Antifreeze proteins (AFPs) are biological antifreezes with unique properties, including thermal hysteresis (TH), ice recrystallization inhibition (IRI), and interaction with membranes and/or membrane proteins. These properties have been utilized in the preservation of biological samples at low temperatures. Here, we review the structure and function of marine-derived AFPs, including moderately active fish AFPs and hyperactive polar AFPs. We also survey previous and current reports of cryopreservation using AFPs. Cryopreserved biological samples are relatively diverse ranging from diatoms and reproductive cells to embryos and organs. Cryopreserved biological samples mainly originate from mammals. Most cryopreservation trials using marine-derived AFPs have demonstrated that addition of AFPs can improve post-thaw viability regardless of freezing method (slow-freezing or vitrification), storage temperature, and types of biological sample type. Antifreeze proteins (AFPs) are biological antifreeze materials originally found in polar fish; AFPs can bind to ice and subsequently inhibit the growth of the ice crystals. Fish can inhabit ice-laden or cold seawater below the freezing point (−0.7 °C) of their blood serum by virtue of AFPs ,2,3,4. HColwellia sp. [Flavobacterium frigoris [Glaciozyma antarctica [Navicula glaciei [Fragilariopsis cylindrus [Chaetocero neoglacile [Marine organisms known to possess or express AFPs, as shown in llia sp. , Flavobafrigoris , Glacioztarctica ,22, Navi glaciei , Fragilaylindrus ,24, and oglacile . The unioglacile ,29,30,31Generally, AFPs can be characterized based on two properties: TH and IRI. However, interaction of AFPs with membranes should not be ruled out. In this section, the unique features of AFPs and their contribution to cryopreservation are discussed briefly .TH refers to the difference between melting and freezing points of a solution. In AFP-containing solution, the temperature gap can be created by irreversible binding of AFPs to ice crystals and subsequent inhibition of their growth until the temperature decreases to the non-equilibrium freezing point ,37,38,39The second function of AFPs, which may be more useful for cryopreservation, is IRI. Ice recrystallization (IR), as depicted in Both TH and IRI properties are based on the affinity of AFPs for ice. Intriguingly, however, TH activity is not always proportional to IRI activity , which rAlong with the IRI feature, the interaction of AFPs with membranes (or proteins in membranes) also may also ameliorate the cryoinjury of cells. In the early study of Rubinsky and his colleagues, fish AFPs were found to protect cell membranes during hypothermic storage . As membPseudopleuronectes americanus [Two scientists, Scholander and DeVries, first observed that some fish inhabiting the polar oceans could survive in cold water that occasionally reached sub-zero temperatures ,3,97. Foericanus . In addiAFGPs contain a three amino-acid (Ala-Ala-Thr) repeating sequence motif with a disaccharide connected to the hydroxyl group of the threonine residue . HoweverMyoxocephalus Scorpius; ss3 AFP), also displaying a high Ala content . The structure of the ss3 AFP (PDB code 1Y03) was determined by NMR spectroscopy. The overall structure of ss3 AFP is similar to that of HPLC6 AFP; however, ss3 AFP contains a Pro residue at position 4, inducing a helix kink. Recently, the four-repeat containing isoform AFP9 and a much larger type I AFP  were discovered in winter flounder (Pseudopleuronectes americanus). These two proteins exhibit significantly higher TH activities than HPLC6 AFP. Moreover, the increased size of the AFP may induce higher antifreeze activity by facilitating binding to multiple ice crystals and increasing coverage of the ice surface. Furthermore, analysis of the AFP Maxi structure (PDB code 4KE2) revealed that this protein folds into a dimeric four-helix bundle and that its ordered water may be involved in ice binding, thereby enhancing its antifreeze activity.Type I AFPs are found in many flounders and sculpins. Type I AFP HPLC6 from winter flounder has been the most extensively studied. This protein possesses 37 amino acids and its sequences are composed of 11 amino acid repeating units ,105. MorPseudopleuronectes americanus) and Alaskan plaice (Pleuronectes quadritaberulatus) adsorb onto the {2 0 −2 1} pyramidal planes of ice, whereas the sculpin  AFP adsorbs onto {2 −1 −1 0}, the secondary prism planes [The ice-binding mechanism of type I AFP was previously investigated through an ice-etching experiment, which is used to identify AFP binding sites. A simple crystal growth and etching technique allows the identification of the crystallographic planes where the binding occurs . Furtherm planes . This fim planes ,110,111.Type II AFPs are found in sea raven, smelt, herring, and long snout poacher. Type II AFPs are globular cysteine-rich fish AFPs with molecular weights ranging from 11 to 24 kDa. The overall structure of type II AFPs shows numerous similarities with C-type lectin-like domains (CTLDs). Type II AFPs have two α-helices and nine β-strands with specific cysteines forming disulfide bonds. Those disulfide bonds are known for their capacity to increase the structural stability of type II AFPs ,115,116.2+-dependent type II AFPs. Herring type II AFP (hAFP) has close structural similarities with lithostathine  and mannose-binding protein . However, these two proteins have no ice-binding activities. Likewise, hAFP has no carbohydrate-binding activity. Thus, this high similarity in carbon backbone structure along with different activities indicates a divergent evolutionary pattern. Another difference between hAFP and C-type lectin protein is the number of cysteine bonds. hAFP has five disulfide bonds, whereas C-type lectin only possesses three or four. Thr96, Leu97, Thr98, and Thr115 residues are important for ice-binding. Interestingly, all of these residues are located near the Ca2+ binding site. Therefore, the results obtained from these investigations suggest that Ca2+ binding in hAFP is critical for forming an ice-binding state structure and increasing ice-binding activity [2+-independent type II AFPs. Structural comparisons between Ca2+-dependent and -independent type II AFPs showed that several residues near the Ca2+ binding site are different. Gln92, Asp94, Glu99, and Asn113 residues of hAFP are substituted with Lys95, Asn97, Asp102, and Asp116 residues, respectively, in long snout poacher AFP (lpAFP). Through these studies, the critical amino acids for Ca2+ binding were identified. These amino acids could be important indicators allowing the distinction between Ca2+-dependent and-independent type II AFPs [Hypomesus nipponensis; HniAFP), which does not inhabit polar regions, but instead is found in fresh waters in regions near the middle latitudes. Interestingly, HniAFP can bind to Ca2+, but its ice binding activity does not depend on this feature; indeed, despite adding ethylenediaminetetraacetic acid (EDTA) to remove Ca2+, its antifreeze activity was not affected [Type II AFPs are distinguished by their dependence on calcium ions to enable their antifreeze activities. Herring and two types of smelts produce Caaffected .Macrozoarces americanus) and wolf fish [http://www.rcsb.org/pdb/) to date. Among these, the three-dimensional structure of HPLC12 AFP, belonging to the QAE1 subgroup, was the first to be determined, showing a globular β-sandwich consisting of two antiparallel triple-stranded β-sheets [Type III AFPs are small globular proteins with an average molecular weight of 6.5 kDa, found in Antarctic eelepout  and therefore have no antifreeze activity [The most widely accepted hypothesis to describe the mechanism through which type III AFPs interact with ice crystals involves the Thr18 residue located on the flat surface; this residue is thought to be responsible for the recognition and interaction with the primary prism planes of ice. AFPs cover water-accessible ice surfaces, thereby inhibiting ice growth. Several reports have shown that putative ice-binding residues  are capable of significantly altering TH activity and ice crystal morphology ,131,132.activity .Rhigophila dearborni [Interestingly, two almost identical type III AFP domains tied by linker residues, designated RD3, were found in nature in the Antarctic eelpout, earborni ,138,139.Typhula ishikariensis), and two yeast organisms (Glaciozyma antarctica and Glaciozyma sp. AY30) have been characterized both genomically and for their antifreeze properties [To date, various mushrooms and Basidiomycetous psychrophilic yeast species have been screened and reported to have antifreeze activities. Only two mushrooms (enoki and shiitake), one snow mold fungus  secretes seven antifreeze protein isoforms composing the TisAFPs [Snow mold fungus ( TisAFPs . Among t TisAFPs . The resGlaciozyma antarctica AFP (Afp1), described by Hashim et al., possesses both TH and RI activities and shows 30% sequence similarity with TisAFPs [Glaciozyma antarctica AFP (Afp4) was identified and characterized [ TisAFPs . Amino-a TisAFPs . In addicterized . The AfpChaetoceros neogracile, Berkeleya sp., Navicula sp., Fragilariopsis sp., and Nitzschia frustulum) have been performed, and further gene expression studies have shown that the expression of AFP genes is regulated in response to stress conditions, such as cold temperature and high salinity [C. neogracile, and characterized its antifreeze activity [Studies aiming to identify new AFP genes from polar sea diatoms , Psychromonas ingrahamii , and marine bacterium Shewanella frididimarina .In 2004, Gilbert et al. published an interesting finding showing bacterial AFP screening results obtained from Antarctic lake bacteria . The autin SLW05 . In 2008in SLW05 . The seqMarinomonas primoryensis; MpAFP) [The first bacterial AFP structure was solved using a protein isolated from an Antarctic lake bacterium  . MpAFP i; MpAFP) . The solF. frigoris PS1 was identified from sea ice on the shore of McMurdo Sound (GenBank accession no. AHKF00000000.1) and characterized [Recently, another IBP (FfIBP) from the Antarctic bacterium cterized ,151. FfIcterized . These sColwellia sp. strain SLW05 (ColAFP) were published [In 2014, structural and biochemical data on an AFP from ublished . Interesublished . One theublished . IBPs seublished .Euplotes focardii revealed two sequences encoding IBPs, designated as EFsymbAFP and EFsymbIBP, obtained from its putative bacterial symbiont [E. coli and characterized [Furthermore, a new bacterial AFP  with higsymbiont ,155. Thesymbiont . Of thesCryopreservation is an important technique used to store various types of cells, tissues, and organs at very low temperature, usually in liquid nitrogen (−196 °C) , and hasDuring the slow-freezing process, since the solute concentration inside a cell is higher than that in the medium, the cell is supercooled and ice forms extracellularly . The groVitrification is a process in which a liquid turns into an amorphous glass solid in the absence of crystallization . VitrifiThe first application of marine AFPs to the protection of membranes at hypothermic temperatures was made in 1990 using AFGP from Antarctic and Arctic fishes . Since tCryopreserved biological samples are relatively diverse ranging from diatoms and reproductive cells to embryos and organs B. Most cE. coli [In the cryopreservation of cell lines, AFPs have been used as additives to conventional freezing medium to reduce the high amount of cytotoxic CPAs and reduce freezing damage . Some ofE. coli , and humE. coli  includinE. coli ,162,164.E. coli ,201. TheE. coli , diatomsE. coli , oocytesE. coli , and mamE. coli , whereasE. coli . Since tEmbryos from fish ,196,202,Lee and colleagues evaluated the beneficial effects of AFPs in vitrification of mouse ovarian tissues ,67. OvarFew studies have evaluated the potential use of AFPs in the hypothermic storage of organs ,184,204.Thanks to their unique properties as biological antifreezes, AFPs have attracted interest from researchers in academia and biomedical fields. In this review, we surveyed the past and current trends in cryopreservation applications of AFPs. The first property of freezing point depression, termed TH, has typically been utilized primarily in the hypothermic storage of tissues and organs. Due to the complexity and size of tissues and organs, more advancement is needed to achieve effective hypothermic storage of these biological materials. The ability to inhibit ice recrystallization is known to neutralize the catastrophic large icy environment for the cryopreserved cells during freezing and/or warming. The third and less characterized function of AFPs is the interaction with cellular membranes and/or integral membrane proteins. It is not likely that these interactions themselves can confer the cryopreserved cells with post-thaw viability. However, AFPs are thought to augment the viability or cryopreservation efficiency of the cells together with the other two features, particularly IRI.Our physicochemical understanding of unique binding of AFPs to ice crystal has been the main focus of scientists within the last five decades ,207. RelGlaciozyma IBP, has been reported [For the application of AFPs to be practical, a few questions should be addressed. First, mass production of AFPs should be established. Currently, only type III AFP has been produced on the industrial scale owing to its use as an ingredient in ice cream. However, advancements in molecular biology and genomics have improved our ability to produce genes and proteins easily, expanding AFP-related research. Indeed, a mass production system for LeIBP, reported . Additioreported ,144,145.reported . Third, reported ,209. In reported ,209. Stureported ,211,212.reported , and Phireported . A few greported ,220,221.Finally, it is encouraging that many research groups studying AFP worldwide have started expanding their research into cryopreservation using AFPs. We hope these concerted efforts will accelerate the development of biomedical application of AFPs.


The effects of rauwolscine, Y-27632, chelerythrine, and ML-7 hydrochloride on the dexmedetomidine-induced phosphorylation of CPI-17 or of the 20-kDa regulatory light chain of myosin (MLC20) were investigated in rat aortic vascular smooth muscle cells. The effects of rauwolscine, Y-27632, and chelerythrine on the membrane translocation of Rho-kinase and protein kinase C (PKC) phosphorylation induced by dexmedetomidine were assessed. Y-27632 and chelerythrine each reduced the slopes of the [Ca2+]i-tension curves of dexmedetomidine-induced contraction, and Y-27632 more strongly reduced these slopes than did chelerythrine. Rauwolscine, Y-27632, chelerythrine, and ML-7 hydrochloride attenuated the dexmedetomidine-induced phosphorylation of CPI-17 and MLC20. Taken together, these results suggest that dexmedetomidine-induced contraction involves calcium sensitization, which appears to be mediated by CPI-17 phosphorylation via Rho-kinase or PKC.Dexmedetomidine, a highly selective α-2 adrenoceptor agonist, produces vasoconstriction, which leads to transiently increased blood pressure. The goal of this study was to investigate specific protein kinases and the associated cellular signal pathways responsible for the increased calcium sensitization induced by dexmedetomidine in isolated rat aortas, with a particular focus on phosphorylation-dependent inhibitory protein of myosin phosphatase (CPI-17). The effect of Y-27632 and chelerythrine on the dexmedetomidine-induced intracellular calcium concentration ([Ca As a highly selective α-2 adrenoceptor agonist, dexmedetomidine induces sedation, reduces the opioid analgesic requirement, and attenuates the hemodynamic response to anesthesia and surgery ,2. Becau2+]i)-tension curve induced by an agonist or drug is higher than that induced by the depolarization evoked by high KCl concentrations [20) and subsequently enhanced contraction [20 [2+]i-tension curves induced by dexmedetomidine are higher than those induced by high KCl in isolated rat aortas, suggesting that dexmedetomidine-induced contraction produces a relatively greater increase in calcium sensitization i-tension curves to the lower-right compared with that of the control, suggesting that Y-27632 decreases Ca2+ sensitization  attenuated dexmedetomidine-induced contraction in a concentration-dependent manner  reduced the slopes of the [Ca2+]i-tension curves induced by dexmedetomidine compared with those of the control  and ML-7 hydrochloride (10−5 and 3 × 10−5 M) attenuated the contraction induced by dexmedetomidine (10−6 M) . The highest concentrations of rauwolscine (10−5 M) and ML-hydrochloride (3 × 10−5 M) nearly abolished the dexmedetomidine (10−6 M)-induced contraction (p < 0.001 versus control at 20 to 60 min). Y-27632 (10−6 and 3 × 10−6 M) also attenuated the dexmedetomidine (10−6 M)-induced contraction (p < 0.001 versus control). The highest concentration of Y-27632 (3 × 10−6 M) nearly abolished the dexmedetomidine (10−6 M)-induced contraction (p < 0.001 versus control at 10 to 60 min), and chelerythrine (5 × 10−6 to 3 × 10−5 M) attenuated the dexmedetomidine (10−6 M)-induced contraction (p < 0.01 versus control at 30 to 60 min). In addition, the highest concentration of chelerythrine (3 × 10−5 M) nearly abolished the dexmedetomidine (10−6 M)-induced contraction (p < 0.001 versus control at 10 to 60 min).Rauwolscine (10−6 M) induced CPI-17 phosphorylation at Thr38 in rat aortic vascular smooth muscle cells (VSMCs) (p < 0.05), whereas pretreatment with chelerythrine (10−5 M) or rauwolscine (10−5 M) attenuated this effect . In addition, Y-27632 (10−5 M) also attenuated the dexmedetomidine-induced CPI-17 phosphorylation . In addition, 90 mM KCl induced CPI-17 phosphorylation in rat aortic VSMCs (p < 0.001 versus control), whereas Y-27632 attenuated the 90 mM KCl-induced CPI-17 phosphorylation (p < 0.001). Dexmedetomidine (10−6 M) induced the phosphorylation of MLC20 at Ser19 in rat aortic VSMCs (p < 0.001 versus control), whereas pretreatment with rauwolscine (10−5 M), Y-27632 (10−6 M), ML-7 hydrochloride (3 × 10−6 M) and chelerythrine (10−5 M) attenuated this effect . Dexmedetomidine (10−6 M) also induced membrane translocation of Rho-kinase (p < 0.001 versus control), and this effect was inhibited by Y-27632 (3 × 10−6 M) and rauwolscine (10−5 M) . Furthermore, dexmedetomidine (10−6 M) induced PKC phosphorylation (p < 0.001), which was inhibited by chelerythrine (10−5 M) and rauwolscine (10−5 M) .Dexmedetomidine  rauwolscine, Y-27632 and chelerythrine attenuated the dexmedetomidine-induced CPI-17 phosphorylation; and (3) rauwolscine, Y-27632, chelerythrine, and ML-7 hydrochloride attenuated the dexmedetomidine-induced MLC20 phosphorylation.This is first study to suggest that dexmedetomidine-induced contraction involves the calcium sensitization mediated by CPI-17 phosphorylation. The major findings of this in vitro study are as follows: (1) the magnitudes of the reduction of the slopes of [Ca2+]i-tension curve induced by dexmedetomidine is steeper than the slope of the [Ca2+]i-tension curve induced by high KCl, suggesting that dexmedetomidine-induced α-2 adrenoceptor-mediated contraction would be mediated by calcium sensitization [2+]i [2+]i-tension curves induced by dexmedetomidine compared with those induced by chelerythrine ; this may be ascribed to an additional inhibitory effect of Y-27632 on the dexmedetomidine-induced phosphorylation of the myosin phosphatase target subunit, leading to enhanced calcium sensitization. Thus, further studies regarding the effect of dexmedetomidine on the phosphorylation of the myosin phosphatase target subunit are needed to elucidate the detailed mechanism. Because cytoskeletal organization, including stress fiber and focal adhesion formation, is controlled by Rho-kinase-induced myosin light chain phosphorylation, the dexmedetomidine-induced increase in the slope of the [Ca2+]i-tension curves may be associated with cytoskeletal rearrangement [A previous study demonstrated that the slope of the [Catization . Howeveron [2+]i ,17. Howeon [2+]i ,18. In ton [2+]i ,19. Therangement .2+]i suggests a contribution to myosin light chain kinase activation through the calcium-calmodulin complex i and tension were performed as described earlier i was measured using the fluorescent Ca2+ indicator fura-2. Vascular smooth muscle strips were preincubated with the acetoxymethyl ester of fura-2  in the presence of 0.02% Cremophor EL for 5–6 h at room temperature. Fura-2-loaded tissues were then transferred to a temperature-controlled 7 mL organ bath in a fluorimeter  and washed with Krebs solution at 37 °C for 20 min to remove uncleaved fura-2/AM. Isometric contraction of the muscle was recorded using a force-displacement transducer . The muscle strips were alternately illuminated (48 Hz) at excitation wavelengths of 340 and 380 nm. The light emitted from the tissue (F340 and F380) was measured by a fluorometer through a 500-nm filter, and the ratio of F340/F380 was used as a measure of [Ca2+]i. The absolute [Ca2+]i was not calculated in this experiment because the dissociation constant of the fluorescence indicator for Ca2+ in cytosol may differ from that measured in vitro [2+]i-tension relationships obtained by cumulative addition of dexmedetomidine (10−9–10−6 M) were generated in the absence or presence of either a Rho-kinase inhibitor  or a PKC inhibitor . Inhibitors were added to the organ bath 15 min before the cumulative application of dexmedetomidine and remained until the end of the measurement.Fura-2 loading and simultaneous measurements of i are expressed as the percentage of the 60 mM KCl-induced maximal contraction and [Ca2+]i, respectively. The contraction or [Ca2+]i found in response to each concentration of dexmedetomidine was analyzed using repeated measures analysis of variance followed by Bonferroni’s post hoc test . p Values less than 0.05 were considered statistically significant.Values are expressed as the means ± SD. The vasodilation induced by rauwolscine, chelerythrine, Y-27632, and ML-7 hydrochloride is expressed as a percentage of the dexmedetomidine (10Taken together, these results suggest that the contraction induced by the α-2 adrenoceptor agonist dexmedetomidine involves calcium sensitization in isolated endothelium-denuded rat aortas, and this effect appears to be mediated by CPI-17 phosphorylation via Rho-kinase or PKC.


Several methods including free-hand technique, fluoroscopic guidance, image-guided navigation, computer-assisted surgery system, robotic platform and patient’s specific templates are being used for pedicle screw placement. These methods have screw misplacements and are not always easy to be applied. Furthermore, it is necessary to expose completely a large portions of the spine in order to access fit entirely around the vertebrae.In this study, a multi-level patient’s specific template with medium invasiveness was proposed for pedicle screw placement in the scoliosis surgery. It helps to solve the problems related to the soft tissues removal. After a computer tomography (CT) scan of the spine, the templates were designed based on surgical considerations. Each template was manufactured using three-dimensional printing technology under a semi-flexible post processing. The templates were placed on vertebras at four points—at the base of the superior-inferior articular processes on both left–right sides. This helps to obtain less invasive and more accurate procedure as well as true-stable and easy placement in a unique position. The accuracy of screw positions was confirmed by CT scan after screw placement.The result showed the correct alignment in pedicle screw placement. In addition, the template has been initially tested on a metal wire series Moulage . The results demonstrated that it could be possible to implement it on a real patient.The proposed template significantly reduced screw misplacements, increased stability, and decreased the sliding & the intervention invasiveness. The pedicle screw placement in scoliosis of the lumbar and thoracic spine has been extensively used in the surgical community . The PedCorrect placement of pedicle screws, in the lumbar and thoracic spine, needs to have a well 3D sensation and perception of the pedicle morphology for accurate identification of the ideal screw axis , 5. It hAt the present time, the pedicle screw placements are applied by free-hand technique –13 or beFree-hand procedure usage avoids any complexity but has error in rang 10–40% –48. ImagThe robotic platform effectively reduces screw misplacement , 54 but Another different approach, which is cheaper with less complexity, could be the use of patient’s specific templates . These tA comprehensive literature review has been performed on various aspects of Patient’s specific templates. It is evident from the literature review that there is approximately no report on the design of templates by considering three factors including stability, medium invasiveness and easy verification, simultaneously. The outcome of our study would be extremely useful as the technology charts for designing medium invasiveness patient’s specific templates which are false-stable and not easily verifiable for pedicle screw placement in the spine.A multi-slice 64 bit 3D CT scan  was performed on 12 scoliosis patients  with 0.625 mm slice thickness and 0.35 mm in-plane resolution. The subjects have been complied by the World Medical Association Declaration of Helsinki regarding ethical conduct of research involving human subjects. The images were stored in DCM format and analyzed in Mango . Mimics Medical 17.0 software  was used to generate a 3D reconstruction model. The 3D vertebral model was exported in STL format for MiniMagics 3.0 software  in order to evaluate file quality, detect bad edges, flipped triangles and multiple shells, and make single point to point measurements , under a transparent post processing, in order to achieve appropriate identification of correct positioning Table .Table 1LFDM is a popular RP technology, widely used in industries, to build complex geometrical functional parts in short time , 60. FDMIn order to consider the strength of the designed template during surgery, the optimal conditions in fabrication parameters of FDM machine have been obtained to improve the strength of shafts. Two variables have remarkable impact on the fabricated product’s strength in the FDM machine. These two variables are Layer thickness and Fill angle .Therefore, before fabricating drill guide templates, different samples regarding various conditions, mentioned in , were faRegarding the considered values, the maximum (UTS) of each shaft is 38.67 MPa and the diameter of each shaft has been considered 5 mm. Therefore, it is obvious that every shaft can tolerate 75.8 N. Note that, 4 shafts were used for every drill guide template.Moreover, based on information associated with force applied by surgeon on the drill guide template during surgery, the applied force would be variable between 30 and 50 N. Therefore, it could be concluded that the designed shafts would not be broken during surgery.In addition, the orientation of shafts has been designed to guarantee drill guide template during surgery. Finally, the experimental condition demonstrated that the drill guide template is stable during surgery and can tolerate surgeon force properly.As mentioned in the introduction section, using free-hand technique for pedicle screw placement is followed by high risk of screws miss positioning –48. ImagBased on these studies, another different method, which is cheaper and easier, could be the use of patient’s specific templates . ApplicaAs it mentioned, the spinous and transverse processes should not be used as the supporting points, due to the problems related to the soft tissues removal. However, these listed points have been mistakenly used in most studies. The supporting points on another spinal level, at the designed multi-level template, has fixed this problem. The use of designed template, in 12 scoliosis surgeries, helped to reduce the rate of screws misplacement, due to template stability. The post-operative CT evaluation showed that 103 screws (94%) were implanted accurately Fig. .Fig. 8ThNote that, 7 out of 103 screws, were implanted correctly but with less accurately (between 1 and 2 mm). This error can be ignored using free-hand procedure that has error in range 10–40% –48. TherThe results of surgery, testing for 110 screws, are given in Tables The statistical analysis were performed using Minitab 17 statistical software. Descriptive statistic were used to analyze the obtained data. The inaccuracy of screws were presented in percent, as mean value and standard deviation (SD). The learning curve for the application of multi-level templates was estimated using Pearson correlation. The analysis showed that the average final position of screws was less lateral and caudal than predicted. The deviation measurements were revealed that the screws were directed less medially and caudally in comparison to the predefined direction Fig. .Fig. 9DiPearson correlation estimated that the smaller group of screws has a larger values of screw displacement at the center of the pedicle Fig. .Fig. 10CAll the multilevel templates, manufactured until now, have been associated to a high error rate due to the changes of vertebral bodies from the CT acquisition to the surgical table , 57, 58.To achieve easy verification of correct positioning, templates has been processed under a transparent post processing. During the in vivo trials, the surgeon found that template alignment was easy, due to the transparency of templates. We have found two previous research which have relatively similar results to us , 63. FerThe anatomical data of spinous process can not be specified due to supra-spinous. Therefore, we think that spinous process can not be used as contact point.It is correct that usage of transverse processes and lamina increases the stability but a considerable amount of soft tissue shall be eliminated. Therefore, these could not be suggested as contact points.We have used inferior articular process as contact point which avoids the above mentioned problems.They have used spinous process, both lamina, and transverse processes as contact points but these points are not suggested due to following reasons:One of the weak points of that work is that the size of the guide is not suitable for multi-level in vivo surgeries. They have mentioned in result section that the guide has hit to L4 screw head due to its big size and they have used fluoroscopy method instead to fix the screws. In general, the big guides normally hit to the screws and other equipment and verification of the process is not easy. Furthermore, the method is not user friendly for surgeons. The big guide leads to elimination of much soft tissues as well.The accuracy of their method for fixing the screws was 84% but our method’s accuracy is 96%.The most important point in design of the guides, which are used in in vivo surgeries, is the geometry and size of the guide. In in vivo surgeries, the expose of soft tissue is an important factor concerning the time of surgery and related infection. Therefore, big size guides are only suitable for single level method not multi-level one. The multi-level guides could lead to minimum tissue expose.Putzier et al. tool for pedicle screw placement in patients with severe scoliosis  was alsoWith respect to the design principles, under the surgical considerations and noting to small and thin-fitting areas, it is feasible to decrease intervention invasiveness.Using additional supporting points on another spinal level, at the multi-level templates, decreases the possibility to have a false-stable template positions.Semi-flexible bridges, between different levels in multi-level templates, significantly reduce error rate due to the changes in each vertebral bodies.Transparent multi-level patient’s specific template is easy to apply and give hint to the surgeon how to use the template correctly.The presented study demonstrated a medium invasiveness multi-level patient’s specific template for pedicle screw placement, which is easy to apply in the scoliosis surgery. The following conclusions are drawn from the present investigation:The outcome of this study would be extremely useful as the technology charts for designing medium invasiveness patient’s specific templates which are false-stable and not easily verifiable for pedicle screw placement in the scoliosis surgery.


Delphinapterus leucas) populations occur between July-November. Our goal was to develop population-specific beluga habitat selection models that quantify relative use of sea ice and bathymetric features related to oceanographic processes, which can provide context to the importance of changing sea ice conditions. We established habitat selection models that incorporated daily sea ice measures  and bathymetric features  to establish quantitative estimates of habitat use for the Eastern Chukchi Sea (‘Chukchi’) and Eastern Beaufort Sea (‘Beaufort’) populations. We applied ‘used v. available’ resource selection functions to locations of 65 whales tagged from 1993–2012, revealing large variations in seasonal habitat selection that were distinct between sex and population groups. Chukchi whales of both sexes were predicted to use areas in close proximity to Barrow Canyon  as well as the continental slope in summer, although deeper water and denser ice were stronger predictors for males than females. Habitat selection differed more between sexes for Beaufort belugas. Beaufort males selected higher ice concentrations (≥40%) than females (0–40%) in July-August. Proximity to shore (<200 km) strongly predicted summer habitat of Beaufort females, while distance to the ice edge was important for male habitat selection, especially during westward migration in September. Overall, our results indicate that sea ice variables were rarely the primary drivers of beluga summer-fall habitat selection. While diminished sea ice may indirectly affect belugas through changes in the ecosystem, associations with bathymetric features that affect prey availability seemed key to habitat selection during summer and fall. These results provide a benchmark by which to assess future changes in beluga habitat use of the Pacific Arctic.There has been extensive sea ice loss in the Chukchi and Beaufort seas where two beluga whale ( Ursus maritimus) and ice-associated pinnipeds such as seals and walruses use sea ice as a platform for foraging, reproduction, and resting . Seas. SeasUrsng  are ice-associated cetaceans that utilize a broad range of habitats from open water, loose annual pack ice, sea ice edge, and multi-year pack ice )Males likely segregate from females as they mature to exploit alternative prey resources and reduce competition with females and calves, as do other socially-structured cetaceans (e.g. ). We fouDue to reductions in sea ice over recent decades as well as projections for continued loss ,81, therAlthough mitigation of sea ice loss primarily requires global reduction of greenhouse gas emissions, habitat models can help inform management of anthropogenic activities and conservation planning efforts that will increasingly need to identify seasonally important areas for this critical species. Our results suggest that belugas select habitat based on a number of factors. Sea ice variables were rarely the most significant factors affecting beluga summer-fall habitat selection compared with bathymetric features. This suggests perhaps sea ice loss may not impact beluga habitat use. However there are other ways changing sea ice cover could affect belugas, and a recent study showed Chukchi belugas shifted fall migration timing as sea ice freeze-up occurred later in the 2000s . Other e


Dryas heath community. We recorded feeding marks on the most common vascular plant species in warmed and control plots at two experimental sites at different elevations and carried out a brief inventory of insect herbivores. Experimental warming increased herbivory on Dryas octopetala and Bistorta vivipara. Dryas octopetala also experienced increased herbivory at the lower and warmer site, indicating an overall positive effect of warming, whereas B. vivipara experienced an increased herbivory at the colder and higher site indicating a mixed effect of warming. The Lepidoptera Zygaena exulans and Sympistis nigrita were the two most common leaf‐chewing insects in the Dryas heath. Based on the observed patterns of herbivory, the insects life cycles and feeding preferences, we argue that Z. exulans is the most important herbivore on B. vivipara, and S. nigrita the most important herbivore on D. octopetala. We conclude that if the degree of insect herbivory increases in a warmer world, as suggested by this study and others, complex interactions between plants, insects, and site‐specific conditions make it hard to predict overall effects on plant communities.Climate warming is predicted to affect species and trophic interactions worldwide, and alpine ecosystems are expected to be especially sensitive to changes. In this study, we used two ongoing climate warming (open‐top chambers) experiments at Finse, southern Norway, to examine whether warming had an effect on herbivory by leaf‐chewing insects in an alpine  Global warming is particularly pronounced in northern areas, and mean temperatures are predicted to increase by 0.3–4.8°C by year 2100 IPCC, . This isMost research on climate warming effects in arctic and alpine regions has focused on plants  in southwestern Norway. The climate is alpine‐oceanic with an annual average temperature of −2.1°C and precipitation of 1030 mm Moen . The proDryas heaths. These are species‐rich plant communities (ca 10 vascular species per 50 × 50 cm plot) dominated by Dryas octopetala, and with other common species, such as Bistorta vivipara, Saussurea alpina, Salix reticulata, Carex rupestris, and C. vaginata. The high site is more exposed to wind, so the snow melts earlier and the growing season is approximately 3 weeks longer  and high site , are both situated in Zygaena exulans larvae crawling on the OTC walls, and they were clearly not limited by the barrier. As for adult insects, they can fly freely in and out the ca 60‐cm open tops of the OTCs. The insects do not live their entire life within the OTCs as do plants. Therefore, we are not able to simulate the full effect of climate change, that is, simultaneous change in insect and plant phenology and dynamics. However, choosing to stay in plots of increased temperature will speed up insect development relative to the colder ambient conditions and it is likely to have a measureable effect on insect life cycles. However, the main effect that we are targeting with the experimental heating is the changes in feeding capacity. However, by simultaneously comparing sites of high and low elevation, we add effects of population dynamics and phenology that are likely to vary between these sites.Open‐top chambers (OTCs) are a common method to study global warming experimentally and are considered to have few undesired side effects  and 1 m (high site). Distance between OTCs and control plots is ≥1 m (low site) and ≥2 m (high site).To detect differences in insect herbivory, we recorded feeding marks of leaf‐chewing insects on leaves of all vascular plant species in all the 40 plots in early (19th to 28th of June) and late (28th of July to 4th of August) summer. The data were collected within a 50 × 50 cm frame placed in the middle of the OTCs and control plots. The frame was divided into 10 × 10 cm subplots to facilitate data collection.In addition to the 50 × 50 cm frame, we searched the whole inside of the OTCs and an additional 15‐cm zone at all sides of the control plots for 15 min and recorded and took pictures of all leaf‐chewing insects observed before analyzing the vegetation for feeding marks. In order not to disturb the experiment, no insects were removed from the plots. Therefore, species identification was performed at the sites or later based on photographs. We recorded feeding marks on all living vascular plants inside the frame in each plot and the amount (%) of each leaf removed. These two estimates represent the total feeding activity of herbivores (number of feeding marks) and to what extend the herbivores utilize each leaf (% of each leaf removed).D. octopetala, B. vivipara, and S. reticulata) was estimated inside the frame at the low site in early and late summer (no significant difference with season). For the high site, we used vegetation data recorded in the late season in 2011  at the low site and 5.0–95.0 (mean 54.7) at the high site. As for B. vivipara, the cover varied from 0.5% to 22.5% (mean 4.4) at the low site and 1% to 4% (mean 2.1) at the high site.The percent cover of plant species with most feeding marks , 28th of July (mid‐summer), and 11th of August (late summer). Data from the five pitfall traps were pooled within each site to minimize workload. The insects were preserved in 70% alcohol, and the leaf‐chewing species were identified in the laboratory to the lowest taxonomic level possible.To get a rough estimate of insect herbivores present at the two sites, we used five pitfall traps  filled half way up with propylene glycol and water  and a few drops of soap. The traps were protected by a plexiglass roof and mounted in the Dryas octopetala and B. vivipara were the only species with enough feeding marks for analyzing statistically, and we ran models for these two species separately. To test the effect of site  and treatment  on the amount of insect herbivory, we used an herbivory index defined as “number of feeding marks divided by percentage cover of the plant fed on” as a response variable in a factorial ANOVA analysis. We also did the analysis with number of feeding marks as a response variable and percent cover of the given plant species as a covariate in an ANCOVA analysis, which gave the same overall results as the index . We also tested whether site and treatment effected the amount (%) of each leaf eaten (response variable) with an ANCOVA analysis, including percentage cover of the particular plant species added as a covariate. Knowing that the experiments had been running for several years and might have affected the plant species cover, we also included the interaction term between cover of the particular species and treatment.The two recordings of feeding marks in time (early and late summer) are highly dependent and could not be included in the same analysis. Thus, linear models were performed with data from early and late summer separately. The response variables were log‐transformed to reach normality if needed.All analyses were carried out in JMP version 11.1.1. .Dryas octopetala (53%) and Bistorta vivipara (41%). The remaining marks were found on S. reticulata, Salix herbacea, Vaccinium uliginosum, Saussurea alpina, Parnassia palustris, Poa alpina, Ranunculus acris, Thalictrum alpinum, Tofieldia pusilla, and Carex sp., but were too few to allow for statistical analysis.As many as 94% of 4744 feeding marks recorded were found on two plant species, D. octopetala mainly at the low site in late summer  comprised 63.4% of the 41 Lepidoptera larvae found in the high site and 62.5% of the 24 larvae found in the low site. The comparable proportion was 12.2% and 20.8% for Sympistis nigrita  in the high and low site, respectively. The other Lepidoptera larvae were a mix of several species but only Erebia pandrose  (one observation at low site in late summer) was identified to species. The pitfall traps and the observations in the control and OTC plots gave similar information of the leaf‐chewing insect fauna: Z. exulans were present in the first part of the summer, whereas S. nigrita and the other unidentified larvae were more common in the later part  experimentally heated plots. Herbivory on B. vivipara, on the other hand, showed the opposite pattern with reduced herbivory in the lower (and warmer) plots relative to the higher (and colder) plots, and the response to experimental warming was mainly present at the upper and colder site.We have shown that both B. vivipara at the colder site. First, timing of snowmelt has been identified as an important factor for insect phenology and activity in the arctic  of the caterpillar Gynaephora groenlandica was found in the lowest (and warmest) sites in Canada, with the highest herbivory at intermediate (medium warm) sites . Parasitism causing more than 50% mortality has been suggested to drive the phenology of G. groenlandica  (Kamil, Krebs and Pulliam Dryas, the herbivory index showed an increasing trend with warming across sites and heat stress is unlikely to be important. Also, Dryas form large mats which indicate very low movements costs between leaves. Dryas is also protected by high concentrations of defensive compounds (Nybakken et al., Dryas octopetala respond to warming by producing larger and heavier leaves (Nybakken et al., More of each Z. exulans are considered a polyphagous herbivore (Naumann et al. Z. exulans to feed mainly on Salix at lower elevations at Finse, but feeding marks on Salix were few in the present study. Generalist herbivores often specialize on the most abundant plant groups (Wilf and Labandeira D. octopetala and B. vivipara are likely to be preferred plants at our sites by any generalist herbivore. Z. exulans is known to feed on B. vivipara (Naumann et al. Dryas in a no‐choice feeding experiment in the laboratory (Hasle Z. exulans and other generalist herbivores not identified (Table The larvae of ry Hasle . Thus, Zed Table  might poDryas, most likely caused by the monophagous S. nigrita feeding primarily on flowers, is likely to increase in importance in a warmer future and might already contribute to Dryas heath community dynamics by reducing Dryas seed production. Herbivory on B. vivipara is likely to be most important in the cooler sites and possibly deceases in importance with increasing temperatures. Clearly, the outcome of plant–insect interactions on plant community level in a warmer world is hard to predict, and more studies confirming possible outcomes are still needed.Our results are in line with recent meta‐analyses on plant community responses to climate change, suggesting that site‐specific conditions, such as ambient temperature, herbivory, moisture, and snow patterns, are important drivers of community dynamics in addition to climate warming. Although we find a general increase in herbivory, we also show complex interactions between plants and insects, possibly linked to phenology and species‐specific food preferences. Herbivory on None declared.Figure S1. Number of feeding marks on D. octopetala in control and OTC plots at a low and a high elevation site recorded in early and late summer in an alpine Dryas heath at Finse, Norway.Click here for additional data file.Figure S2. Number of feeding marks on B. vivipara in control and OTC plots at a high and a low elevation site recorded in early and late summer in an alpine Dryas heath at Finse, Norway.Click here for additional data file.Table S1. ANCOVA results with number of feeding marks on D. octopetala and B. vivipara as response variables explained by site, treatment and the percentage cover of these species within the plots.Click here for additional data file.


Recent studies have shown synergistic cytotoxic effects of simultaneous Chk1- and Wee1-inhibition. However, the mechanisms behind this synergy are not known. Here, we present a flow cytometry-based screen for compounds that cause increased DNA damage in S-phase when combined with the Wee1-inhibitor MK1775. Strikingly, the Chk1-inhibitors AZD7762 and LY2603618 were among the top candidate hits of 1664 tested compounds, suggesting that the synergistic cytotoxic effects are due to increased S-phase DNA damage. Combined Wee1- and Chk1-inhibition caused a strong synergy in induction of S-phase DNA damage and reduction of clonogenic survival. To address the underlying mechanisms, we developed a novel assay measuring CDK-dependent phosphorylations in single S-phase cells. Surprisingly, while Wee1-inhibition alone induced less DNA damage compared to Chk1-inhibition, Wee1-inhibition caused a bigger increase in S-phase CDK-activity. However, the loading of replication initiation factor CDC45 was more increased after Chk1- than Wee1-inhibition and further increased by the combined treatment, and thus correlated well with DNA damage. Therefore, when Wee1 alone is inhibited, Chk1 suppresses CDC45 loading and thereby limits the extent of unscheduled replication initiation and subsequent S-phase DNA damage, despite very high CDK-activity. These results can explain why combined treatment with Wee1- and Chk1-inhibitors gives synergistic anti-cancer effects. Inhibitors of Wee1 kinase are currently in clinical trials for cancer treatment as single agents and in combination with radiation or chemo-therapy . Wee1 isHowever, in addition to its role in G2 checkpoint regulation, Wee1 also plays a major role in suppressing DNA breakage during S-phase –8. InhibSimilar as for Wee1, inhibition of Checkpoint kinase 1 (Chk1) causes both G2 checkpoint abrogation and replication catastrophe , 13. Chkin vitro and in vivo by combined Wee1 and Chk1 inhibition, as compared to inhibition of each of these kinases alone pyrene dihydrodiol epoxide . Recent Interestingly, the combined treatment with Wee1 and Chk1 inhibitors caused massive DNA damage in S-phase without causing premature mitosis Figure . In contClinical.Trials.Gov). Moreover, three anti-folates were among the candidate hits  and 1% Penicillin/Streptomycin (Life Technologies). All cell lines (except Reh) were verified by STR (short tandem repeat) technology as described previously . Thereafter, we calculated the Z score for each set of paired plates separately . Samples with higher Z´ score values than 2.5 were considered as candidate hits in the screen  equipped with a BD High Throughput Sampler using the FACS Diva Software version 6.1.3 (BD Biosciences) during acquisition. The FlowJo software  was used during analysis. A region in S-phase was defined based on the Hoechst signal, and the median γH2AX signal within this region was obtained for all samples. To evaluate effects of the drug libraries alone , we calcn Figure .™ Far Red (200 nM FxCycle and 0.1 mg/ml RNase A) (Thermo Fisher Scientific) was used in barcoding experiments, and Hoechst 33258 (1.5 μg/ml) in other experiments. The TUNEL TdT kit from Roche, Biotin-16-dUTP (Roche) and Streptavidin-Cy5  were used according to the manufacturer's instruction, combined with antibody staining of anti-phospho-H2AX(Ser139). Flow cytometry analysis was performed on a LSRII flow cytometer (BD Biosciences) using FACS Diva software. The Pearsons correlation coefficient was calculated to quantify the degree of correlation between parameters.For analysis of protein phosphorylation, cells were fixed with 70% ethanol and stained with antibodies as described previously . The priBetween 150 and 300 U2OS cells were seeded in 6 cm culture dishes (BD Biosciences) in triplicate with medium containing various concentrations of Wee1 and/or Chk1 inhibitors. After 24 hours, the medium was replaced by 4 ml fresh medium without inhibitors. Cells were then cultured for an additional 13 days, fixed in 70% ethanol and stained with methylene blue. Colonies of 50 or more cells were counted as survivors. Survival fractions were calculated in each experiment as the average cloning efficiency  after treatment with the inhibitors, divided by the average cloning efficiency for non-treated cells.3VO4), and immunoblotting was performed as described previously [2, 300 mM Sucrose) was added for 5 minutes whilst the cells were kept on ice, before one wash in ice-cold PBS, lysis and immunoblotting.Cells were lysed in SDS boiling buffer (2% SDS, 10 mM Tris-HCl pH 7.5, 100 μM Naeviously . The fol


PICK1), dpy 19-like 2 (DPY19L2), and spermatogenesis associated 16 (SPATA16). Although mouse models have clarified the physiological functions of Pick1 and Dpy19l2 during spermatogenesis, Spata16 remains to be determined. Globozoospermic patients carried a homozygous point mutation in SPATA16 at 848G→A/R283Q. We generated CRISPR/Cas9-mediated mutant mice with the same amino acid substitution in the fourth exon of Spata16 to analyze the mutation site at R284Q, which corresponded with R283Q of mutated human SPATA16. We found that the point mutation in Spata16 was not essential for male fertility; however, deletion of the fourth exon of Spata16 resulted in infertile male mice due to spermiogenic arrest but not globozoospermia. This study demonstrates that Spata16 is indispensable for male fertility in mice, as well as in humans, as revealed by CRISPR/Cas9-mediated mouse models.A recent genetic analysis of infertile globozoospermic patients identified causative mutations in three genes: a protein interacting with C kinase 1 ( PICK1), dpy 19-like 2 (DPY19L2), and spermatogenesis associated 16 (SPATA16) , as described previously [https://crispr.dbcls.jp/) [NciI digestion following PCR. Eight clones contained a disruption at the NciI restriction site and were also confirmed by direct sequencing. Four out of eight clones introduced the targeted point mutation with other indels (insertion/deletion). Three correctly targeted clones were used to make chimeric mice by a microinjection. The mutant ES cell clones were injected into eight-cell stage ICR embryos, and chimeric blastocysts were transferred into the uterine horns of pseudopregnant ICR females the next day. To confirm germline transmission, chimeric males were mated with B6D2F1 females. Spata16 point mutant mice were genotyped by NciI restriction enzyme  digestion following PCR. The NciI recognition site (5′-CCCGG-3′) was disrupted by the point mutation (G to A) at the last nucleotide of the fourth exon. Spata16 781-bp deleted mice were genotyped by a 2.9 kb band as the wild-type allele and a 2.2 kb band as the mutant allele.eviously ,39. A secls.jp/) . The sgRSpata16 mutant mouse lines, STOCK-Spata16 <em1(R284Q)Osb)> and STOCK-Spata16 <em2Osb>, were deposited to the RIKEN BioResource Center (http://mus.brc.riken.jp/en/) and the Center for Animal Resources and Development (CARD), Kumamoto University (http://card.medic.kumamoto-u.ac.jp/card/english/).The Sexually mature mutant male mice were caged with two-month-old B6D2F1 or mutant females for several months, and the number of pups in each cage was counted within a week of birth. Average litter sizes are presented as the number of total pups born divided by the number of litters for each genotype.After breeding studies, males were sacrificed by cervical dislocation following anesthesia. Testes were weighed individually. Testes were fixed in 4% paraformaldehyde in PBS and were processed for paraffin embedding or frozen section. Five micron paraffin sections were stained with periodic acid-Schiff (PAS) and then counterstained with Mayer hematoxylin solution . Ten micron frozen sections were stained with antibodies for immunostaining. Round spermatids and cauda epididymal spermatozoa were dispersed in PBS, observed under phase-contrast to assess morphology.The monoclonal antibodies used here were described previously: KS64-10 for SLC2A3 . PolycloImmunostaining was performed as described previously ,43. BrieImmunoblot analysis was performed as described previously . Brieflyt-test inserted in Excel 2016  after the data were tested for the normality of distribution. Differences were considered significant at p < 0.005 (**).Statistical analyses were performed using a Student’s SPATA16 point mutation found in globozoospermic patients. Spata16 point mutant mice were fertile suggesting that this mutation  is dispensable for sperm function in mice. This mutation was localized to the fourth exon of the SPATA16 gene in humans and mice. The replacement of the mouse fourth intron with that of the human SPATA16 fourth intron by CRISPR/Cas9 may provide the ideal model for the point mutation seen in human globozoospermia (Spata16 caused male infertility with a spermiogenic arrest. Therefore, Spata16 itself is indispensable for spermiogenesis and male fertility in mice. Further investigations will be required to clarify the mechanism of SPATA16 during spermiogenesis and the differences in phenotype between humans and mice. These investigations have been aided by CRISPR/Cas9-mediated genome editing, which has emerged as a powerful and efficient method in the biomedical sciences and holds enormous promise for genetic research and therapy in human infertility.We have generated a CRISPR/Cas9-mediated mouse model of an ospermia . Deletio


Cdc20) to delay anaphase onset. Using in vitro reconstitution, we show that Mps1 promotes APC/C inhibition by MCC components through phosphorylating Bub1 and Mad1. Phosphorylated Bub1 binds to Mad1–Mad2. Phosphorylated Mad1 directly interacts with Cdc20. Mutations of Mps1 phosphorylation sites in Bub1 or Mad1 abrogate the spindle checkpoint in human cells. Therefore, Mps1 promotes checkpoint activation through sequentially phosphorylating Knl1, Bub1, and Mad1. This sequential multi-target phosphorylation cascade makes the checkpoint highly responsive to Mps1 and to kinetochore-microtubule attachment.The master spindle checkpoint kinase Mps1 senses kinetochore-microtubule attachment and promotes checkpoint signaling to ensure accurate chromosome segregation. The kinetochore scaffold Knl1, when phosphorylated by Mps1, recruits checkpoint complexes Bub1–Bub3 and BubR1–Bub3 to unattached kinetochores. Active checkpoint signaling ultimately enhances the assembly of the mitotic checkpoint complex (MCC) consisting of BubR1–Bub3, Mad2, and Cdc20, which inhibits the anaphase-promoting complex or cyclosome bound to Cdc20 (APC/CDOI:http://dx.doi.org/10.7554/eLife.22513.001 Cdc20)  . Inhibitdiseases .Cdc20 throughout the cell.The kinetochore-microtubule attachment is closely monitored by the evolutionarily conserved checkpoint kinase Mps1 . Mps1 reArtificial tethering of Mad1–Mad2 to attached kinetochores causes prolonged activation of the spindle checkpoint and delays anaphase onset in the absence of spindle poisons . This arCdc20 inhibition by MCC components, which requires a functional phospho-Bub1–Mad1 scaffold. Furthermore, we have found that Mps1 directly phosphorylates Mad1 in its CTD. Phosphorylated Mad1 CTD directly binds to the N-terminal region of Cdc20 and contributes to APC/CCdc20 inhibition presumably through stimulating MCC assembly. Therefore, Mps1 activates the spindle checkpoint through a three-step phosphorylation cascade. Phosphorylation of Knl1 by Mps1 enables Bub1 recruitment. Phosphorylation of Bub1 by Mps1 establishes the Bub1–Mad1 interaction. Phosphorylation of Mad1 by Mps1 positions Cdc20 for MCC assembly. We propose that this sequential multi-target phosphorylation cascade makes the checkpoint highly responsive to Mps1 whose function is directly regulated by kinetochore-microtubule attachment.Here, we report a direct phosphorylation-dependent interaction between human Bub1 and Mad1 in vitro. This interaction requires phosphorylation of Bub1 by both Cdk1 and Mps1, and is critical for spindle checkpoint activation in human cells. We have in vitro reconstituted checkpoint-dependent APC/CBub1 is a spindle checkpoint kinase conserved from yeast to man. Aside from its C-terminal kinase domain, the N-terminal non-kinase region of Bub1 contains multiple functional domains or motifs, including the tetratricopeptide repeat (TPR) domain for Knl1 binding, the Gle2-binding sequence (GLEBS) for Bub3 binding, the conserved middle region , and the Cdc20-binding phenylalanine box  and lysine-glutamate-asparagine (KEN) box  Figure . Bub1 isΔK–Bub3 was strongly phosphorylated at S459 by recombinant Cdk1, but not Mps1, in vitro (We generated a phospho-specific antibody against Bub1 phospho-S459 (pS459). Recombinant Bub1in vitro . This fiin vitro . InteresWe then tested whether this phosphorylation site was required for the spindle checkpoint. HeLa cells depleted of endogenous Bub1 with a small interfering RNA (siRNA) had reduced mitotic index in the presence of taxol, indicative of checkpoint defects . This miIn mitosis, Bub1 phosphorylates histone H2A and Cdc20 . While HΔK) at higher levels restored the taxol-induced mitotic arrest of Bub1-depleted cells , as compared to cells expressing Bub1ΔK (47 min)  . CollectSaccharomyces cerevisiae Bub1 (scBub1), when phosphorylated by Mps1, binds to Mad1–Mad2 and that this phosphorylation-dependent Bub1–Mad1 interaction is critical for the spindle checkpoint (ΔK–Bub3 phosphorylated by Cdk1 in vitro. We decided to first examine whether phosphorylation of T453 of scBub1 (which corresponds to S459 in human Bub1) was required for binding to scMad1. Because the full-length Mad1 was difficult to express, we used a C-terminal fragment of Mad1 (Mad1E) that contains both the Mad2-interacting motif (MIM) and the C-terminal domain (CTD)  analysis showed that the smaller phosphorylated scBub1449–530 fragment containing the CM bound to scMad1–Mad2 with a moderate affinity of 2.2 µM (It has been reported previously that the middle region of eckpoint . An attrin (CTD) . Co-exprfragment . Consistfragment , only thf 2.2 µM . Interes449–530 co-expressed with scMps1. We next synthesized scBub1 CM phospho-peptides containing pT453 or pT455 or both. We could not test the contribution of scBub1 pS458 to scMad1 binding, because the synthesis of pS458-containing peptides failed for unknown reasons. Both the singly phosphorylated pT455 peptide and the doubly phosphorylated pT453-pT455 peptide, but not the singly phosphorylated pT453 peptide, bound efficiently to scMad1 CTD  was more critical for scMad1 binding than phospho-T453 (which corresponds to S459 in human Bub1), we wondered if phosphorylation of T461 in human Bub1 was similarly required for human Mad1 binding. We synthesized several phospho-peptides of human Bub1 CM and tested their binding to human Mad1. The pT461 peptide exhibited weak binding to human Mad1 CTD, and the doubly phosphorylated pS459-pT461 peptide had stronger binding . ITC assΔK–Bub3 with both Cdk1 and Mps1 led to efficient phosphorylation of both residues, whereas incubation with either kinase did not (ΔKS459A/T461A (SATA) mutant treated with both Cdk1 and Mps1, demonstrating the specificity of the antibody. The weak signal seen with Cdk1 alone was likely due to cross-reactivity of the pSpT antibody with Bub1 singly phosphorylated at S459. We then tested the order of these two Cdk1- and Mps1-mediated phosphorylation events. When we first incubated Bub1ΔK with Cdk1 to phosphorylate S459 and then inhibited Cdk1 with RO3306, subsequent addition of Mps1 led to efficient phosphorylation at T461, as detected by the pSpT antibody  in Bub1. Incubation of recombinant Bub1 did not . The pSpantibody . In contentially . Cdk1 phentially .ΔK–Bub3 and GST-Mad1E–Mad2 in vitro  was similarly required for the spindle checkpoint. Because we could not deplete Mad1 to sufficiently low levels to reveal a defect in nocodazole-dependent mitotic arrest, we adopted a previously reported strategy that involved the artificial targeting of Mad1 to kinetochores through a fusion to Mis12, a subunit of the outer kinetochore Mis12 complex  Maldona. ExpressNext, we investigated the mechanism by which the Bub1–Mad1 interaction contributed to checkpoint activation. Although neither Bub1 nor Mad1 is a component of MCC, they both directly interact with MCC components. The constitutive Mad1–C-Mad2 complex interacts with O-Mad2 and converts it to I-Mad2, which can bind to Cdc20 to form the Cdc20–C-Mad2 complex. Furthermore, several previous studies have characterized the direct, checkpoint-relevant binding of Bub1 to Cdc20 . These rCdc20 inhibition using a pre-assembled phospho-Bub1–Mad1 complex (N) has been shown previously to support the formation of functional MCC (Mono) is mostly the inactive O-Mad2, while the dimeric Mad2 (Mad2Di) contains a mixture of dimers of active C-Mad2 and asymmetric dimers between C-Mad2 and I-Mad2  were mixed with BubR1N, Cdc20ΔN60, and Mad2Mono (referred to as the MCC mixture) to allow MCC formation (ΔN60 as opposed to Cdc20FL will become apparent later.) The entire reaction mixture was then assayed for its ability to inhibit APC/CCdc20. Strikingly, securin ubiquitination by APC/CCdc20 was strongly inhibited when the MCC mixture containing Mad2Mono was pre-incubated with the WT Bub1–Mad1 mixture , Mps1 stimulated APC/CCdc20 inhibition by the MCC mixture containing Cdc20FL    might be the binding site for Mad1 pT716. Because I-Mad2 binds to the Mad1-bound C-Mad2, this phosphorylation-dependent binding between Mad1 CTD and Cdc20N might place the MIM of Cdc20 in the vicinity of I-Mad2, promoting the formation of Cdc20–C-Mad2 complex, which can further bind BubR1 to form MCC.As shown in and BM2) . Indeed, Cdc20N) . Inhibit binding , suggestMps1 is a master kinase for spindle checkpoint activation. How Mps1 regulates checkpoint signaling is not fully understood. It is well established that Mps1 can phosphorylate Knl1 at multiple MELT motifs to recruit the Bub1–Bub3 complex to kinetochores . Mps1-deThe phosphorylated region of Bub1 and the RLK motif in Mad1 involved in phospho-peptide binding are both conserved from yeast to man, indicating a conserved binding mode. The checkpoint-dependent Bub1–Mad1 interaction is readily detectable in the budding yeast . On the 641IDIT644) identified in this study matches the substrate consensus of Mps1. Furthermore, phosphorylation of T461 in human Bub1 by Mps1 is enhanced by prior phosphorylation at S459 by Cdk1. The negatively charged phospho-S459 can serve the same purpose as the −2 acidic residue in the substrate recognition by Mps1. This type of Cdk1-primed Mps1 phosphorylation consensus likely exists in other Mps1 substrates. To our surprise, Mad1 T716 and adjacent residues do not match the consensus of Mps1 substrates. The MELT motifs of Knl1, Bub1 T461, and Mad1 T644 are all located in loops or flexible regions. In contrast, although it is surface exposed, Mad1 T716 resides in a well-folded α helix. We speculate that Mps1 might recognize Mad1 T716 through tertiary contacts, instead of a linear peptide motif. On the other hand, we cannot completely rule out the possibility that T716 is phosphorylated by a kinase other than Mps1 in vivo.Mps1 prefers to phosphorylate serine and threonine residues with a negatively charged residue at the −2 position . SimilarWe do not know how phosphorylation of Mad1 T644 by Mps1 contributes to the spindle checkpoint, as it is not required for the reconstituted APC/C inhibition by MCC components in vitro. This phosphorylation does not appear to enhance the binding of phospho-Bub1 CM to Mad1. One possibility is that this phosphorylation is required for Mad1 CTD to interact with other regions of Bub1.Cdc20. We have reconstituted Bub1–Mad1- and Cdk1/Mps1-dependent checkpoint inhibition of APC/CCdc20. Although we did not directly monitor the kinetics of MCC formation, it is very likely that the effects of Bub1–Mad1 and Mps1 on APC/CCdc20 inhibition are through promoting the assembly of MCC. We note that Bub1 has been reported to directly interact with BubR1 , and were cultured in Dulbecco’s Modified Eagle Medium  supplemented with 10% fetal bovine serum and 2 mM L-glutamine. The cells were authenticated by genome sequencing and metaphase chromosome spread, and were routinely checked by 4',6-diamidino-2-phenylindole (DAPI) staining to ascertain that they were free of mycoplasma contamination. For G1/S synchronization, cells were treated with 2 mM thymidine  for 14–16 hr. For mitotic arrest, cells were treated with 330 nM or 1 µM nocodazole (Sigma) or 100 nM taxol (Sigma) for 12–14 hr. Other chemicals used in this study were: RO3306 (Sigma) at 20 µM, BI 2536  at 1 µM, MG132  at 10 µM, okadaic acid  at 500 nM, and reversine  at 1 µM.For RNAi experiments, cells were transfected with siRNA oligonucleotides  using Lipofectamine RNAiMAX (Invitrogen) and harvested at 24–72 hr after transfection. The sequences of the siRNAs were (from 5’ to 3’): siBub1-c, CCCAUUUGCCAGCUCAAGC ; siBub1-Internal deletion mutants were constructed with overlapping extension polymerase chain reaction (PCR). Point mutations were introduced with QuikChange Lightening Site-Directed Mutagenesis kit . All plasmids were verified by sequencing. Plasmid transfection was performed with the Effectene reagent  according to the manufacturer’s instructions.ΔK or GFP-Bub1 transgenes. Cells were treated with 200–300 μg/ml hygromycin (Clontech). Surviving clones were screened for Bub1 expression in the presence of 1 μg/ml doxycycline (Sigma). Clones that expressed Bub1ΔK at a level similar to that of endogenous Bub1 were chosen for further analysis. Stable cell line clones were maintained with 100 μg/ml hygromycin.For the generation of stable cell lines, HeLa Tet-On cells were transfected with pTRE2 vectors encoding siRNA-resistant wild-type or mutant Myc-Bub1The following antibodies were used for immunoblotting and immunoprecipitation: anti-Tubulin , anti-BubR1 , anti-Bub3 , anti-Bub3 , anti-GAPDH , anti-GFP (Roche), anti-Myc (Roche), and anti-Cdc20 . Fluorescent secondary antibodies were purchased from Life Technologies. Antibodies against Mad2, APC3, Mad1, Cdc20 (pS153), and Bub1 were previously described . The BubCells were synchronized at G1/S with thymidine and released into fresh medium containing 330 nM nocodazole for 13 hr before being harvested. For Plk1 inhibition, BI 2536 was added 1 hr prior to harvest. Collected cells were washed with PBS and fixed with pre-chilled 70% ethanol at −20°C overnight. After being washed with PBS, the fixed cells were permeabilized with 0.2% Triton X-100 in PBS for 20 min. Cells were then incubated with the anti-MPM2 antibody  in PBS containing 1% BSA for 2 hr. After being washed with PBS, cells were incubated with fluorescent secondary antibodies (Invitrogen) in PBS containing 1% BSA for 30 min. After being washed with PBS, cells were stained with 20 μg/ml propidium iodide (Sigma) in PBS containing 200 μg/ml RNase (Qiagen). All staining steps were performed at room temperature. The samples were analyzed on a FACSCalibur flow cytometer (BD Biosciences). Data were processed with the FlowJo software. The mitotic index was defined as the percentage of cells that had 4N DNA content and were also positive for MPM2 staining. The graphs and statistic analysis were generated with the Prism software (GraphPad).2) and a CoolSNAP HQ2 camera (Roper Scientific). Differential interference contrast and fluorescent images were taken with a 40X objective (Olympus) at 10 min intervals for taxol-treated cells and at 5 min intervals for asynchronous cells. For taxol-treated cells, the mitotic duration was defined as the time from nuclear envelope breakdown (NEBD) to mitotic cell death or slippage. For asynchronous cells, the mitotic duration was defined as the time from NEBD to anaphase onset. Images were processed with ImageJ, and the graphs were generated with the Prism software (GraphPad).Cells stably expressing Bub1 transgenes were passaged onto four-well chambered coverglass (Lab-Tek) and transfected with the Bub1 siRNA. For visualization of chromosomes, Hoechst 33342  was added at 1 hr before imaging. In the case of taxol treatment, cells were treated with thymidine for 16 hr, and then released into fresh medium for 6 hr before taxol addition. Imaging was performed at 3 hr after taxol addition. Live-cell imaging was performed with a DeltaVision microscope (Applied Precision) equipped with an environmental chamber  supplemented with complete EDTA-free protease inhibitor cocktail (Roche), 500 nM okadaic acid, and 10 units/ml TurboNuclease (Accelagen). Cell lysates were cleared by centrifugation. Supernatants were incubated with antibody-coupled Protein A beads (Bio-Rad) at 4°C for 2 hr. Proteins bound to beads were released with the SDS loading buffer, and analyzed by Western blotting. For quantitative Western blots, immunoglobulin G (IgG) (H+L) conjugated with Dylight 680 or Dylight 800  was used as the secondary antibody. The membrane strips were scanned with the Odyssey Infrared Imaging System .Mitotic cells were harvested by shake-off and washed with PBS once. Cell pellets were lysed with the wash buffer , GST-Mad2, GST-scBub1 fragments, GST-Mad1, and GST-scMad1 fragments in BL21(DE3) was induced by 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 16°C overnight, after the OD600 reached 0.8. Mad1 and scMad1 fragments (Mad1E) containing the Mad2-interacting motif (MIM) were co-expressed with Mad2 and scMad2, respectively. Fragments of scBub1 were co-expressed with His6-scMps1 . Harvested pellets were lysed in the wash buffer I  supplemented with a protease inhibitor cocktail. After sonication, lysates were cleared by centrifugation at 4°C. Supernatants were filtered by 0.45 µm filter and incubated with pre-equilibrated Glutathione Sepharose 4B beads . Protein-bound beads were washed with 40 volumes of the wash buffer I. The proteins were then eluted with reduced glutathione (Sigma) or cleaved with the PreScission protease. Eluates were dialyzed to the QA buffer , loaded onto a Resource Q column , and then eluted with the QB buffer . Peak fractions were pooled and further purified with a Superdex 200 size exclusion column . The relevant protein fractions were pooled, aliquoted, and snap-frozen for future experiments.Expression of GST-BubR16 proteins containing the N-terminal regions of Cdc20 was induced by 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37 ˚C for 6 h. Harvested pellets were lysed in denaturing lysis buffer , and incubated overnight by gentle rocking at room temperature. After centrifugation, the supernatants were incubated with pre-equilibrated Ni2+-NTA beads (Qiagen). Protein-bound beads were washed with 15 volumes of denaturing wash buffer . The proteins were then eluted with denaturing elution buffer . The eluted proteins were then dialyzed into the storage buffer . The soluble proteins were snap-frozen for future experiments.Expression of Cdc20-His6-Strep-Bub1 (full-length or Bub1ΔK containing residues 1–723), His6-Bub3, His6-Strep-Mps1 (full-length or the kinase domain containing residues 516–794), GST-Cdk1 (containing constitutively active mutations T14A/Y15F), His6-Cyclin B1, or His6-Cdc20 (full-length or the △N60 mutant lacking residues 1–60) were generated with the Bac-to-Bac system (Invitrogen). Bub1 and Cdk1 were co-expressed with Bub3 and Cyclin B1, respectively. Insect cells were infected with the appropriate viruses for 50 hr before being harvested. Cells expressing Bub1–Bub3, Mps1, or Cdk1–Cyclin B were lysed in the wash buffer I supplemented with a protease inhibitor cocktail. Cleared lysates were incubated with pre-equilibrated Strep-Tactin Superflow (Qiagen) or Glutathione Sepharose 4B beads. The protein-bound beads were washed and eluted with dethiobiotin (Sigma) or reduced glutathione. Eluates were dialyzed into the storage buffer I  and snap-frozen. Cells expressing His6-Cdc20 were lysed in the wash buffer II  Triton X-100, 5 mM β-mercaptoethanol and 10 mM imidazole) supplemented with a protease inhibitor cocktail and 10 units/ml TurboNuclease. Cdc20 proteins were purified with Ni2+-NTA beads (Qiagen) and eluted with 250 mM imidazole. The eluates were exchanged into the storage buffer II  with PD-10 columns .For expression of Bub1–Bub3, Mps1, Cdk1–Cyclin B, and Cdc20 in insect cells, baculoviruses encoding HisUba1, UbcH10, and Myc-Securin were purified as described previously . Ube2S pΔN60, 40 μM Mad1E–Mad2, or 400 nM Bub1ΔK–Bub3 was incubated with 40 nM Bub1, 500 nM Mps1, and/or 50 nM Cdk1–Cyclin B1 at room temperature for 30 min in the kinase buffer  supplemented with 1 mM ATP. The reaction mixtures were stopped with the appropriate kinase inhibitors for further protein-binding assays, or quenched with the SDS loading buffer and analyzed by Coomassie blue staining or Western blotting.For kinase assays, 1 μM Cdc20Recombinant bait proteins or bacteria lysates containing bait proteins were incubated with the appropriate affinity beads. Bait-bound beads were then blocked with 2% fat-free milk when necessary before being mixed with 100 µl diluted prey proteins. After incubation, beads were washed and eluted with the SDS loading buffer. The bead-bound proteins were analyzed by Coomassie blue staining or Western blotting. For the quantification of band intensities, Coomassie-stained gels were scanned and quantified with the Odyssey Infrared Imaging System . In cases that synthesized peptides were used as bait, peptides were immobilized on SulfoLink resin  according to the manufacturer’s instructions. The synthetic scBub1 peptides contained residues 449–473 of scBub1, various phospho-residues, and a cysteine at the N-terminus. All synthetic human Bub1 peptides, except the pS459 peptide, contained residue 455–477 of human Bub1, the appropriate phospho-residues, and a cysteine at the N-terminus. The Bub1 pS459 peptide contained residues 439–480, pT452/pS459, and a cysteine at the C-terminus.CTD protein or scMad1–scMad2 complexes were dialyzed into the HEPES Buffer I . Recombinant human Mad1CTD protein was dialyzed into the HEPES Buffer II . For each titration, 300 μl of scMad1 proteins (20 µM) or human Mad1 (50 µM) were added to the calorimeter cell. The scBub1 phospho-peptides or purified scMps1-phosphorylated scBub1449–530 fragment (300 µM) in the HEPES Buffer I or the human Bub1 phospho-peptides (500 µM) in the HEPES Buffer II were injected with an injection syringe in 19 2-μl portions. The raw data were processed and fitted with the NITPIC software package  assays were performed with a MicroCal Omega ITC200 titration calorimeter (GE Life Sciences) at 20°C. Recombinant scMad1 package .Xenopus egg extracts with the anti-APC3/Cdc27 antibody-coupled Protein A beads (Bio-Rad). For each reaction, 5 μl anti-APC3 beads were incubated with 60 μl extracts. The APC/C-bound beads were incubated with 1 μg recombinant human Cdc20. After washing away the unbound Cdc20, the APC/CCdc20 beads were incubated with the MCC mixture containing BubR1N, Mad2, and Cdc20 with or without Bub1ΔK–Bub3 or Mad1E–Mad2, and then washed before being added to ubiquitination reactions. The ubiquitination reaction mixture was prepared with the XB buffer  containing 150 μM bovine ubiquitin (Sigma), 5 μM Uba1, 750 nM UbcH10, 3 μM Ube2S, and 5 μM Myc-tagged human Securin, supplemented with 1X energy mixture . After incubation at room temperature with gentle shaking for 30 min, the reactions were quenched with the SDS loading buffer and analyzed by Western blotting.APC/C was isolated from ΔK–Bub3 WT or SATA mutant was incubated with 75 nM Cdk1–Cyclin B1 and 900 nM Mps1 in the presence or absence of 10 μM reversine. After incubation at room temperature, the reactions were stopped by 20 μM RO3306 and 10μM reversine.  The inhibited kinase reactions were mixed with Mad1E–Mad2 WT or RLK/AAA, resulting in the Bub1–Mad1 mixture (1.77 μM Bub1ΔK–Bub3 and 21 μM Mad1E–Mad2). Then the Bub1–Mad1 mixture was diluted and supplemented with BubR1N, Mad2Mono, and Cdc20ΔN60, resulting in the MCC mixture . After a 20-min incubation, 8 μl MCC mixture (unless otherwise indicated) was diluted with 17 μl XB buffer and applied to the 5-μl APC/CCdc20 beads.To prepare the Bub1–Mad1 mixtures for reconstitution, 2.2 μM Bub1E–Mad2 was incubated with 1 µM Mps1 in the presence or absence of reversine. After incubation at room temperature, the reactions were stopped by 10 μM reversine.  Then the Mad1–Mps1 mixtures were combined with BubR1N, Mad2Mono, and Cdc20ΔN60, resulting in the MCC mixture . After a 20-min incubation, 8 μl MCC mixture was diluted with 17 μl buffer and applied to the 5-μl APC/CCdc20 beads.To prepare the Mad1–Mps1 mixture for Bub1-independent APC/C inhibition, 8 µM WT or mutant Mad1 In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been favorably evaluated by Ivan Dikic (Senior Editor) and three reviewers, one of whom is a member of our Board of Reviewing Editors. The reviewers have opted to remain anonymous.Thank you for submitting your article "A sequential multi-target Mps1 phosphorylation cascade promotes spindle checkpoint signaling" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:In the past few years, the players of the mitotic checkpoint and their hierarchy of action have been well established. However, a mechanistic understanding of the catalytic assembly of the MCC at the kinetochore is still missing. In this study the authors provide important insights in the role of the kinases Cdk1 and Mps1 in promoting the interaction between Bub1 and Mad1 in human cells. In addition, they reveal a new function of MAD1 in recruiting Cdc20. Through in vitro reconstitution, they demonstrate that these interactions help to bring together the core components of the MCC, enabling its assembly. The work is of high quality and the findings are of great interest for the SAC field.Essential revisions:1) One concern is with the data in 2) It seems that experiments in  Essential revisions:1) One concern is with the data in The original experiments (three independent repeats) in fact included samples without Plk1 inhibition. These samples were not shown in the original submission to avoid repetition. As requested by the reviewers, we have now shown the entire dataset in 2) It seems that experiments in The original experiment with mCherry-Bub1 stable cell lines was performed only once by a former graduate student Luying Jia in the lab. Because these cell lines did not behave properly after long-term storage in liquid nitrogen, we have performed new experiments in triplicates using stable cell lines expressing GFP-Bub1 proteins . The res