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• W1916209382 abstract "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. 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. INCENP 6The abbreviations used are: INCENPinner centromere proteinSAHsingle α-helixCPCchromosome passenger complexGgGallus gallusHsHomo sapiensTrAPtriple affinity purificationMyoMmyosin MTRITCtetramethylrhodamine isothiocyanateCENPcentromere proteinpNpiconewtonsH3histone H3phphosphorylation. is the scaffolding protein upon which the chromosomal passenger complex (CPC) assembles (1Cooke C.A. Heck M.M. Earnshaw W.C. The inner centromere protein (INCENP) antigens: movement from inner centromere to midbody during mitosis.J. Cell Biol. 1987; 105: 2053-2067Crossref PubMed Scopus (318) Google Scholar, 2Earnshaw W.C. Bernat R.L. Chromosomal passengers: toward an integrated view of mitosis.Chromosoma. 1991; 100: 139-146Crossref PubMed Scopus (162) Google Scholar, 3Adams R.R. Wheatley S.P. Gouldsworthy A.M. Kandels-Lewis S.E. Carmena M. Smythe C. Gerloff D.L. Earnshaw W.C. INCENP binds the Aurora-related kinase AIRK2 and is required to target it to chromosomes, the central spindle and cleavage furrow.Curr. Biol. 2000; 10: 1075-1078Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). The N-terminal region of INCENP assembles a three-helix bundle with Survivin and Borealin (4Jeyaprakash A.A. Basquin C. Jayachandran U. Conti E. Structural basis for the recognition of phosphorylated histone H3 by the survivin subunit of the chromosomal passenger complex.Structure. 2011; 19: 1625-1634Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) that contributes to targeting the CPC to inner centromeres via haspin-mediated phosphorylation of histone H3 (5Kelly A.E. Ghenoiu C. Xue J.Z. Zierhut C. Kimura H. Funabiki H. Survivin reads phosphorylated histone H3 threonine 3 to activate the mitotic kinase Aurora B.Science. 2010; 330: 235-239Crossref PubMed Scopus (352) Google Scholar, 6Wang F. Dai J. Daum J.R. Niedzialkowska E. Banerjee B. Stukenberg P.T. Gorbsky G.J. Higgins J.M. Histone H3 Thr-3 phosphorylation by Haspin positions Aurora B at centromeres in mitosis.Science. 2010; 330: 231-235Crossref PubMed Scopus (339) Google Scholar, 7Yamagishi Y. Honda T. Tanno Y. Watanabe Y. Two histone marks establish the inner centromere and chromosome bi-orientation.Science. 2010; 330: 239-243Crossref PubMed Scopus (336) Google Scholar). This region of INCENP also contributes to CPC localization by binding to HP1 and to microtubules. The IN box, a conserved motif near the C terminus of INCENP, is responsible for binding and activating Aurora B kinase (3Adams R.R. Wheatley S.P. Gouldsworthy A.M. Kandels-Lewis S.E. Carmena M. Smythe C. Gerloff D.L. Earnshaw W.C. INCENP binds the Aurora-related kinase AIRK2 and is required to target it to chromosomes, the central spindle and cleavage furrow.Curr. Biol. 2000; 10: 1075-1078Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 8Bishop J.D. Han Z. Schumacher J.M. The Caenorhabditis elegans Aurora B kinase AIR-2 phosphorylates and is required for the localization of a BimC kinesin to meiotic and mitotic spindles.Mol. Biol. Cell. 2005; 16: 742-756Crossref PubMed Scopus (50) Google Scholar). The central portion of INCENP is predicted to form a coiled coil spacer between the localization and activation modules (3Adams R.R. Wheatley S.P. Gouldsworthy A.M. Kandels-Lewis S.E. Carmena M. Smythe C. Gerloff D.L. Earnshaw W.C. INCENP binds the Aurora-related kinase AIRK2 and is required to target it to chromosomes, the central spindle and cleavage furrow.Curr. Biol. 2000; 10: 1075-1078Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar) and has been proposed to act as a “dog leash,” allowing Aurora B tethered to chromatin to phosphorylate substrates within a constrained region (9Santaguida S. Musacchio A. The life and miracles of kinetochores.EMBO J. 2009; 28: 2511-2531Crossref PubMed Scopus (367) Google Scholar). inner centromere protein single α-helix chromosome passenger complex Gallus gallus Homo sapiens triple affinity purification myosin M tetramethylrhodamine isothiocyanate centromere protein piconewtons histone H3 phosphorylation. This tethering of Aurora B is critical for the regulation of chromosome alignment and the correction of kinetochore attachment errors. Spindle tension causes the elastic chromatin of the inner centromere to stretch, allowing sister kinetochores to move away from the inner centromere where INCENP is concentrated during prometaphase and metaphase. As first proposed for budding yeast and later confirmed in mammalian cells, this centromere stretch moves kinetochore targets of Aurora B away from regions of high kinase concentration and decreases their phosphorylation, thereby stabilizing kinetochore-microtubule interactions (10Tanaka T.U. Rachidi N. Janke C. Pereira G. Galova M. Schiebel E. Stark M.J. Nasmyth K. Evidence that the Ipl1-Sli15 (Aurora kinase-INCENP) complex promotes chromosome bi-orientation by altering kinetochore-spindle pole connections.Cell. 2002; 108: 317-329Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar, 11Liu D. Vader G. Vromans M.J. Lampson M.A. Lens S.M. Sensing chromosome bi-orientation by spatial separation of aurora B kinase from kinetochore substrates.Science. 2009; 323: 1350-1353Crossref PubMed Scopus (416) Google Scholar, 12Carmena M. Wheelock M. Funabiki H. Earnshaw W.C. The chromosomal passenger complex (CPC): from easy rider to the godfather of mitosis.Nat. Rev. Mol. Cell Biol. 2012; 13: 789-803Crossref PubMed Scopus (563) Google Scholar). In misattached chromosomes, which do not exhibit comparable centromere stretch, Aurora B remains in closer proximity to the outer kinetochore. This allows the kinase to phosphorylate key kinetochore components, causing them to release microtubules (13Cheeseman I.M. Chappie J.S. Wilson-Kubalek E.M. Desai A. The conserved KMN network constitutes the core microtubule-binding site of the kinetochore.Cell. 2006; 127: 983-997Abstract Full Text Full Text PDF PubMed Scopus (721) Google Scholar, 14DeLuca J.G. Gall W.E. Ciferri C. Cimini D. Musacchio A. Salmon E.D. Kinetochore microtubule dynamics and attachment stability are regulated by hec1.Cell. 2006; 127: 969-982Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar, 15DeLuca K.F. Lens S.M. DeLuca J.G. Temporal changes in Hec1 phosphorylation control kinetochore-microtubule attachment stability during mitosis.J. Cell Sci. 2011; 124: 622-634Crossref PubMed Scopus (174) Google Scholar). This correction mechanism is now well accepted, but what is less clear is how exactly INCENP achieves the dynamic flexibility to allow Aurora B to extend into the outer kinetochore and to track with kinetochore components at the dynamic microtubule interface. For example, because the Ndc80 and Ska complexes are composed of relatively rigid helical bundles (16Ciferri C. De Luca J. Monzani S. Ferrari K.J. Ristic D. Wyman C. Stark H. Kilmartin J. Salmon E.D. Musacchio A. Architecture of the human ndc80-hec1 complex, a critical constituent of the outer kinetochore.J. Biol. Chem. 2005; 280: 29088-29095Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 17Jeyaprakash A.A. Santamaria A. Jayachandran U. Chan Y.W. Benda C. Nigg E.A. Conti E. Structural and functional organization of the Ska complex, a key component of the kinetochore-microtubule interface.Mol. Cell. 2012; 46: 274-286Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar), it is not clear how Aurora B is able to associate with them if they undergo conformational changes on the dynamically growing and shrinking kinetochore-associated microtubules. Here we show that the central region of INCENP is not a coiled coil but instead is a single α-helix (SAH) domain similar to that found in myosin 10 and many other proteins (18Knight P.J. Thirumurugan K. Xu Y. Wang F. Kalverda A.P. Stafford 3rd, W.F. Sellers J.R. Peckham M. The predicted coiled-coil domain of myosin 10 forms a novel elongated domain that lengthens the head.J. Biol. Chem. 2005; 280: 34702-34708Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 19Peckham M. Knight P.J. When a predicted coiled coil is really a single α-helix, in myosins and other proteins.Soft Matter. 2009; 5: 2493-2503Google Scholar, 20Wolny M. Batchelor M. Knight P.J. Paci E. Dougan L. Peckham M. Stable single α-helices are constant force springs in proteins.J. Biol. Chem. 2014; 289: 27825-27835Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 21Batchelor M. Wolny M. Dougan L. Paci E. Knight P.J. Peckham M. Myosin tails and single α-helical domains.Biochem. Soc. Trans. 2015; 43: 58-63Crossref PubMed Scopus (8) Google Scholar). The N-terminal portion of this SAH is capable of binding directly to microtubules. Furthermore, SAH domains are highly extensible, and by analogy with the myosin SAH domain (20Wolny M. Batchelor M. Knight P.J. Paci E. Dougan L. Peckham M. Stable single α-helices are constant force springs in proteins.J. Biol. Chem. 2014; 289: 27825-27835Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), it is likely that extension of the relatively lengthy INCENP SAH might allow the IN box with its bound Aurora B to undergo excursions of up to ∼80 nm under relatively light loads. These data support the suggestion that the INCENP coil functions as a dog leash that allows Aurora B to “wander” across a substantial target area to reach its substrates (9Santaguida S. Musacchio A. The life and miracles of kinetochores.EMBO J. 2009; 28: 2511-2531Crossref PubMed Scopus (367) Google Scholar). By using an SAH rather than a coiled coil to achieve this flexibility, INCENP avoids the requirement for dimerization, which would significantly complicate the currently accepted mechanism of proximity/clustering-induced activation of the CPC (12Carmena M. Wheelock M. Funabiki H. Earnshaw W.C. The chromosomal passenger complex (CPC): from easy rider to the godfather of mitosis.Nat. Rev. Mol. Cell Biol. 2012; 13: 789-803Crossref PubMed Scopus (563) Google Scholar, 22Bishop J.D. Schumacher J.M. Phosphorylation of the carboxyl terminus of inner centromere protein (INCENP) by the Aurora B kinase stimulates Aurora B kinase activity.J. Biol. Chem. 2002; 277: 27577-27580Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 23Kelly A.E. Sampath S.C. Maniar T.A. Woo E.M. Chait B.T. Funabiki H. Chromosomal enrichment and activation of the aurora B pathway are coupled to spatially regulate spindle assembly.Dev. Cell. 2007; 12: 31-43Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 24Tseng B.S. Tan L. Kapoor T.M. Funabiki H. Dual detection of chromosomes and microtubules by the chromosomal passenger complex drives spindle assembly.Dev. Cell. 2010; 18: 903-912Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). All proteins were expressed in Escherichia coli BL21 Rosetta 2 (Novagen) and purified using a nickel-nitrilotriacetic acid affinity chromatography column. Proteins were dialyzed against 150 mm NaCl, 20 mm Tris, 1 mm DTT, pH 8.0 and proteolyzed for 2 h at room temperature using ULP1 recombinant small ubiquitin-like modifier protease in a substrate to enzyme ratio of 100:1. Next, proteins were purified on ion-exchange columns using an ÄKTA system. The purest fractions were combined and concentrated, resulting in a 1–2 mg/ml protein solution. Purified protein was dialyzed against 100 mm NaCl, 10 mm sodium phosphate, pH 7.4 and snap frozen in liquid nitrogen for long term storage at −80 °C. Protein samples (∼0.2 ml; 20 μm) were dialyzed (G-Biosciences dialyzers 2-kDa-molecular mass cutoff) overnight against 50 mm ammonium acetate, pH 7.4 and analyzed by TOF MS analysis (The University of Leeds Mass Spectrometry Facility). CD measurements were performed on an Applied Photo Physics Chirascan CD spectropolarimeter with a 0.1-cm-path length quartz cuvette in 0.1 m NaCl, 10 mm sodium phosphate, pH 7.4 buffer. Data were collected every 1 nm with 30-s averaging time, each measurement being an average of two repeated scans. Data presented are averaged from at least two separate measurements of different protein preparations. Thermal measurements were performed in a temperature range from 10 to 85 °C with a 0.7 °C/min heating rate with data acquisition every 1 °C and 20-s averaging time. The sample cooling rate prior to measurement of refolded protein was ∼ 2 °C/min. The mean residue molar ellipticity of proteins was calculated as described (25Greenfield N. Fasman G.D. Computed circular dichroism spectra for the evaluation of protein conformation.Biochemistry. 1969; 8: 4108-4116Crossref PubMed Scopus (3318) Google Scholar). The helical content of proteins was calculated from values of the amide nπ* transition at 222 nm ([MRE222]) as described previously (25Greenfield N. Fasman G.D. Computed circular dichroism spectra for the evaluation of protein conformation.Biochemistry. 1969; 8: 4108-4116Crossref PubMed Scopus (3318) Google Scholar). Protein concentration was measured by absorption at 280 nm. Absorption coefficients were obtained from ProtParam software. Standard concentrations were in the range of 10–20 μm. In the salt dependence experiments, stock buffer (5 m NaCl, 10 mm sodium phosphate, pH 7.4) was mixed with stock protein solution to obtain desired salt and protein (10 μm) concentration. Sequences encoding putative full-length INCENP SAH domain (UniProt ID P53352; Gallus gallus; residues 503–715) and its N-terminal (residues 503–597) and C-terminal (residues 598–715) fragments were subcloned into the pET28a SUMO vector (26Wolny M. Colegrave M. Colman L. White E. Knight P.J. Peckham M. Cardiomyopathy mutations in the tail of β-cardiac myosin modify the coiled-coil structure and affect integration into thick filaments in muscle sarcomeres in adult cardiomyocytes.J. Biol. Chem. 2013; 288: 31952-31962Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) to introduce an N-terminal His tag and small ubiquitin-like modifier protein for increased expression and solubility. For all constructs, a tryptophan residue was added to the C terminus to enable A280 concentration measurements. INCENP SAH mutant constructs were based on triple affinity purification (TrAP)-tagged INCENPWT class I under control of an SV40 promoter that is insensitive to doxycycline repression (27Xu Z. Ogawa H. Vagnarelli P. Bergmann J.H. Hudson D.F. Ruchaud S. Fukagawa T. Earnshaw W.C. Samejima K. INCENP-aurora B interactions modulate kinase activity and chromosome passenger complex localization.J. Cell Biol. 2009; 187: 637-653Crossref PubMed Scopus (62) Google Scholar). The TrAP tag incorporates His, streptavidin-binding peptide, and S tags and can be monitored by immunoblotting and immunofluorescence using a monoclonal antibody recognizing the streptavidin-binding peptide tag (28Hudson D.F. Ohta S. Freisinger T. Macisaac F. Sennels L. Alves F. Lai F. Kerr A. Rappsilber J. Earnshaw W.C. Molecular and genetic analysis of condensin function in vertebrate cells.Mol. Biol. Cell. 2008; 19: 3070-3079Crossref PubMed Scopus (48) Google Scholar, 29Samejima K. Ogawa H. Cooke C.A. Hudson D.F. Macisaac F. Ribeiro S.A. Vagnarelli P. Cardinale S. Kerr A. Lai F. Ruchaud S. Yue Z. Earnshaw W.C. A promoter-hijack strategy for conditional shutdown of multiply spliced essential cell cycle genes.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 2457-2462Crossref PubMed Scopus (19) Google Scholar). GFP was inserted in front of the TrAP tag to visualize the mutants. Silent mutations were introduced into INCENP cDNA to create BamHI, EcoRI, and HindIII sites around SAH domain so that the SAH region can be easily modified. Wild type SAH, half-SAH, double SAH, and double MyoM SAH (30Baboolal T.G. Sakamoto T. Forgacs E. White H.D. Jackson S.M. Takagi Y. Farrow R.E. Molloy J.E. Knight P.J. Sellers J.R. Peckham M. The SAH domain extends the functional length of the myosin lever.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 22193-22198Crossref PubMed Scopus (62) Google Scholar) cassettes were synthesized at Geneart (Life technologies) and cloned into the GFP-TrAP-INCENP constructs. DT40 cells were grown in RPMI 1640 medium supplemented with 10% FBS, 1% chicken serum and maintained in 5% CO2 at 39 °C. Doxycycline at a final concentration of 500 ng/ml was added to the culture medium to repress transcription of the promoter-hijacked endogenous INCENP locus (29Samejima K. Ogawa H. Cooke C.A. Hudson D.F. Macisaac F. Ribeiro S.A. Vagnarelli P. Cardinale S. Kerr A. Lai F. Ruchaud S. Yue Z. Earnshaw W.C. A promoter-hijack strategy for conditional shutdown of multiply spliced essential cell cycle genes.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 2457-2462Crossref PubMed Scopus (19) Google Scholar). HeLa Kyoto cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 0.2 mm l-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. Whole cell lysates were prepared, and the equivalent of 0.5–1 × 106 cells was loaded onto a polyacrylamide gel. SDS-PAGE and immunoblotting were performed following standard procedures. Donkey anti-mouse or -rabbit IRdye 800CW was used for analysis using a LI-COR Biosciences Odyssey quantitative fluorescence imager. All fixation, permeabilization, and immunostaining were performed at room temperature as described previously (31Platani M. Santarella-Mellwig R. Posch M. Walczak R. Swedlow J.R. Mattaj I.W. The Nup107–160 nucleoporin complex promotes mitotic events via control of the localization state of the chromosome passenger complex.Mol. Biol. Cell. 2009; 20: 5260-5275Crossref PubMed Scopus (63) Google Scholar). Cells attached on polylysine-coated coverslips were fixed in a 3.7% formaldehyde, PBS solution for 10 min and permeabilized in PBS, 0.15% Triton X-100 for 4 min. Cells were blocked in 10% normal donkey serum for 1 h at room temperature prior to antibody incubations. Antibodies used were α-tubulin antibody (B512 or DMIA, Sigma-Aldrich); anti-H3Ser10ph (Millipore); anti-GFP (Life Technologies); anti-HEC1 mouse monoclonal (Abcam); anti-DSN1ph (32Welburn J.P. Vleugel M. Liu D. Yates 3rd, J.R. Lampson M.A. Fukagawa T. Cheeseman I.M. Aurora B phosphorylates spatially distinct targets to differentially regulate the kinetochore-microtubule interface.Mol. Cell. 2010; 38: 383-392Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar); anti-H3Ser28ph (33Hayashi-Takanaka Y. Stasevich T.J. Kurumizaka H. Nozaki N. Kimura H. Evaluation of chemical fluorescent dyes as a protein conjugation partner for live cell imaging.PLoS One. 2014; 9e106271Crossref PubMed Scopus (35) Google Scholar); and rabbit polyclonal (WCE1186), anti-INCENP (3D3), anti-Aurora B, and anti-CENP-T, which were described previously (1Cooke C.A. Heck M.M. Earnshaw W.C. The inner centromere protein (INCENP) antigens: movement from inner centromere to midbody during mitosis.J. Cell Biol. 1987; 105: 2053-2067Crossref PubMed Scopus (318) Google Scholar, 27Xu Z. Ogawa H. Vagnarelli P. Bergmann J.H. Hudson D.F. Ruchaud S. Fukagawa T. Earnshaw W.C. Samejima K. INCENP-aurora B interactions modulate kinase activity and chromosome passenger complex localization.J. Cell Biol. 2009; 187: 637-653Crossref PubMed Scopus (62) Google Scholar, 34Earnshaw W.C. Cooke C.A. Analysis of the distribution of the INCENPs throughout mitosis reveals the existence of a pathway of structural changes in the chromosomes during metaphase and early events in cleavage furrow formation.J. Cell Sci. 1991; 98: 443-461Crossref PubMed Google Scholar). All affinity-purified donkey secondary antibodies (labeled with either FITC, Alexa Fluor 488, TRITC, Alexa Fluor 594, or Cy5) were purchased from Jackson ImmunoResearch Laboratories. RNAi experiments were performed using annealed siRNA oligos (Qiagen) diluted in serum-free Opti-MEM and transfected using HiPerFect reagent (Qiagen) according to the manufacturer's protocol. HeLa cells were seeded on coverslips at a concentration of 1 × 105 cells/ml, and diluted siRNA was added to cells so that the final concentration of siRNA was 40 nm. Plasmids encoding either GFP-TrAP-GgINCENPWT SAH, GFP-TrAP-GgINCENPDouble SAH, or GFP-TrAP-GgINCENPN-half SAH were transfected for 24 h prior to fixation. Coverslips were fixed at 30–34 h. INCENP siRNA oligo was 5′-AGATCAACCCAGATAACTA-3′ (35Carmena M. Pinson X. Platani M. Salloum Z. Xu Z. Clark A. Macisaac F. Ogawa H. Eggert U. Glover D.M. Archambault V. Earnshaw W.C. The chromosomal passenger complex activates Polo kinase at centromeres.PLoS Biol. 2012; 10e1001250Crossref PubMed Scopus (86) Google Scholar). For control transfections, non-silencing random scrambled siRNA oligos were used at the same concentration. Quantifications of H3Ser28ph, H3Ser10ph, and DSN1ph were carried out as follows. Deconvolved images were imported into OMERO (36Swedlow J.R. Goldberg I.G. Eliceiri K.W. OME ConsortiumBioimage informatics for experimental biology.Annu. Rev. Biophys. 2009; 38: 327-346Crossref PubMed Scopus (81) Google Scholar), and segmentation of centromere foci (anti-centromere antibodies, Cy5, reference channel) or chromatin (DAPI, 435 reference channel) performed using Otsu segmentation implemented in Matlab. Masks stored in OMERO were then used to calculate background-corrected intensities, which were output into a comma-separated value file for plotting in Excel. Growth curves were generated by seeding cells at a concentration of 2 × 105 cells/ml at 39 °C (unless otherwise stated). Cell counting was performed every 24 h for a total of 96 h. To avoid overgrowth, cells were diluted to 2 × 105 cells/ml every 24 h. The cell number at each time point was multiplied by the appropriate dilution factor to get a true count. Tubulin (Cytoskeleton Inc.) was used for the generation of polymerized microtubules according to the manufacturer's instructions. Taxol-stabilized microtubules (18 μm tubulin dimer) were incubated at room temperature for 10 min with 1 μm protein in a 50-μl reaction volume in BRB80 buffer (80 mm PIPES, pH 6.9, 1 mm EGTA, 1 mm MgCl2) with 100 mm NaCl and 4 mm DTT in the presence of 20 μm Taxol. The reaction was then layered onto a 250-μl glycerol cushion buffer (BRB80 buffer, 50% glycerol, 4 mm DTT) and ultracentrifuged for 10 min at 434,400 × g in a Beckman TLA 100.3 rotor at 25 °C. Pellets and supernatants were analyzed by SDS-PAGE. Gels were stained with Coomassie Blue, and protein quantification was performed with NIH ImageJ. Normalized binding data were obtained by dividing the values of the pellet fraction by the sum of pellet and supernatant. The middle region of INCENP links the N-terminal centromere/microtubule-targeting domains with the C-terminal Aurora B regulatory domain. This middle region, GgINCENP503–715, has been predicted to form a coiled coil structure that is required for microtubule binding and microtubule-induced activation of Aurora B (24Tseng B.S. Tan L. Kapoor T.M. Funabiki H. Dual detection of chromosomes and microtubules by the chromosomal passenger complex drives spindle assembly.Dev. Cell. 2010; 18: 903-912Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 37Mackay A.M. Eckley D.M. Chue C. Earnshaw W.C. Molecular analysis of the INCENPs (inner centromere proteins): separate domains are required for association with microtubules during interphase and with the central spindle during anaphase.J. Cell Biol. 1993; 123: 373-385Crossref PubMed Scopus (89) Google Scholar). Subsequent detailed analysis of the INCENP sequence revealed numerous charged residues at positions of the heptad repeat that would disrupt coiled coil formation. We predicted that GgINCENP503–715 is not a coiled coil but might instead form a stable SAH domain (Fig. 1A) (19Peckham M. Knight P.J. When a predicted coiled coil is really a single α-helix, in myosins and other proteins.Soft Matter. 2009; 5: 2493-2503Google Scholar, 38Peckham M. Coiled coils and SAH domains in cytoskeletal molecular motors.Biochem. Soc. Trans. 2011; 39: 1142-1148Crossref PubMed Scopus (26) Google Scholar). SAH domains are characterized by a highly helical secondary structure, non-cooperative thermal unfolding, and the ability of the peptide to completely refold after thermal denaturation. In addition, SAH domains remain helical up to fairly high salt concentrations (18Knight P.J. Thirumurugan K. Xu Y. Wang F. Kalverda A.P. Stafford 3rd, W.F. Sellers J.R. Peckham M. The predicted coiled-coil domain of myosin 10 forms a novel elongated domain that lengthens the head.J. Biol. Chem. 2005; 280: 34702-34708Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). To test this hypothesis, we prepared recombinant proteins containing the full-length SAH (INCENP503–715), the N-terminal SAH (INCENP503–597), and the C-terminal SAH (INCENP598–715). The exact position of the break in the sequence was based on analysis of potential ionic interactions between charged amino acid residues so that it did not disturb any of these potential bonds. We then performed CD measurements to investigate the secondary structure of these protein fragments over a range of temperature and salt conditions and looked at their ability to form monomers or dimers by measuring their molecular weight by mass spectrometry. CD spectra revealed that all three proteins were highly helical at 10 °C at 0.1 m NaCl (Fig. 1, B–D). According to this analysis, INCENP503–715 was 85% helical, whereas INCENP503–597 and INCENP598–715 were ∼90 and 88% helical, respectively. All three constructs melted non-cooperatively as expected for SAH domains (Fig. 1E) and refolded after cooling to at least 90% of their initial helical content measured at 10 °C (Fig. 1, B–D). All three constructs remained highly helical up to 2 m NaCl, and then helical content decreased to ∼60% at 4 m NaCl (Fig. 1F), demonstrating the salt-resistant nature expected of an SAH domain (18Knight P.J. Thirumurugan K. Xu Y. Wang F. Kalverda A.P. Stafford 3rd, W.F. Sellers J.R. Peckham M. The predicted coiled-coil domain of myosin 10 forms a novel elongated domain that lengthens the head.J. Biol. Chem. 2005; 280: 34702-34708Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). The helical nature of INCENP503–597 was slightly more resistant to increasing salt concentrations compared with INCENP503–715 and INCENP598–715. Mass spectrometry analysis confirmed that all of the studied constructs are monomeric with molecular masses of 12, 15.2, and 26.9 kDa for INCENP503–597, INCENP598–715, and INCENP503–715, respectively (data not shown). All of the above strongly suggest that the middle region of INCENP is an SAH domain and not a coiled coil as proposed previously. Consequently, intact INCENP is likely a monomer and not a dimer, which has implications for its mech" @default.
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• W1916209382 date "2015-08-01" @default.
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• W1916209382 title "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" @default.
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