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1.  Drosophila melanogaster γ-TuRC is dispensable for targeting γ-tubulin to the centrosome and microtubule nucleation 
The Journal of Cell Biology  2006;172(4):517-528.
In metazoans, γ-tubulin acts within two main complexes, γ-tubulin small complexes (γ-TuSCs) and γ-tubulin ring complexes (γ-TuRCs). In higher eukaryotes, it is assumed that microtubule nucleation at the centrosome depends on γ-TuRCs, but the role of γ-TuRC components remains undefined.
For the first time, we analyzed the function of all four γ-TuRC–specific subunits in Drosophila melanogaster: Dgrip75, Dgrip128, Dgrip163, and Dgp71WD. Grip-motif proteins, but not Dgp71WD, appear to be required for γ-TuRC assembly. Individual depletion of γ-TuRC components, in cultured cells and in vivo, induces mitotic delay and abnormal spindles. Surprisingly, γ-TuSCs are recruited to the centrosomes. These defects are less severe than those resulting from the inhibition of γ-TuSC components and do not appear critical for viability. Simultaneous cosilencing of all γ-TuRC proteins leads to stronger phenotypes and partial recruitment of γ-TuSC. In conclusion, γ-TuRCs are required for assembly of fully functional spindles, but we suggest that γ-TuSC could be targeted to the centrosomes, which is where basic microtubule assembly activities are maintained.
doi:10.1083/jcb.200511071
PMCID: PMC2063672  PMID: 16476773
2.  Mitosis-specific Anchoring of γ Tubulin Complexes by Pericentrin Controls Spindle Organization and Mitotic Entry 
Molecular Biology of the Cell  2004;15(8):3642-3657.
Microtubule nucleation is the best known function of centrosomes. Centrosomal microtubule nucleation is mediated primarily by γ tubulin ring complexes (γ TuRCs). However, little is known about the molecules that anchor these complexes to centrosomes. In this study, we show that the centrosomal coiled-coil protein pericentrin anchors γ TuRCs at spindle poles through an interaction with γ tubulin complex proteins 2 and 3 (GCP2/3). Pericentrin silencing by small interfering RNAs in somatic cells disrupted γ tubulin localization and spindle organization in mitosis but had no effect on γ tubulin localization or microtubule organization in interphase cells. Similarly, overexpression of the GCP2/3 binding domain of pericentrin disrupted the endogenous pericentrin–γ TuRC interaction and perturbed astral microtubules and spindle bipolarity. When added to Xenopus mitotic extracts, this domain uncoupled γ TuRCs from centrosomes, inhibited microtubule aster assembly, and induced rapid disassembly of preassembled asters. All phenotypes were significantly reduced in a pericentrin mutant with diminished GCP2/3 binding and were specific for mitotic centrosomal asters as we observed little effect on interphase asters or on asters assembled by the Ran-mediated centrosome-independent pathway. Additionally, pericentrin silencing or overexpression induced G2/antephase arrest followed by apoptosis in many but not all cell types. We conclude that pericentrin anchoring of γ tubulin complexes at centrosomes in mitotic cells is required for proper spindle organization and that loss of this anchoring mechanism elicits a checkpoint response that prevents mitotic entry and triggers apoptotic cell death.
doi:10.1091/mbc.E03-11-0796
PMCID: PMC491825  PMID: 15146056
3.  CDK5RAP2 stimulates microtubule nucleation by the γ-tubulin ring complex 
The Journal of Cell Biology  2010;191(6):1089-1095.
A conserved γ-tubulin complex–binding domain in CDK5RAP2 stimulates the microtubule-nucleating activity of γ-TuRC.
CDK5RAP2 is a human microcephaly protein that contains a γ-tubulin complex (γ-TuC)–binding domain conserved in Drosophila melanogaster centrosomin and Schizosaccharomyces pombe Mto1p and Pcp1p, which are γ-TuC–tethering proteins. In this study, we show that this domain within CDK5RAP2 associates with the γ-tubulin ring complex (γ-TuRC) to stimulate its microtubule-nucleating activity and is therefore referred to as the γ-TuRC–mediated nucleation activator (γ-TuNA). γ-TuNA but not its γ-TuC–binding-deficient mutant stimulates microtubule nucleation by purified γ-TuRC in vitro and induces extensive, γ-TuRC-dependent nucleation of microtubules in a microtubule regrowth assay. γ-TuRC bound to γ-TuNA contains NME7, FAM128A/B, and actin in addition to γ-tubulin and GCP2–6. RNA interference–mediated depletion of CDK5RAP2 impairs both centrosomal and acentrosomal microtubule nucleation, although γ-TuRC assembly is unaffected. Collectively, these results suggest that the γ-TuNA found in CDK5RAP2 has regulatory functions in γ-TuRC–mediated microtubule nucleation.
doi:10.1083/jcb.201007030
PMCID: PMC3002024  PMID: 21135143
4.  Centrosomal Proteins CG-NAP and Kendrin Provide Microtubule Nucleation Sites by Anchoring γ-Tubulin Ring Complex 
Molecular Biology of the Cell  2002;13(9):3235-3245.
Microtubule assembly is initiated by the γ-tubulin ring complex (γ-TuRC). In yeast, the microtubule is nucleated from γ-TuRC anchored to the amino-terminus of the spindle pole body component Spc110p, which interacts with calmodulin (Cmd1p) at the carboxy-terminus. However, mammalian protein that anchors γ-TuRC remains to be elucidated. A giant coiled-coil protein, CG-NAP (centrosome and Golgi localized PKN-associated protein), was localized to the centrosome via the carboxyl-terminal region. This region was found to interact with calmodulin by yeast two-hybrid screening, and it shares high homology with the carboxyl-terminal region of another centrosomal coiled-coil protein, kendrin. The amino-terminal region of either CG-NAP or kendrin indirectly associated with γ-tubulin through binding with γ-tubulin complex protein 2 (GCP2) and/or GCP3. Furthermore, endogenous CG-NAP and kendrin were coimmunoprecipitated with each other and with endogenous GCP2 and γ-tubulin, suggesting that CG-NAP and kendrin form complexes and interact with γ-TuRC in vivo. These proteins were localized to the center of microtubule asters nucleated from isolated centrosomes. Pretreatment of the centrosomes by antibody to CG-NAP or kendrin moderately inhibited the microtubule nucleation; moreover, the combination of these antibodies resulted in stronger inhibition. These results imply that CG-NAP and kendrin provide sites for microtubule nucleation in the mammalian centrosome by anchoring γ-TuRC.
doi:10.1091/mbc.E02-02-0112
PMCID: PMC124155  PMID: 12221128
5.  Dgp71WD is required for the assembly of the acentrosomal Meiosis I spindle, and is not a general targeting factor for the γ-TuRC 
Biology Open  2012;1(5):422-429.
Summary
Dgp71WD/Nedd1 proteins are essential for mitotic spindle formation. In human cells, Nedd1 targets γ-tubulin to both centrosomes and spindles, but in other organisms the function of Dgp71WD/Nedd1 is less clear. In Drosophila cells, Dgp71WD plays a major part in targeting γ-tubulin to spindles, but not centrosomes, while in Xenopus egg extracts, Nedd1 acts as a more general microtubule (MT) organiser that can function independently of γ-tubulin. The interpretation of these studies, however, is complicated by the fact that some residual Dgp71WD/Nedd1 is likely present in the cells/extracts analysed. Here we generate a Dgp71WD null mutant lacking all but the last 12 nucleotides of coding sequence. The complete loss of Dgp71WD has no quantifiable effect on γ-tubulin or Centrosomin recruitment to the centrosome in larval brain cells. The recruitment of γ-tubulin to spindle MTs, however, is severely impaired, and spindle MT density is reduced in a manner that is indistinguishable from cells lacking Augmin or γ-TuRC function. In contrast, the absence of Dgp71WD leads to defects in the assembly of the acentrosomal female Meiosis I spindle that are more severe than those seen in Augmin or γ-TuRC mutants, indicating that Dgp71WD has additional functions that are independent of these complexes in oocytes. Moreover, the localisation of bicoid RNA during oogenesis, which requires γ-TuRC function, is unperturbed in Dgp71WD120 mutants. Thus, Dgp71WD is not simply a general cofactor required for γ-TuRC and/or Augmin targeting, and it appears to have a crucial role independent of these complexes in the acentrosomal Meiosis I spindle.
doi:10.1242/bio.2012596
PMCID: PMC3507215  PMID: 23213433
Dgp71WD; Centrosome; Mitosis; Meiosis
6.  The Nup107-160 complex and γ-TuRC regulate microtubule polymerization at kinetochores 
Nature cell biology  2010;12(2):164-169.
The metazoan nuclear pore complex (NPC) disassembles during mitosis, and many of its constituents distribute onto spindles and kinetochores, including the Nup107-160 sub-complex1,2. We have found that Nup107-160 interacts with the γ-tubulin ring complex (γ-TuRC), an essential and conserved microtubule (MT) nucleator3,4, and recruits γ-TuRC to unattached kinetochores. Unattached kinetochores nucleate MTs in a manner that is regulated by the Ran GTPase5; such MTs contribute to the formation of kinetochore fibers (k-fibers)6, MT bundles connecting kinetochores to spindle poles. Our data indicate that Nup107-160 and γ-TuRC act cooperatively to promote spindle assembly through MTs nucleation at kinetochores: HeLa cells lacking Nup107-160 or γ-TuRC were profoundly deficient in kinetochore-associated MT nucleation. Moreover, co-precipitated Nup107-160/γ-TuRC complexes nucleate MT formation in assays using purified tubulin. While Ran did not regulate MTs nucleation by γ-TuRC alone, Nup107-160/γ-TuRC complexes required Ran-GTP for MT nucleation. Our observations collectively show that Nup107-160 promotes spindle assembly through Ran-GTP-regulated nucleation of MT by γ-TuRC at kinetochores, and reveal a novel relationship between nucleoporins and the MT cytoskeleton.
doi:10.1038/ncb2016
PMCID: PMC2859955  PMID: 20081840
7.  Kinesin-14 and kinesin-5 antagonistically regulate microtubule nucleation by γ-TuRC in yeast and human cells 
Nature Communications  2014;5:5339.
Bipolar spindle assembly is a critical control point for initiation of mitosis through nucleation and organization of spindle microtubules and is regulated by kinesin-like proteins. In fission yeast, the kinesin-14 Pkl1 binds the γ-tubulin ring complex (γ-TuRC) microtubule-organizing centre at spindle poles and can alter its structure and function. Here we show that kinesin-14 blocks microtubule nucleation in yeast and reveal that this inhibition is countered by the kinesin-5 protein, Cut7. Furthermore, we demonstrate that Cut7 binding to γ-TuRC and the Cut7 BimC domain are both required for inhibition of Pkl1. We also demonstrate that a yeast kinesin-14 peptide blocks microtubule nucleation in two human breast cancer cell lines, suggesting that this mechanism is evolutionarily conserved. In conclusion, using genetic, biochemical and cell biology approaches we uncover antagonistic control of microtubule nucleation at γ-TuRC by two kinesin-like proteins, which may represent an attractive anti-mitotic target for cancer therapies.
Mitotic spindle assembly requires strict control of microtubule nucleation by γ-tubulin ring complexes. Olmsted et al. report that the kinesin-like proteins Pkl1 and Cut7 antagonistically regulate nucleation in fission yeast, and show that a Pkl1 peptide blocks spindle assembly in human cancer cells.
doi:10.1038/ncomms6339
PMCID: PMC4220466  PMID: 25348260
8.  γ-Tubulin ring complexes regulate microtubule plus end dynamics 
The Journal of Cell Biology  2009;187(3):327-334.
Independently of their nucleation activity, γ-tubulin ring complex proteins localize along microtubules, limiting catastrophe events during interphase.
γ-Tubulin is critical for the initiation and regulation of microtubule (MT) assembly. In Drosophila melanogaster, it acts within two main complexes: the γ-tubulin small complex (γ-TuSC) and the γ-tubulin ring complex (γ-TuRC). Proteins specific of the γ-TuRC, although nonessential for viability, are required for efficient mitotic progression. Until now, their role during interphase remained poorly understood. Using RNA interference in Drosophila S2 cells, we show that the γ-TuRC is not critical for overall MT organization. However, depletion of any component of this complex results in an increase of MT dynamics. Combined immunofluorescence and live imaging analysis allows us to reveal that the γ-TuRC localizes along interphase MTs and that distal γ-tubulin spots match with sites of pause or rescue events. We propose that, in addition to its role in nucleation, the γ-TuRC associated to MTs may regulate their dynamics by limiting catastrophes.
doi:10.1083/jcb.200905060
PMCID: PMC2779254  PMID: 19948476
9.  The centriolar satellite protein SSX2IP promotes centrosome maturation 
The Journal of Cell Biology  2013;202(1):81-95.
SSX2IP promotes centrosome maturation and maintenance at the onset of vertebrate development, preserving centrosome integrity and mitosis during rapid cleavage divisions and in somatic cells.
Meiotic maturation in vertebrate oocytes is an excellent model system for microtubule reorganization during M-phase spindle assembly. Here, we surveyed changes in the pattern of microtubule-interacting proteins upon Xenopus laevis oocyte maturation by quantitative proteomics. We identified the synovial sarcoma X breakpoint protein (SSX2IP) as a novel spindle protein. Using X. laevis egg extracts, we show that SSX2IP accumulated at spindle poles in a Dynein-dependent manner and interacted with the γ-tubulin ring complex (γ-TuRC) and the centriolar satellite protein PCM-1. Immunodepletion of SSX2IP impeded γ-TuRC loading onto centrosomes. This led to reduced microtubule nucleation and spindle assembly failure. In rapidly dividing blastomeres of medaka (Oryzias latipes) and in somatic cells, SSX2IP knockdown caused fragmentation of pericentriolar material and chromosome segregation errors. We characterize SSX2IP as a novel centrosome maturation and maintenance factor that is expressed at the onset of vertebrate development. It preserves centrosome integrity and faithful mitosis during the rapid cleavage division of blastomeres and in somatic cells.
doi:10.1083/jcb.201302122
PMCID: PMC3704989  PMID: 23816619
10.  CEP192 interacts physically and functionally with the K63-deubiquitinase CYLD to promote mitotic spindle assembly 
Cell Cycle  2012;11(19):3555-3558.
CEP192 is a centrosome protein that plays a critical role in centrosome biogenesis and function in mammals, Drosophila and C. elegans.1-6 Moreover, CEP192-depleted cells arrest in mitosis with disorganized microtubules, suggesting that CEP192’s function in spindle assembly goes beyond its role in centrosome activity and pointing to a potentially more direct role in the regulation of the mitotic microtubule landscape.7 To better understand CEP192 function in mitosis, we used mass spectrometry to identify CEP192-interacting proteins. We previously reported that CEP192 interacts with NEDD1, a protein that associates with the γ-tubulin ring complex (γ-TuRC) and regulates its phosphorylation status during mitosis.8 Additionally, within the array of proteins that interact with CEP192, we identified the microtubule binding K63-deubiquitinase CYLD. Further analyses show that co-depletion of CYLD alleviates the bipolar spindle assembly defects observed in CEP192-depleted cells. This functional relationship exposes an intriguing role for CYLD in spindle formation and raises the tantalizing possibility that CEP192 promotes robust mitotic spindle assembly by regulating K63-polyubiquitin-mediated signaling through CYLD.
doi:10.4161/cc.21574
PMCID: PMC3478306  PMID: 22895009
CEP192; CYLD; mitosis; spindle; microtubules; ubiquitination; K63; centrosome; proteomics
11.  Kinesin-14 Pkl1 targets γ-tubulin for release from the γ-tubulin ring complex (γ-TuRC) ‬‬‬‬‬‬‬ 
Cell Cycle  2013;12(5):842-848.
The γ-tubulin ring complex (γ-TuRC) is a key part of microtubule-organizing centers (MTOCs) that control microtubule polarity, organization and dynamics in eukaryotes. Understanding regulatory mechanisms of γ-TuRC function is of fundamental importance, as this complex is central to many cellular processes, including chromosome segregation, fertility, neural development, T-cell cytotoxicity and respiration. The fission yeast microtubule motor kinesin-14 Pkl1 regulates mitosis by binding to the γ-tubulin small complex (γ-TuSC), a subunit of γ-TuRC. Here we investigate the binding mechanism of Pkl1 to γ-TuSC and its functional consequences using genetics, biochemistry, peptide assays and cell biology approaches in vivo and in vitro. We identify two critical elements in the Tail domain of Pkl1 that mediate γ-TuSC binding and trigger release of γ-tubulin from γ-TuRC. Such action disrupts the MTOC and results in failed mitotic spindle assembly. This study is the first demonstration that a motor protein directly affects the structural composition of the γ-TuRC, and we provide details of this mechanism that may be of broad biological importance.
doi:10.4161/cc.23822
PMCID: PMC3610732  PMID: 23388459
MTOC; microtubule motor; spindle assembly; γ-TuSC
12.  Bub3 reads phosphorylated MELT repeats to promote spindle assembly checkpoint signaling 
eLife  2013;2:e01030.
Regulation of macromolecular interactions by phosphorylation is crucial in signaling networks. In the spindle assembly checkpoint (SAC), which enables errorless chromosome segregation, phosphorylation promotes recruitment of SAC proteins to tensionless kinetochores. The SAC kinase Mps1 phosphorylates multiple Met-Glu-Leu-Thr (MELT) motifs on the kinetochore subunit Spc105/Knl1. The phosphorylated MELT motifs (MELTP) then promote recruitment of downstream signaling components. How MELTP motifs are recognized is unclear. In this study, we report that Bub3, a 7-bladed β-propeller, is the MELTP reader. It contains an exceptionally well-conserved interface that docks the MELTP sequence on the side of the β-propeller in a previously unknown binding mode. Mutations targeting the Bub3 interface prevent kinetochore recruitment of the SAC kinase Bub1. Crucially, they also cause a checkpoint defect, showing that recognition of phosphorylated targets by Bub3 is required for checkpoint signaling. Our data provide the first detailed mechanistic insight into how phosphorylation promotes recruitment of checkpoint proteins to kinetochores.
DOI: http://dx.doi.org/10.7554/eLife.01030.001
eLife digest
The cell cycle is the process by which a cell divides to produce two near-identical daughter cells. Two crucial parts of the cell cycle are the duplication of the chromosomes in the original cell, and the segregation of these chromosomes between the two daughter cells. These and other parts of the cell cycle are strictly regulated to prevent errors, which can lead to cancer and other diseases.
After chromosome duplication has taken place, the pairs of identical chromosomes, known as sister chromatids, remain tightly bound to each other. These sister chromatids line up in the middle of the cell, with protein filaments called microtubules connecting them to a bipolar structure called the spindle. For the cell to divide correctly, the sister chromatids in each pair must be connected to opposite poles of the spindle. A signalling network known as the spindle assembly checkpoint (SAC) ensures that the sister chromatids have enough time to line up correctly and to correct possible problems. Once everything is in place, the SAC releases its ‘break’, and the microtubules then pull the sister chromatids away from each other. This way, each daughter cell receives the same complement of chromosomes that was present in the mother cell.
The microtubules are not directly attached to the sister chromatids but to protein complexes called kinetochores that assemble on each sister chromatid. In particular, each microtubule binds to a very large protein complex called the KMN network. Knl1, which is part of this network, recruits two SAC proteins–Bub1 and Bub3–to the kinetochore. It is known that a phosphate group is added to Knl1 when the SAC is active, and that Knl1 can only recruit Bub1 and Bub3 after it has been phosphorylated. However, the details of the interactions between Knl1, Bub1 and Bub3 are not understood, and it is not clear whether these interactions are essential for the SAC.
Now Primorac et al. have shown that Bub3 binds directly to Knl1 through a region that contains multiple MELT motifs (where M, E, L and T are all amino acids), and that this interaction only happens if these ‘MELT repeats’ have been phosphorylated. Moreover, once bound to the Knl1, Bub3 then recruits Bub1 to the kinetochore. By showing that the recognition of phosphorylated Knl1 by the Bub1-Bub3 complex has a central role in the spindle assembly checkpoint, these results highlight the importance of phosphorylation as a way of regulating the timing of events during the cell cycle.
DOI: http://dx.doi.org/10.7554/eLife.01030.002
doi:10.7554/eLife.01030
PMCID: PMC3779320  PMID: 24066227
spindle assembly checkpoint; kinetochore; Bub3; Bub1; Mad3; Knl1; S. cerevisiae
13.  BRCA1 Regulates γ-Tubulin Binding to Centrosomes 
Cancer biology & therapy  2007;6(12):1853-1857.
Centrosomes are the cellular organelles that nucleate microtubules (MTs) via the activity of gamma-tubulin ring complex(s) (γTuRC) bound to the pericentriolar material of the centrosomes. BRCA1, the breast and ovarian cancer specific tumor suppressor, inhibits centrosomal MT nucleation via its ubiquitin ligase activity, and one of the known BRCA1 substrates is the key γTuRC component, γ-tubulin. We analyzed the mechanism by which BRCA1 regulates centrosome function using an in vitro reconstitution assay, which includes separately staged steps. Our results are most consistent with a model by which the BRCA1 ubiquitin ligase modifies both γ-tubulin plus a second centrosomal protein that controls localization of γTuRC to the centrosome. We suggest that this second protein is an adapter protein or protein complex that docks γ-TuRC to the centrosome. By controlling γ-TuRC localization, BRCA1 appropriately inhibits centrosome function, and loss of BRCA1 would result in centrosome hyperactivity, supernumerary centrosomes and, possibly, aneuploidy.
PMCID: PMC2643382  PMID: 18087219
BRCA1; breast cancer; centrosome; ubiquitin; microtubules; γ-tubulin; microtubule nucleation
14.  p38α MAPK is a MTOC-associated protein regulating spindle assembly, spindle length and accurate chromosome segregation during mouse oocyte meiotic maturation 
Cell Cycle  2010;9(20):4130-4143.
P38αMAPK (p38α) is usually activated in response to various stresses and plays a role in the inhibition of cell proliferation and tumor progression, but little is known about its roles in meiotic spindle assembly. In this study, we characterized the dynamic localization of p38α and explored its function in mouse oocyte meiotic maturation. P38α specifically colocalized with γ-tubulin and Plk1 at the center of MTOCs and spindle poles. Depletion of p38α by specific morpholino injection resulted in severely defective spindles and misaligned chromosomes probably via MK2 dephosphorylation. Notably, depletion of p38α led to significant spindle pole defects, spindle elongation, non-tethered kinetochore microtubules and increased microtubule tension. The disruption of spindle stability was coupled with decreased γ-tubulin and Plk1 at MTOCs. Overexpression of Eg5, a conserved motor protein, also caused spindle elongation and its morpholino injection almost completely rescued spindle elongation caused by p38α depletion. In addition, p38α-depletion decreased BubR1 and interfered with spindle assembly checkpoint (SAC), which resulted in aneuploid oocytes. Together, these data indicate that p38α is an important component of MTOCs, which regulates spindle assembly and spindle length, as well as stabilizes the spindle and spindle poles. Perturbed SAC and abnormal microtubule tension may be responsible for the misaligned chromosomes and high aneuploidy in p38α-depleted mouse oocytes.
doi:10.4161/cc.9.20.13389
PMCID: PMC3055197  PMID: 20948319
p38α; meiosis; mouse oocyte; spindle assembly; microtubule organization center (MTOC); Eg5; spindle assembly checkpoint
15.  Drosophila CENP-A Mutations Cause a BubR1- Dependent Early Mitotic Delay without Normal Localization of Kinetochore Components 
PLoS Genetics  2006;2(7):e110.
The centromere/kinetochore complex plays an essential role in cell and organismal viability by ensuring chromosome movements during mitosis and meiosis. The kinetochore also mediates the spindle attachment checkpoint (SAC), which delays anaphase initiation until all chromosomes have achieved bipolar attachment of kinetochores to the mitotic spindle. CENP-A proteins are centromere-specific chromatin components that provide both a structural and a functional foundation for kinetochore formation. Here we show that cells in Drosophila embryos homozygous for null mutations in CENP-A (CID) display an early mitotic delay. This mitotic delay is not suppressed by inactivation of the DNA damage checkpoint and is unlikely to be the result of DNA damage. Surprisingly, mutation of the SAC component BUBR1 partially suppresses this mitotic delay. Furthermore, cid mutants retain an intact SAC response to spindle disruption despite the inability of many kinetochore proteins, including SAC components, to target to kinetochores. We propose that SAC components are able to monitor spindle assembly and inhibit cell cycle progression in the absence of sustained kinetochore localization.
Synopsis
Normal inheritance of genetic traits from one cell or organismal generation to the next depends on accurate chromosome replication and segregation. Defective chromosome segregation is associated with birth defects and cancer. The centromere is a single site on the chromosome that is responsible for assembling the kinetochore, which mediates chromosome attachment to the microtubule spindle and all chromosome movements. In addition, the spindle assembly checkpoint (SAC) ensures normal inheritance by delaying entry into anaphase when chromosome–spindle attachments are defective. Previous studies suggested that SAC function required kinetochore localization of key components. This study shows that elimination of a centromere-specific histone (CID) results in an early mitotic delay. Although this delay occurs earlier than the established time of SAC function (at the metaphase–anaphase transition), it depends on the presence of an essential SAC protein (BUBR1). Furthermore, the CID-mediated early mitotic delay occurs in the absence of kinetochore formation or localization of key SAC proteins. These results suggest that the fidelity of kinetochore–microtubule attachment is also monitored early in mitosis, and in the absence of kinetochore formation and localization of SAC components.
doi:10.1371/journal.pgen.0020110
PMCID: PMC1500813  PMID: 16839185
16.  Pericentrin and γ-Tubulin Form a Protein Complex and Are Organized into a Novel Lattice at the Centrosome  
The Journal of Cell Biology  1998;141(1):163-174.
Pericentrin and γ-tubulin are integral centrosome proteins that play a role in microtubule nucleation and organization. In this study, we examined the relationship between these proteins in the cytoplasm and at the centrosome. In extracts prepared from Xenopus eggs, the proteins were part of a large complex as demonstrated by sucrose gradient sedimentation, gel filtration and coimmunoprecipitation analysis. The pericentrin–γ-tubulin complex was distinct from the previously described γ-tubulin ring complex (γ-TuRC) as purified γ-TuRC fractions did not contain detectable pericentrin. When assembled at the centrosome, the two proteins remained in close proximity as shown by fluorescence resonance energy transfer. The three- dimensional organization of the centrosome-associated fraction of these proteins was determined using an improved immunofluorescence method. This analysis revealed a novel reticular lattice that was conserved from mammals to amphibians, and was organized independent of centrioles. The lattice changed dramatically during the cell cycle, enlarging from G1 until mitosis, then rapidly disassembling as cells exited mitosis. In cells colabeled to detect centrosomes and nucleated microtubules, lattice elements appeared to contact the minus ends of nucleated microtubules. Our results indicate that pericentrin and γ-tubulin assemble into a unique centrosome lattice that represents the higher-order organization of microtubule nucleating sites at the centrosome.
PMCID: PMC2132723  PMID: 9531556
17.  Regulation of Microtubule Assembly and Organization in Mitosis by the AAA+ ATPase Pontin 
Molecular Biology of the Cell  2008;19(7):3097-3110.
To identify novel proteins important for microtubule assembly in mitosis, we have used a centrosome-based complementation assay to enrich for proteins with mitotic functions. An RNA interference (RNAi)-based screen of these proteins allowed us to uncover 13 novel mitotic regulators. We carried out in-depth analyses of one of these proteins, Pontin, which is known to have several functions in interphase, including chromatin remodeling, DNA repair, and transcription. We show that reduction of Pontin by RNAi resulted in defects in spindle assembly in Drosophila S2 cells and in several mammalian tissue culture cell lines. Further characterization of Pontin in Xenopus egg extracts demonstrates that Pontin interacts with the gamma tubulin ring complex (γ-TuRC). Because depletion of Pontin leads to defects in the assembly and organization of microtubule arrays in egg extracts, our studies suggest that Pontin has a mitosis-specific function in regulating microtubule assembly.
doi:10.1091/mbc.E07-11-1202
PMCID: PMC2441676  PMID: 18463163
18.  DNA Damage Activates the SAC in an ATM/ATR-Dependent Manner, Independently of the Kinetochore 
PLoS Genetics  2008;4(2):e1000015.
The DNA damage checkpoint and the spindle assembly checkpoint (SAC) are two important regulatory mechanisms that respond to different lesions. The DNA damage checkpoint detects DNA damage, initiates protein kinase cascades, and inhibits the cell cycle. The SAC relies on kinetochore-dependent assembly of protein complexes to inhibit mitosis when chromosomes are detached from the spindle. The two checkpoints are thought to function independently. Here we show that yeast cells lacking the DNA damage checkpoint arrest prior to anaphase in response to low doses of the DNA damaging agent methyl methane sulfonate (MMS). The arrest requires the SAC proteins Mad1, Mad2, Mad3, Bub1, and Bub3 and works through Cdc20 and Pds1 but unlike the normal SAC, does not require a functional kinetochore. Mec1 (ATR) and Tel1 (ATM) are also required, independently of Chk1 and Rad53, suggesting that Mec1 and Tel1 inhibit anaphase in response to DNA damage by utilizing SAC proteins. Our results demonstrate cross-talk between the two checkpoints and suggest that assembling inhibitory complexes of SAC proteins at unattached kinetochores is not obligatory for their inhibitory activity. Furthermore, our results suggest that there are novel, important targets of ATM and ATR for cell cycle regulation.
Author Summary
Genome integrity is assured, in part, by regulatory systems called “checkpoints” that assure that cells do not improperly progress through the cell cycle. The DNA damage checkpoint assesses the status of DNA replication and inhibits cell cycle progression when the cell makes mistakes in DNA replication or when the cell has been assaulted by a DNA damaging agent from the environment. The checkpoint allows the cell time to repair the DNA and then permits the cell cycle to resume. There is a separate “spindle checkpoint” that monitors whether chromosomes are properly attached to the spindle and if so, allows cells to proceed through mitosis. The DNA damage checkpoint and the spindle checkpoint assure that daughter cells receive the correct number of chromosomes that are identical in DNA sequence. Here we show that the two checkpoints are not independent but that they cooperate to restrict mitotic progression in the face of DNA damage. We show that the spindle checkpoint can be induced by DNA damage and that there is a novel kinetochore independent mechanism to activate the spindle checkpoint proteins. In addition, we implicate the ATM and ATR kinases as kinetochore-independent activators of the spindle checkpoint.
doi:10.1371/journal.pgen.1000015
PMCID: PMC2265443  PMID: 18454191
19.  Caenorhabditis elegans Cyclin B3 Is Required for Multiple Mitotic Processes Including Alleviation of a Spindle Checkpoint–Dependent Block in Anaphase Chromosome Segregation 
PLoS Genetics  2010;6(11):e1001218.
The master regulators of the cell cycle are cyclin-dependent kinases (Cdks), which influence the function of a myriad of proteins via phosphorylation. Mitotic Cdk1 is activated by A-type, as well as B1- and B2-type, cyclins. However, the role of a third, conserved cyclin B family member, cyclin B3, is less well defined. Here, we show that Caenorhabditis elegans CYB-3 has essential and distinct functions from cyclin B1 and B2 in the early embryo. CYB-3 is required for the timely execution of a number of cell cycle events including completion of the MII meiotic division of the oocyte nucleus, pronuclear migration, centrosome maturation, mitotic chromosome condensation and congression, and, most strikingly, progression through the metaphase-to-anaphase transition. Our experiments reveal that the extended metaphase delay in CYB-3–depleted embryos is dependent on an intact spindle assembly checkpoint (SAC) and results in salient defects in the architecture of holocentric metaphase chromosomes. Furthermore, genetically increasing or decreasing dynein activity results in the respective suppression or enhancement of CYB-3–dependent defects in cell cycle progression. Altogether, these data reveal that CYB-3 plays a unique, essential role in the cell cycle including promoting mitotic dynein functionality and alleviation of a SAC–dependent block in anaphase chromosome segregation.
Author Summary
Every time a cell divides in two, the genetic material, DNA, is copied; each copied chromosome is referred to as a pair of sister chromatids. Each chromatid must be cleanly separated from its sister so that each daughter cell inherits the same DNA complement as the starting cell. The mitotic spindle is a cellular machine that physically separates the sister chromatids from one another. The chromatids are attached to the spindle at kinetochores, which are structures built at specific sites (centromeres) on each chromatid. The cell monitors the attachment of each chromatid and blocks their separation until they are all properly attached. This process is called the spindle assembly checkpoint (SAC). Here we report that loss of an evolutionarily conserved cell cycle regulator, Cyclin B3/CYB-3, results in an unusual and strikingly persistent SAC–dependent delay in sister chromatid separation. Furthermore, CYB-3 promotes the activity of a cellular motor, dynein, in this and other mitotic processes. Altogether, our results indicate that Cyclin B3 genetically interacts with mitotic dynein and is absolutely required to satisfy a SAC–dependent inhibition in sister chromatid separation.
doi:10.1371/journal.pgen.1001218
PMCID: PMC2991249  PMID: 21124864
20.  Microtubule stabilization in vivo by nucleation-incompetent γ-tubulin complex 
Journal of Cell Science  2011;124(8):1207-1213.
Although the fission yeast Schizosaccharomyces pombe contains many of the γ-tubulin ring complex (γ-TuRC)-specific proteins of the γ-tubulin complex (γ-TuC), several questions about the organizational state and function of the fission yeast γ-TuC in vivo remain unresolved. Using 3×GFP-tagged γ-TuRC-specific proteins, we show here that γ-TuRC-specific proteins are present at all microtubule organizing centers in fission yeast and that association of γ-TuRC-specific proteins with the γ-tubulin small complex (γ-TuSC) does not depend on Mto1, which is a key regulator of the γ-TuC. Through sensitive imaging in mto1Δ mutants, in which cytoplasmic microtubule nucleation is abolished, we unexpectedly found that γ-TuC incapable of nucleating microtubules can nevertheless associate with microtubule minus-ends in vivo. The presence of γ-TuC at microtubule ends is independent of γ-TuRC-specific proteins and strongly correlates with the stability of microtubule ends. Strikingly, microtubule bundles lacking γ-TuC at microtubule ends undergo extensive treadmilling in vivo, apparently induced by geometrical constraints on plus-end growth. Our results indicate that microtubule stabilization by the γ-TuC, independently of its nucleation function, is important for maintaining the organization and dynamic behavior of microtubule arrays in vivo.
doi:10.1242/jcs.083741
PMCID: PMC3065382  PMID: 21444751
Fission yeast; Gamma-tubulin; Microtubules
21.  Mad2 prolongs DNA damage checkpoint arrest caused by a double-strand break via a centromere-dependent mechanism 
Current biology : CB  2010;nihpa168171.
Eukaryotic cells employ a suite of replication and mitotic checkpoints to ensure the accurate transmission of their DNA. In budding yeast, both the DNA damage checkpoint and the spindle assembly checkpoint (SAC) block cells prior to anaphase [1-5]. The presence of a single unrepaired double-strand break (DSB) activates ATR and ATM protein kinase homologs, Mec1 and Tel1, which then activate downstream effectors to trigger G2/M arrest and also phosphorylate histone H2A (creating γ-H2AX) in chromatin surrounding the DSB [6-8]. The SAC monitors proper attachment of spindle microtubules to the kinetochore formed at each centromere and the biorientation of sister centromeres toward opposite spindle pole bodies. Although these two checkpoints sense quite different perturbations, recent evidence has demonstrated both synergistic interactions and cross-talk between them [9-11]. Here we report that Mad2 and other SAC proteins play an unexpected role in prolonging G2/M arrest after induction of a single DSB. This function of the SAC depends not only on Mec1 and other components of the DNA damage checkpoint but also on the presence of the centromere located ≥ 90kb from the DNA damage. DNA damage induces epigenetic changes at the centromere, including the γ-H2AX modification, that appear to alter kinetochore function, thus triggering the canonical spindle assembly checkpoint. Thus, a single DSB triggers a response by both checkpoints to prevent the segregation of a damaged chromosome.
doi:10.1016/j.cub.2009.12.033
PMCID: PMC2811853  PMID: 20096585
22.  Spindle checkpoint–independent inhibition of mitotic chromosome segregation by Drosophila Mps1 
Molecular Biology of the Cell  2012;23(12):2275-2291.
The conserved protein kinase Mps1 is required for the spindle assembly checkpoint (SAC). It is also involved in correction of erroneous attachments of kinetochores to the mitotic spindle before anaphase onset. Characterization of Drosophila Mps1 reveals yet another function: SAC-independent inhibition of sister chromatid separation.
Monopolar spindle 1 (Mps1) is essential for the spindle assembly checkpoint (SAC), which prevents anaphase onset in the presence of misaligned chromosomes. Moreover, Mps1 kinase contributes in a SAC-independent manner to the correction of erroneous initial attachments of chromosomes to the spindle. Our characterization of the Drosophila homologue reveals yet another SAC-independent role. As in yeast, modest overexpression of Drosophila Mps1 is sufficient to delay progression through mitosis during metaphase, even though chromosome congression and metaphase alignment do not appear to be affected. This delay in metaphase depends on the SAC component Mad2. Although Mps1 overexpression in mad2 mutants no longer causes a metaphase delay, it perturbs anaphase. Sister kinetochores barely move apart toward spindle poles. However, kinetochore movements can be restored experimentally by separase-independent resolution of sister chromatid cohesion. We propose therefore that Mps1 inhibits sister chromatid separation in a SAC-independent manner. Moreover, we report unexpected results concerning the requirement of Mps1 dimerization and kinase activity for its kinetochore localization in Drosophila. These findings further expand Mps1's significance for faithful mitotic chromosome segregation and emphasize the importance of its careful regulation.
doi:10.1091/mbc.E12-02-0117
PMCID: PMC3374747  PMID: 22553353
23.  Microtubule assembly in meiotic extract requires glycogen 
Molecular Biology of the Cell  2011;22(17):3139-3151.
We identified a clarified extract containing the soluble factors for microtubule assembly. We found that microtubule assembly does not require ribosomes, mitochondria, or membranes. Our clarified extracts will provide a powerful tool for activity-based biochemical fractionations for microtubule assembly.
The assembly of microtubules during mitosis requires many identified components, such as γ-tubulin ring complex (γ-TuRC), components of the Ran pathway (e.g., TPX2, HuRP, and Rae1), and XMAP215/chTOG. However, it is far from clear how these factors function together or whether more factors exist. In this study, we used biochemistry to attempt to identify active microtubule nucleation protein complexes from Xenopus meiotic egg extracts. Unexpectedly, we found both microtubule assembly and bipolar spindle assembly required glycogen, which acted both as a crowding agent and as metabolic source glucose. By also reconstituting microtubule assembly in clarified extracts, we showed microtubule assembly does not require ribosomes, mitochondria, or membranes. Our clarified extracts will provide a powerful tool for activity-based biochemical fractionations for microtubule assembly.
doi:10.1091/mbc.E11-02-0158
PMCID: PMC3164461  PMID: 21737678
24.  The Chromosomal Passenger Complex Controls Spindle Checkpoint Function Independent from Its Role in Correcting Microtubule–Kinetochore Interactions 
Molecular Biology of the Cell  2007;18(11):4553-4564.
The chromosomal passenger complex (CPC) is a critical regulator of chromosome segregation during mitosis by correcting nonbipolar microtubule-kinetochore interactions. By severing these interactions, the CPC is thought to create unattached kinetochores that are subsequently sensed by the spindle assembly checkpoint (SAC) to prevent premature mitotic exit. We now show that spindle checkpoint function of the CPC and its role in eliminating nonbipolar attachments can be uncoupled. Replacing the chromosomal passenger protein INCENP with a mutant allele that lacks its coiled-coil domain results in an overt defect in a SAC-mediated mitotic arrest in response to taxol treatment, indicating that this domain is critical for CPC function in spindle checkpoint control. Surprisingly, this mutant could restore alignment and cytokinesis during unperturbed cell divisions and was capable of resolving syntelic attachments. Also, Aurora-B kinase was localized and activated normally on centromeres in these cells, ruling out a role for the coiled-coil domain in general Aurora-B activation. Thus, mere microtubule destabilization of nonbipolar attachments by the CPC is insufficient to install a checkpoint-dependent mitotic arrest, and additional, microtubule destabilization–independent CPC signaling toward the spindle assembly checkpoint is required for this arrest, potentially through amplification of the unattached kinetochore-derived checkpoint signal.
doi:10.1091/mbc.E07-04-0328
PMCID: PMC2043551  PMID: 17699588
25.  Systematic Characterization of Human Protein Complexes Identifies Chromosome Segregation Proteins 
Science (New York, N.Y.)  2010;328(5978):593-599.
Chromosome segregation and cell division are essential, highly ordered processes that depend on numerous protein complexes. Results from recent RNA interference (RNAi) screens indicate that the identity and composition of these protein complexes is incompletely understood. Using gene tagging on bacterial artificial chromosomes, protein localization and tandem affinity purification-mass spectrometry, the MitoCheck consortium has analyzed about 100 human protein complexes, many of which had not or only incompletely been characterized. This work has led to the discovery of previously unknown, evolutionarily conserved subunits of the anaphase-promoting complex (APC/C) and the γ-tubulin ring complex (γ-TuRC), large complexes which are essential for spindle assembly and chromosome segregation. The approaches we describe here are generally applicable to high throughput follow-up analyses of phenotypic screens in mammalian cells.
doi:10.1126/science.1181348
PMCID: PMC2989461  PMID: 20360068

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