) Google Scholar, Howell et al., 2001Howell B.J. McEwen B.F. Canman J.C. Hoffman D.B. Farrar E.M. Rieder C.L. Salmon E.D. J. Cell Biol. 2001; 155: 1159-1172Crossref PubMed Scopus (400) Google Scholar, Dong et al., 2007Dong Y. Vanden Beldt K.J. Meng X. Khodjakov A. McEwen B.F. Nat. Cell Biol. 2007; 9: 516-522Crossref PubMed Scopus (100) Google Scholar). Interestingly, Xenopus kinetochores contain approximately 800 copies of KMN, which should be sufficient to bind 100 microtubules (Emanuele et al., 2005Emanuele M.J. McCleland M.L. Satinover D.L. Stukenberg P.T. Mol. Biol. Cell. 2005; 16: 4882-4892Crossref PubMed Scopus (61) Google Scholar). Therefore, vertebrates have an additional requirement to remove checkpoint proteins, not only from binding sites with microtubules attached but potentially from unbound sites as well (Figure 3). Mutually exclusive binding of microtubules or checkpoint proteins to KMN may happen in vertebrate kinetochores but would not effectively silence the checkpoint. This may explain why higher cells evolved an additional dynein-dependent mechanism to strip checkpoint proteins in order to silence the checkpoint. This is supported by the observation that Mad2 is reduced at kinetochores where microtubules are bound and dynein is inhibited as compared with kinetochores without bound microtubules (Howell et al., 2001Howell B.J. McEwen B.F. Canman J.C. Hoffman D.B. Farrar E.M. Rieder C.L. Salmon E.D. J. Cell Biol. 2001; 155: 1159-1172Crossref PubMed Scopus (400) Google Scholar). KMN as a dual site for microtubule binding and checkpoint protein binding accommodates the popular idea that the role of the kinetochore is to assemble MCC (Cleveland et al., 2003Cleveland D.W. Mao Y. Sullivan K.F. Cell. 2003; 112: 407-421Abstract Full Text Full Text PDF PubMed Scopus (793) Google Scholar, Musacchio and Salmon, 2007Musacchio A. Salmon E.D. Nat. Rev. Mol. Cell Biol. 2007; 8: 379-393Crossref PubMed Scopus (1616) Google Scholar). However, this model is not necessarily correct. It is clear in yeast that Mad2-Cdc20 and MCC are essential for the checkpoint (Hardwick et al., 2000Hardwick K.G. Johnston R.C. Smith D.L. Murray A.W. J. Cell Biol. 2000; 148: 871-882Crossref PubMed Scopus (207) Google Scholar, Fraschini et al., 2001Fraschini R. Beretta A. Sironi L. Musacchio A. Lucchini G. Piatti S. EMBO J. 2001; 20: 6648-6659Crossref PubMed Scopus (154) Google Scholar, Poddar et al., 2005Poddar A. Stukenberg P.T. Burke D.J. Eukaryot. Cell. 2005; 4: 867-878Crossref PubMed Scopus (40) Google Scholar). However, Mad2-Cdc20 complexes form in mitosis independently of both the checkpoint and the kinetochore in yeast. MCC also exists in the absence of kinetochores in mitotic Xenopus egg extracts, although the amount of the complex increases in checkpoint signaling conditions (Chen, 2002Chen R.H. J Cell Biol. 2002; 158: 487-496Crossref PubMed Scopus (155) Google Scholar). MCC has also been purified from HeLa cells arrested in S phase (thymidine starved), but the interpretation is potentially complicated by a possible lack of synchrony (Sudakin et al., 2001Sudakin V. Chan G.K. Yen T.J. J. Cell Biol. 2001; 154: 925-936Crossref PubMed Scopus (629) Google Scholar). If MCC assembly is not integral to spindle checkpoint signaling, then what role does KMN play in signaling? Perhaps KMN acts as a platform to localize and activate kinases to generate a phosphorylation cascade. In checkpoint signaling Xenopus extracts, Bub1 and BubR1 are highly phosphorylated on chromatin while soluble protein is not phosphorylated (Chen, 2004Chen R.H. EMBO J. 2004; 23: 3113-3121Crossref PubMed Scopus (57) Google Scholar), strongly suggesting that there are local phosphorylation events. The spindle checkpoint requires a large number of protein kinases, many of which have recently been identified. These include Bub1, BubR1, Mps1, Aurora B, Tao1, Nek2a, p38 MAP kinase, Prp4, CDK1, and possibly Plk1 (Minshull et al., 1994Minshull J. Sun H. Tonks N.K. Murray A.W. Cell. 1994; 79: 475-486Abstract Full Text PDF PubMed Scopus (349) Google Scholar, Kallio et al., 2002Kallio M.J. McCleland M.L. Stukenberg P.T. Gorbsky G.J. Curr. Biol. 2002; 12: 900-905Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, Lew and Burke, 2003Lew D.J. Burke D.J. Annu. Rev. Genet. 2003; 37: 251-282Crossref PubMed Scopus (222) Google Scholar, D'Angiolella et al., 2003D'Angiolella V. Mari C. Nocera D. Rametti L. Grieco D. Genes Dev. 2003; 17: 2520-2525Crossref PubMed Scopus (118) Google Scholar; Cleveland et al., 2003Cleveland D.W. Mao Y. Sullivan K.F. Cell. 2003; 112: 407-421Abstract Full Text Full Text PDF PubMed Scopus (793) Google Scholar, Hauf et al., 2003Hauf S. Cole R.W. LaTerra S. Zimmer C. Schnapp G. Walter R. Heckel A. van Meel J. Rieder C.L. Peters J.M. J. Cell Biol. 2003; 161: 281-294Crossref PubMed Scopus (942) Google Scholar, Lou et al., 2004Lou Y. Yao J. Zereshki A. Dou Z. Ahmed K. Wang H. Hu J. Wang Y. Yao X. J. Biol. Chem. 2004; 279: 20049-20057Crossref PubMed Scopus (107) Google Scholar, Qi et al., 2006Qi W. Tang Z. Yu H. Mol. Biol. Cell. 2006; 17: 3705-3716Crossref PubMed Scopus (106) Google Scholar, Baumann et al., 2007Baumann C. Korner R. Hofmann K. Nigg E.A. Cell. 2007; 128: 101-114Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, Montembault et al., 2007Montembault E. Duterte S. Prigent C. Giet R. J. Cell Biol. 2007; 179: 601-609Crossref PubMed Scopus (49) Google Scholar). The role of kinases in the checkpoint has been disputed because kinase-inactive forms of Bub1 and BubR1 still generate signals (Sharp-Baker and Chen, 2001Sharp-Baker H. Chen R.H. J. Cell Biol. 2001; 153: 1239-1250Crossref PubMed Scopus (183) Google Scholar, Fernius and Hardwick, 2007Fernius J. Hardwick K.G. PLoS. Genet. 2007; 3: e213Crossref PubMed Scopus (81) Google Scholar), although the requirement for BubR1 kinase is disputed (Mao et al., 2005Mao Y. Desai A. Cleveland D.W. J. Cell Biol. 2005; 170: 873-880Crossref PubMed Scopus (115) Google Scholar). However, when one considers that several kinases are involved, it is reasonable that a signal may still be produced after the loss of some kinases because of redundancy. An exciting model that requires additional experimentation involves Mad1 binding bringing Nek2a and CDK1 to kinetochores (Figure 2B), thus perhaps completing a signal transduction circuit to inhibit mitosis in response to a lack of microtubule occupancy in the kinetochore. Mad1 is a long coiled-coil protein that interacts tightly with Mad2 and recruits it to kinetochores. Mad1 also binds the Nek2a kinase that is required for signaling (Lou et al., 2004Lou Y. Yao J. Zereshki A. Dou Z. Ahmed K. Wang H. Hu J. Wang Y. Yao X. J. Biol. Chem. 2004; 279: 20049-20057Crossref PubMed Scopus (107) Google Scholar) and has a CDK1 binding site in its N-terminal region that is required for signaling as well (J. Pines, personal communication). We suggest that Mad1 recruitment initiates three independent pathways that inhibit Cdc20 (Figure 2B). Bub1 kinase phosphorylates and inhibits Cdc20 directly (Tang et al., 2004Tang Z. Shu H. Oncel D. Chen S. Yu H. Mol. Cell. 2004; 16: 387-397Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). Moreover, CDK1 priming phosphorylation on Bub1 targets Polo kinase to kinetochores, an event that allows Mad2 and BubR1 localization and thus concentrates all of the components of the MCC (Qi et al., 2006Qi W. Tang Z. Yu H. Mol. Biol. Cell. 2006; 17: 3705-3716Crossref PubMed Scopus (106) Google Scholar). Moreover, after phosphorylation by CDK1 in vitro, Cdc20 interacts with Mad2 rather than APC (D'Angiolella et al., 2003D'Angiolella V. Mari C. Nocera D. Rametti L. Grieco D. Genes Dev. 2003; 17: 2520-2525Crossref PubMed Scopus (118) Google Scholar). This model for the spindle checkpoint is appealing because it uses kinase cascades as the mechanism to amplify the signal from a single unattached kinetochore and uses redundant pathways that converge to inhibit Cdc20. Our goal is to point out potential shortcomings of current thinking and stimulate new directions. Our model extends many of the key aspects of the old model. Both envision that the spindle checkpoint is a simple two-state switch. When microtubules are absent the Mad1 scaffold assembles on KMN-RZZ and the checkpoint is on. When microtubules bind, RZZ is removed, the scaffold is displaced, and the checkpoint is off. However, by incorporating KMN as the Mad1 docking site, our model needs only a slight modification for yeast where single microtubules bind kinetochores and RZZ is not present. It has been largely assumed that the rapid half-lives of checkpoint proteins argue that the proteins are being modified at kinetochores and soluble active complexes are released. Our model provides a simpler explanation for why some checkpoint proteins have short half-lives in kinetochores. If they associated tightly to kinetochores, then microtubules would be precluded from binding. Although most parts of our model are based on experimental evidence, there are some untested elements. This includes biochemical evidence that Mad1 acts as a scaffold to activate kinases and brings Nek2a and CDK to kinetochores. It is critical to move beyond simply localizing proteins to the kinetochore, and to continue to move toward mechanistic dissection of kinase activation and substrate identification. A beautiful example of such lines of experimentation can be found in the elegant experiments in Xenopus that established that p38 MAP kinase phosphorylation of Mps1 is required for its localization to the kinetochore (Zhao and Chen, 2006Zhao Y. Chen R.H. Curr. Biol. 2006; 16: 1764-1769Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Similarly, CENP-E binds to BubR1 only under checkpoint signaling conditions, and this activates kinase activity (Mao et al., 2005Mao Y. Desai A. Cleveland D.W. J. Cell Biol. 2005; 170: 873-880Crossref PubMed Scopus (115) Google Scholar). The last several years have witnessed an explosion in identifying and characterizing kinetochore and checkpoint proteins. We are only beginning to understand how the checkpoint is organized in the kinetochore. The future is very promising for the spindle checkpoint and kinetochore fields, and the prospect for understanding, in molecular terms, the role of the kinetochore in the spindle checkpoint is exceedingly bright.}, number={4}, journal={Developmental Cell}, publisher={Elsevier BV}, author={Burke, Daniel J. and Stukenberg, P. Todd}, year={2008}, month={Apr}, pages={474–479} }
 @article{lew_burke_dutta_2008, title={The Immortal Strand Hypothesis: How Could It Work?}, volume={133}, ISSN={0092-8674}, url={http://dx.doi.org/10.1016/j.cell.2008.03.016}, DOI={10.1016/j.cell.2008.03.016}, abstractNote={The “immortal strand” hypothesis was proposed by John Cairns as a strategy for stem cells to avoid retaining mutations introduced during DNA replication (Cairns, 1975Cairns J. Nature. 1975; 255: 197-200Crossref PubMed Scopus (1198) Google Scholar). The hypothesis holds that when stem cells undergo asymmetric cell division, one daughter (the self-renewing stem cell) selectively retains the older template DNA strand from each chromosome. Recent successes in detecting nonrandom sister chromatid segregation in various types of stem cells have rekindled interest in the hypothesis. Another conceptually related situation involves cell type-specific selective segregation of chromosome 7 sister chromatids in certain mouse cells, which requires a dynein motor (Armakolas and Klar, 2007Armakolas A. Klar A.J. Science. 2007; 315: 100-101Crossref PubMed Scopus (63) Google Scholar). However, the occurrence of nonrandom sister chromatid segregation and its purpose are still under debate, as discussed in a pair of recent Essays in Cell (Lansdorp, 2007Lansdorp P.M. Cell. 2007; 129: 1244-1247Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, Rando, 2007Rando T.A. Cell. 2007; 129: 1239-1243Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). In this debate, both sides concede that some new cell biology would have to be invoked to provide a mechanistic basis for the proposed segregation. In this Correspondence, we suggest that recent advances in unrelated research areas provide a simple framework of documented phenomena that could, if suitably combined, systematically cause nonrandom sister chromatid segregation during mitosis. Sister chromatid segregation by the mitotic spindle involves the “search and capture” of kinetochores by microtubules emanating from the two spindle poles. For nonrandom sister chromatid segregation to occur, distinct (nonequivalent) spindle poles would have to preferentially connect (presumably via kinetochore microtubules) with nonequivalent sister centromeres. The direction in which a replication fork moves over a stretch of DNA differentiates the sister chromatids over that stretch because one sister is replicated by the leading-strand apparatus, and the other by the lagging-strand apparatus (Figure 1A). Lagging-strand synthesis involves multiple PCNA molecules (one for each Okazaki fragment), multiple RNA primers, and multiple RNase and ligation steps. After the ligation steps, PCNA rings have been proposed to remain on the double-stranded DNA until they are actively removed by the RF-C complex (Bylund and Burgers, 2005Bylund G.O. Burgers P.M. Mol. Cell. Biol. 2005; 25: 5445-5455Crossref PubMed Scopus (95) Google Scholar, Shibahara and Stillman, 1999Shibahara K. Stillman B. Cell. 1999; 96: 575-585Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar). Moreover, recent findings indicate that PCNA can bind to numerous factors and recruit them to chromatin (Moldovan et al., 2007Moldovan G.L. Pfander B. Jentsch S. Cell. 2007; 129: 665-679Abstract Full Text Full Text PDF PubMed Scopus (1222) Google Scholar), making it plausible to speculate that the sister stretch produced by lagging-strand synthesis would attract specific epigenetic marks that could then persist until mitosis. Indeed, the epigenetic imprint in the fission yeast Schizosaccharomyces pombe that distinguishes two chromatids during mating type switching is related to lagging-strand DNA synthesis (Dalgaard and Klar, 1999Dalgaard J.Z. Klar A.J. Nature. 1999; 400: 181-184Crossref PubMed Scopus (111) Google Scholar). If the direction of fork movement is essentially constant across the centromere, then sister centromeres could acquire epigenetic differences that make them segregate asymmetrically during mitosis. Because centromeres are large (thousands of kilobases), they must be replicated using multiple replication origins. Despite this, centromere replication could be heavily biased toward one direction of fork movement (Figure 1B). First, the distribution of replication origins and fork-blocking terminators could be nonrandom, with terminators occurring close to and on one side of the origins so that forks moving in one direction travel much farther than those traveling in the other direction from the origin. Second, forks moving in different directions could travel at different rates so that a large fraction of the centromere sequence is replicated by forks moving in a specific direction. A close examination of time-of-replication profiles from different stretches of yeast and human chromosomes indicates that there are occasional origins with different rates of fork progression on either side of the origin (Karnani et al., 2007Karnani N. Taylor C. Malhotra A. Dutta A. Genome Res. 2007; 17: 865-876Crossref PubMed Scopus (87) Google Scholar, Raghuraman et al., 2001Raghuraman M.K. Winzeler E.A. Collingwood D. Hunt S. Wodicka L. Conway A. Lockhart D.J. Davis R.W. Brewer B.J. Fangman W.L. Science. 2001; 294: 115-121Crossref PubMed Scopus (587) Google Scholar). Third, centromeres may contain a high density of unidirectional replication terminators. Termination signals that allow fork movement in one direction but not the other have been found in bacteria (Bussiere and Bastia, 1999Bussiere D.E. Bastia D. Mol. Microbiol. 1999; 31: 1611-1618Crossref PubMed Scopus (70) Google Scholar) and in the ribosomal DNA (rDNA) locus of some eukaryotes (reviewed in Rothstein et al., 2000Rothstein R. Michel B. Gangloff S. Genes Dev. 2000; 14: 1-10PubMed Google Scholar). Clearly, these strategies could also be used in combination to bias the directionality of centromere replication. The signals causing slowed fork progression or termination could be sequence based, with either the DNA structure or specific binding proteins influencing fork progression. They could also be dynamic, like the transcription of genes or noncoding RNAs that make it difficult for a replication fork to pass through while transcription is in progress. In contrast to centromeres, the existence of nonequivalent spindle poles has been well documented in several fungal and animal cells. Replication of spindle pole bodies (fungi) and centrioles (in animal centrosomes) occurs in a partially conservative manner, generating “old” and “new” poles (Lange and Gull, 1995Lange B.M. Gull K. J. Cell Biol. 1995; 130: 919-927Crossref PubMed Scopus (174) Google Scholar, Pereira et al., 2001Pereira G. Tanaka T.U. Nasmyth K. Schiebel E. EMBO J. 2001; 20: 6359-6370Crossref PubMed Scopus (219) Google Scholar). In budding yeast, the old pole preferentially recruits a cytoplasmic protein, Kar9p, that then moves out along microtubules from that pole through the action of a kinesin family motor (Maekawa et al., 2003Maekawa H. Usui T. Knop M. Schiebel E. EMBO J. 2003; 22: 438-449Crossref PubMed Scopus (133) Google Scholar). Near the cortex, Kar9p binds to a myosin that moves the microtubule tip along bud-directed actin fibers, thereby delivering the microtubule and its attached old pole into the bud (Hwang et al., 2003Hwang E. Kusch J. Barral Y. Huffaker T.C. J. Cell Biol. 2003; 161: 483-488Crossref PubMed Scopus (153) Google Scholar). In some animal stem cells, the old centrosomes are retained in the self-renewing stem cell during asymmetric mitosis (Yamashita et al., 2007Yamashita Y.M. Mahowald A.P. Perlin J.R. Fuller M.T. Science. 2007; 315: 518-521Crossref PubMed Scopus (402) Google Scholar). If this centrosome were to traffic unique factors along kinetochore microtubules to the spindle midzone, then the asymmetric spindle would be poised to interact with asymmetric sister kinetochores. We envisage that the old spindle pole recruits a cytoplasmic kinetochore detachment factor (blue cannon, Figure 1C, left panel) that is shipped out along microtubules from the minus ends at the centrosome toward the plus ends at kinetochores by a plus-end-directed kinesin motor. Upon reaching the kinetochore, this factor would trigger detachment of the kinetochore microtubule (Figure 1C, middle), unless it was antagonized by some protective factor already present at that kinetochore (Figure 1C, right). Sister centromeres, replicated unidirectionally during S phase, would generate asymmetric kinetochores such that the lagging strand-replicated centromere (blue dot) loads the protective factor. Thus, kinetochore microtubules linking the old pole to lagging strand-replicated centromeres would be stably attached (Figure 1C, right), whereas those linking the old pole to leading strand-replicated centromeres would not (Figure 1C, middle). The result would be a systematic segregation of sister chromatids containing the immortal strand to one spindle pole, which in turn is attached to the cell cortex in a manner that allows the resulting daughter cell to remain in the stem cell niche, promoting self-renewal. An attractive candidate for a factor that moves specifically from one spindle pole to affect sister kinetochore behavior is an Aurora-family kinase (or an Aurora regulator). At kinetochores, Aurora B is known to promote detachment of kinetochore microtubules (Tanaka et al., 2002Tanaka T.U. Rachidi N. Janke C. Pereira G. Galova M. Schiebel E. Stark M.J. Nasmyth K. Cell. 2002; 108: 317-329Abstract Full Text Full Text PDF PubMed Scopus (550) Google Scholar). This action of Aurora B is normally regulated by tension between sister kinetochores, which is generated when sister centromeres are pulled toward opposite poles of the spindle (Tanaka et al., 2002Tanaka T.U. Rachidi N. Janke C. Pereira G. Galova M. Schiebel E. Stark M.J. Nasmyth K. Cell. 2002; 108: 317-329Abstract Full Text Full Text PDF PubMed Scopus (550) Google Scholar), but we suggest that additional forms of regulation may occur in stem cells. Aurora A is recruited to spindle poles during mitosis (Ducat and Zheng, 2004Ducat D. Zheng Y. Exp. Cell Res. 2004; 301: 60-67Crossref PubMed Scopus (154) Google Scholar), and Aurora B has been shown to associate with plus-end kinesin motors during telophase (Gruneberg et al., 2004Gruneberg U. Neef R. Honda R. Nigg E.A. Barr F.A. J. Cell Biol. 2004; 166: 167-172Crossref PubMed Scopus (217) Google Scholar). We speculate that in stem cells, a specific pool of Aurora kinase might traffic from the old spindle pole to kinetochores in prometaphase, resulting in detachment of kinetochores. For the centromeres containing the immortal strand, a protective factor, such as a phosphatase that antagonizes Aurora action, could stabilize kinetochore-microtubule attachments, leading to asymmetric strand segregation. This specific model can be tested by visualizing the behavior of Aurora kinases and their regulators in stem cells to determine whether or not they concentrate specifically at one of the two spindle poles. The idea that replication direction is biased in centromeres can be tested using techniques such as Fiber-FISH (fluorescence in situ hybridization of extended chromatin fibers). Sequential labeling with nucleotide derivatives like bromodeoxyuridine and chlorodeoxyuridine followed by two-color immunofluorescence detection of the derivatives yields the direction of movement of a specific fork. FISH with centromere-specific probes would identify specific DNA fragments from centromeres. Applying these two methods on stretched DNA molecules will be sufficient to test whether centromeric DNA is indeed replicated primarily by forks moving in one direction. Our model does not predict whether such biased replication of DNA occurs generally or only in relevant stem cell populations. Finally, immunofluorescence studies on dividing stem cells can test for the persistence of PCNA on centromeric DNA after the completion of S phase. Such studies may also reveal asymmetric association of PCNA on sister centromeres. Of course, if PCNA is replaced rapidly in S phase by an asymmetric epigenetic mark, then the epigenetic mark, and not PCNA, will be seen to persist until mitosis.}, number={1}, journal={Cell}, publisher={Elsevier BV}, author={Lew, Daniel J. and Burke, Daniel J. and Dutta, Anindya}, year={2008}, month={Apr}, pages={21–23} }
 @article{emanuele_burke_stukenberg_2007, title={A Hec of a microtubule attachment}, volume={14}, ISSN={1545-9993 1545-9985}, url={http://dx.doi.org/10.1038/nsmb0107-11}, DOI={10.1038/nsmb0107-11}, number={1}, journal={Nature Structural & Molecular Biology}, publisher={Springer Science and Business Media LLC}, author={Emanuele, Michael and Burke, Daniel J and Stukenberg, P Todd}, year={2007}, month={Jan}, pages={11–13} }
 @article{chi_huttenhower_geer_coon_syka_bai_shabanowitz_burke_troyanskaya_hunt_2007, title={Analysis of phosphorylation sites on proteins from Saccharomyces cerevisiae by electron transfer dissociation (ETD) mass spectrometry}, volume={104}, ISSN={0027-8424 1091-6490}, url={http://dx.doi.org/10.1073/pnas.0607084104}, DOI={10.1073/pnas.0607084104}, abstractNote={We present a strategy for the analysis of the yeast phosphoproteome that uses endo-Lys C as the proteolytic enzyme, immobilized metal affinity chromatography for phosphopeptide enrichment, a 90-min nanoflow-HPLC/electrospray-ionization MS/MS experiment for phosphopeptide fractionation and detection, gas phase ion/ion chemistry, electron transfer dissociation for peptide fragmentation, and the Open Mass Spectrometry Search Algorithm for phosphoprotein identification and assignment of phosphorylation sites. From a 30-μg (≈600 pmol) sample of total yeast protein, we identify 1,252 phosphorylation sites on 629 proteins. Identified phosphoproteins have expression levels that range from <50 to 1,200,000 copies per cell and are encoded by genes involved in a wide variety of cellular processes. We identify a consensus site that likely represents a motif for one or more uncharacterized kinases and show that yeast kinases, themselves, contain a disproportionately large number of phosphorylation sites. Detection of a pHis containing peptide from the yeast protein, Cdc10, suggests an unexpected role for histidine phosphorylation in septin biology. From diverse functional genomics data, we show that phosphoproteins have a higher number of interactions than an average protein and interact with each other more than with a random protein. They are also likely to be conserved across large evolutionary distances.}, number={7}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Chi, A. and Huttenhower, C. and Geer, L. Y. and Coon, J. J. and Syka, J. E. P. and Bai, D. L. and Shabanowitz, J. and Burke, D. J. and Troyanskaya, O. G. and Hunt, D. F.}, year={2007}, month={Feb}, pages={2193–2198} }
 @article{devasahayam_ritz_helliwell_burke_sturgill_2006, title={Pmr1, a Golgi Ca2+/Mn2+-ATPase, is a regulator of the target of rapamycin (TOR) signaling pathway in yeast}, volume={103}, ISSN={0027-8424 1091-6490}, url={http://dx.doi.org/10.1073/pnas.0604303103}, DOI={10.1073/pnas.0604303103}, abstractNote={
            The rapamycin·FKBP12 complex inhibits target of rapamycin (TOR) kinase in TORC1. We screened the yeast nonessential gene deletion collection to identify mutants that conferred rapamycin resistance, and we identified
            PMR1
            , encoding the Golgi Ca
            2+
            /Mn
            2+
            -ATPase. Deleting
            PMR1
            in two genetic backgrounds confers rapamycin resistance. Epistasis analyses show that Pmr1 functions upstream from Npr1 and Gln-3 in opposition to Lst8, a regulator of TOR. Npr1 kinase is largely cytoplasmic, and a portion localizes to the Golgi where amino acid permeases are modified and sorted. Nuclear translocation of Gln-3 and Gln-3 reporter activity in
            pmr1
            cells are impaired, but expression of functional Gap1 in the plasma membrane of a
            pmr1
            strain in response to nitrogen limitation is enhanced. These two phenotypes suggest up-regulation of Npr1 function in the absence of Pmr1. Together, our results establish that Pmr1-dependent Ca
            2+
            and/or Mn
            2+
            ion homeostasis is necessary for TOR signaling.
          }, number={47}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Devasahayam, G. and Ritz, D. and Helliwell, S. B. and Burke, D. J. and Sturgill, T. W.}, year={2006}, month={Nov}, pages={17840–17845} }
 @article{jiang_niu_clines_burke_sturgill_2004, title={Analyses of the effects of Rck2p mutants on Pbs2pDD-induced toxicity in Saccharomyces cervisiae identify a MAP kinase docking motif, and unexpected functional inactivation due to acidic substitution of T379}, volume={271}, ISSN={1617-4615 1617-4623}, url={http://dx.doi.org/10.1007/s00438-003-0972-6}, DOI={10.1007/s00438-003-0972-6}, abstractNote={Rck2p is a Ser/Thr kinase that binds to, and is activated by, Hog1p. Expression of the MAP kinase kinase Pbs2pDD from a GAL1-driven plasmid hyperactivates the HOG MAP kinase pathway, and leads to cessation of growth. This toxic effect is reduced by deletion of RCK2. We studied the structural and functional basis for the role of Rck2p in mediating the growth arrest phenotype associated with overexpression of Pbs2pDD. Rck2p kinase activity is required for the effect, because Rck2p(Delta487-610), as well as full-length Rck2p, is toxic with Pbs2pDD, but kinase-defective versions of either protein with a K201R mutation are not. Thus, the C-terminal portion of Rck2p is not required provided the protein is activated by removal of the autoinhibitory domain. Relief of inhibition in Rck2p normally requires phosphorylation by Hog1p, and Rck2p contains a putative MAP kinase docking site (TILQR589R590KKVQ) in its C-terminal segment. The Rck2p double mutant R589A/R590A expressed from a centromeric plasmid did not detectably bind Hog1p-GFP and was functionally inactive in mediating the toxic effect of Pbs2pDD, equivalent to an RCK2 deletion. However, overexpression of Rck2p R589A/R590A from a multicopy plasmid restored function. In contrast, RCK2-K201R acted as a multicopy suppressor of PBS2DD, markedly reducing its toxicity. This suppressor activity required the K201R mutation, and the effect was largely lost when the docking site was mutated, suggesting suppression by inhibition of Hog1p functions. We also studied the effect of replacing the predicted T379 and established S520 phosphorylation sites in Rck2p by glutamic acid. Surprisingly, the T379E mutant markedly reduced Pbs2pDD toxicity, and toxicity was only partially rescued by S520E. Rck2 T379E was sufficiently inactive in an rck2Delta strain to allow some cells to survive PBS2DD toxicity even when overexpressed. The significance of these findings for our understanding of Rck2p function is discussed.}, number={2}, journal={Molecular Genetics and Genomics}, publisher={Springer Science and Business Media LLC}, author={Jiang, L. and Niu, S. and Clines, K. L. and Burke, D. J. and Sturgill, T. W.}, year={2004}, month={Jan}, pages={208–219} }