A serological method to simultaneously detect antibodies against infectious laryngotracheitis virus (ILTV) and infectious bronchitis virus (IBV) is imperative for the differential diagnosis and evaluation of antibodies titers after vaccination.The microspheres coated with purified recombinant glycoprotein D (gD) of ILTV or nucleocapsid (N) protein of IBV were incubated with serum samples. The simultaneous quantification of ILTV and IBV antibodies were achieved through the interrogation of microspheres by Luminex 200 detection system. .This xMAP detection demonstrated no nonspecific reactions with avian influenza virus (AIV), avian leukosis virus (ALV), newcastle disease virus (NDV), and Marek’s disease virus (MDV). The results also demonstrated that the xMAP assay was four times more sensitive than the enzyme-linked immunosorbent assay (ELISA) for ILTV detection and two times more sensitive for IBV detection. A total of 90 chicken serum samples from a chicken farm were tested by xMAP and ELISA assays. The results showed that the coincidence rates were 84.44 and 100% for ILTV and IBV detection, respectively.This study exhibited an opportunity for the differential diagnosis through simultaneous detection of multiplex antibodies in serum and can be used for the multiplex antibodies evaluation after vaccination. Infectious laryngotracheitis (ILT) and infectious bronchitis (IB) are common respiratory diseases in poultry and are currently present at epidemic levels around the world, including in China, the United States, and India. Because ILT and IB have high incidence and infectivity in chickens at different ages (in days), they have caused huge economic losses to the poultry industry. Both diseases have similar clinical symptoms and pathological changes, leading to significant difficulties in clinical differential diagnosis, especially in cases of mixed infection , 2. In aInfectious laryngotracheitis virus (ILTV), an alphaherpes virus, possesses at least 10 envelope glycoprotein, two main proteins of which are the glycoprotein B (gB) and gD, respectively, which are highly conserved herpesvirus structural glycoproteins . The gD At present, the methods used for the diagnosis and effect evaluation of vaccine immunity of avian respiratory diseases primarily include virology detection, serological detection, and molecular biological detection. Traditional methods, such as virus isolation, animal inoculation experiments, ELISA, hemagglutination (HA), and hemagglutination inhibition (HI) assays are characterized by complex procedures and long periods of time required for diagnosis. Despite its unique, sensitive, simple features and rapidity, polymerase chain reaction (PCR) is unable to meet the requirements for high-flux quarantine and is not favorable for the urgent screening of bulk samples. Therefore, it is imperative to establish a new technology for the effect evaluation of vaccine immunity and the rapid differentiation or correct identification of important avian respiratory diseases.,s antibodies, with simple operation and high accuracy that are superior to conventional methods. In this study, a method employing Luminex xMAP technology to simultaneously detect ILTV and IBV antibodies in serum was established, optimized and used for the differential diagnosis of IBV and ILTV. This assay can also be used to simultaneous monitoring of IBV and ILTV antibody levels for the evaluation of immunity after vaccination.A new high-flux detection technology, flexible xMAP  assay, uses variously colored polystyrene beads that is carboxylated to allow covalent coupling of protein. Conjugated beads can be used to capture specific antibodies in serum, and a fluorescent secondary antibody is incubated to bind to the captured serum antibodies. A red laser is used to determine the color of the bead and a green laser to detect fluorescence intensity of bound secondary antibodies through the Luminex 200 detection system . This me5 magbeads), seen in Table 5 magbeads for singleplex xMAP detection of ILTV. The mean P/N value under the condition was 10.18.A singleplex xMAP for ILTV was established by testing six concentrations of the ILTV gD envelope protein , seen in Table 5 magbeads for singleplex xMAP detection of IBV. The mean P/N value under the condition was 17.16.A singleplex xMAP for IBV was established by testing six concentrations of the IBV N envelope protein  was 5.09% for ILTV and 8.05% for IBV. Inter-assay repeatability experiments showed that the CV was 8.11% for ILTV and 9.48% for IBV. All CVs were less than 10%, indicating that the method has high repeatability. The results were shown in Tables The sensitivities of xMAP and ELISA were compared using 1:50 to 1:3200 ILTV-positive sera and 1:100 to 1:6400 IBV-positive sera. The results showed that xMAP detected ILTV-positive sera at 1:1600 and IBV-positive sera at 1:3200, while ELISA detected ILTV-positive sera at 1:400 and IBV-positive sera at 1:1600 , with an optimal serum dilution of 1:100. However, it is not ideal to diagnose only one type of pathogen antibody because more than one disease often affects the respiratory tract in poultry. Given the similar clinical symptoms of ILT and IBV, the differential diagnosis of these two pathogens with two single xMAP assays using one sample wastes time and energy. To achieve the rapid differential diagnosis of clinical samples, it is necessary to establish a duplex xMAP. In addition to ILTV and IBV, AIV and NDV are also common respiratory viruses for poultry. These four diseases have high incidences, and it is difficult to make a differential diagnosis based on clinical symptoms, which are all similar. Figures This study describes the establishment and validation of single xMAP and duplex xMAP assays. First, to achieve effective and rapid clinical diagnosis of ILT and IBV, single xMAPs were established for the separate diagnosis of these two diseases after screening to identify the best concentrations of microsphere antigens for ILTV gD and IBV N  cells [The antigens used for ILTV were a recombinant gD , express3) cells . The negPrior to coupling, the recombinant proteins was desalted by gel filtration using MicroBio-Spin 6 columns  based on the manufacture’s protocol to exchange the buffer from sodium azide or imidazole to PBS. All the antigens were quantified using Pierce BCA protein Quantification Kit . Coupling of proteins to fluorescent microspheres was performed as described by Karanikola et al. . MAP MC15 magbeads to determine the optimum antigen concentration for singleplex xMAP assay. A serious dilution of sera at the range from 1:25 to 1:200 have been made to determine the optimal sample dilution. Cross-reactivity or specificity was evaluated by making the assay with sera from chicken infected with AIV, ALV, NDV, and MDV. Then, a duplex xMAP assay was established based on the result of two separated singleplex xMAP assay. In briefly, a 50 μL aliquot of the simplex beads (50 beads/μL) or the duplex beads  was transferred into the wells, added in 50 μL of diluted sera. Incubation was conducted on a plate shaker (800 rpm) at RT for 60 min. The centrifugation was conducted in the magnetic separator for 60 s. The supernatant was removed from each well. Beads were washed 5 times in 100 μL PBS. Each well was added with 100 μL of a biotinylated secondary antibody  at 2 μg/mL. The plate was shaken (800 rpm) for 30 min at RT and washed as described above. Finally, 2 μg/mL of streptavidin-phycoerythrin  was added to each well at 100 μL/well. The plate was shaken and the supernatant was removed. Finally, 100 μL of assay buffer was added to each well. The plate was shaken for approximately 15 s and analyzed according to the manufacturer’s protocol. All samples were conducted in triplicates in each assay.The xMAP assays were carried out in 96-well polystyrene microplates using Luminex 200 detection system . Relevant positive and negative sera have been prepared for the establishment of the singleplex xMAP assay to detect ILTV antibody or IBV antibody in the serum. ILTV gD protein at the following concentrations  and IBV N protein at the following concentrations  were coupled with 5 × 10Commercial ILTV and IBV ELISA kit  were used to detect presence of ILTV or IBV antibodies in poultry sera. The assay was performed according to the manufacturer’s protocols.


Alloying on the cation (A) site is a frequently used approach to optimize the thermoelectric transport properties of AMgA) site. We show that the small ionic radius of Mg The str cations , as showM site and the octahedrally coordinated A site, has attracted a great deal of attention. Unlike other compounds with this structure type, MgMgn-type doped, leading to the highest thermoelectric figure of merit, In recent years, the binary compound MgMg to date ,9,10,11. to date ,13, band to date ,14,15, a to date ,17,18 inn-type MgMgA = Mg, Ca, Sr, Ba, Sm, Eu, Yb) was frequently used in earlier work on p-type A = Ca, Yb, and Eu in the AZnACdAMgn-type MgMgn-type doping can still be achieved for A≠ Mg.)Thus far, much of the optimization of  AZn2Sb2 ,20,21 an systems ,23,24, astigated ,25,26. Astigated ,26. Equastigated ,28. AlthA, M or X site [A cation site in AMgA site seems to be an applicable approach to further optimize the thermoelectric properties of n-type MgMgTo date, complete solubility has been reported in most investigations of alloys between different r X site ,24,25,26x = 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 1), , , and  were synthesized via direct ball-milling of elements followed by spark plasma sintering (SPS). The corresponding stoichiometric elements  were cut into small pieces in an argon filled glove box, loaded into stainless steel vials with two stainless steel balls, and milled for one hour using a SPEX mill, SPEX SamplePrep LLC., Metuchen, NJ, USA. The ball-milled powder was then loaded into graphite dies with 10 mm inner diameter. The powder was heated to the target temperatures shown in Samples of . The ionic radius of MgA site in AMgA site, being too large to occupy the tetrahedrally coordinated M site in A = Mg site only, not on the M = Mg site.Among (1.35 Å) ,30. The x = 0–1  solubility for Mg on the Sr site, but no solubility of Sr on the Mg site. In the Ba-Mg alloy, no solubility of Ba on the Mg site, or of Mg on the Ba site was observed. Please note that the lattice parameters shown in For the  to formA = Mg site. However, no superstructure formation was observed after quenching from high temperature for any of our alloyed samples. The upper size limit established here may provide guidance for doping on the Mg site in MgMgn-type dopants [n-type dopants will not be a primary factor limiting their solubility. We note, however, that complete solid solubility would never be expected for alio-valent dopants in Zintl phases. Furthermore, in alio-valent doping, the size of the dopant is only a minor factor. Other factors controlling solubility include the valence of the dopant and the impact that the dopant has on the chemical potential of other types of defects.One notable exception in the literature is the Mga et al. . Dependi dopants ,37. The A site in In


In vivo, the primary molecular mechanotransductive events mechanically initiating cell differentiation remain unknown. Here we find the molecular stretching of the highly conserved Y654-β-catenin-D665-E-cadherin binding site as mechanically induced by tissue strain. It triggers the increase of accessibility of the Y654 site, target of the Src42A kinase phosphorylation leading to irreversible unbinding. Molecular dynamics simulations of the β-catenin/E-cadherin complex under a force mimicking a 6 pN physiological mechanical strain predict a local 45% stretching between the two α-helices linked by the site and a 15% increase in accessibility of the phosphorylation site. Both are quantitatively observed using FRET lifetime imaging and non-phospho Y654 specific antibody labelling, in response to the mechanical strains developed by endogenous and magnetically mimicked early mesoderm invagination of gastrulating Drosophila embryos. This is followed by the predicted release of 16% of β-catenin from junctions, observed in FRAP, which initiates the mechanical activation of the β-catenin pathway process. Such deformations can subsequently lead to changes in the molecular biochemical reactivity and states, thereby initiating the activation or modulation of biochemical signal-transduction pathways  dependent phosphorylation of the highly conserved β-cat Y654 site  pathway is mechanotransductively involved in major biochemical patterning processes in both developmental and tumorous contexts . Initialretching , in tumoretching , or in fretching . In both654 site , a major654 site . This ch654 site . The molin vivo the hypothesis that the major Y654-β-cat/E-cad-D665 interaction site of the AJ complex  simulations of the β-cat-E-cad complex submitted to a stretching uniaxial force .The force is applied on E-Cad by both the cell’s and the neighbouring cell’s apical constrictions that drive Drosophila embryos mesoderm invagination . It thusa posteriori the biological force that is mimicked by our procedure as follows. The mean elongation of the simulated complex showing the opening of the Y654-D665 site on the timescale of 12 ns is of 6 nm  on a protein complex of significant size (more than 500 residues). Our simulations under strain employed a constant force of 150 pN in order to observe relevant conformational changes on the 30-ns timescale of the simulation . We esti of 6 nm . The pro 1 pN/nm . Therefo 1 pN/nm .After a global extension of 8 nm of the overall complex, a local and fine stretching deformation is observed between the A648 site of the α-helix HII-E-cad and the C-terminus of the α-helix H3-β-cat, which are linked by the Y654-β-cat-D665-E-cad (Y654-D665) interacting site, of 1.25 ± 0.6 nm (45% strain). This change led, at 6 nm of extension mimicking an actual strain of 6 pN, to a discrete opening of the Y654-D665 interacting site of 0.42 ± 0.08 nm in 3 simulations out of 20, namely in 15% of cases .in vivo the prediction of the existence of a strain deformation in the β-cat-E-cad complex of 1.25 nm between the A648-E-cad and the β-cat-C-terminus, due to mesoderm invagination in Drosophila embryos. In this case, mechanical induction of the Y654-β-cat phosphorylation by mesoderm invagination is specifically required for mesoderm specification  neighbouring directly the alpha helix of region II. The latter contains the D665 (D1170 in Drosophila), which interacts with the Y654 site of β-cat (Y667 in Drosophila), and is directly labelled with Alexa 555 (in orange in Drosophila proteome (taxid: 7227)  see . The GFP227) see   see .At the time of antibody injection, the cells in the embryo still form a syncytium, that is they are open to the same internal yolk volume, which allows the antibody to diffuse into any cell. Afterwards, cells are closed in a process called cellularization followed by the first embryonic morphogenetic movement, the invagination of the mesodermal tissue during gastrulation . FRET exFLIM measurements were performed during the two following different developmental stages during gastrulation: just before mesoderm invagination , and during invagination (stage 6)  when the‘Calculation of the strain from FRET efficiency variations’ in the Materials and methods section) between the GFP tagged β-cat and the fluorescently tagged E-cad antibody. Indeed, such ~1 nm stretching is quantitatively predicted by the MD simulations , as well as in embryos injected with PBS alone in the absence of the antibody acceptor -right. Sulations . The lifin cellula, namely of 2.6 ns coupled to Histones in the nucleus in the absence of acceptor and of 2.4 ns in the presence of an acceptor 2 being the cross-section surface of the molecular complex.Note that the lifetime of the controls with GFP alone in the embryo junctions and with PBS injection without antibody, of 2.60 ± 0.04 ns, , was conacceptor . Furtheracceptor , modulatacceptor . Indeed,These experiments thus demonstrate the existence of a mechanical deformation involving a specific site connecting two interacting proteins. The molecular detail of this deformation involves the stretching of the β-cat and E-cad helices which interact through their respective Y654 and D665 residues, during mesoderm invagination.sna and twi siRNAs for RNAi (sna twi RNAi mesoderm invagination defective embryos. This was achieved by injecting ultramagnetic liposomes (UMLs) into mesoderm cells , in contrast to the increase observed in the WT  and stage 5 in n the WT . This obsna twi RNAi embryos is rescued mechanically. Consequently, it is associated to the lack of mechanical strains in mesoderm defective embryo. These observations demonstrate that the mechanical strains developed by mesoderm invagination induce a mechanical stretching deformation in the order of ~1 nm in the molecular hetero-complex between the HII-E-cad and H3-β-cat helices, which are linked by the Y654-D665 site. These results confirm quantitatively the 1.25 ± 0.6 nm deformation predicted in our MD simulations.Thus, the defect of molecular mechanical strain deformation between the fluorescently labelled linear domain II of E-cad and the C-terminus of β-cat in in vivo before gastrulation, immuno-fluorescent staining was performed on fixed embryos using the ultra-rapid MeOH heat procedure that maintains junctional labelling only . To prevent disturbing competition between the Y654-β-cat antibody and E-cad on the Y654 site by injection of the E-cad anti-body ing only , at stagWe found an increase in the signal for the un-phosphorylated site of β-cat at AJ relative to the junctional β-cat signal during gastrulation in the invaginating tissue. The increase is specific of gastrulation early stage 6 and late-stage 6 compared to pre-gastrulation stage 5, of 19.6 ± 6.2% and 23.0 ± 5.7% respectively . As a coThe mechanically induced opening of the Y654-D665 binding site should thus favour the accessibility of the Y654 tyrosine to Src42A and thereby its phosphorylation by Src42A which leads to the release of the β-cat from AJ . Howeversrc42A RNAi embryos , and of about 25 ± 5% compared to wild type, both at early stage 6 and late-stage 6 . As a coo the WT . Consisto the WT . These rTherefore, we conclude that during mesoderm invagination, the accessibility of the site of Y654 β-cat to Src42A increases due to its mechanical opening. At the same time, the Y654 β-cat residue becomes specifically more available for Src42A phosphorylation at AJ, as the strain builds up at the Y654 β-cat-D665-E-cad molecular complex interaction site. The mechanical induction of the increase in accessibility of the Y654 site under strain for Src42A phosphorylation should thus lead to the release of an amount of junctional β-cat into the cytosol proportional to the subset of β-cat/E-cad complex that opens at Y654-D665 under strain .Drosophila embryo mesodermal AJs under strain during gastrulation. This was achieved by fluorescent recovery after photobleaching (FRAP) experiments at three different developmental stages: just before invagination , at the onset of invagination (early 6) and during invagination (mid-stage 6)  .We found upon photobleaching that the fluorescent recovery amplitude, the so-called mobile fraction, of β-cat upon photobleaching is increased by 16 ± 6% when mesoderm invagination has built up at mid-stage 6. This observation indicated that about 16% more molecules at the AJ are able to be recycled and exchanged at stage 6 compared to stage 5 . This reThis relationship shows that the morphogenetic movements of invagination locally cause a mechanical stretch at the Y654-D665 interaction site in the β-cat/E-cad molecular complex. It indeed statistically weakens the interaction of β-cat with E-cad in AJ of the mesoderm cells and thereby favours cytosolic β-cat enrichment.Drosophila embryo, the Src42A kinase required for the mechanical induction of Y654-β−cat phosphorylation is already activated (phosphorylated on Y400) before tissue deformation. Therefore, Src42A is surprisingly not mechanically ‘over-activated’ by tissue deformation during the morphogenetic movements of gastrulation . Chain B was truncated at residue 648 on the N-terminal side, as the corresponding portion of the chain would be extended in vivo and not interacting with β-cat . The system was solvated in a water box of 7.2 * 7.2 * 21 nm, so that the protein long axis  was aligned with the z direction. The system was then minimized using a steepest-descent algorithm, and was progressively heated to 300 K during 120 ps. The bond between Y654 and D665 was constrained to its value in the crystal structure to ensure that the heating toward room temperature did not dissociate the complex. The system was then equilibrated for 2 ns at ambient pressure (1 bar) and temperature (300 K) using Langevin thermostats and barostats, and keeping the constraint between the two residues. The constraint was then progressively relaxed during 2 ns, and the system was subsequently equilibrated for 10 ns under the same conditions. 10 control trajectories in the absence of force were propagated for 30 ns. To ensure that the protein complex stayed aligned to the z axis in the elongated simulation box, and could not interact with its images by rotating around the z direction, a light harmonic constraint  was applied to the projection of the end-to-end axis of β-cat in the (xy) plane. This constraint did not affect the interaction between β-cat and E-cad, as we verified that the same simulations (but for a shorter simulation time of 10 ns so that the protein could not significantly tumble) in the absence of such constraint lead to quantitatively similar results. For the simulations under force, we fixed the alpha carbon of Y142 of β-cat, and we applied a constant force of 150 pN in the z direction to the alpha carbon of A648  of E-cad. 10 simulations were propagated for 30 ns. Analysis of interatomic distances were performed using in-house Fortran 90 codes, and visualization was done using the VMD software.We used the NAMD 2.10 software on GPU/CPU nodes to run the molecular dynamics (MD) simulations in explicit solvent . The CHABDSC_7063) and β-cat-GFP  were provided by Bloomington. Homozygous UAS-src42A RNAi was produced by crossing 7873 R-2 src42A RNAi with the 17643 UAS-src42A-RNAi (II) (RRID:FlyBase_FBst0452809) provided by the NIG Stock Centre and VDRC Stock Centre, respectively. UAS*Gal4 crosses were stored 2 hr at 28°C prior to experiments.Stocks were maintained at room temperature . Oregon-R (used for WT), Mat-Gal4 (III)  antibody RRID:AB_528089, DSHB (dilution 1:200). Secondary antibodies labelled with Alexa 488 or Alexa 633 were used from Invitrogen (dilution 1:200). Embryos were mounted vertically in Vectashield with DAPI for confocal observation. Images were obtained with a Zeiss 510 inverted confocal microscope. Images were processed and analysed with Fiji software. The intensity of the non-phospho-Y654 antibody staining in the mesoderm was either normalised to the β-cat intensity within the same ROI or to the intensity of the non-phospho-Y654 antibody staining in the ectoderm, all after background substraction (measured outside the embryo).Embryos were dechorionated and fixed according to Muller, H. A. and Wieschaus J. Cell Biol. 134, 149–163 (1996) . ProteinDrosophila Hybridoma bank was used in a 1:500 dilution, RRID:AB_528120.Western blots were conducted as described in D Mitrossilis et al. Nat Commun 8, 13883. 2017 Jan 23 with the following specifications: the JCAb20 was used in a 1:500 dilution and the anti-DE-cad DCAD2from 70–80 β-cat-GFP embryos at stage five were lysed in 50 mM Tris pH 8.0, 150 mM NaCl, 1% Triton X-100, 1 mM MgCl2 buffer plus a protein inhibitor cocktail (Sigma). After centrifugation at 14000 rpm for 20 min, protein lysate was pre-cleared with 50 μl of protein A-agarose beads , incubated with 10 μg of the JCAb20 antibody over night at 4°C, and subsequently incubated with 50 μl of protein A-agarose beads for 3 hr at 4°C. After washing, bound proteins were eluted with gel sample buffer and analyzed by Western-blot using the anti-DE-cad DCAD2 antibody (dilution 1:500).twi and sna defective non gastrulating Arm-GFP embryos, we injected siRNAs for twi and sna RNAi into Arm-GFP embryos. twi sna RNAi embryos have been described to phenocopy twi sna double zygotic mutants, and do not gastrulate twi-siRNAs Conditions of injection with the UMLs and siRNA are described in reference .An injector (Eppendorf Femtojet) was fixed on the microscope to inject magnetic particles or/and siRNA. A x,y,z piezo-electric system (Princeton Instruments) was mounted on the microscope to control the position of the embryo mounted on a coverslip relative to the injecting needles, the micromagnets, or the indent needle, with the 100 nm resolution.UML injections. Using a Femtojet Eppendorf injector ~0.05 µl of 0.1 µm/g UML were injected into the ventral cells and the basal domain of the ventral cells of the embryo from 20 to 40 min before the end of ventral cellularisation. Injections were performed at constant pressure, with the visualisation of UML labelled with Rhodamine observed in red in spinning disc. Prior to initiation of the sna-dependent pulsating phase, all blastoderm cells are under cellularisation and are basally open to the yolk. Ultra-magnetic liposomes (UML), composed of a high concentration of 9 nm Fe-oxide superparamagnetic nanoparticles (20% v:v) encapsulated in 200 nm fluorescently labelled phospholipid membranes . This results in a correction factor C with Cn=I0/InCn=I0. Bleached cell boundaries were tracked and kymographs were created using Packing Analyzer.FRAP experiments were performed on an inverted confocal Zeiss 510 microscope. Arm-GFP expressing embryos were imaged using the 488 nm laser line of the microscope. Bleaching was performed using the 488 nm and 514 nm laser lines at 100% for three seconds. Recovery of bleached areas was monitored with a frame rate of 6 s for 3 min after bleaching. Cell boundaries were identified and kymographs of bleached boundaries were produced using Packing Analyzer v2.0 . To corrFRET experiments were performed on an inverted 2-photon Leica TSC SP8 NLO SMD (Picoquant module) microscope system or on a spinning disc CSU10 head (Yokogawa) mounted on a Nikon TE2000 microscope, both equipped with a Lifa module (Lambert Instruments) by measuring the lifetime of the donor, so called FLIM-FRET. The FRET efficiency was thereby measured as a shortening of the donor lifetime. Lifetime measurements were analysed using either the SymphoTime software (Leica/Picoquant system) or the FLIMfit 4.12.1 , with a monoexponential fit. For this, the lifetime of the donor alone was first determined for calibration of the system. Afterwards the lifetime of the donor in the presence of the acceptor in the mesoderm and ectoderm region was analysed at the level of the adherens junctions in the lifetime images.Drosophila embryo before cellularization using an Eppendorf Transjector as described in Misquitta L et al πW^2L)Fluorescently labelled antibodies were injected into the  L et al . with thTA et al , which gThe antibody was purified from rabbit that was immunized with the following peptide: H-CALQGNMRDPNDIPDI-NH2 (16AA) corresponding to the AA 1143–1158 of the DE-cad protein in Drosophila. The sequence was chosen based on the position of it’s corresponding mouse sequence in the crystal structure of β-cat in complex with the cytoplasmic part of the E-cad protein (PDB 1i7x). The DE-cad sequence corresponds to a loop in Region II adjacent to the alpha-helical stretch that contains the D1170 (D665 mice), which interacts with the Y667 (Y654 mice) phosphorylation site of β-cat in the complex. Peptide synthesis and antibody production was carried out by eurogentech. Received antibody solutions were stored in in glycerol (1:2) at −20°C. The antibody was concentrated using an antibody concentration kit from abcam, labelled with Alexa 555 using the Alexa Fluor 555 Antibody Labeling Kit from lifetechnologies and stored in PBS at 4°C.The FRET efficiency was calculated using the E = 1 tda/td with tda being the lifetime of the donor in the presence of the acceptor, while td denotes the lifetime of the donor alone. The distances r are then calculated as r = R0((1/E)−1)^1/6 with R0 being the Förster Radius, in the approximation that there are no cross-FRET between adjacent molecules . The Föra posteriori once the p-value showed statistically significant results enough, relative to the difficulty and time consuming of individual experiments and simulations. Statistics are based on the ANOVA test , the Introduction is extremely brief consisting of 3 sentences followed by only 29 references. Also, the manuscript requires major editing.The authors describe a mechanotransduction process that induces a conformational change of a fraction of AJ-localized β-catenin leading to Src-mediated phosphorylation of β-catenin at Y654, release of pY654-β-catenin from E-cadherin, translocation into the nucleus and gene transcription. The findings are supported by molecular dynamics simulation studies and a series of in vivo experiments. These findings are significant but the manuscript lacks several important controls. Furthermore, the text of the paper is difficult to read. Sentences are fusions of several statements, figures are described in an unusual order , manipulations are not sufficiently explained  The lifetime differences of the antibody injected embryos are surprisingly small. As it is very difficult to control the amount of antibody (and thus FRET acceptor) being present at different locations in the syncytial blastoderm one would expect much larger variations. To ensure that the reported values are reproducible the number of injected embryos should be increased (from N=8 embryos). Furthermore, an injection control is necessary, as uninjected embryos are not sufficient to control the experiment. Does water/PBS injection have an effect? Does this vary between the stages?How was the lifetime fit done? Is it a mono-exponential fit? Is it not surprising that the GFP donor lifetime is <2.6 ns in the absence of an acceptor? This is impressively short!twi and sna injected instead of using zygotic double mutants? The small differences the authors observe upon RNAi injections could be due to differential knock-down efficiencies, particularly if the number of treated embryos is so low (N=8).2) Why are double RNAi against 3) The rescue with the magnet should also be controlled with water injected embryos.4) Quantification of the Y654 antibody stainings also requires a control experiment. The authors observe an increase in Y654 non-phospho antibody staining during gastrulation or upon Src knock-down. The authors seem to have normalized the Y654 non-phospho staining to general β-catenin antibody staining. An additional control, just outside of the mesoderm from the same embryo should be included, as the forces on the lateral cells should be less, the difference should also be less. Since the antibody signals can vary between embryos, such an internal standard makes it easier to compare different embryos.5) Is it possible to design a control experiment with mutant, stretch-resistant DE-cadherin and/or β-catenin and show that if the sites are made resistant to stretching, accessibility of the Y654 site is unaffected by the application of strain?6) The JCAb20 antibody recognises numerous bands in addition to the DE-cadherin (peptide). Based on the unspecific bindings it is not clear how valid the findings with this antibody are?7) Do the authors have an explanation why 85% of β-catenin does not alter the association with DE-cadherin during mechanotransduction? How do these 85% of β-catenin escape the dissociation and phosphorylation by Src? Summary:The authors describe a mechanotransduction process that induces a conformational change of a fraction of AJ-localized β-catenin leading to Src-mediated phosphorylation of β-catenin at Y654, release of pY654-β-catenin from E-cadherin, translocation into the nucleus and gene transcription. The findings are supported by Molecular Dynamics Simulation studies and a series of in vivo experiments. These findings are significant but the manuscript lacks several important controls.We thank the reviewers for all of their stimulating comments and questions on the work, and for the additional important controls suggested, which have all been added into the new version of the manuscript.Furthermore, the text of the paper is difficult to read. Sentences are fusions of several statements, figures are described in an unusual order , manipulations are not sufficiently explained , the Introduction is extremely brief consisting of 3 sentences followed by only 29 references. Also, the manuscript requires major editing.twi sna siRNA injections). And the Introduction was lengthened to introduce the context of the present finding in more detail.We also have followed all reviewers' advice regarding editing. The long sentences that contained several statements were shortened and cut into distinct sentences. The inverted Figure numbers were reordered . Manipulations were further explained when required  The lifetime differences of the antibody injected embryos are surprisingly small.“Calculation of the strain from FRET efficiency variations” in the Materials and methods section).Indeed, the lifetime differences of the antibody-injected embryos are small (~0.1 ns), but significant . This is in fact in agreement with the molecular dynamics simulation, in which the strain induces small conformation changes on the order of 1 nm for the distance between the GFP and A555 two chromophores attached to the a-helixes connected by the β-cat Y654/E-Cad D665 link, at the opening of the β-cat Y654/E-Cad D665 site . Such 1nm fine stretching leads to a life-time difference on the order of 0.1ns. This is given by the FRET efficiency equation r=R0((1/E)-1)^1/6 that links the distances r between the chromophores, and the donor lifetime tda . The Förster Radius for this particular FRET pair (GFP and Alexa 555) is R0=6.3nm (Thermofisher datasheet)  . […] The lifetime increase observed specifically in the mesoderm at gastrulation thus shows the existence of a mechanical stretching strain between the fluorescently labelled linear domain II of E-cad, and the C-terminus of β-cat, respectively, in the invaginating mesoderm.”In addition, given these small but significant differences, we increased the number of experiments (N=11), as suggested by the review below, to further test the robustness of the results. The results remained robust, with an increase of the statistical significance from p=0.0120 to 0.004 for the lifetime difference of the mesoderm at stage 6 compared to stage 5 . This is newly added in Figure 2C legend:“c Left – Quantification of the lifetime difference at different stages and different tissues. […] No significant change was observed in the ectoderm at stage 6 (N=10) compared to stage 5 (N=8) (p=0.74), and between the mesoderm and the ectoderm at stage 5 (p=0.33). In some embryos, the ectoderm could not be imaged properly."As it is very difficult to control the amount of antibody (and thus FRET acceptor) being present at different locations in the syncytial blastoderm one would expect much larger variations.The reviewers are correct that fluctuations could be expected to be large, due to putative inhomogeneities in the labelling of the acceptor at junctions into the mesoderm. Indeed, fluctuations are relatively large, on the order 0.5 ns compared to the 0.1 ns of the mean values in Figure 2C and 3D.However, we have verified that all junctions are labelled with the acceptor antibody . This observation indicates that fluctuations in the labelling, with variations in the FRET lifetime, should be smaller than under conditions including a mixture of non-labelled and labelled junctions. This possible origin of the observed fluctuations is now implemented in the Results section:“Furthermore, despite the fact that all junctions are labelled with the E-cad Alexa 555 acceptor , modulations in the labelling efficiency may be at the origin of variations observed in the lifetime.”To ensure that the reported values are reproducible the number of injected embryos should be increased (from N=8 embryos).1, Figure 4 of ref2 and Figure 4D of ref3). In the first version of the manuscript, we had generated 8 embryos for each condition. However, despite the significant p values of the results with 8 embryos, we agree that the fluctuations in the results discussed above suggested the conduction of additional experiments. We now have a total of N=11 embryos in which the lifetime increase was measured at stage 6 in the mesoderm during its invagination.In fact, the number of embryos tested for single embryonic mechanical manipulation is generally of 5-8 for each condition in mechanobiology, because of the time consuming nature of the experiments . Statistical tests are t-student.”Furthermore, an injection control is necessary, as uninjected embryos are not sufficient to control the experiment. Does water/PBS injection have an effect? Does this vary between the stages?We agree with the review comment. We have thus conducted new experiments in which embryos were injected with PBS alone. As expected, PBS injection without the antibody lead to a lifetime of 2.6 +/- 0.039 ns, larger compared to 2.47 +/- 0.074 ns with the antibody at stage 5 ((p=0.00016) see Figure 2—source data 1). In addition, PBS injected embryos did not show any variation of the lifetime during gastrulation stage 6, compared to before at stage 5 . Together with the non-injected β-cat-GFP control embryos, these new controls further confirm the presence of the acceptor labelled antibody as required for the observed increase in GFP life-time in the mesoderm during its invagination compared to before. This new control is now shown as new Figure 2C-right, and described as follows:“As a control, no change in the GFP donor lifetime was observed during gastrulation in the absence of injected antibody acceptor (E-cad Alexa 555 labelled AB), as well as in embryos injected with PBS alone in the absence of the antibody acceptor .”and in the Figure 2C legend:“As expected, PBS injection without the antibody acceptor lead to a lifetime of 2.6 +/- 0.039 ns, higher compared to 2.473 +/- 0.074 ns with the antibody at stage 5 . Statistical tests are t-student.”How was the lifetime fit done? Is it a mono-exponential fit?The lifetime was fitted with a mono-exponential. This information, that was effectively missing, is now added in the related Materials and methods section.Is it not surprising that the GFP donor lifetime is <2.6 ns in the absence of an acceptor? This is impressively short!In the absence of acceptor, we measured a GFP lifetime of 2.6 +/- 0.04 ns at any stage , which is consistently higher than the lifetime measured at stage 5 before the mechanical strains of mesoderm invagination, of 2.473 +/- 0.074 ns . Moreover, these values are on the order of magnitude of the lifetime of GFP tagged proteins measured in cellula, for instance of 2.6 ns coupled to Histone in the nucleus in the absence of an acceptor, and of 2.4 ns in the presence of an acceptor .This information has been added:“Note that the lifetime of the controls with GFP alone in the embryo junctions and with PBS injection without antibody, of 2.60 +/- 0.04 ns, , was consistently higher than in the presence of the acceptor before stretching, of 2.473 +/- 0.074 ns . These values are similar to the lifetime of GFP tagged proteins measured in cellula, namely of 2.6ns coupled to Histones in the nucleus in the absence of acceptor and of 2.4ns in the presence of an acceptor .”2) Why are double RNAi against twi and sna injected instead of using zygotic double mutants?twi sna zygotic mutants do not include the Arm-GFP (β-cat-GFP). To generate the cross between the sna twi /Cyo double zygotic mutants and Arm-GFP flies would have significantly delayed the project. Indeed, the twi sna RNAi treatment has already been described as efficient in blocking gastrulation just like twi sna double zygotic mutants . Furthermore, injecting siRNA for twi and sna to induce sna twi RNAi in Arm-GFP embryos leads directly to sna twi gastrulation defective embryos in the presence of Arm-GFP.Zygotic double mutants were not used because existing This information is now newly detailed with the RNAi sequences:“RNAitwi and sna defective non gastrulating Arm-GFP embryos, we injected siRNAs for twi and sna RNAi into Arm-GFP embryos. twi sna RNAi embryos have been described to phenocopy twi sna double zygotic mutants, and do not gastrulate6,7. 3 siRNA sequences were designed and produced by Eurogentec, and already used in Mitrossilis et al, 2017:To produce sna-siRNAs twi-siRNAs ”As well as, following, the conditions of injection with the UMLs described in Mitrossilis et al, 2017, and the characteristics of the magnetic conditions used to produce the deformation that mimics the initiation of apex constrictions initiating mesoderm invagination.The small differences the authors observe upon RNAi injections could be due to differential knock-down efficiencies, particularly if the number of treated embryos is so low (N=8).twi sna RNAi compared to the WT embryos . Despite the small number of embryos of the twi sna RNAi condition of the previous version of the manuscript (N=4), this in fact already showed a full inhibition of the increase of the lifetime in the twi sna RNAi injected embryos compared to the WT embryos of Figure 2c, at stage 6 compared to stage 5, with a statistically relevant difference between the two conditions at stage 6 with a p-value of p=0.016.There was effectively an inhibition of the lifetime increase observed at stage 6 compared to stage 5 embryos, in the This p-value was effectively not provided in the previous version of the manuscript. It is now present in the new version of the manuscript .twi sna RNAi without magnet, and N=9 in the twi sna RNAi in the presence of the magnet), which were effectively relatively low in the previous version of the manuscript. The legend of Figure 3D thus now states:This p value is now upgraded to p=0.0003 by following the reviewer’s advice to increase the number of experiments .”3) The rescue with the magnet should also be controlled with water injected embryos.twi sna RNAi non-gastrulating embryos. It is like the requirement of the acceptor to observe an increase in the lifetime in the mesoderm under endogenous gastrulation constraints in the WT, at stage 6 ). This information was added to Figure 3D:We agree that this control was missing. The magnet experiment was thus performed in the absence of the antibody, with PBS and UML injection alone. No increase or modification in the lifetime was observed . This observation confirmed the requirement of the acceptor to observe an increase in the lifetime in the mesoderm under magnetically controlled forces mimicking the apex constriction at the onset of gastrulation in sna twi RNAi non-invaginating conditions without magnet between stage 5 and stage 6 is characterized by the p-value p=0.0003 compared to the increase between stage 5 and 6 in the invaginating WT of Figure 2C-left, and of p=0.75 compared to the lifetime difference in the invaginating WT injected with PBS of Figure 2C-right (t-students test).”“d Lifetime difference of stage 6 to the average life-time before magnetic field application of stage 5, of N=9 experiments with magnet applied and N=8 without magnet applied as well as N=6 experiments with PBS instead of antibody injected and magnet applied. […] The absence of increase of the life-time difference within And in the Results:“As controls, no increase in the lifetime between stage 5 and stage 6 was observed after injection of the UMLs and RNAi without magnet , or with magnets after UML and PBS injection without the E-cadh Alexa 555 acceptor .”4) Quantification of the Y654 antibody stainings also requires a control experiment. The authors observe an increase in Y654 non-phospho antibody staining during gastrulation or upon Src knock-down. The authors seem to have normalized the Y654 non-phospho staining to general β-catenin antibody staining. An additional control, just outside of the mesoderm from the same embryo should be included, as the forces on the lateral cells should be less, the difference should also be less. Since the antibody signals can vary between embryos, such an internal standard makes it easier to compare different embryos.We agree that an internal control measurement in the ectoderm should re-enforce the significance of the results. We have thus measured Y654 non-phospho relative to general β-cat into the ectoderm at stage 6 compared to stage 5. Indeed, consistent with the force being less important in the ectoderm than in the mesoderm at gastrulation, we found no significant increase in both the WT and Src42A knock-down conditions . We additionally normalized the mesoderm Y654 antibody staining with the ectoderm Y654 antibody staining outside of the mesoderm. This procedure consistently showed an increase of the signal in both the WT and Src42A knock-down conditions . This, with associated p-values, is now implemented in Figure 4:src42A RNAi early stage 6 p=0.049, late stage 6 p=0.063, difference between the two curves p=0.021.”“Figure 4: Increase of accessibility of Y654-β-cat in the mesoderm during invagination. […] WT compared to 5) Is it possible to design a control experiment with mutant, stretch-resistant DE-cadherin and/or β-catenin and show that if the sites are made resistant to stretching, accessibility of the Y654 site is unaffected by the application of strain?We thank the reviewers for this suggestion. Its implementation, however, would have been excessively difficult to achieve in 3 months. Based on such a suggestion, we would propose, for future investigations, the design of relatively soft E-cad/β-cat proteins, for instance by adding soft coiled elements in the most linear domain of the E-cadherin between A648 and the membrane. All of the mechanical work energy being first of all stored in the coiled protein domain conformation change, this design would prevent the force to apply efficiently at the Y654-D655 site and to open it.6) The JCAb20 antibody recognises numerous bands in addition to the DE-cadherin (peptide). Based on the unspecific bindings it is not clear how valid the findings with this antibody are?We agree that the multiple non-specific bands of the western blot may suggest the possibility that the findings might have been independent on the specific labelling of E-cadherins. However, immunofluorescence labelling clearly shows a labelling specific of junctions, fully co-localized with β-catenin, and measurements were realised focused on the junctions plan only.To fully exclude the labelling of another junctional protein, that would only have been stabilized and observed if it was concentrated close enough to the β-catenin-GFP fluorophore  to show a FRET effect, we performed an immunoprecipitation assay with the JCAb20 antibody acting as trap by being attached to protein-A agarose beads. We found a single band in western blot recognized by the DE-cadherin antibody at the 140kDa molecular weight, which corresponds to the full length DE-cadherin protein, as newly introduced in Figure 2—figure supplement 1A . Note that we could properly synchronize 70 embryos at stage 5 for the immunoprecipitation experiments, which represents a relatively low amount of biological materials, and explains the relatively slight band of immunoprecipitated material.In contrast to the western blot procedure, immuno-precipitation uses non-de-naturating procedures for proteins, therefore the additional bands observed in the western blot are non-specifically associated to denatured proteins. This new result definitively demonstrates the high specificity of the DE-cadherin labelling by the JCAb20 antibody. It is now added to Figure 2—figure supplement 1A:“Figure 2—figure supplement 1: Specific labeling of junctional E-cad with JCAb20. […] Right: Immunoprecipitation by using the JCAb20 antibody as a trap, and the DE-cad antibody for revelation. The band is at the 140kDA weight of the mature DE-cad protein. Other bands around 40kDA are presumably immunoglobulins. Colored bands are pre-stained markers.”as well as newly described as follows:Drosophila proteome (taxid: 7227) .”“The antibody was tested for its specificity by immuno-staining, western-blot analysis and immunoprecipitation . The peptide sequence of the immunogen was checked for possible similarities with other proteins by a standard protein-protein BLAST search against the with the experimental procedure described in the Materials and methods section.7) Do the authors have an explanation why 85% of β-catenin does not alter the association with DE-cadherin during mechanotransduction? How do these 85% of β-catenin escape the dissociation and phosphorylation by Src?Our interpretation is that, once under tension, the deformation of the E-cadherin/β-cat complex can a priori statistically occur at different locations all along the complex structure, including at the Y654-D665 site. We further assume that in 15% of cases, the deformation occurs at the Y654-D655 site and opens it. Future investigations on the simulations would be required to quantitatively test this hypothesis.This interpretation is now added to the Discussion:“A possible interpretation for the observed 15% efficiency of site opening is that, once under tension, the deformation of the β-cat/E-cad complex can a priori statistically occur at different locations all along the complex structure. […] Future molecular dynamics investigations will be required to quantitatively test this hypothesis.”1) Hutson, M. S. et al. Forces for morphogenesis investigated with laser microsurgery and quantitative modeling. Science 300, 145-149 (2003).2) Fernandez-Gonzalez, R., Simoes Sde, M., Roper, J. C., Eaton, S. and Zallen, J. A. Myosin II dynamics are regulated by tension in intercalating cells. Dev Cell 17, 736-743 (2009).eLife 5, doi:10.7554/eLife.10757 (2016).3) Yu, J. C. and Fernandez-Gonzalez, R. Local mechanical forces promote polarized junctional assembly and axis elongation in Drosophila.


Proper chromosome segregation depends upon kinetochore phosphorylation by the Chromosome Passenger Complex (CPC). Current models suggest the activity of the CPC decreases in response to the inter-kinetochore stretch that accompanies the formation of bi-oriented microtubule attachments, however little is known about tension-independent CPC phosphoregulation. Microtubule bundles initially lie in close proximity to inner centromeres and become depleted by metaphase. Here we find these microtubules control kinetochore phosphorylation by the CPC in a tension independent manner via a microtubule-binding site on the Borealin subunit. Disruption of Borealin-microtubule interactions generates reduced phosphorylation of prometaphase kinetochores, improper kinetochore-microtubule attachments and weakened spindle checkpoint signals. Experimental and modeling evidence suggests that kinetochore phosphorylation is greatly stimulated when the CPC binds microtubules that lie near the inner centromere, even if kinetochores have high inter-kinetochore stretch. We propose the CPC senses its local environment through microtubule structures to control phosphorylation of kinetochores. How the chromosome passenger complex (CPC) phosphorylates the kinetochores that can be a micron away to control mitotic events is unknown. Here the authors find that the CPC directly binds microtubules near inner centromeres, which controls its ability to phosphorylate kinetochores independently of tension generated by kinetochore microtubule attachments. The inability to obtain biorientation is a major source of chromosomal instability in tumors3. The Chromosome Passenger Complex (CPC), a four-protein complex consisting of chromatin targeting subunits Survivin and Borealin, the scaffold INCENP and a kinase Aurora-B, controls biorientation as well as other mitotic events by phosphorylating kinetochore substrates and destabilizing kinetochore-microtubule attachments4. The majority of the CPC (~75%) is localized to the inner-centromere, which is the chromatin between kinetochores on mitotic chromosomes, during prometaphase and metaphase6. Inner centromere localization is believed to concentrate the protein to enable kinase auto-activation7. CPC recognizes the inner centromere via two distinct histone phosphorylation marks, Histone H3 phosphorylated on T3 (H3pT3)10 and Histone H2A phosphorylated on T120 (H2ApT120)14. The CPC phosphorylates kinetochore substrates that are greater than 500 nm away from inner centromeres16. Phosphorylation of kinetochore substrates such as the Ndc80 complex, by Aurora-B, is higher on unaligned kinetochores than metaphase-aligned kinetochores17, which may regulate many events including the maturation of kinetochore-microtubule attachments18. This is caused in part by recruitment of phosphatases to kinetochores after they obtain proper kinetochores attachments21, but most models suggest that the CPC’s ability to phosphorylate kinetochores is also decreased in metaphase24.Human kinetochores bind ~20 microtubules and faithful chromosome segregation requires that the majority of the microtubules attached to one sister kinetochore orient towards one spindle pole, while those of its sister orient towards the opposite pole (biorientation)23. Upon biorientation the pulling force exerted by the kinetochore bound microtubules increases the distance between the CPC and its kinetochore-localized substrates thus reducing the INCENP’s reach and therefore phosphorylation of kinetochore substrates. Another model suggests that the centromeric pool of the CPC activates soluble CPC that propagates to kinetochores via a reaction-diffusion mechanism that involves chromatin-bound CPC25. A pool of the CPC may directly localize to kinetochores26, however, direct binding of kinetochores is unlikely to be the only mechanism because depletion of the centromere-bound pool or expression of CPC mutants that do not localize to inner centromeres compromises the ability of Aurora-B to phosphorylate distant substrates27. Budding yeast and chicken DT40 cells do not require centromere localization for biorientation30, but the CPC in yeast require the ability to bind microtubules29.How the CPC phosphorylates kinetochores and why kinetochore phosphorylation is higher in unaligned chromosomes than aligned chromosome is a matter of intense research. It has been proposed that centromere anchored CPC uses an extended single alpha-helix (SAH) on the INCENP subunit to reach the kinetochore substrates and phosphorylate them32. Apart from tension sensitive mechanisms, the tension-independent mechanisms are also likely to be involved since some pro-metaphase kinetochores may also become stretched due to kinetochore localized motor activity on microtubule bundles that lie in close proximity to inner centromeres34.Many of these models suggest that the CPC is regulated by changes to the inner centromeric chromatin that results from the pulling forces exerted by microtubules bound to the kinetochores (inter-kinetochore stretch or centromeric tension)33. These observations suggested that there is distinct prometaphase state when inner centromeres are in close proximity with spindle microtubules that span from inner-centromeres to kinetochores and beyond. These inner centromere proximal microtubules are largely reduced by metaphase33 when they are replaced by the “end-on” attachments of mature kinetochore fibers (K-fibers). Moreover, the CPC was also shown to localize specifically to these centromere proximal microtubules in prometaphase35. Microtubules stimulate the CPC activity and auto-activation in vitro, and they are required for proper localization of the CPC to the inner-centromere37. Microtubules are also required for full activation of the CPC in a Xenopus extract system where the clustering of CPC by chromatin is replaced by activation by dimerizing antibodies38. The SAH domain of INCENP binds microtubules and is important for the maintenance of the paclitaxel-dependent SAC arrest40. However, it is unclear whether this region is required to correct improper kinetochore-microtubule attachments41.It was recently shown that the initial kinetochore-microtubule attachments in prometaphase place the inner-centromere regions adjacent to large bundles of microtubules that also run adjacent to sister kinetochoresHere, we investigated the role of CPC-microtubule interaction in regulation of kinetochore phosphorylation. Specifically, we have identified a microtubule-binding site in the Borealin subunit of the CPC, and show that this interaction is important for kinetochore phosphorylation, proper error-correction and for maintenance of paclitaxel-induced SAC-dependent arrest. Using theoretical approaches, we demonstrate that microtubules near the inner centromere stimulate kinase activity at adjacent kinetochores. Interestingly, microtubules extend CPC activity even further than kinetochores at their most stretched state, suggesting that the regulation by microtubules is independent of chromatin changes by tension. Our model also demonstrates that end-on attached K-fiber microtubules do not stimulate phosphorylation of kinetochores to the same extent. Together these findings uncover a tension-independent layer of kinetochore regulation where local changes to microtubules near inner centromeres stimulate kinetochore phosphorylation by the CPC.E. coli. Our preparations contained the first 48 amino acids of INCENP tagged on the N-terminus with 6-histidines, and full-length Survivin and Borealin (ISB)). ISB was mixed with paclitaxel-stabilized microtubules and the protein that remained bound to microtubules after sedimentation was quantified by immunoblot. The ISB sub-complex bound taxol-stabilized microtubules with an apparent Kd of ~164 nM   also reduced the affinity for microtubule binding  Fig. . DeletinWT complex also bundled paclitaxel-stabilized microtubules in a concentration-dependent manner  than LAP-BorealinWT cells -tagged BorealinUTR Fig.  and imaglls Fig. , demonstlls Fig. . We alsonWT Fig. .Fig. 2In43. Cells expressing the BorealinMTBM or BorealinΔ20 doubled the number of anaphases with lagging chromatids over controls demonstrating a requirement of the Borealin MBD in error correction  or the INCENP (INCENPΔSAH) MBD or both and determined the percent of cells undergoing anaphase with lagging chromosomes. Cells lacking either the Borealin or the INCENP MBD had two-fold more anaphases with lagging chromosomes (~40%)  Fig. . Cells l0%) Fig. . These c0%) Fig. . We concWT, BorealinMTBM or BorealinΔ20 by live imaging upon treatment with 100 nM paclitaxel , the concentration of chromatin-bound CPC exceeds activation threshold, so kinase activity is high at the Ndc80 site of the kinetochore  was reduced when Borealin-microtubule interaction was disrupted  compared to either mutant alone   Fig. . In contμM) Fig. . These dMTBM, since the CPC controls the H2ApT120 pathway by phosphorylation of kinetochore substrates MPS1 and the Ndc80 complex49.To gain insight into molecular mechanism of the CPC localization defect, we examined whether the Borealin MBD controls either of the two-histone phosphorylation feedback loops that localize the centromeric CPC. Both the H2A pT120 phospho-histone mark and the Sgo1 levels at the kinetochores are both reduced about 25% in the microtubule-binding mutants Fig. . However747-918 with the DNA binding domain of the CENP-B, which recognizes alpha satellite DNA. This chimeric protein should be incapable of stretching significant distances and binding microtubules since it cannot bind Borealin and Survivin and lacks the SAH domain of INCENP .40. Simultaneous depletion of microtubule binding activities on INCENP and borealin results in an increase in cells with anaphase lagging chromosomes compared to removing either alone. The CPC also binds other microtubule interacting proteins like kinesin MKLP-2, EB1, and GTSE1, suggesting that indirect interactions may also regulate the CPC54. Microtubules regulate Aurora kinase activity but it is unclear if there are additional reasons why the CPC requires these multiple microtubule interactions. For example, the CPC may play a structural role in building microtubule structures. Consistent with this idea the ISB complex built microtubule bundles in vitro and the CPC is intimately tied to microtubule bundle structures in vivo, as it both localizes to and coordinates the assembly of midzone microtubules in anaphase and preformed K-fibers in prometaphase cells57. In fact, a similar mutant in the borealin MTBD has been shown to have reduced localization to midzone microtubule bundles42.The ISB MTBD described here is the third direct microtubule-binding region on the CPC along with the PR/SAH domain on INCENP and a microtubule binding region on Aurora-B/IN-Box37, and why multiple-microtubule binding domains that are required for viability in yeast29. How the CPC in the inner centromere controls phosphorylation of adjacent kinetochores is an important unanswered question. We provide mathematical modeling and in vivo evidence that the inner centromere pool of the CPC is required to generate active kinase, but the spreading of kinase activity to kinetochores is enhanced by its ability to interact with microtubules. Moreover, we show that the CPC that phosphorylates kinetochores needs to interact with microtubule but not chromatin.The MTBD of Borealin is important for full kinetochore phosphorylation and mitotic functions. This finding explains recent results demonstrating that the centromere-targeting domain of the CPC has roles independent of centromere targeting during paclitaxel-dependent checkpoint arrest59. Second, merotelic attachments bring the merotelic K-fiber microtubules adjacent to inner centromeres60. Third, electron micrographs have suggested that the microtubule density in the center of the prometaphase spindle excludes chromosomes so that the chromosome arms fold back so that telomeres are pointed away from the spindle and inner centromeres are proximal to the spindle microtubules33 . Here we provide evidence that the CPC can also regulate kinetochores by a “tension-independent” mechanism, where microtubules adjacent to inner-centromeres enable robust phosphorylation even if kinetochores are fully stretched. Since CPC binding to microtubules stimulates its kinase activity in vitro, the idea that specific microtubule structures stimulate CPC phosphorylation of kinetochores is appealing. There are at least three chromosome configurations on the mitotic spindle that would place inner centromeres adjacent to microtubules to stimulate robust kinetochore phosphorylation. First, they may be generated by preformed K-fibers, which are microtubule bundles that specifically bind the CPC and emanate from kinetochores before they generate end-on microtubule attachmentss33 Fig. . In thes24. In the previous model, the chromatin-bound pool of the CPC in the inner centromere strongly activates Aurora-B kinase because this is the place of the highest Aurora-B concentration. This pool is in a constant exchange with the soluble CPC pool, which sustains high kinase activity of the chromatin-bound CPC where it is less abundant  were grown in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum (Gibco) in a humidified incubator at 37 °C in presence of 5% COWT, LAP-BorealinMTBM, LAP-BorealinΔ20, LAP-PRC1MBD-BorealinΔ20, LAP-BorealinT230E and LAP-BorealinMTBM/T230E, the BorealinWT transgene fragment was sub-cloned in to pCDNA5/FRT vector (Invitrogen) containing N-terminal LAP (GFP and S-peptide) tag sequence. QuickChange II XL site-directed mutagenesis kit (Agilent) was used to generate all the point mutations and deletions constructs. All primers used for site-directed mutagenesis and cloning are described in Supplementary Table DBD-INCENP747–918 and mCherry-BorealinWT or mCherry-BorealinMTBM transgenes, the GFP-CENP-BDBD-INCENP747-918 was sub-cloned downstream of Tet-operator binding site. CMV promoter containing fragment of mCherry-BorealinWT or mCherry-BorealinMTBM was cloned at the 3′ end of GFP-CENP-BDBD-INCENP747-918. This whole cassette of GFP-CENP-BDBD-INCENP747-918 and mCherry-BorealinWT or mCherry-BorealinMTBM was then cloned into pCDNA5/FRT vector (Invitrogen). The resulting plasmids were co-transfected with the pOG44 plasmid (Invitrogen) with Lipofectamine 2000 (Invitrogen). Hygromycin B (Invitrogen) 200 μg/ml was added one-day post transfection and the cells were selected for 15 days. After the selection period, the surviving colonies were pooled and FACS sorted for GFP  or mCherry  expression to get cells expressing equal amount of the transgene.In order to generate HeLa T-REx cells (HeLa-TREx cells were a gift from Dan Foltz lab) stably expressing LAP-BorealinWT-GFP or vsv-INCENPΔSAH-GFP with mCherry-BorealinWT or mCherry-BorealinMTBM transgenes. The Tet-inducible HeLa T-REx cells expressing vsv-INCENPWT-GFP or vsv-INCENPΔSAH-GFP  were infected with virus carrying mCherry-BorealinWT or mCherry-BorealinMTBM transgenes in the presence of 8 µg/ml polybrene (Sigma). The double stable cells were then selected for 10–12 days in presence of Puromycin (Invitrogen) at 1 μg/ml and Hygromycin B at 200 μg/ml. The surviving colonies were pooled and FACS sorted for mCherry expression to obtain double stable cell lines.For generating cells expressing vsv-INCENPWT or mCherry-BorealinMTBM transgenes were cloned into pBABE-Puro retrovirus vector using cold fusion cloning kit (System Biosciences). HEK-293GP cells were co-transfected with pBABE-Puro-mCherry-Borealin (WT or MTBM) and VSVG plasmid in order to package pseudotyped MULV viruses. The viruses were collected 3 days post transfection by filtering the media through 0.45 μm syringe filter.For making retrovirus, mCherry-BorealinFor plasmid transfection cells were grown to 80–90% confluence followed by plasmid transfection using Lipofectamine 2000 (Invitrogen) according to the manufacture’s protocol.In case of siRNA transfection in order to avoid the indirect effect we analyzed the first mitosis after depletion of the target proteins. To achieve this, cells were plated in presence of 2 mM thymidine, 24 h after plating, cells were released into fresh media and siRNA was transfected using RNAiMAX (Invitrogen) according to the manufacturer’s protocol. Another round of siRNA transfection was done after 10–12 h of 1st siRNA treatment and at the same time 2 mM Thymidine was added. Cells were released into fresh media 12h after the second siRNA treatment. For immunofluorescence analysis cells were fixed after 8–10h of second thymidine release or after the indicated treatment. Depletion of the protein upon siRNA treatment was assessed by western blotting. Uncropped scan of all the blots are shown in Supplementary Figs. 2. Cells were fixed and stained with DAPI and mounted on coverslips using ProlongGold antifade (Invitrogen). Sequences for all the siRNA used in this study are in Supplementary Table For STLC washout assay 6–7h after the second thymidine release 5 μM STLC was added and cells were incubated for 2h. After 2h of incubation STLC was washed out of the cells by washing with PBS followed by 1.5h of incubation at 37 °C in presence of 5% CO2.For live cell imaging, cells were plated in the 4-well Lab-Tek II chambered coverglass (Thermo Fisher Scientific) in presence 2 mM thymidine followed by siRNA treatment. After 3–4 h of the 2nd thymidine release 200 nM SiR-DNA (Cytoskeleton Inc.) dye was added to the cells. One and a half hour after SiR-DNA treatment time-lapse images were taken at 5 min interval for 15 h on a Zeiss Axio-observer-Z1 in a humidified environmental chamber maintained at 37 °C in presence of 5% COg for 10 min and the supernatants was incubated with GFP antibody (a kind gift from Dan Foltz) for 3 h at 4 °C. Equilibrated protein-A beads (GE Life Sciences) were then incubated with the antibody lysate mixture for an additional hour. The beads were washed with lysis buffer 3 times. The washed beads were re-suspended in 2× sample buffer and loaded on to SDS–PAGE gel after brief boiling at 95 °C, desired proteins were detected in the immune-precipitate by western blotting. Uncropped images of the western blots are shown in Supplementary Fig.For immunoprecipitation, HeLa cells were synchronized to mitosis with 0.33 μM nocodazole for 16h. Mitotic cells were collected and lysed in CPC lysis buffer  and sonicated using Bioruptor-300 (Diagenode) for 30 cycles with 30s on and 30s off at 4 °C. The whole-cell lysate was cleared by centrifugation at 14,000 × l-Lysine (Sigma) and indicated siRNA transfection and subsequent indicated treatment were done. The cells were then co-fixed with 4% paraformaldehyde in PHEM buffer  supplemented with 0.5% Triton-X 100 for 20 min at room temperature. The cells were then washed 3 times with Tris buffered saline (TBS), followed by 1 h blocking with 3% BSA at room temperature. Fixed cells were then incubated with indicated primary antibodies for 1 h at room temperature. Concentration and source of all the antibodies used in this study are in Supplementary Table 35. Images were processed and analyzed using Volocity . To quantify fluorescence levels at centromeres, we used an intensity thresholding algorithm to mark all centromeres on the basis of ACA or GFP  and box representing 25–75 percentile and median is the line in the boxes) using Prism software (GraphPad) and indicated two tailed statistical tests where applied. For all the immunofluorescence experiments data from one out of atleast 2 repeats is shown.HeLa T-REx cells were seeded onto coverslips coated with poly-for Fig.  intensit1-58-Survivin-Borealin complex was expressed in BL21-pLysS (DE3) cells from a tri-cistronic pET28a vector containing 6XHis-INCENP1-58-Survivin-Borealin sequence. ISBMTBM and ISBT230E were generated by site directed mutagenesis. For GFP-ISB construct, the ISB construct was modified by sub-cloning GFP between 6His tag and INCENP1-58. Cells were grown in 2XYT media in presence of 30 μg/ml Kanamycin. Protein expression was induced at O.D. 0.6 by addition of 0.45 mM IPTG and the media was supplemented with 0.2% glucose 60 μg/ml ZnCl2, protein expression was carried out for 16–18 h at 18 °C. Cells were subsequently pelleted and lysed in buffer containing 50 mM Tris, pH 7.5; 500 mM NaCl; 0.5 mM TCEP; 5 mM Imidazole; 5% glycerol and protease inhibitor cocktail (Roche) using EmulsiFlex-C3 Homogenizer. Lysate was cleared by centrifugation at 95,834 × g for 1 h. Cleared lysate was then mixed with Ni-NTA beads (Qiagen) for 4 h at 4 °C. Ni-NTA beads (Qiagen) where then washed with 200 ml buffer containing 25 mM Imidazole, 50 mM Tris pH 7.5, 500 mM NaCl, 0.5 mM TCEP and 5% glycerol. The Protein was then eluted with buffer containing 250 mM Imidazole, 50 mM Tris pH 7.5, 500 mM NaCl, 0.5 mM TCEP and 5% glycerol. Upon elution the proteins were gel filtered on Superdex-200 column 10/300 GL size-exclusion column (GE Life Sciences). Gel filtration was done in buffer containing 50 mM Tris pH 7.5, 500 mM NaCl, 0.5 mM TCEP and 5% glycerol. Upon gel filtration the desired fractions were pooled and concentrated with Amicon Ultra-4 Centrifugal Filter Unit with 3 kDa cutoff.INCENP2, 1 mM EGTA, pH 6.8 with NaOH), 50 mM NaCl, 1 mM DTT and 1 mM GTP with increasing concentration of taxol, taxol-stabilized microtubules were then separated from the un-polymerized tubulin dimers by centrifuging through a 40% glycerol cushion at 137,000 × g. Various concentrations of taxol-stabilized microtubules were mixed with 100 nM of ISB  in BRB80, 1 mM DTT, 50 mM NaCl and 20 μM paclitaxel. Samples were allowed to equilibrate at room temperature for 15 min. Samples were then layered onto a 50% glycerol cushion and centrifuged at 279,000 × g for 10 min, and both the supernatant (S) and pellet (P) were collected and resuspended in SDS sample buffer, and equal amounts of supernatant and pellet were run on 15% SDS-PAGE gels followed by western blotting. Quantification of the relative amounts of ISB in supernatants and pellets was performed using ImageJ . The dissociation constants measured by fitting the data from three separate experiments to the one-site specific binding equation using Prism software (GraphPad).Taxol-stabilized microtubules were prepared by polymerizing bovine brain tubulin dimers in BRB80  biochemical reactions of Aurora B phosphorylation-dependent auto-activation (in cis and in trans) and its phosphatase-dependent inactivation, (2) the reactions of Aurora B kinase binding/unbinding to the centromere-localized binding sites and (3) diffusion of the soluble kinase and phosphatase pools.General model framework: Quantitative analysis of the spatial distribution of Aurora B kinase activity was carried out based on our previously published mathematical model24.Briefly, the model is built upon the fact that Aurora-B kinase can activate itself, so its activation is strongly concentration-dependent Fig. , in the 5. To model the metaphase configuration, tension is applied to stretch the concentration profile of the chromatin-bound CPC, reducing its concentration everywhere along the centromere–kinetochore axis. This shifts the kinase activity boundary slightly closer to the centromere relative to the Ndc80 site, where CPC concentration drops below the threshold. Thus, the model recapitulates lower kinase activity at the Ndc80 site of the stretched vs. unstretched kinetochores , concentration of chromatin-bound CPC is higher than the activation threshold everywhere throughout the centromere, leading to a high level of Aurora B kinase activity at the Ndc80 site of the kinetochore Fig. . Due to res Fig. , providiAdditionally, we have now incorporated into this model (4) the interactions between kinase and microtubules. Two configurations were compared, in one microtubules are bound to the kinetochore end-on (bi-oriented) and in another they run adjacent to the centromere .We assume a rapid equilibrium in the binding-unbinding of Aurora B kinase to chromatin-localized and microtubule-localized binding sites. In our previous model, rate of kinase binding to chromatin was estimated to exceed the rate of formation of the enzyme-substrate complexes by more than three orders of magnitude. Much faster rate of kinetic binding/unbinding reactions allows considering these reactions at steady-state. After the steady-state kinase pools are defined , equations for biochemical reactions of phosphorylation/dephosphorylation are solved to determine the fraction and concentration of active kinase within each pool and the diffusion-mediated spatial profiles for these different kinase forms depending on tension or microtubule geometry.5. Specifically, in all calculations concentration of soluble kinase is set at 10 nM. Also, the total number of chromatin-binding and microtubule-binding sites is chosen such that the soluble pool represents ~ 17% of the total kinase , similar to the 25% fraction estimated in cells5.To simplify calculations, we used one-dimensional model representation along the centromere–kinetochore axis. Model parameters . The bound forms of kinase can phosphorylate soluble kinase molecules with similar enzymatic rate constants, but the catalytic rate constant for microtubule-bound kinase is assumed to be 2-fold higher . Parameter s = 6/µM defines steepness of this profile, matching experimental distribution. Parameters cent and k are used to scale the profile in response to tension: with no tension k=1.1 and cent = 0.55 (corresponding to NDC80-NDC80 distance 1.02 µm), while for the fully stretched centromere k =2.3 and cent = 0.8 (corresponding to NDC80-NDC80 distance 1.64 µm). These NDC80 distances and therefore NDC80 locations on all graphs are based on previous findings63.Spatial distribution profiles of kinase-binding sites: Spatial distribution of the chromatin-localized CPC-binding sites along centromere–kinetochore axis, 64. Also, the ends of kinetochore microtubules within one kinetochore fiber do not end in the same location, spreading up to ~200 nm along this axis, so some microtubule ends appear to be surrounded by chromatin. It is unknown whether such geometry results in a significant presence of chromatin-bound CPC near the NDC80 substrate. It is also unclear whether activity of the chromatin-bound kinase toward NDC80 is similar to that of the microtubule-bound and soluble kinase forms. We note, however, that presence of the chromatin-bound kinase at the NDC80 site in our model does not affect its major conclusions. This is illustrated with Supplemental Fig. This profile of the chromatin-localized CPC-binding sites is represented with a smoothly decaying (differentiable) function, simplifying model calculations and resulting in the “chromatin” slightly extending beyond the NDC80 site. It is unclear whether this feature of the employed profile is physiological. High resolution electron microscopy images of the kinetochore-microtubule interface in human cells show that chromatin threads often extend along the walls of the end-on attached microtubulesMTtotal is concentration of CPC binding sites on microtubules, q = 100/µm is parameter that defines shape of the microtubule ends distribution, and x0 = 0.8 µm is position of the microtubule ends at the stretched kinetochore, coinciding with NDC80 location. Parameter α describes microtubule abundance. Because our model is one-dimensional, it is difficult to ascribe specific number of microtubules to this parameter. However, this approach allows examining the relative effects of different number of kinetochore microtubules attached end-on vs. centromere-proximal. Unless stated differently, parameter α = 1.The profiles of the CPC-binding sites on microtubules were different for different microtubule geometry. For the end-on attached microtubules we used:For centromere-proximal microtubules, we used constant level of binding sites:For kinetochore with both end-on and merotelic microtubules, the end-on microtubules were simulated as described above. Merotelic microtubules were described using the following profile:Asol – soluble concentration of Aurora B kinase ; MTProfile(x) – steady-state concentration profile of the microtubule-bound kinase ; Profile(x) – steady-state concentration profile of the chromatin-bound kinase ; Calculation of the steady-state distributions of the bound kinase pools: Bound kinase pool is the sum of all chromatin- and microtubule-bound kinase forms, including active and partially active kinases, kinase complexed with phosphatase and the enzyme-substrate complexes. Steady-state one-dimensional distributions for bound kinase pools were determined using the following expressions:To examine model predictions in the absence of CPC-microtubule binding, calculations were carried out as in the full model but with Choice of model parameters: All model parameters and their values are listed in Supplementary Table 24. Parameter B0, the maximum concentration of chromatin kinase binding sites at x=0, was set at 30 µM to match the peak concentration of chromatin-bound kinase in previous model: 20 µM. For simplicity, the association rate constant kon was assumed the same for microtubules and chromatin, so 62.Binding constants. For Aurora B kinase interaction with chromatin, we used same parameters as used previouslysee Fig. . With th65.Enzymatic constants: Characteristics of cytoplasmic phosphatase toward CPC are not known, so its catalytic activity was free parameter, which was adjusted to optimize model behavior. The catalytic rate of active Aurora B kinase is based on previous findings2/s. The diffusion rate of the full CPC, which has additional two microtubule binding sites and is likely to be dimeric, could be lower. For main model calculations we used DMT = 0.1 µm2/s. Additionally, we varied this coefficient to examine how the rate of CPC diffusion affects the distribution profile of the microtubule-bound active kinase. Importantly, the model remains sensitive to microtubule configuration when this coefficient ranges from 0.005 and 0.7 µm2/s  but measurements for human CPC complex were lacking. Therefore, to justify our assumption about microtubule diffusion of CPC, we examined behavior of purified ISB-GFP protein on taxol-stabilized microtubules labeled with HiLyte-645. Single molecule TIRF visualization and data analysis were carried out as previously mentioned, except motility buffer was prepared with 10mM DTT in place of beta-mercaptoethanol. Figure Full set of biochemical equations: The following system of differential reaction-diffusion equations was used:x = 0 for the left boundary and x = R for the right boundary of the simulated spatial segment. Calculations here were carried out for R = 10 µm.Boundary conditions were chosen to avoid the flow of soluble components:Following initial conditions were used:Additionally, the sums of all bound and soluble kinase forms and soluble phosphatase forms were constrained:Equations were solved numerically using Mathematica software (Wolfram Research) with total simulation time 50,000s using automatic time and space step size option to ensure convergence.Further information on experimental design is available in the Supplementary InformationPeer Review FileReporting Summary