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William C. Earnshaw - One of the best experts on this subject based on the ideXlab platform.
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the inner centromere protein INCENP coil is a single α helix sah domain that binds directly to microtubules and is important for chromosome passenger complex cpc localization and function in mitosis
Journal of Biological Chemistry, 2015Co-Authors: Kumiko Samejima, Hiromi Ogawa, Melpomeni Platani, Marci Wolny, Giulia Vargiu, Pete J Knigh, Michelle Peckham, William C. EarnshawAbstract:The chromosome passenger complex (CPC) is a master regulator of mitosis. Inner centromere protein (INCENP) acts as a scaffold regulating CPC localization and activity. During early mitosis, the N-terminal region of INCENP forms a three-helix bundle with Survivin and Borealin, directing the CPC to the inner centromere where it plays essential roles in chromosome alignment and the spindle assembly checkpoint. The C-terminal IN box region of INCENP is responsible for binding and activating Aurora B kinase. The central region of INCENP has been proposed to comprise a coiled coil domain acting as a spacer between the N- and C-terminal domains that is involved in microtubule binding and regulation of the spindle checkpoint. Here we show that the central region (213 residues) of chicken INCENP is not a coiled coil but a ∼ 32-nm-long single α-helix (SAH) domain. The N-terminal half of this domain directly binds to microtubules in vitro. By analogy with previous studies of myosin 10, our data suggest that the INCENP SAH might stretch up to ∼ 80 nm under physiological forces. Thus, the INCENP SAH could act as a flexible "dog leash," allowing Aurora B to phosphorylate dynamic substrates localized in the outer kinetochore while at the same time being stably anchored to the heterochromatin of the inner centromere. Furthermore, by achieving this flexibility via an SAH domain, the CPC avoids a need for dimerization (required for coiled coil formation), which would greatly complicate regulation of the proximity-induced trans-phosphorylation that is critical for Aurora B activation.
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targeting the INCENP in box aurora b interaction to inhibit cpc activity in vivo
Open Biology, 2014Co-Authors: Florence H Gohard, Daniel J Stcyr, Mike Tyers, William C. EarnshawAbstract:1. Summary The chromosome passenger complex (CPC) is an essential regulator of mitosis and cytokinesis. The CPC consists of Aurora B kinase, inner centromere protein (INCENP), and the targeting subunits survivin and borealin/Dasra B. INCENP is a scaffolding subunit for the CPC and activates Aurora B via its conserved IN-box domain. We show that overexpression of soluble IN-box in HeLa cells affects endogenous CPC localization and produces a significant increase in multinucleated and micronucleated cells consistent with CPC loss of function. The dominant-negative effect of soluble IN-box expression depends on residues corresponding to hINCENP W845 and/or F881, suggesting that these are essential for Aurora B binding in vivo. We then screened a targeted library of small (five to nine residues long) circular peptide (CP) IN-box fragments generated using split intein circular ligation of proteins and peptides (SICLOPPS) methodology. We identified a number of CPs that caused modest but reproducible increases in rates of multinucleated and micronucleated cells. Our results provide proof of concept that inhibition of the Aurora B–IN-box interaction is a viable strategy for interfering with CPC function in vivo.
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INCENP aurora b interactions modulate kinase activity and chromosome passenger complex localization
Journal of Cell Biology, 2009Co-Authors: Hiromi Ogawa, Paola Vagnarelli, William C. Earnshaw, Damien F. Hudson, Jan H Bergmann, Sandrine Ruchaud, Tatsuo Fukagawa, Kumiko SamejimaAbstract:Dynamic localization of the chromosomal passenger complex (CPC) during mitosis is essential for its diverse functions. CPC targeting to centromeres involves interactions between Survivin, Borealin, and the inner centromere protein (CENP [INCENP]) N terminus. In this study, we investigate how interactions between the INCENP C terminus and aurora B set the level of kinase activity. Low levels of kinase activity, seen in INCENP-depleted cells or in cells expressing a mutant INCENP that cannot bind aurora B, are sufficient for a spindle checkpoint response when microtubules are absent but not against low dose taxol. Intermediate kinase activity levels obtained with an INCENP mutant that binds aurora B but cannot fully activate it are sufficient for a robust response against taxol, but cannot trigger CPC transfer from the chromosomes to the anaphase spindle midzone. This transfer requires significantly higher levels of aurora B activity. These experiments reveal that INCENP interactions with aurora B in vivo modulate the level of kinase activity, thus regulating CPC localization and functions during mitosis.
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dual roles of INCENP crucial to the assembly of the acentrosomal metaphase spindle in female meiosis
Development, 2008Co-Authors: Nathalie Colombie, Mar Carmena, William C. Earnshaw, Fiona C Cullen, Amy L Brittle, Janet K Jang, Kim S Mckim, Hiroyuki OhkuraAbstract:Spindle formation in female meiosis differs from mitosis in many animals, as it takes place independently of centrosomes, and the molecular requirements of this pathway remain to be understood. Here, we report two crucial roles of INCENP, an essential subunit of the chromosomal passenger complex (the Aurora B complex), in centrosome-independent spindle formation in Drosophila female meiosis. First, the initial assembly of spindle microtubules is drastically delayed in an INCENP mutant. This clearly demonstrates, for the first time, a crucial role for INCENP in chromosome-driven spindle microtubule assembly in living oocytes. Additionally, INCENP is necessary to stabilise the equatorial region of the metaphase I spindle, in contrast to mitosis, where the equivalent function becomes prominent after anaphase onset. Our analysis suggests that Subito, a kinesin-6 protein, cooperates with INCENP for this latter function, but not in microtubule assembly. We propose that the two functions of INCENP are part of the mechanisms that compensate for the lack of centrosomes during meiotic spindle formation.
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INCENP and aurora b promote meiotic sister chromatid cohesion through localization of the shugoshin mei s332 in drosophila
Developmental Cell, 2006Co-Authors: Tamar D Resnick, Terry L Orrweaver, William C. Earnshaw, David L Satinover, Fiona Macisaac, Todd P Stukenberg, Mar CarmenaAbstract:The chromosomal passenger complex protein INCENP is required in mitosis for chromosome condensation, spindle attachment and function, and cytokinesis. Here, we show that INCENP has an essential function in the specialized behavior of centromeres in meiosis. Mutations affecting Drosophila INCENP profoundly affect chromosome segregation in both meiosis I and II, due, at least in part, to premature sister chromatid separation in meiosis I. INCENP binds to the cohesion protector protein MEI-S332, which is also an excellent in vitro substrate for Aurora B kinase. A MEI-S332 mutant that is only poorly phosphorylated by Aurora B is defective in localization to centromeres. These results implicate the chromosomal passenger complex in directly regulating MEI-S332 localization and, therefore, the control of sister chromatid cohesion in meiosis.
Mar Carmena - One of the best experts on this subject based on the ideXlab platform.
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Aurora B activity is required for the activation of Polo kinase at the inner centromere.
2012Co-Authors: Mar Carmena, Fiona Macisaac, Xavier Pinson, Melpi Platani, Zeina Salloum, Anthony Clark, Hiromi Ogawa, Ulrike Eggert, David M. GloverAbstract:(A) Aurora B phosphorylates Polo kinase in vitro. Bacterially expressed HIS-Polo or HIS-PoloT182A (which is catalytically inactive and therefore unable to autophosphorylate) were incubated with (or without) Drosophila Aurora B in complex with a fragment of INCENP (residues 654–755) in presence of 32P-g-ATP, in triplicate. Reaction products were resolved by SDS-PAGE transferred to nitrocellulose and analyzed by autoradiography (AR) and anti-Polo Western blot (WB). Quantitative measurements of signals were obtained (see Materials and Methods), and the ratios were calculated for each reaction (AR/WB, A.U.: arbitrary units). Right, average values for the relative phosphorylatin of PoloWT and PoloT182A by Aurora B. Error bars, SEM. (B–D) DMel-2 cells stably expressing Polo-GFP treated with (B) DMSO or (C–D) Binucleine-2, immunostained for INCENP, Polo, and PoloT182Ph (insets: zoomed images of kinetochores). In (C–D) asterisks indicate centrosomes. Merged images show INCENP/Polo/DNA. Zoomed images in (C–D) insets show examples of kinetochore pairs showing decreased levels of PoloT182Ph. (E) Dot plot showing the quantification of INCENP/Polo/PoloT182Ph signal intensity at the kinetochore (t test: *** p
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dual roles of INCENP crucial to the assembly of the acentrosomal metaphase spindle in female meiosis
Development, 2008Co-Authors: Nathalie Colombie, Mar Carmena, William C. Earnshaw, Fiona C Cullen, Amy L Brittle, Janet K Jang, Kim S Mckim, Hiroyuki OhkuraAbstract:Spindle formation in female meiosis differs from mitosis in many animals, as it takes place independently of centrosomes, and the molecular requirements of this pathway remain to be understood. Here, we report two crucial roles of INCENP, an essential subunit of the chromosomal passenger complex (the Aurora B complex), in centrosome-independent spindle formation in Drosophila female meiosis. First, the initial assembly of spindle microtubules is drastically delayed in an INCENP mutant. This clearly demonstrates, for the first time, a crucial role for INCENP in chromosome-driven spindle microtubule assembly in living oocytes. Additionally, INCENP is necessary to stabilise the equatorial region of the metaphase I spindle, in contrast to mitosis, where the equivalent function becomes prominent after anaphase onset. Our analysis suggests that Subito, a kinesin-6 protein, cooperates with INCENP for this latter function, but not in microtubule assembly. We propose that the two functions of INCENP are part of the mechanisms that compensate for the lack of centrosomes during meiotic spindle formation.
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INCENP and aurora b promote meiotic sister chromatid cohesion through localization of the shugoshin mei s332 in drosophila
Developmental Cell, 2006Co-Authors: Tamar D Resnick, Terry L Orrweaver, William C. Earnshaw, David L Satinover, Fiona Macisaac, Todd P Stukenberg, Mar CarmenaAbstract:The chromosomal passenger complex protein INCENP is required in mitosis for chromosome condensation, spindle attachment and function, and cytokinesis. Here, we show that INCENP has an essential function in the specialized behavior of centromeres in meiosis. Mutations affecting Drosophila INCENP profoundly affect chromosome segregation in both meiosis I and II, due, at least in part, to premature sister chromatid separation in meiosis I. INCENP binds to the cohesion protector protein MEI-S332, which is also an excellent in vitro substrate for Aurora B kinase. A MEI-S332 mutant that is only poorly phosphorylated by Aurora B is defective in localization to centromeres. These results implicate the chromosomal passenger complex in directly regulating MEI-S332 localization and, therefore, the control of sister chromatid cohesion in meiosis.
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drosophila INCENP is required for cytokinesis and asymmetric cell division during development of the nervous system
Journal of Cell Science, 2006Co-Authors: Chihjui Chang, Richard R Adams, William C. Earnshaw, Sarah E Goulding, Mar CarmenaAbstract:The chromosomal passenger protein complex has emerged as a key player in mitosis, with important roles in chromatin modifications, kinetochore-microtubule interactions, chromosome bi-orientation and stability of the bipolar spindle, mitotic checkpoint function, assembly of the central spindle and cytokinesis. The inner centromere protein (INCENP; a subunit of this complex) is thought to regulate the Aurora B kinase and target it to its substrates. To explore the roles of the passenger complex in a developing multicellular organism, we have performed a genetic screen looking for new alleles and interactors of Drosophila INCENP. We have isolated a new null allele of INCENP that has allowed us for the first time to study the functions of the chromosomal passengers during development. Homozygous INCENP(EC3747) embryos show absence of phosphorylation of histone H3 in mitosis, failure of cytokinesis and polyploidy, and defects in peripheral nervous system development. These defects are consistent with depletion of Aurora B kinase activity. In addition, the segregation of the cell-fate determinant Prospero in asymmetric neuroblast division is abnormal, suggesting a role for the chromosomal passenger complex in the regulation of this process.
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INCENP at the kinase crossroads
Nature Cell Biology, 2006Co-Authors: Mar Carmena, William C. EarnshawAbstract:INCENP — the targeting and activating subunit of Aurora B kinase— has several key mitotic functions. Now, INCENP emerges as a nodal point of the network of kinases that regulate mitotic events. Phosphorylation of INCENP by CDK1 is required for the kinetochore localization of Plk1 kinase, and this in turn is necessary for a timely metaphase–anaphase transition.
Erich A. Nigg - One of the best experts on this subject based on the ideXlab platform.
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structure of a survivin borealin INCENP core complex reveals how chromosomal passengers travel together
Cell, 2007Co-Authors: Arockia A Jeyaprakash, Erich A. Nigg, Ulf R Klein, Doris Lindner, Judith Ebert, Elena ContiAbstract:The chromosomal passenger complex (CPC) is a key regulator of chromosome segregation and cytokinesis. CPC functions are connected to its localization. The complex first localizes to centromeres and later associates with the central spindle and midbody. Survivin, Borealin, and INCENP are the three components of the CPC that regulate the activity and localization of its enzymatic component, the kinase Aurora B. We determined the 1.4 A resolution crystal structure of the regulatory core of the CPC, revealing that Borealin and INCENP associate with the helical domain of Survivin to form a tight three-helical bundle. We used siRNA rescue experiments with structure-based mutants to explore the requirements for CPC localization. We show that the intertwined structural interactions of the core components lead to functional interdependence. Association of the core "passenger" proteins creates a single structural unit, whose composite molecular surface presents conserved residues essential for central spindle and midbody localization.
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centromere targeting of the chromosomal passenger complex requires a ternary subcomplex of borealin survivin and the n terminal domain of INCENP
Molecular Biology of the Cell, 2006Co-Authors: Ulf R Klein, Erich A. Nigg, Ulrike GrunebergAbstract:The chromosomal passenger complex (CPC), consisting of the serine/threonine kinase Aurora B, the inner centromere protein INCENP, Survivin, and Borealin/DasraB, has essential functions at the centromere in ensuring correct chromosome alignment and segregation. Despite observations that small interfering RNA-mediated knockdown of any one member of the CPC abolishes localization of the other subunits, it remains unclear how the complex is targeted to the centromere. We have now identified a ternary subcomplex of the CPC comprising Survivin, Borealin, and the N-terminal 58 amino acids of INCENP in vitro and in vivo. This subcomplex was found to be essential and sufficient for targeting to the centromere. Notably, Aurora B kinase, the enzymatic core of the CPC, was not required for centromere localization of the subcomplex. We demonstrate that CPC targeting to the centromere does not depend on CENP-A and hMis12, two core components for kinetochore/centromere assembly, and provide evidence that the CPC may be directed to centromeric DNA directly via the Borealin subunit. Our findings thus establish a functional module within the CPC that assembles on the N terminus of INCENP and controls centromere recruitment.
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complex formation of plk1 and INCENP required for metaphase anaphase transition
Nature Cell Biology, 2006Co-Authors: Hidemasa Goto, Erich A. Nigg, Tohru Kiyono, Yasuko Tomono, Aie Kawajiri, Takeshi Urano, Koichi Furukawa, Masaki InagakiAbstract:Mitotic chromosomal dynamics is regulated by the coordinated activities of many mitotic kinases1, such as cyclin-dependent kinase 1 (Cdk1)2,3, Aurora-B4 or Polo-like kinase 1 (Plk1)5, but the mechanisms of their coordination remain unknown. Here, we report that Cdk1 phosphorylates Thr 59 and Thr 388 on inner centromere protein (INCENP), which regulates the localization4 and kinase activity6,7,8 of Aurora-B from prophase to metaphase. INCENP depletion disrupts Plk1 localization specifically at the kinetochore. This phenotype is rescued by the exogenous expression of INCENP wild type and INCENP mutated at Thr 59 to Ala (T59A), but not at Thr 388 to Ala (T388A). The replacement of endogenous INCENP with T388A resulted in the delay of progression from metaphase to anaphase. We propose that INCENP phosphorylation by Cdk1 is necessary for the recruitment of Plk1 to the kinetochore, and that the complex formation of Plk1 and Aurora-B on INCENP may play crucial roles in the regulation of chromosomal dynamics.
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borealin a novel chromosomal passenger required for stability of the bipolar mitotic spindle
Journal of Cell Biology, 2004Co-Authors: Reto Gassmann, Reiko Honda, Erich A. Nigg, Damien F. Hudson, Dietlind L Gerloff, Ana Carvalho, Sandrine Ruchaud, Alexander J Henzing, William C. EarnshawAbstract:The chromosomal passenger complex of Aurora B kinase, INCENP, and Survivin has essential regulatory roles at centromeres and the central spindle in mitosis. Here, we describe Borealin, a novel member of the complex. Approximately half of Aurora B in mitotic cells is complexed with INCENP, Borealin, and Survivin; and Borealin binds Survivin and INCENP in vitro. A second complex contains Aurora B and INCENP, but no Borealin or Survivin. Depletion of Borealin by RNA interference delays mitotic progression and results in kinetochore–spindle misattachments and an increase in bipolar spindles associated with ectopic asters. The extra poles, which apparently form after chromosomes achieve a bipolar orientation, severely disrupt the partitioning of chromosomes in anaphase. Borealin depletion has little effect on histone H3 serine10 phosphorylation. These results implicate the chromosomal passenger holocomplex in the maintenance of spindle integrity and suggest that histone H3 serine10 phosphorylation is performed by an Aurora B–INCENP subcomplex.
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borealin a novel chromosomal passenger required for stability of the bipolar mitotic spindle
Journal of Cell Biology, 2004Co-Authors: Reto Gassmann, Reiko Honda, Erich A. Nigg, Damien F. Hudson, Dietlind L Gerloff, Ana Carvalho, Sandrine Ruchaud, Alexander J Henzing, William C. EarnshawAbstract:The chromosomal passenger complex of Aurora B kinase, INCENP, and Survivin has essential regulatory roles at centromeres and the central spindle in mitosis. Here, we describe Borealin, a novel member of the complex. Approximately half of Aurora B in mitotic cells is complexed with INCENP, Borealin, and Survivin; and Borealin binds Survivin and INCENP in vitro. A second complex contains Aurora B and INCENP, but no Borealin or Survivin. Depletion of Borealin by RNA interference delays mitotic progression and results in kinetochore-spindle misattachments and an increase in bipolar spindles associated with ectopic asters. The extra poles, which apparently form after chromosomes achieve a bipolar orientation, severely disrupt the partitioning of chromosomes in anaphase. Borealin depletion has little effect on histone H3 serine10 phosphorylation. These results implicate the chromosomal passenger holocomplex in the maintenance of spindle integrity and suggest that histone H3 serine10 phosphorylation is performed by an Aurora B-INCENP subcomplex.
Richard R Adams - One of the best experts on this subject based on the ideXlab platform.
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drosophila INCENP is required for cytokinesis and asymmetric cell division during development of the nervous system
Journal of Cell Science, 2006Co-Authors: Chihjui Chang, Richard R Adams, William C. Earnshaw, Sarah E Goulding, Mar CarmenaAbstract:The chromosomal passenger protein complex has emerged as a key player in mitosis, with important roles in chromatin modifications, kinetochore-microtubule interactions, chromosome bi-orientation and stability of the bipolar spindle, mitotic checkpoint function, assembly of the central spindle and cytokinesis. The inner centromere protein (INCENP; a subunit of this complex) is thought to regulate the Aurora B kinase and target it to its substrates. To explore the roles of the passenger complex in a developing multicellular organism, we have performed a genetic screen looking for new alleles and interactors of Drosophila INCENP. We have isolated a new null allele of INCENP that has allowed us for the first time to study the functions of the chromosomal passengers during development. Homozygous INCENP(EC3747) embryos show absence of phosphorylation of histone H3 in mitosis, failure of cytokinesis and polyploidy, and defects in peripheral nervous system development. These defects are consistent with depletion of Aurora B kinase activity. In addition, the segregation of the cell-fate determinant Prospero in asymmetric neuroblast division is abnormal, suggesting a role for the chromosomal passenger complex in the regulation of this process.
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essential roles of drosophila inner centromere protein INCENP and aurora b in histone h3 phosphorylation metaphase chromosome alignment kinetochore disjunction and chromosome segregation
Journal of Cell Biology, 2001Co-Authors: Richard R Adams, Helder Maiato, William C. Earnshaw, Mar CarmenaAbstract:We have performed a biochemical and double-stranded RNA-mediated interference (RNAi) analysis of the role of two chromosomal passenger proteins, inner centromere protein (INCENP) and aurora B kinase, in cultured cells of Drosophila melanogaster. INCENP and aurora B function is tightly interlinked. The two proteins bind to each other in vitro, and DmINCENP is required for DmAurora B to localize properly in mitosis and function as a histone H3 kinase. DmAurora B is required for DmINCENP accumulation at centromeres and transfer to the spindle at anaphase. RNAi for either protein dramatically inhibited the ability of cells to achieve a normal metaphase chromosome alignment. Cells were not blocked in mitosis, however, and entered an aberrant anaphase characterized by defects in sister kinetochore disjunction and the presence of large amounts of amorphous lagging chromatin. Anaphase A chromosome movement appeared to be normal, however cytokinesis often failed. DmINCENP and DmAurora B are not required for the correct localization of the kinesin-like protein Pavarotti (ZEN-4/CHO1/MKLP1) to the midbody at telophase. These experiments reveal that INCENP is required for aurora B kinase function and confirm that the chromosomal passengers have essential roles in mitosis.
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human INCENP colocalizes with the aurora b airk2 kinase on chromosomes and is overexpressed in tumour cells
Chromosoma, 2001Co-Authors: Richard R Adams, Paola Vagnarelli, Alastair M Mackay, Sally P Wheatley, Dietlind L Gerloff, D M Eckley, Phyllis A Svingen, Scott H Kaufmann, William C. EarnshawAbstract:The inner centromere protein (INCENP), which has previously been described in chicken, frog and mouse, is required for correct chromosome segregation and cytokinesis. We have identified the human INCENP gene by library screening and reverse transcription-polymerase chain reaction (RT-PCR) and localized it to chromosomal region 11q12. HsINCENP is a single-copy gene that consists of 17 exons and covers 25 kb of genomic DNA. The gene is expressed at highest levels in the colon, testis and prostate, consistent with its likely role in cell proliferation. HsINCENP encodes a highly basic protein of 915 amino acids that localizes to metaphase chromosomes and to the mitotic spindle and equatorial cortex at anaphase. Recently we showed that INCENP is stockpiled in a complex with the Aurora-B/XAIRK2 kinase in Xenopus eggs. Here we demonstrate that, consistent with such an interaction, the two proteins colocalize on human metaphase chromosomes. Levels of Aurora-B are increased in several human cancers, and we show here that HsINCENP protein levels are also significantly increased in several colorectal cancer cell lines.
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chromosomal passengers and the aurora abcs of mitosis
Trends in Cell Biology, 2001Co-Authors: Richard R Adams, Mar Carmena, William C. EarnshawAbstract:Chromosomal passengers are proteins that move from centromeres to the spindle midzone during mitosis. Recent experiments show that the passengers inner centromere protein (INCENP) and aurora-B kinase are in a macromolecular complex that might also contain a third passenger, survivin. The chromosomal passenger complex functions throughout mitosis in chromosome condensation and segregation, and at the end of mitosis, in the completion of cytokinesis.
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INCENP binds directly to tubulin and requires dynamic microtubules to target to the cleavage furrow
Experimental Cell Research, 2001Co-Authors: Sally P Wheatley, Richard R Adams, Alexandra M Ainsztein, Stefanie Kandelslewis, William C. EarnshawAbstract:Inner centromere protein (INCENP) is a chromosomal passenger protein with an essential role in mitosis. At the metaphase/anaphase transition, some INCENP transfers from the centromeres to the central spindle; the remainder then transfers to the equatorial cortex prior to cleavage furrow formation. The molecular associations dictating INCENP behavior during mitosis are currently unknown. Here we show that targeting INCENP to the cleavage plane requires dynamic microtubules, but not F-actin. When microtubules are eliminated, INCENP is dispersed across the entire cell cortex. Yeast two-hybrid and in vitro binding data demonstrate that INCENP binds directly to β-tubulin via a conserved domain encompassing residues 48–85. Furthermore, INCENP binds to microtubules polymerized from purified tubulin in vitro and appears to bundle microtubules when expressed in the interphase cytoplasm. These data indicate that INCENP is a microtubule-binding protein that targets to the equatorial cortex through interactions requiring microtubules.
Ana Carvalho - One of the best experts on this subject based on the ideXlab platform.
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borealin a novel chromosomal passenger required for stability of the bipolar mitotic spindle
Journal of Cell Biology, 2004Co-Authors: Reto Gassmann, Reiko Honda, Erich A. Nigg, Damien F. Hudson, Dietlind L Gerloff, Ana Carvalho, Sandrine Ruchaud, Alexander J Henzing, William C. EarnshawAbstract:The chromosomal passenger complex of Aurora B kinase, INCENP, and Survivin has essential regulatory roles at centromeres and the central spindle in mitosis. Here, we describe Borealin, a novel member of the complex. Approximately half of Aurora B in mitotic cells is complexed with INCENP, Borealin, and Survivin; and Borealin binds Survivin and INCENP in vitro. A second complex contains Aurora B and INCENP, but no Borealin or Survivin. Depletion of Borealin by RNA interference delays mitotic progression and results in kinetochore-spindle misattachments and an increase in bipolar spindles associated with ectopic asters. The extra poles, which apparently form after chromosomes achieve a bipolar orientation, severely disrupt the partitioning of chromosomes in anaphase. Borealin depletion has little effect on histone H3 serine10 phosphorylation. These results implicate the chromosomal passenger holocomplex in the maintenance of spindle integrity and suggest that histone H3 serine10 phosphorylation is performed by an Aurora B-INCENP subcomplex.
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borealin a novel chromosomal passenger required for stability of the bipolar mitotic spindle
Journal of Cell Biology, 2004Co-Authors: Reto Gassmann, Reiko Honda, Erich A. Nigg, Damien F. Hudson, Dietlind L Gerloff, Ana Carvalho, Sandrine Ruchaud, Alexander J Henzing, William C. EarnshawAbstract:The chromosomal passenger complex of Aurora B kinase, INCENP, and Survivin has essential regulatory roles at centromeres and the central spindle in mitosis. Here, we describe Borealin, a novel member of the complex. Approximately half of Aurora B in mitotic cells is complexed with INCENP, Borealin, and Survivin; and Borealin binds Survivin and INCENP in vitro. A second complex contains Aurora B and INCENP, but no Borealin or Survivin. Depletion of Borealin by RNA interference delays mitotic progression and results in kinetochore–spindle misattachments and an increase in bipolar spindles associated with ectopic asters. The extra poles, which apparently form after chromosomes achieve a bipolar orientation, severely disrupt the partitioning of chromosomes in anaphase. Borealin depletion has little effect on histone H3 serine10 phosphorylation. These results implicate the chromosomal passenger holocomplex in the maintenance of spindle integrity and suggest that histone H3 serine10 phosphorylation is performed by an Aurora B–INCENP subcomplex.
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survivin is required for stable checkpoint activation in taxol treated hela cells
Journal of Cell Science, 2003Co-Authors: Ana Carvalho, Mar Carmena, William C. Earnshaw, Clara Sambade, Sally P WheatleyAbstract:Survivin is an essential chromosomal passenger protein whose function remains unclear. Here, we have used RNA interference to specifically repress Survivin in cultured HeLa cells. Immunoblot analysis showed that Survivin was no longer detectable in cultures 60 hours after transfection with Survivin-specific siRNA. Live cell analysis showed that many Survivin-depleted cells were delayed in mitosis, and immunofluorescence analysis of fixed specimens revealed that Survivin-depleted cells accumulated in prometaphase with misaligned chromosomes. The chromosomal passenger proteins, INCENP and Aurora-B, which can interact directly with Survivin, were absent from the centromeres of Survivin-depleted cells. These data contribute to the emerging picture that Survivin operates together with INCENP and Aurora-B to perform its mitotic duties. Some Survivin-depleted cells eventually exited mitosis without completing cytokinesis. This resulted in a gradual increase in the percentage of multinucleated cells in the culture. Time-lapse imaging of synchronized cultures revealed that control and Survivin-depleted cells arrested in mitosis in the presence of nocodazole; however, the latter failed to arrest in mitosis when treated with taxol. Immunofluorescence studies revealed that Survivin-depleted cells were unable to stably maintain BubR1 at the kinetochores in the presence of either taxol or nocodazole. Our data reveal that Survivin is not required for the spindle assembly checkpoint when it is activated by the loss of microtubules. However, Survivin is required for the maintenance of the checkpoint when it is activated by taxol, which is generally thought to cause a loss of spindle tension.
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INCENP is required for proper targeting of survivin to the centromeres and the anaphase spindle during mitosis
Current Biology, 2001Co-Authors: Sally P Wheatley, Paola Vagnarelli, Ana Carvalho, William C. EarnshawAbstract:Abstract Three lines of investigation have suggested that interactions between Survivin and the chromosomal passenger proteins INCENP and Aurora-B kinase may be important for mitotic progression. First, interference with the function of Survivin/BIR1, INCENP, or Aurora-B kinase leads to similar defects in mitosis and cytokinesis [1–7] (see [8] for review). Second, INCENP and Aurora-B exist in a complex in Xenopus eggs [9] and in mammalian cultured cells [7]. Third, interference with Survivin or INCENP function causes Aurora-B kinase to be mislocalized in mitosis in both C. elegans and vertebrates [5, 7, 9]. Here, we provide evidence that Survivin, Aurora-B, and INCENP interact physically and functionally. Direct visualization of Survivin-GFP in mitotic cells reveals that it localizes identically to INCENP and Aurora-B. Survivin binds directly to both Aurora-B and INCENP in yeast two-hybrid and in vitro pull-down assays. The in vitro interaction between Survivin and Aurora-B is extraordinarily stable in that it resists 3 M NaCl. Finally, Survivin and INCENP interact functionally in vivo; in cells in which INCENP localization is disrupted, Survivin adheres to the chromosomes and no longer concentrates at the centromeres or transfers to the anaphase spindle midzone. Our data provide the first biochemical evidence that Survivin can interact directly with members of the chromosomal passenger complex.
