CCAN subunits with biochemical and functional assays in the model system budding yeast, we demonstrate that Cdk1 phosphorylation activates phospho-degrons on the essential subunit Ame1CENP-U, which are recognized by the E3 ubiquitin ligase complex SCF-Cdc4. Gradual phosphorylation of degron motifs culminates in M-phase and targets the protein for degradation. Binding of the Mtw1Mis12 complex shields the proximal phospho-degron, protecting kinetochore-bound Ame1 from the degradation machinery. Artificially increasing degron strength partially suppresses the temperature sensitivity of a cdc4 mutant, while overexpression of Ame1-Okp1 is toxic in SCF mutants, demonstrating the physiological importance of this mechanism. We propose that phospho-regulated clearance of excess CCAN subunits facilitates efficient centromere-dependent kinetochore assembly. Our results suggest a novel strategy for how phospho-degrons can be used to regulate the assembly of multi-subunit complexes.Kinetochores are multi-subunit protein assemblies that link chromosomes to microtubules of the mitotic and meiotic spindle. It is still poorly understood how efficient, centromere-dependent kinetochore assembly is accomplished from hundreds of individual protein building blocks in a cell cycle-dependent manner. Here, by combining comprehensive phosphorylation analysis of native Ctf19 Kinetochores form a dynamically regulated binding interface between chromosomes and the mitotic spindle, a connection that is essential to partition the genetic material equally during cell division . As chroCCAN in budding yeast). The CCAN contains the essential protein Mif2CENP-C, which contacts and organizes many other subunits of the CCAN , Mis12c (Mtw1c in yeast), and Ndc80c subcomplexes are necessary to efficiently couple dynamic microtubules to centromeres  and alsoCENP-A. High levels of Cse4CENP-A lead to its mis-incorporation into non-centromeric loci  from log-phase yeast extracts and identified phosphorylation sites by mass spectrometry. In total, this analysis detected more than 70 phosphorylation sites on nine different CCAN subunits  followed the full Cdk1 consensus (S/TP_K/R). While Cdk1 phosphorylation of Cnn1 has been described before  simulations of Ame1 phospho-peptide binding to the WD40 domain of Cdc4 using as template for initial coordinates the published crystal structure of a cyclin E-derived model peptide associated with Cdc4 . The anaThe simulations also indicated that the doubly phosphorylated Ser41/Ser45 peptide establish more interactions with Cdc4 with respect to the singly phosphorylated variants –3. Furthts alleles were used at the permissive temperature, ensuring progression through the cell cycle. Western blotting showed that Ame1-WT expressed from the GAL promoter strongly accumulated in the skp1-3 mutant relative to a wild-type background  in a deletion mutant of the cytoplasmic F-box protein Grr1  subcomplexes was tolerated in a wild-type strain background, overexpression of the full four-protein COMA complex was toxic, indicating that maintenance of the proper protein level of this complex is critical for viability . The analysis of the resulting trajectories indicated that the peptide establishes conserved interactions at the binding pocket of the protein and remains bound  of Ame1 into a stronger Cdc4 phospho-degron (ILTPP), which was shown in the context of the Cdk1 inhibitor Sic1 to provide the highest binding affinity for Cdc4  did not stabilize the protein . The serial dilution assays also showed that differences in benomyl hypersensitivity between Ame1 wild-type and Ame1-CPDILTPP became apparent at 15 and 20 µg/ml benomyl and were minor compared to the effect of a ctf19 deletion  and the kinase complex Cdc28-Clb2 used in this study were created by amplification of the DNA for the respective genes from yeast genomic DNA and cloning into pETDuet-1, pET3aTr/pST39, pST44 plasmids  following the protocol for restriction-free cloning or pESC two-micron plasmids (yeast expression) using classical cloning methods. Restriction-free cloning was also used to produce vectors encoding phospho-eliminating mutants in Ame1. Site-directed mutagenesis (Agilent Technologies) was applied for introduction of amino acid substitutions. A list of all vectors used for protein production and purification in bacteria or yeast cells can be found in 600 of 0.6 and subsequently induced with 0.5 mM IPTG. Expressions were conducted overnight at 18–20 °C. Expression of Mtw1c and MN constructs was performed in BL21  and AO was expressed in Rosetta 2(pLys) (DE3) cells (EMD Millipore). Cdc28-Clb2 was overexpressed in the protease-deficient yeast strain DDY1810 overnight at 30°C using 2% galactose. Lysis, wash, and elution buffers as well as chromatography steps varied for the different protein complexes and are described in the individual sections. The Poly-Histidine fusion proteins were isolated with HisTrap HP 5 ml columns. The kinase complex was isolated using IgG Sepharose 6 Fast Flow and calmodulin sepharose 4B affinity resin . In the last step of all bacterially expressed proteins, the protein was passed over a size-exclusion chromatography (SEC) column that was appropriate for the proteins size while elution of proteins was measured by absorbance at 280 nm using an Äkta FPLC System connected to a Windows-based laptop running the UNICORN control software. All proteins were concentrated to the desired concentration except the kinase complex and flash frozen in liquid nitrogen before stored at −80°C until usage.The following conditions were used for protein production and purification unless indicated otherwise: Competent bacterial cells were grown at 37°C until ODThe Mtw1 complex (Mtw1c) used in this study includes an N-terminal 171 amino acid truncation of the Dsn1 subunit and an N-terminal 6xHis tag at the same protein. Lysis buffer for Mtw1c was 50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 30 mM imidazole, 10% glycerol, and 5 mM β-mercaptoethanol as described previously . LysatesBacterial pellets expressing the Ame1-Okp1 complex (AO) were resuspended in lysis buffer . AO loaded on the HisTrap HP 5 ml column was washed with 30 CV lysis buffer before eluted with 300 mM imidazole in the otherwise same buffer or using an imidazole gradient from 0% to 60%. AO eluted between 150 mM and 300 mM imidazole. Subsequently, the complex was purified over gel filtration Superdex 200 10/300 GL or HiLoad Superdex 200 16/600 pg columns  in 30 mM HEPES, pH 7.5, 250 mM NaCl, 5% glycerol, and 2 mM TCEP. The complex eluted in two peaks from the column, of which the later eluting peak was used for subsequent experiments. All used amino acid substitutions were purified in the same way as described above.4OAc pH 3.4) for 4 hr at 4°C and the salt concentration was increased to 300 mM KCl. Beads were washed with 50 bead volumes 1× Hyman buffer and proteins were eluted with TEV-protease overnight at 4°C. TEV-Eluate (with increase CaCl2) was further incubated using pre-equilibrated calmodulin sepharose 4B affinity resin in calmodulin binding buffer  for 1–2 hr at 4°C. Beads were washed in 40 bead volumes calmodulin binding buffer and eluted in small fractions (300 µl) using calmodulin elution buffer . Single fractions were flash frozen in liquid nitrogen and stored at −80°C until further usage in in vitro kinase assays.Yeast cells were incubated in minimal medium under constant selective pressure (SD or S-RG doURA). Expression was induced by adding 2% galactose to S-R doURA medium containing 2% raffinose and additionally 10× YEP solution to boost cell growth. Yeast pellets were flash frozen in liquid nitrogen as droplets and grind into powder using a freezer mill (SPEX SamplePrep 6870). Powder was lysed in 1× Hyman buffer  with 1% Triton X-100 using sonification. The lysate after high-speed centrifugation was incubated with pre-equilibrated IgG Sepharose 6 Fast Flow affinity resin  background. A list of all yeast strains used in this study can be found in 600 of 0.4 for anchor-away approach or 0.5 for overexpression approach. The dilutions were spotted on YPD medium with and without rapamycin or on minimal medium with either glucose or raffinose + galactose (2% each) and grown at 30°C for 2–3 days. To confirm phenotypes observed in the serial dilution assays for Ame1 mutants, Ame1 hemizygous deletion strains were used to introduce Ame1 wild-type or phospho-mutants at an exogenous locus before haploid spores were produced. Pds1-13xMyc was integrated exogenously into haploid strains for cell cycle experiments. For overexpression of proteins, pESC two-micron plasmids were transformed into haploid wild-type or SCF mutant strains without integration into the genome and clone pools were used for further analyses. Selective pressure was used for maintenance of the plasmids. Expression of integrated proteins was checked for all created yeast strains by protein extraction from yeast  and incubated in YEP + R for 3 hr at 30°C. Overexpression was induced with the addition of 2% galactose and timepoints were taken after 0, 3, and 5 hr in YEP + RG. Protein extracts  for west600 = 0.2 and incubated for 1 hr at 30°C. Next, cells were arrested in a G1-like state with alpha-factor for 2 hr at 30°C. Afterwards, cells were washed extensively in YEPD + pronase (100 µg/ml) and in YEPD to remove the alpha-factor. Cells were then grown in fresh YEPD at 30°C and protein extracts were prepared immediately and in intervals of 15 min using the method described in For cell cycle experiments, cells were diluted in 80 ml of liquid YEPD with a starting ODTo quantify the signal intensity of Ame1 phosphorylation isoforms, strains that carry two-micron plasmids were overexpressed by adding galactose to the medium and whole-cell extracts were prepared as follows: cells were incubated overnight in YEPD and diluted back into YEP + RG medium to induce overexpression. After 5 hr in YEP + RG, a 50 ml logarithmic growing culture was pelleted, washed once in 1× PBS, and the pellet was taken up in lysis buffer . Lysis was generated through mechanic disruption using acid-washed glass beads and vortexing for 1.5 min at 4°C, 1 mM DTT in the lysis buffer, and additionally 0.1% NP-40. The supernatant was spun at high speed and protein concentration was measured using Bradford Reagent. 30 µl lysate with 2 mg/ml protein concentration was treated with 1 µl of lambda phosphatase (NEB) for 30 min at room temperature. Reaction was stopped by adding 6× loading dye and boiling. Phosphatase treatment was followed by western blotting.2, 40 mM MnCl2, 100 mM β-glycerolphosphate, 4 mM DTT) in a final volume of 20 µl. The reaction was incubated for 30 min at 30°C and stopped by adding 6× SDS loading dye. The whole reaction was loaded on a SDS-PAGE and stained with Coomassie Brilliant Blue. After destaining the gel overnight, an X-ray film was exposed for 5–20 min and developed with CAWOMAT 200 IR machine.For in vitro kinase reactions, purified AO complexes (wild-type or different phospho-mutants in Ame1) were phosphorylated using Cdc28-Clb2. AO with a concentration of 5 µM was incubated with recombinant Cdc28-Clb2, 1 µl 1 mM cold ATP, 1 µl radioactive labeled ATP (10 µCi/µl), and 4× kinase buffer . The reaction was stopped at 10 min  by addition of SDS loading dye. The samples loaded on SDS-PAGE and γ‐32P phosphorylation signals were detected using Amersham Typhoon 5 Biomolecular Imager .For the quantitative phosphorylation analysis presented in The crystal structure of Cdc4 with a phosphopeptide (PDB ID: 1NEX  resoluti+/Cl-) to ensure the overall electroneutrality of the system. The parameters of the ions were taken from the literature  and underwent equilibration (5 ns). Finally, GaMD simulations were performed for 100 ns . The trajectories were processed using the Cpptraj code  feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Acceptance summary:This paper presents convincing genetic and biochemical evidence that the kinetochore protein Ame1(CENP-U) contains a phospho-degron which regulates its stability, and that phosphorylation of this degron is reduced when Ame1 is bound to another kinetochore protein, Mtw1(Mis12). These findings led the authors to an interesting model: they suggest that cell cycle-dependent phosphorylation of phosphodegrons in kinetochore proteins may allows cells to have an abundance of kinetochore proteins during kinetochore assembly in S-phase, ensuring efficient assembly, but avoid any excess kinetochore proteins during mitosis, when they could be detrimental. This is an attractive model for kinetochore biology that merits further exploration.Decision letter after peer review:CCAN kinetochore subunit stability in mitosis" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Anna Akhmanova as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Differentially accessible Cdc4 phospho-degrons regulate Ctf19The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:1) The conclusion that the binding of the Mtw1 complex shields the Ame1 phosphodegron is arguably one of the most significant and interesting claims made in this paper. However, the evidence presented to support this claim relies exclusively on in vitro data. Thus, this part is out of balance with other parts of the paper where some in vivo correlations are attempted/made. All reviewers felt that this finding requires additional confirmation in vivo.This could take several forms. For example:– Determining the phosphorylation status of Ame1 at kinetochores (or on chromatin) versus in the soluble fraction. This could be done in skp1-3 mutants in order to avoid degradation of the phosphorylated Ame1.– Using mutants in which Mtw1c or Ame1 cannot bind to one another.– Depletion of Mtw1c, measuring Ame1 amount and phosphorylation.2) The simplification of the phosphorylation patterns of Ame1 to CPD-only and CPD-null for tracking throughout the cell cycle  is logical, but it would be good to see the data for WT Ame1.3) Some additional evidence on the functional consequences of the phosphoregulation is required: does a failure to remove the phosphorylated pool indeed lead to mitotic defects, or does precocious phosphorylation/degradation lead to kinetochore assembly defects? Some experimental possibilities are listed below. We do not expect all to be addressed, but some evidence beyond of what is currently shown is needed.– Figure 1 shows that expressing Ame1 phosphomutants has no impact on general viability. However, it would be interesting to characterize whether these mutants result in any modest mitotic phenotypes or cell cycle delays, including in a colony sectoring assay for mini-chromosome loss or other sensitized assays.– If Ame1 is precociously degraded in S-phase, kinetochore assembly should be impaired. It may be possible to test this with the Ame1-CPD-ILTPP mutant, or by finding another way to induce precocious phosphorylation or degradation.– The model predicts changes in Ame1 abundance throughout the cell cycle. Whether this is consistent with the data in Figure 6 is not entirely clear. Do the authors think that only a minor pool is affected? In the absence of clearer data, the authors should use caution in their wording throughout the paper.– The model strongly predicts that the mitotic degradation of Ame1 does not impact its abundance at centromeres. Some measurement (fluorescence of a tagged Ame1 or a ChIP on centromere DNA) of Ame1 at centromeres before and through mitosis would help instill confidence in the proposal.4) The reviewers suggest several modifications to the text that should make it easier to follow the authors' arguments:4a) Discussion of the results in Figure 8:The section 'overexpression of COMA is toxic to cells' reads a bit like an afterthought, or perhaps an attempt to address the critique that the paper focuses on Ame1 at the expense of other COMA/CCAN components. This section should either be experimentally strengthened or could be incorporated into previous sections. With respect to strengthening: the authors argue that expressing proteins of the COMA complex in excess could result in ectopic kinetochore assembly. If maintaining this claim, this should be assessed by looking at the localization of COMA proteins following overexpression.The growth assay shows that overexpression of COMA is toxic, but this toxicity does not seem to be aggravated by making COMA non-phosphorylatable – suggesting that phosphorylation is largely irrelevant in the overexpression toxicity. Why the phosphomutants show less accumulation than the WT in this assay remained unclear.4b) More consistent use in the Results section of a summary sentence stating the conclusions of the experiments described, prior to moving on to the next set of experiments, would make it much easier to follow the text.The text as a whole could use copyediting, in particular a few commas need to be added to separate clauses and make sentences clearer.4c) Citations:Some citations in the introduction (particularly the review articles) are a bit dated, and more updated citations could be added instead of or in addition to what is already cited.The discussion and citations could include papers that have touched on connections between SCF and the centromere/kinetochore in human cells, for example Davies/Kaplan JCB 2010 and Gascoigne/Cheeseman JCB 2013, and the results by Au/Basrai PLoS Genetics 2020 maybe deserve a little more discussion with respect to the influence of SCF on budding yeast mitosis. In line 1016, the paper cited may not be the one that the authors intended to cite.4d) Title and abstract:– The title does not reflect the central model. As it stands, it is about 'subunit' stability with no context or proposal for what this would accomplish for chromosome segregation to occur with high fidelity.– Abstract lines 30-32 are hard to understand. The version of the same sentence in the introduction paragraph to the main text works better.– Abstract lines 42/43: "…phospho-regulated clearance of excess CCAN subunits protects against ectopic kinetochore assembly and contributes to mitotic checkpoint silencing". This sentence would benefit from rewording for two reasons:(i) The authors suggest that ectopic kinetochores may form if the CCAN phospho-regulation fails. This seems unlikely, since the excess kinetochore subunits will not have a binding platform on chromatin. However, an excess of soluble kinetochore proteins may compete for binding partners with the kinetochore-bound pool, and this may result in some mitotic defects.(ii) It is unclear how the authors imagine that the observed phosphoregulation contributes to checkpoint silencing. The fact that SCF mutants arrest with an active checkpoint does not necessarily mean that the SCF or Ame1 degradation contributes actively to checkpoint silencing.4e) Legends:Generally, please provide more information in the legends. For example, in Figure 2, explain YEPD and YEPR; in Figure 4A/B mention that overexpression is used; in Figure 6A, explain how the different arrests were obtained. The diagram in the upper-right corner of Figure 1 appears to be intended as part of Figure 1a; this could be made clearer both visually and in the figure legend. In the legend to Supplementary Figure 2, A and B are reversed compared to the actual figure.4f) Main text:Line 65: the term 'inner centromere' is used for where the CCAN is located. 'Inner centromere' generally refers to the inter-kinetochore space on the chromatin between kinetochore-forming chromatin where the CPC is located in many species. I think 'inner kinetochore' is better to use here.Lines 68-72: the wording suggests that Mif2/CENP-C and COMA make up the entirety of the CCAN. Reword to make it clear that you are merely highlighting these two components. As it is, this is likely to cause confusion later when you mention other CCAN subunits and list representatives of each major subcomplex (lines 126-127) without introducing these other components. Similarly, the introduction of the KMN network should more clearly explain the relationship between this network and the CCAN.Lines 85-93: The introduction of the concept that CENP-A nucleosome assembly can lead to a new kinetochore is not clearly written. It starts with the idea of CENP-A overexpression in experiments and in the context of cancer. It then goes to tether-experiments of some CCAN components to chromosome arms sites in human cells, and then back to mechanisms in budding yeast for degrading overexpressed CENP-A. This is difficult to follow. In budding yeast, CEN DNA seems to drive the show. That being said, excess components not at centromeres would titrate out other components or otherwise poison faithful kinetochore assembly. The point that there is danger to kinetochores can be made without invoking ectopic kinetochore formation.Line 87: one example of many in which the authors use a human gene name without the yeast equivalent, then soon thereafter (ex: line 93) use the yeast version. This should be standardized across the manuscript (or at least in the introduction/discussion) to avoid causing confusion.Lines 130-136: The explanation for why the authors chose to pursue Ame1 phosphorylation and exclude other CCAN subunits with promising phosphorylation motifs  is incomplete/insufficient.Line 146 "Okp1 is a better substrate for.… Cdk1-Clb5" – this seems to depend on what one compares to. Compared to Ame1, yes, but even Okp1 seems a better substrate for Cdk1-Clb2 than for Cdk1-Clb5. Please re-phrase. The following sentence only mentions hp-dependence of Okp1, although Ame1 phosphorylation also seems to depend on the hp.Line 184: The graph in Figure 2C lacks the quantification of the 7E mutant. Either only cite 2B, or add the 7E data to 2C.Line 189: first example of a few in which a yeast mutant is given by name without explanation of what it represents. This example in particular is the first mention of mad1 in the text and so a reader who is unfamiliar with the field is unlikely to understand the relevance of the experiment presented. In some cases, better explanations of yeast strains are provided in the figure legends than in the text. A brief explanation of what mad1 is (in this example) would be very helpful to a non-expert reader.Line 194: Please define the abbreviation AO-or avoid it altogether.Lines 390-401: This paragraph is a bit hard to follow without the context provided in the last sentence. Perhaps it could be reorganized to make it easier for the reader to follow and understand the significance of the results described.On line 422, the authors refer to Figure 3 a,b as showing stabilization of Ame1 in the absence of SCF. However, it is actually Figure 4 that shows this.Line 443 "other kinases must contribute": There are likely other phosphorylation site, but it seems formally possible that those are CDK1 sites – albeit not fitting the consensus.Line 519: "does not fully prevent SCF regulation of the inner kinetochore". It may be better to say "does not fully prevent the mitotic effects of SCF" or something along these lines, leaving out "inner kinetochore"-since SCF may well act on other mitotic structures than the inner kinetochore.Table 2: The phosphoglycerate kinase antibody may not have been used in this paper?(5) Other technical suggestions:– Multiple phosphorylated forms of Ame1 exist and run differently on a gel, which may influence the protein level comparisons in Figure 2B/E and S2. At least WT and 7A from Figure 2B should be treated with phosphatase  before comparing the levels on an immunoblot.– Figure 8A shows conservation of the Mcm21 phosphorylation site in closely related yeast species. Are the Ame1 sites conserved as well? Please add this information.–  Figure 1F: For the longest of the deletions , a sequence-unrelated, flexible spacer could be inserted in order to address whether these mutants fail to function because specific sequences in this region are now missing, or because they have been shortened. Essential revisions:1) The conclusion that the binding of the Mtw1 complex shields the Ame1 phosphodegron is arguably one of the most significant and interesting claims made in this paper. However, the evidence presented to support this claim relies exclusively on in vitro data. Thus, this part is out of balance with other parts of the paper where some in vivo correlations are attempted/made. All reviewers felt that this finding requires additional confirmation in vivo.This could take several forms. For example:– Determining the phosphorylation status of Ame1 at kinetochores (or on chromatin) versus in the soluble fraction. This could be done in skp1-3 mutants in order to avoid degradation of the phosphorylated Ame1.– Using mutants in which Mtw1c or Ame1 cannot bind to one another.– Depletion of Mtw1c, measuring Ame1 amount and phosphorylation.7D). Using this anchor-away setting we also analyzed the stability of phospho-forms of Ame1 in presence or absence of the binding partner Mtw1c. Upon inhibition of protein synthesis by cycloheximide, slowly migrating forms of Ame1 disappeared when Mtw1 was removed from the nucleus, but persisted when Mtw1c was still present (7E). Taken together, these in-vivo results complement the in vitro analysis on degron shielding by the Mtw1 complex.We have constructed several new yeast strains to address this important point. In particular, we have combined Ame1-3A and -4A mutants with Mtw1-FRB in an anchor-away strain, which allows removal of the Mtw1 complex from the nucleus and follow Ame1 phosphorylation specifically at the identified motifs 1 and 2. The results of the experiment are shown in the new Figure 7C-E. Upon removal of Mtw1c from the nucleus, phosphorylation at motif 1 and 2 gradually increases over time (2) The simplification of the phosphorylation patterns of Ame1 to CPD-only and CPD-null for tracking throughout the cell cycle  is logical, but it would be good to see the data for WT Ame1.As requested, we have included the cell cycle experiment also for Ame1-WT in the new Figure 6—figure supplement 1A. The blot shows a dynamic phosphorylation pattern with phosphorylated Ame1 forms accumulating and some of them disappearing around min 60. The complexity of the pattern justifies the use of the Ame1-3A mutant to deconvolve the phosphorylation events and specifically analyze motif 1 and 2 phosphorylation.3) Some additional evidence on the functional consequences of the phosphoregulation is required: does a failure to remove the phosphorylated pool indeed lead to mitotic defects, or does precocious phosphorylation/degradation lead to kinetochore assembly defects? Some experimental possibilities are listed below. We do not expect all to be addressed, but some evidence beyond of what is currently shown is needed.– Figure 1 shows that expressing Ame1 phosphomutants has no impact on general viability. However, it would be interesting to characterize whether these mutants result in any modest mitotic phenotypes or cell cycle delays, including in a colony sectoring assay for mini-chromosome loss or other sensitized assays.ctf19 deletion strain serves as a positive control in this experiment.We have performed a colony sectoring assay  to analyze differences in chromosome segregation fidelity between the Ame1 mutants. We find a modest increase in mis-segregation for the Ame1-7A mutant versus wild-type, the We have performed additional cell cycle experiments with Ame1 phosphorylation mutants to see if we can detect mitotic delays. Generally, there are no strong delays detectable over a single cell cycle. We see a subtle delay when phosphorylation is prevented at motif 2 (S41 S45), based on Pds1 degradation kinetics and in the FACS plots , C, althWe also noticed that in the strains with C-terminally tagged Ame1-6xFlag variants and Pds1-myc, we sometimes observe >2C DNA signals in FACS, which complicates the analysis. Because of the subtlety of the effect, we show this additional analysis only here. In the manuscript, we have added additional explanations to the discussion, why the described Ame1 phospho-null mutant do not show stronger phenotypes, and how additional degron sequences can be identified and analyzed in future studies.– If Ame1 is precociously degraded in S-phase, kinetochore assembly should be impaired. It may be possible to test this with the Ame1-CPD-ILTPP mutant, or by finding another way to induce precocious phosphorylation or degradation.ILTPP mutant further by crossing it to selected inner KT mutants predicted to be impaired in kinetochore assembly. We show that Ame1-CPDILTPP aggravates the growth defect of ctf19 deletion mutants at 20 °C and at low benomyl concentrations .We have characterized the Ame1-CPD– The model predicts changes in Ame1 abundance throughout the cell cycle. Whether this is consistent with the data in Figure 6 is not entirely clear. Do the authors think that only a minor pool is affected? In the absence of clearer data, the authors should use caution in their wording throughout the paper.We have adjusted the wording throughout the text and indeed think that only a fraction of Ame1 is subjected to the type of phospho-regulation described in this study. We have improved the Discussion section to better cover this point.– The model strongly predicts that the mitotic degradation of Ame1 does not impact its abundance at centromeres. Some measurement (fluorescence of a tagged Ame1 or a ChIP on centromere DNA) of Ame1 at centromeres before and through mitosis would help instill confidence in the proposal.We have constructed the respective strains yeast and quantified the fluorescent signal of Ame1-WT-GFP and Ame1-7A-GFP at kinetochore clusters in metaphase and anaphase cells see . We find4) The reviewers suggest several modifications to the text that should make it easier to follow the authors' arguments:4a) Discussion of the results in Figure 8:The section 'overexpression of COMA is toxic to cells' reads a bit like an afterthought, or perhaps an attempt to address the critique that the paper focuses on Ame1 at the expense of other COMA/CCAN components. This section should either be experimentally strengthened or could be incorporated into previous sections. With respect to strengthening: the authors argue that expressing proteins of the COMA complex in excess could result in ectopic kinetochore assembly. If maintaining this claim, this should be assessed by looking at the localization of COMA proteins following overexpression.The growth assay shows that overexpression of COMA is toxic, but this toxicity does not seem to be aggravated by making COMA non-phosphorylatable – suggesting that phosphorylation is largely irrelevant in the overexpression toxicity. Why the phosphomutants show less accumulation than the WT in this assay remained unclear.We agree that this section felt somewhat disconnected from the rest of the paper. We have removed the original Figure 8 and the corresponding section from the revised manuscript and include the information on the toxicity of overexpressing the entire COMA complex now as a separate panel in the new Figure 4D instead. We feel that this experiment is necessary to demonstrate the overall importance of COMA level regulation.4b) More consistent use in the Results section of a summary sentence stating the conclusions of the experiments described, prior to moving on to the next set of experiments, would make it much easier to follow the text.The text as a whole could use copyediting, in particular a few commas need to be added to separate clauses and make sentences clearer.We have edited the Results section to improve readability.4c) Citations:Some citations in the introduction (particularly the review articles) are a bit dated, and more updated citations could be added instead of or in addition to what is already cited.The discussion and citations could include papers that have touched on connections between SCF and the centromere/kinetochore in human cells, for example Davies/Kaplan JCB 2010 and Gascoigne/Cheeseman JCB 2013, and the results by Au/Basrai PLoS Genetics 2020 maybe deserve a little more discussion with respect to the influence of SCF on budding yeast mitosis. In line 1016, the paper cited may not be the one that the authors intended to cite.Thank you for pointing this out. We have removed the “Daniel et al.” citation from line 1016. We have added the citations Kaplan JCB2010 and Gascoigne/Cheeseman 2013 to the Discussion section (line 519 ff.) We also updated some of the citations in the introduction .4d) Title and abstract:– The title does not reflect the central model. As it stands, it is about 'subunit' stability with no context or proposal for what this would accomplish for chromosome segregation to occur with high fidelity.As suggested, we have used the sentence from the introduction in the revised abstract (line 30). We have rephrased the title to make it more specific for the findings regarding Ame1 and to avoid too general claims.– Abstract lines 30-32 are hard to understand. The version of the same sentence in the introduction paragraph to the main text works better.– Abstract lines 42/43: "…phospho-regulated clearance of excess CCAN subunits protects against ectopic kinetochore assembly and contributes to mitotic checkpoint silencing". This sentence would benefit from rewording for two reasons:(i) The authors suggest that ectopic kinetochores may form if the CCAN phospho-regulation fails. This seems unlikely, since the excess kinetochore subunits will not have a binding platform on chromatin. However, an excess of soluble kinetochore proteins may compete for binding partners with the kinetochore-bound pool, and this may result in some mitotic defects.(ii) It is unclear how the authors imagine that the observed phosphoregulation contributes to checkpoint silencing. The fact that SCF mutants arrest with an active checkpoint does not necessarily mean that the SCF or Ame1 degradation contributes actively to checkpoint silencing.We thank the reviewer for these valuable points. We note that excess inner kinetochore proteins that contain DNA or chromatin-binding domains could be more susceptible to induce ectopic kinetochore formation than excess outer kinetochore components. We fully agree that titrating away subunits from the kinetochore-bound pool by excess unbound subunits is another important problem. We now mention both of these possibilities in the revised discussion (line 478 ff). We have removed the point on checkpoint silencing from the revised abstract.4e) Legends:Generally, please provide more information in the legends. For example, in Figure 2, explain YEPD and YEPR; in Figure 4A/B mention that overexpression is used; in Figure 6A, explain how the different arrests were obtained. The diagram in the upper-right corner of Figure 1 appears to be intended as part of Figure 1a; this could be made clearer both visually and in the figure legend. In the legend to Supplementary Figure 2, A and B are reversed compared to the actual figure.We have corrected these points in the legends to Figures 2, 4 and 6 of the revised manuscript. We have corrected the numbering in the new Figure 2—figure supplement 1C and D.4f) Main text:Line 65: the term 'inner centromere' is used for where the CCAN is located. 'Inner centromere' generally refers to the inter-kinetochore space on the chromatin between kinetochore-forming chromatin where the CPC is located in many species. I think 'inner kinetochore' is better to use here.We have changed the wording and now use the term “inner kinetochore”Lines 68-72: the wording suggests that Mif2/CENP-C and COMA make up the entirety of the CCAN. Reword to make it clear that you are merely highlighting these two components. As it is, this is likely to cause confusion later when you mention other CCAN subunits and list representatives of each major subcomplex (lines 126-127) without introducing these other components. Similarly, the introduction of the KMN network should more clearly explain the relationship between this network and the CCAN.We have rephrased the respective section to improve readability.Lines 85-93: The introduction of the concept that CENP-A nucleosome assembly can lead to a new kinetochore is not clearly written. It starts with the idea of CENP-A overexpression in experiments and in the context of cancer. It then goes to tether-experiments of some CCAN components to chromosome arms sites in human cells, and then back to mechanisms in budding yeast for degrading overexpressed CENP-A. This is difficult to follow. In budding yeast, CEN DNA seems to drive the show. That being said, excess components not at centromeres would titrate out other components or otherwise poison faithful kinetochore assembly. The point that there is danger to kinetochores can be made without invoking ectopic kinetochore formation.We have re-organized this section starting at line 85, and have removed the CCAN tethering experiments to streamline the text.Line 87: one example of many in which the authors use a human gene name without the yeast equivalent, then soon thereafter (ex: line 93) use the yeast version. This should be standardized across the manuscript (or at least in the introduction/discussion) to avoid causing confusion.CENP-A.As standard, upon first mention we use the respective yeast name with the superscripted human/general name e.g Cse4Lines 130-136: The explanation for why the authors chose to pursue Ame1 phosphorylation and exclude other CCAN subunits with promising phosphorylation motifs  is incomplete/insufficient.We have added an additional explanatory sentence, see line 136 f.Line 146 "Okp1 is a better substrate for.… Cdk1-Clb5" – this seems to depend on what one compares to. Compared to Ame1, yes, but even Okp1 seems a better substrate for Cdk1-Clb2 than for Cdk1-Clb5. Please re-phrase. The following sentence only mentions hp-dependence of Okp1, although Ame1 phosphorylation also seems to depend on the hp.We have rephrased this point in the revised manuscript to make our statement more precise (line 143 ff). The respective section now reads: “Quantitative phosphorylation analysis confirmed that in the Ame1-Okp1 complex, Clb2-Cdk1 preferentially phosphorylated Ame1, whereas Clb5-Cdk1 preferred Okp1. The phosphorylation of the Ame1-Okp1 complex was dependent on the hydrophobic patch, a known substrate docking region in Clb5 and Clb2 cyclins, and a slightly stronger docking potentiation was seen in case of Okp1.”Line 184: The graph in Figure 2C lacks the quantification of the 7E mutant. Either only cite 2B, or add the 7E data to 2C.We have changed this and now only cite 2B in the text.Line 189: first example of a few in which a yeast mutant is given by name without explanation of what it represents. This example in particular is the first mention of mad1 in the text and so a reader who is unfamiliar with the field is unlikely to understand the relevance of the experiment presented. In some cases, better explanations of yeast strains are provided in the figure legends than in the text. A brief explanation of what mad1 is (in this example) would be very helpful to a non-expert reader.mad1 deletion (line 197 ff).We have now included an explanation for the Line 194: Please define the abbreviation AO-or avoid it altogether.We introduce this in the revised manuscript in line 139.Lines 390-401: This paragraph is a bit hard to follow without the context provided in the last sentence. Perhaps it could be reorganized to make it easier for the reader to follow and understand the significance of the results described.skp1-3 allele at different temperatures and then describe the results of the experiment (see line 409 ff).We agree and have reorganized this section to start with the description of the On line 422, the authors refer to Figure 3 a,b as showing stabilization of Ame1 in the absence of SCF. However, it is actually Figure 4 that shows this.We thank the reviewer for spotting this mistake, we have removed this section on overexpression of COMA from the revised manuscript.Line 443 "other kinases must contribute": There are likely other phosphorylation site, but it seems formally possible that those are CDK1 sites – albeit not fitting the consensus.We agree, although the observation that the combination of Ame1-7A and Okp1-S26A fully prevents Cdk1 phosphorylation in vitro , makes this relatively unlikely. We have cut this section of the manuscript, but mention the possibility of non-consensus Cdk1 sites in the revised discussion. See line 532 ff.Line 519: "does not fully prevent SCF regulation of the inner kinetochore". It may be better to say "does not fully prevent the mitotic effects of SCF" or something along these lines, leaving out "inner kinetochore"-since SCF may well act on other mitotic structures than the inner kinetochore.We agree and have rephrased this point (line 526).Table 2: The phosphoglycerate kinase antibody may not have been used in this paper?We have removed this from the materials section.(5) Other technical suggestions:– Multiple phosphorylated forms of Ame1 exist and run differently on a gel, which may influence the protein level comparisons in Figure 2B/E and S2. At least WT and 7A from Figure 2B should be treated with phosphatase  before comparing the levels on an immunoblot.We have added the requested lambda phosphatase experiment as new Figure 2—figure supplement 1 in the revised manuscript. Level differences between Ame1-WT and Ame1-7A are confirmed after phosphatase treatment.– Figure 8A shows conservation of the Mcm21 phosphorylation site in closely related yeast species. Are the Ame1 sites conserved as well? Please add this information.We detect a conservation of the Cdk phosphorylation sites, especially T31, S41 S45 and S52/53 only in the most closely related Saccharomycetes strains see . In more–  Figure 1F: For the longest of the deletions , a sequence-unrelated, flexible spacer could be inserted in order to address whether these mutants fail to function because specific sequences in this region are now missing, or because they have been shortened.Due to time constraints, we have not constructed these variants, yet. We have added a sentence to the result section acknowledging the different possibilities for the observed defects (line 174 ff).


We demonstrated by immunohistochemistry of cortical neuronal cultures that the expression of Arc, a known plasticity-related gene, requires triple phosphorylation of CREB at Ser133, Ser142, and Ser143. Moreover, we recorded visually-evoked field potentials in awake mice before and after a 7-d period of monocular deprivation (MD) to show that, in addition to CREB phosphorylation at Ser133, ocular dominance plasticity (ODP) in the visual cortex also requires CREB phosphorylation at Ser142/143. Our findings suggest that Ser142/143 phosphorylation is an additional post-translational modification of CREB that triggers the expression of specific target genes and activity-dependent neuronal plasticity processes.The transcription factor cAMP response element-binding protein (CREB) is involved in a myriad of cellular functions in the central nervous system. For instance, the role of CREB via phosphorylation at the amino-acid residue Serine (Ser)133 in expressing plasticity-related genes and activity-dependent neuronal plasticity processes has been extensively demonstrated. However, much less is known about the role of CREB phosphorylation at Ser142 and Ser143. Here, we employed a viral vector containing a dominant negative form of CREB, with serine-to-alanine mutations at residue 142 and 143 to specifically block phosphorylation at both sites. We then transfected this vector into primary neurons  The transcription factor cAMP response element-binding protein (CREB) triggers the expression of numerous different gene clusters in response to different cellular stimuli. Previous studies have shown that CREB can be activated by phosphorylation at several of its serine residues. We discovered that ocular dominance plasticity (ODP), a type of activity-dependent plasticity in the visual cortex, requires the phosphorylation of three different serine residues on CREB . The expression of the critical early gene Arc also requires this triple phosphorylation pattern. Elucidating such phosphorylation patterns of CREB required for activity-dependent gene expression could help us better understand the mechanisms of neuronal plasticity.cAMP response element-binding protein (CREB) has long been known as a master regulator of neuronal plasticity . It has in vitro by CaMKII 20 were used to make cortical cultures for the in vitro experiments. All animals were used in accordance with the protocols of the University of Maryland School of Medicine Institutional Animal Care and Use Committee.Wild-type C57BL/6 mice between the ages of postnatal day (P)25 and P35 were used for the 7 transducing units/ml, and titer of CREBdn-S142A/S143A was 3 × 108 transducing units/ml.Two different CREB dominant-negative (CREBdn) plasmid constructs were packaged in HSV vectors. These constructs contain a GFP tag and have serine to alanine point mutations at the residue 142 and 143 (CREBdn-S142A/S143A) or 133 (CREBdn-S133A), which prevents phosphorylation of CREB at these sites. The control viral construct (HSV-GFP) expressed GFP alone. All the HSV constructs were generated by Rachel Neve at the Massachusetts Institute of Technology Viral Core. Virus titer of GFP and CREBdn-S133A was 6 × 102 on coverslips coated with poly-L-lysine (Sigma). The next day, the media was changed to Neurobasal medium (Sigma) supplemented with B27, glutamax, and 1 μg/ml gentamycin. FUDR (10 μm) was added 1–3 d after plating. Cultures were grown at 37°C and in 5% CO2.Cortical cultures were prepared in the lab of our collaborator, Thomas Blanpied, at the University of Maryland School of Medicine. Briefly, primary cultures of cortical neurons were obtained from E20 rat embryos and dissociated with trypsin. On the day of dissection, the cells were grown in Neurobasal Medium (Sigma) supplemented with 5% bovine serum supplemented with B27, glutamax and 50 U/ml gentamicin. Cells were plated at 50,000 cells/cmm KCl and 9.2 mm CaCl2 as previously described , 10 μm DNQX (to block AMPA receptors), and 10 μm APV  was added to each well. Plates were returned to the 37°C incubator overnight, and virus infection was confirmed the following day by visualizing GFP expression in the culture plate. Cells were stimulated for 20 min either in a potassium chloride (KCl)-rich media containing artificial CSF (ACSF) with 50 mescribed  or normaescribed  as the cKCl-stimulated cells used to stain for phospho-CREB (pCREB) were fixed immediately after stimulation with 4% paraformaldehyde (PFA) in 20% EGTA. However, stimulated cells used to stain for CREB target genes were switched back into culture media for 20 min to allow for gene expression, before subsequent fixation.SCR_003070) was used for densitometry and all OD values were normalized by loading control. Cyclophilin B  was used as the loading control in all cases. As soon as the imaging of pCREB-133 was complete, the membranes were washed in stripping buffer for 15 min at room temperature and blotted again for total CREB  using the same blotting procedure described above. Arc expression was assessed by similar procedures, antibody used was catalog #66550-1-ig from proteintech at 1:5000 concentration.The HSV construct was intracortically administered into Layer IV of the visual cortex of mice via stereotaxic injection . The animals were killed after 2–4 d  with isoflurane followed by decapitation. The visual cortex was dissected out on ice and GFP fluorescence was confirmed by microscopy. The GFP-positive tissue was isolated and homogenized in RIPA lysis buffer (Millipore 20-188) with protease/phosphatase inhibitors . Protein concentrations were determined by Bradford assay and samples were run on 15% TGX Protean gels (Bio-Rad), in the mini-protean Bio-Rad Tetracell electrophoresis chamber. Gels were transferred to PVDF membranes using the Bio-Rad Trans-blot turbo transfer system. Membranes were blocked for at least 90 min using 2–4% non-fat blotting-grade blocker (Bio-Rad) in 1× Tris-buffered saline with 0.1% Tween (1× TBST), then incubated overnight at 4°C with a 1:1000 dilution of rabbit anti-pCREB-133 . After washing three times in 1× TBST, membranes were incubated for 1 h in horseradish peroxidase-conjugated anti-rabbit IgG at  at a 1:3000 dilution. ECL reagents (Bio-Rad CLARITY) were used to chemiluminescently visualize the protein on an imaging system (ProteinSimple FluorChem HD2). ImageJ (RRID: m glycine (PBS/Gly), and then blocked in 10% normal goat serum (NGS) in PBS/Gly with 0.1% Triton X-100 for 60 min at 37°C. They were then incubated at 4°C overnight with the following primary antibodies in PBS/Gly/0.1% Triton X-100 in 5% NGS: rabbit anti-pCREB Ser133 , rabbit anti-pCREB Ser142/143 (1:500), rabbit anti-Arc , rabbit anti-CaMKII conjugated to Alexa Fluor 647 . Each coverslip of cells was stained for either pCREB Ser133, pCREB Ser142/143, or Arc, together with CaMKII as an excitatory neuronal marker. The next day, the cells were washed in PBS/Gly, incubated for 1 h at room temperature with fluorescent secondary antibody anti-rabbit Alexa Fluor 568 (1:500), washed again in PBS/Gly, and then mounted on glass slides with mounting media . The pCREB Ser142/143 antibody was generously provided to us by Michael Greenberg of Harvard University.Fixed cells were washed with 1× PBS and 100 mConfocal imaging was performed at the University of Maryland School of Medicine Confocal Microscopy Core Facility, on a point-scanning confocal (Zeiss LSM 510 Meta) microscope, with a 40×/1.3 NA oil-immersion objective. Specifications for data collection on each fluorescent track were as follows: for GFP, 488-nm laser excitation was bandpassed from 500 to 550 nm; for Alexa Fluor 594, laser excitation at 543 nm was set to a long-pass filter at 560 nm; and for DAPI, 730-nm pulsed two-photon laser excitation was bandpassed at 380–550 nm. Colocalization analysis was performed on maximum projection images. Using the Grid and Cell Counter tools in ImageJ software, all GFP-positive cells (GFP = virus expression) that co-expressed CamKII  were manually selected and counted. Only virus-infected, excitatory neurons with clear somatic boundaries and a visible nucleus were included in this analysis, determined by colocalization of GFP and CaMKII. The localization of CamKII in the cytosol and dendrites allowed us to clearly delineate the nucleus of every cell analyzed. To determine nuclear staining, we used ImageJ to specifically define the nuclear region as the region of interest (ROI), and then quantified the CREB fluorescence within each ROI. The average optical density (OD) of the ROI was determined, and the background fluorescence was subtracted from the OD value of each cell.Surgery was conducted on mice under isoflurane anesthesia. Burr holes were drilled in the skull at 0.5 mm rostral to λ and 3 mm lateral to the midline, which corresponds to the binocular zone of the visual cortex (V1B) in mice. A microsyringe pump-controller (World Precision Instruments Micro4) was used to bilaterally deliver 1 μl of the desired HSV construct into Layer IV of the cortex at an infusion rate of 1 nl/s, with a diffusion time of 2–3 min. Tungsten electrodes (FHC) with tip impendences between 0.3 and 0.55 MΩ were stereotaxically implanted bilaterally in the same location as the injection, at a depth of 450–480 μm, to target Layer IV cells as previously described . ReferenSurgery was conducted under isoflurane anesthesia, following the baseline VEP recording. Ophthalmic proparacaine  was applied topically, and the edges of the upper and lower eyelids trimmed. The lids were stitched together using 7–0 prolene suture , and Gluture tissue glue (Abbott Laboratories) sealed the lids together. A thin film of cyanoacrylate (tissue glue) was used to cover the area to protect the surgical site during the 7-d period of MD.This VEP recording procedure was performed the same way for both pre-MD and post-MD recording sessions. The animals’ heads were immobilized so that movement artifact was kept to a minimum during electrophysiological recordings. Mice were then presented with a visual stimulus to each eye independently, during which visually-evoked local field potentials were recorded. Recordings were conducted using XCell-3 amplifiers (FHC), a 1401 digitizer (Cambridge Electronics Design), and Spike 2 software (Cambridge Electronics Design). XCell-3 amplifiers were set at a low-frequency cutoff of 0.1–10 Hz and a high-frequency cutoff of 100 Hz. The visual stimulus consisted of a full-field, phase-reversing, ordinal sine grating at 0.5 Hz with 100% contrast. The stimulus was controlled by a custom program written in MATLAB (The MathWorks) and presented at a distance of 21 cm from the animal. The angle of the stimulus grating was changed (45° to 135°) for sessions before and after MD, to avoid any confounding results with respect to stimulus-selective response potentiation . Recordit tests because this in vivo technique allows for within-subject controls. One-way ANOVA was used to analyze differences in VEP amplitudes and CBI between naive mice and each virus-infected group. For immunocytochemistry, pCREB was compared using two-tail Student’s t tests, whereas Arc and CaMKII were compared using one-way ANOVA. Western blottings were analyzed by two-tail Student’s t tests. All statistical tests were performed on IBM SPSS (v23) and statistical significance was set to p ≤ 0.05, or p < 0.03 (with Bonferroni correction), and denoted by an asterisk (*). For clarity, statistical details are reported in the figure legends.VEP experiments assessing ODP (CBI or VEP amplitudes) were analyzed using directional paired J Neurosci  or at Ser142/143 (pCREB142/143). We observed that phosphorylation at Ser133 and Ser142/143 (nuclear staining) was increased in cells exposed to KCl-rich ACSF compared with cells exposed to control ACSF  or a virus that blocked phosphorylation at Ser133 (CREBdn-S133A) or phosphorylation at Ser142/143 (CREBdn-S142A/S143A). After confirming successful virus expression by GFP visualization, we depolarized these cells with KCl to measure the change in activity-dependent Arc expression. Since mechanistic differences do exist with respect to cell type, we refined our experimental design by co-labeling these cells with CaMKII. As CaMKII is expressed only in glutamatergic neurons and not inhibitory neurons in the cortex , the colin vivo. Mice received intracortical injections of CREBdn-S142A/S143A (n = 7) or control GFP (n = 9) and were monocularly deprived for 3 d to induce Arc expression . In summary, blockade of CREB phosphorylation at Ser142/143 reduced Arc expression both in vitro and in vivo.To confirm our findings observed in the aforementioned experiments in culture we also tested whether blocking CREB phosphorylation at Ser142/143 would reduce Arc expression pression  and tissin vivo VEP recordings in virus-injected mice before and after 7 d of MD during the critical period of visual cortex plasticity. The chronic implantation of the recording electrodes allowed for a within-subject, before-and-after comparison of evoked field potential amplitudes in the visual cortex. We tested whether ODP is affected by blocking CREB phosphorylation at Ser142/143 using This method was similar to what we used in a recent study, where we showed that virus injections targeted to cortical Layer IV resulted in robust infection of cells in Layers IV and II/III, and few cells in deeper Layers V and VI . GFP-marHSV-GFP control animals copied the expected pattern in naive mice , with a Taken together, the novel results presented here reveal the phosphorylation of CREB at Ser142/143 as a novel mechanism of ODP during the critical period, specifically necessary for both components of activity-dependent neuronal plasticity in the visual cortex.In this study, we confirmed previous evidence  showing Although other studies have shown that CREB induces Arc expression , the mecIt was previously reported that phosphorylation at Ser142 alone can be inhibitory and attenuated CREB-dependent transcription . KornhauSince the CREB-dependent transcriptome is vast in number and diverse in purpose, the search for which genes CREB expresses to fulfill different cellular needs has been a long-standing question. We showed that triple phosphorylation at Ser133, Ser142, and Ser143 is required for both components of ODP, Dc-ODP and Pc-ODP. Future studies could use transcriptome profiling with these phosphorylation sites blocked, to identify CREB-dependent genes required for both components of activity-dependent plasticity. It would also be important to test whether individually blocking phosphorylation at Ser142 or Ser143 might differentially affect Dc-ODP and Pc-ODP.


Figures 3, 4 as published. The legends for those figures were swapped. The corrected legends appear below.In the original article, there was a mistake in the legend for FIGURE 3 | Underlying network connection can render some neurons more susceptible to GABAergic disinhibition. Excitatory neuron A is reciprocally connected with inhibitory neuron B to form a small feedback loop. With a reduction in KCC2 function , in excitatory neuron A (below), Cl− will accumulate leading to a faster rate of collapse in the Cl− driving force subsequently weakening GABAergic inhibition. As neuron A experiences less inhibition, it will fire more, leading to greater excitation of the inhibitory neuron B. As a result of increased excitatory input, the inhibitory neuron will then increase inhibitory input back onto the excitatory neuron A, which will further enhance the Cl− load in the excitatory neuron to create a deleterious loop. Created with BioRender.com.FIGURE 4 | Synaptic changes in the basal ganglia circuitry in HD. In the healthy brain, activation of D1-MSNs of the direct pathway (blue) leads to disinhibition of the thalamus, which increases excitatory feedback to the motor cortex. Activation of the D2-MSNs of the indirect pathway (purple) leads to inhibition of the thalamus, which decreases excitatory feedback to the motor cortex. Dopaminergic inputs from the SNc produces different outcomes in D1- and D2- MSNs; inhibiting D2-MSNs, while exciting D1-MSNs. Early in HD, there is a preferential loss of D2-MSNs leading to increased output along the direct pathway, mediated by D1- MSNs. The decreased inhibitory output through the indirect pathway and increased output through the direct pathway, lead to the development of unwanted, hyperkinetic movements called chorea . Template taken and modified with BioRender.com.The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Introduction: Asthma and allergy occur frequently among seafood processing workers, with the highest prevalence seen in the crustacean processing industry. In this study we established for the first time the prevalence of allergic sensitization in the Norwegian king- and edible crab processing industry and characterized the IgE-reactive proteins.Materials and Methods: Two populations of crab processing workers participated; 119 king crab and 65 edible crab workers. The investigation included information on work tasks and health through a detailed questionnaire. Allergic sensitization was investigated by crab-specific IgE quantification and skin prick tests (SPT) to four in-house prepared crab extracts; raw meat, cooked meat, raw intestines and raw shell. Allergen-specific IgE binding patterns were analyzed by IgE immunoblotting to the four allergen extracts using worker serum samples. Total proteins in crab SPT extracts and immunoblot-based IgE binding proteins were identified by mass spectrometric analysis.Results: Positive SPTs were established in 17.5% of king- and 18.1% of edible crab workers, while elevated IgE to crab were demonstrated in 8.9% of king- and 12.2% of edible crab processing workers. There was no significant difference between the king and edible crab workers with respect to self-reported respiratory symptoms, elevated specific IgE to crab or SPT results. Individual workers exhibited differential IgE binding patterns to different crab extracts, with most frequent binding to tropomyosin and arginine kinase and two novel IgE binding proteins, hemocyanin and enolase, identified as king- and edible crab allergens.Conclusions: Occupational exposure to king- and edible crabs may frequently cause IgE mediated allergic sensitization. Future investigations addressing the diagnostic value of crab allergens including tropomyosin and arginine kinase and the less well-known IgE-binding proteins hemocyanin and enolase in a component-resolved diagnostic approach to crab allergy should be encouraged. According to the Food and Agriculture Organization about 59.51 million people were engaged in seafood and aquaculture production in 2018 –8. Snow Paralithodes camtschaticus) workers from four plants, and 83 edible crab (Cancer pagurus) workers from one plant participated in one or more parts of this study. The inclusion criterion was their work in the processing plants at the time of the study. The study was conducted in two production seasons; from September 2009 to January 2010, and in September and October 2011. Written informed consent was obtained from all participants of this study. The study was conducted with the approval of the Regional Committee for Medical Research Ethics in Northern Norway (2009/1037/REK nord).The population in this study has previously been investigated in a study of lung function and prevalence of respiratory symptoms . A totalDo you have asthma/allergy?” and “was your asthma/allergy diagnosed by a doctor?” Smoking habits were categorized as never smokers (persons who had never smoked regularly) or ever smokers (persons who were current or former smokers). Self-reported respiratory symptoms were defined as positive if the workers answered “yes” to one or more of the following questions; “Have you experienced wheezing in the last 12 months?” and “Do you usually cough or hem in the morning?” and “Do you cough daily/almost daily for, on average, 3 months or more throughout the year?”A questionnaire was distributed and completed by 119 king crab workers and 65 edible crab workers containing questions on personal background, health and occupational characteristics. This questionnaire was previously developed and published  as well Cancer pagurus code f23) and shrimp (f24) using the ImmunoCAP system . The detection of specific IgE ≥ 0.35 kU/L on one or both of the inhalation panels was used as a positive result for atopy. Workers with specific IgE ≥ 0.35 kU/L to crab were defined as having elevated IgE to crab.Blood samples of 113 king crab workers and 78 edible crab workers were collected in Vacutainer separation tubes. The samples were centrifuged and stored refrigerated until arrival at the laboratory after 2–4 days, and stored at −70°C until further analyses in the department of Laboratory Medicine at the University Hospital of North Norway. The atopy status of the workers was established by quantifying specific IgE antibody to common inhalant allergens on inhalation panels  in addition to sensitization to crab , pH 7.4, 4°C, in a Waring blender for 90 s (1 min low and 30 s high intensity). The crab mass to buffer volume ratio was kept constant for each protein extract. The slurry was further stirred (Heidolph RZR 2100 Electronic) in a refrigerated room for 2 h, and then centrifuged at 10,000 g for 1 h to remove insoluble shell debris. The supernatant was centrifuged at 80,000 g for 1 h, and the protein content in this supernatant assayed using the Bradford method . These sSkin prick tests (SPT) were performed on 40 king crab workers and 83 edible crab workers. In-house generated crab extracts were used, king crab extracts on king crab production workers and edible crab extracts on edible crab production workers. Standard commercial SPT solutions for positive control (1% histamine) and negative control  were used . The SPT was performed on the ventral forearm, and reactions were read after 15 min. Positive results were identified as a wheal of ≥3 mm in the presence of a positive control of ≥3 mm, and no response to the negative control.Tables 2, 3. Briefly, 10 μg of the crab extracts were separated on a 12% SDS-PAGE gel and transferred to a polyvinylidene difluoride (PVDF) membrane using a semi-dry western blotting apparatus . After blocking the unbound regions of the membrane using 5% skimmed milk in phosphate buffered saline with 0.05% Tween-20 (PBS-T), the membrane was incubated with 1:10 diluted worker sera for 16 h at 4°C using a slot-blot apparatus . The blot was subsequently incubated with rabbit anti-human IgE antibody diluted 1:10,000  in 2% blocking buffer and goat anti-rabbit IgG-HRP . After washing the blots with PBS-T, the IgE binding was visualized based on enhanced chemiluminescence (ECL). The IgE binding was semi-quantified using densitometric analysis as described earlier  as described previously , 33. Tryhttps://doi.org/10.25903/qdmh-4r06), Sequest search engine, trypsin as enzyme with maximum 2 missed cleavage, oxidation at methionine as variable modification, carbamidomethyl at cysteine as fixed modification, 10 ppm and 0.2 Da for MS and MS/MS mass tolerance. At least 2 peptides were required for each protein group.The spray voltage, temperature of ion transfer tube and S-lens of the Q Exactive Plus Orbitrap mass spectrometer were set at 1.8 kV, 250°C and 50%, respectively. The full MS scans were acquired at m/z 375–1,400, a resolving power of 70,000, an AGC target value of 3.0 × 106 and a maximum injection time of 50 ms. The top 15 most abundant ions in the MS spectra was subjected to higher-energy collisional dissociation (HCD) at a resolving power of 17,500, AGC target value of 5 × 104, maximum injection time of 50 ms, isolation window of m/z 1.2 and normalized collision energy (NCE) of 30%. Dynamic exclusion of 30 s was enabled. Raw data was analyzed through Proteome Discoverer 2.3  using the Crustacean database   in all king and edible crab extracts near the 32–44 kDa region, as well as to the dimeric form at 75 kDa as shown previously , 35. IgEin vitro IgE binding to crab allergens using in-house immunoblots. Several workers who reacted to only some of the SPT extracts show IgE binding to all four extracts in the allergograms. King crab worker number 5 who did not report respiratory symptoms, asthma or allergy was SPT positive to cooked crabmeat and intestines and had IgE binding to several proteins in all four extracts. In contrast, in king crab worker number 2, an elevated IgE in the commercial test to crab was not reflected by respiratory symptoms, asthma, allergy or the SPT, and only a low binding intensity was identified to two proteins in raw meat and one in intestines in the in-house-immunoblot assay. Edible crab workers 2 and 3 had elevated specific IgE to crab by ImmunoCAP, but did not have positive SPT results, nor did they show any IgE binding to cooked crabmeat by immunoblotting. In contrast, edible crab worker number 10 had a high specific IgE to cooked edible crabmeat by ImmunoCAP, positive SPT to cooked meat extract, and strong binding to almost all proteins in the cooked meat extract by immunoblotting. This worker also showed strong IgE binding to proteins in the three other extracts, however SPT was negative to these extracts.When comparing the results of the SPT to the allergograms of allergen reactivity of the same worker, there are several similarities, but also some discrepancies. All workers with elevated specific IgE to crab extracts by ImmunoCap demonstrated also The aim of this study was to establish the prevalence of allergic sensitization among crab processing workers and identify the sensitizing allergens in these two different crab species. Furthermore, we explored the associations between positive IgE to crab proteins, positive SPT and reported respiratory symptoms.Both king crab and edible crab workers were predominantly male with mean age of approximately 35 years, and with the mean time of employment being 1.5 years. Significantly more king crab workers were smokers compared to the edible crab workers. Atopic workers have been found to have an increased risk of developing IgE sensitization . In our However, 36.6% of king crab workers and 29.3% of edible crab workers reported respiratory symptoms, which is higher than what has been found in previous studies including a population study in southern Norway  as well In the king crab plants participating in this study, most of the workers were processing raw crab. However, an open plant layout without shielding of the cooking process, resulted in exposure to bioaerosols from both raw and cooked production to all processing workers . In the The prevalence of SPT positive workers were 17.5 and 18.1% in king crab and edible crab workers, respectively. This is lower than previous studies among snow crab processing workers , 46, 47 Our study highlighted the difference in protein and allergen composition in the different extracts as well as between two crab species. Using proteomic analysis, the presence of a unique set of crab proteins (including hemocyanin and enolase) was shown predominantly in the raw meat, intestine or shell components; all of which were not thermally processed, indicating the heat-labile nature of these proteins. Yet, immunoblot analysis with worker sera showed strong IgE binding to these proteins, and may play an important role in allergic sensitization among the crab workers. The abundance of heat stable allergen such as tropomyosin was found in all different protein extracts.IgE binding analysis conducted in this study have shown that workers are sensitized to several and different proteins in the crabs. These reactivities do however not necessarily match with the results of the skin prick tests. A comparison of the allergograms of IgE reactivity between the same proteins in raw and cooked meat indicates a higher number of IgE binding proteins in the raw meat in both crab species. However, king crab processing workers demonstrated stronger IgE binding to the raw meat while edible crab processing workers had strong IgE binding to the cooked meat. The current study compared for the first time differential IgE binding analysis of two processed crab species as well as between raw meat, cooked meat and in particular intestine and crab shell extract that were used for the skin prick testing analysis. It was evident from the experimental data that the different processing methods as well as isolated crab sections resulted in altered IgE binding to the various allergens. As shown previously, tropomyosin and arginine kinase were identified in the raw and cooked meat, shell and intestines extracts. However, we have shown for the first time that hemocyanin is present in crab intestines and shell. It has been previously identified as an ingestion related allergen in crab roe  and shriThe commercial IgE test (ImmunoCAP) used in this study was made from cooked edible crabmeat and may therefore not include important allergens such as hemocyanin and enolase. This is also illustrated through comparing the specific IgE to SPT and allergograms. Three king crab workers and six edible crab workers did not have elevated specific IgE to crab, but were SPT positive. Several of the workers who were not SPT positive to one or more of the extracts did show however IgE binding to allergens in the immunoblots. Sensitized crab processing workers may not be diagnosed correctly with the current methods available. A detailed proteomic analysis of all the IgE binding proteins was conducted in this study. IgE binding analysis using a bigger worker cohort to purified novel allergens identified in this study will provide an insight into the presence of novel air-borne food allergens as well as cross reactive allergens responsible for occupational asthma and allergy.This study is a cross-sectional study design where it is not possible to determine how many workers have left the processing plants due to occupational health problems before the start of the study. The short duration of employment among the crab processing workers and low prevalence of asthma may indicate a healthy worker effect where workers who develop health problems are selected out –61. ThisCrab processing workers in Norway report respiratory symptoms and are sensitized to specific crab allergens as demonstrated by commercial IgE test, SPT and immunoblotting, and thus are at risk of developing asthma and allergy to crab. Through SPT and immunoblot analysis, we demonstrate that workers in both king crab and edible crab processing are sensitized to at least four allergenic proteins present in both raw and cooked meat, intestines and shell. Two novel occupational crab allergens, enolase and hemocyanin, were identified for the first time. There are several additional unidentified sensitizing allergens to be characterized in the future, enabling better component resolved diagnosis of occupational allergies. Our previous study on air-borne exposure to crab allergens  and highhttps://doi.org/10.25903/qdmh-4r06; https://doi.org/10.25903/78fb-f688.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: The studies involving human participants were reviewed and approved by Regional Committee for Medical Research Ethics in Northern Norway. The patients/participants provided their written informed consent to participate in this study.MT helped in conceptualizing the research project, experimental design, data collection in the field, data analysis, manuscript writing, and editing. SK helped in experimental design, data analysis, manuscript writing, and editing. BB was central in conceptualizing the research project, experimental design, data analysis, reviewing, and editing the manuscript. SK and RN helped with the immunoblotting analysis, in gel tryptic digestion, mass spectrometric data analysis, and methods writing. SN and NW performed the proteomic analysis, including experimental work, raw data analysis, writing methods, and provided resources from Bio21 Proteomic Facility. AL helped in conceptualizing the research project, experimental design, reviewing, and editing the manuscript. LA is project manager and was central in conceptualizing the research project, experimental design, reviewing, and editing the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Vitamin D is critical for bone physiology. In this study, we quantified Vitamin D Binding Protein (VitDBP) levels in saliva as a measure of Vitamin D during orthodontic tooth movement.In this longitudinal study, saliva samples were collected from 73 orthodontic patients for 4 timepoints for the first six months of orthodontic treatment, along with dental casts at the beginning and the end of the study period. The saliva was measured for VitDBP as a biological marker for bone apposition and clinical tooth movement. We used the absolute change in Little's Irregularity Index as a quantitative measure for alignment. In addition, we measured the levels of alkaline phosphatase (ALP) in saliva as a marker of bone turnover.p < 0.05) seasonal changes in VitDBP using a two-season year model were found with lower levels observed in the summer (Apr–Sept) than in the winter (Oct–Mar).Both low (< 2.75 ng/ml) and high (> 6.48 ng/ml) VitDBP levels were associated with reduced tooth movement. Significant , the predictive value or salivary VitDBP for tooth movement should be studied in larger cohorts in future studies. Vitamin D (VitD) plays a significant role in bone biology and remodeling . It regu−6 M) is substantially higher than the concentration of 25-OHD with more stability (4.10−8 M) [VitD Binding Protein (VitDBP), a polymorphic single-chain serum glycoprotein ubiquitously found in body fluids and organs, has a vital role in VitD metabolism , binds t.10−8 M) . Therefo. [While several studies have focused on the association between orthodontic tooth movement and VitD levels, this study addresses the critical gap in knowledge that the orthodontic tooth movement has not been correlated with salivary levels of vitamin D binding protein as a measure of circulating vitamin D. , 18, 19.A total of 127 patients were recruited between April 2017 and March 2018 from the Department of Orthodontics and Dentofacial Orthopedics clinics at the Boston University's Henry M. Goldman School of Dental Medicine after the IRB approval (# H-34695). Seventy-three subjects were included in the final analysis. All subjects were provided with written and oral consent before being included in the study. Participants with any medical conditions that might affect bone metabolism or those taking any VitD supplements were excluded. The treatment plans included extraction and non-extraction cases, braces, clear aligner therapy, and others such as expanders or headgears.Impressions and orthodontic records were taken before treatment and 6 months after the start of the study period. Plaster casts were scanned using the Ortho Insight 3D from Motion View LLC.,  scanner and software. Incisor teeth were identified using the software. The mesial and distal contact points were located manually, and Little's Irregularity Index (II)  was measSaliva samples were collected each month for the first 6 months of orthodontic treatment. Of those, four time-points were chosed for analysis representing the the leveling and aligning stage of orthodontic treatment. The first sample was taken during the treatment planning appointment. The last sample was obtained at the end of the 6-month study phase with two samples approximately 2 and 4 months from the beginning of orthodontic treatment. Whole unstimulated saliva was collected in 15 ml polypropylene Falcon tubes at each morning appointment and immediately placed in a − 80 °C freezer. Once the collection was completed, samples were thawed and centrifuged at 12,000 rpm for 10 min, and the supernatants were aliquoted into 2 ml Eppendorf tubes. The samples collected between April and September were considered to represent "summer," and those between October and March represent "winter."For the VitDBP measurement, a commercially available kit was used from EMD Millipore  , and samples were run on Luminex® 200™ . As a marker of bone-associated changes, we measured the ALP levels in saliva using a colorimetric ALP kit   according to the manufacturer's instructions. Optical density (OD) was measured at 405 nm by spectrophotometry. Enzyme activity was calculated as the OD of the reaction product multiplied by the reaction volume and normalized to the reaction time and the total protein. All samples were run using the same kit and the same batch simultaneously to reduce possible variation, with 10% of the samples repeated to ensure the reliability of measurements.Statistical analyses were performed using SAS 9.4 software. Before statistical modeling, data were thoroughly checked for potential data entry errors and outliers. Descriptive statistics were summarized using mean and standard deviation for continuous variables and frequency for categorical variables. Mixed linear regression models with random intercept were used to assess the relationship between VitDBP and season. A multiple linear regression model was used to assess the relationship between baseline VitDBP and absolute difference in tooth irregularity measures between baseline and end-of-treatment time-points.n = 36) to child  ratio in the study was 0.97. In the child group, the mean age was 13.2 years, and, in the adults, the mean age was 30.2 years. Racial ethnicity was self-reported, as African American (n = 27), Asian (n = 6), Caucasian (n = 17), and Hispanic (n = 23). The Angle classification of subjects was Class I (n = 48), Class II, including both divisions 1 and 2 (n = 18), and Class III (n = 7), which was consistent with the typical ratios seen in a university-based orthodontic clinic. No statistically significant differences were noted considering age, gender, race, or malocclusion.A total of 127 patients were recruited for the study; 84 patients completed the study, and 73 of them were included in the final analysis due to multiple missing samples and casts. There were 43 females and 30 males with an age range of 8–63 years and a mean age of 21.5 ± 11.1 years. The adult  than the "low" or "high" groups (mean = 1.22 ± 0.31). Figure Demographic information by normal vs. "low" and "high" VitDBP groups is shown in Table p < 0.05). Overall, however, the VitDBP levels stayed in the normal range for both seasons.As VitD can be strongly impacted by seasonal changes due to the availability or lack of sunlight exposure, we then used a mixed regression model with a random intercept to assess the seasonal effects on VitDBP. The samples collected between April and September were considered to represent "summer," and those between October and March represent "winter." The average monthly temperature and average sunshine index were considered to distinguish between “summer” and “winter” of that year samples collected in the winter season (4.47 ± 0.30) had significantly higher VitDBP than those collected in the summer season  and the absolute change in ALP (p = 0.0018). Neither ALP concentration nor ALP activity was significantly related to the change in tooth movement.We also examined the patterns of change in VitDBP and ALP levels across the four treatment time points. Subjects were grouped into four groups according to whether their VitDBP or ALP levels changed from quartile to quartile over various time points into changed (fluctuating), increasing, decreasing and no change. Comparing subjects with any changing patterns in VitDBP  to those in the no change group, we observed that the "no change" VitDBP group showed overall smaller mean absolute differences in irregularity index than those in the changed groups as shown in Table In this study, we aimed to determine whether salivary levels of VitDBP were associated with the rate of orthodontic tooth movement. We also identified the impact of confounding factors such as the seasonal effects on VitDBP levels, measured the change in VitDBP and ALP levels in response to the first six months of orthodontic treatment , and determined the correlation between salivary VitDBP and ALP levels during orthodontic tooth movement. The results demonstrated that "normal" levels of VitDBP in saliva were associated with a greater tooth alignment rate and resolution of crowding during orthodontic tooth movement. On the other hand, "low" and "high" levels of VitDBP were associated with a reduced orthodontic tooth movement rate. These data suggested that optimal levels of VitD may be required for orthodontic therapy.3 [While the role of VitD in bone metabolism is well-established , 13, 21,3 –28. Bein3 –32. Seru3 . However3 [p = 0.0748). While our study demonstrated a trend, the predictive value of salivary VitDBP for tooth movement was not statistically significant. Yet, given the string trend that is independent of the confounding factors  this issue should be studied further in larger cohorts in future studies.VitD is considered as an "accelerator" and a stabilizing agent in the orthodontic tooth movement , 34, 36.3 . In huma3 , 38. As As there were no normal levels of VitDBP available for saliva, we also ranked the subjects by their VitDBP levels. Measurements were grouped according to the average VitDBP levels of the subjects rather than the time-points measured. The subjects whose levels were within the middle 50% were considered “normal” and those above or below were considered as “high” or “low”. The subjects in the “normal’ range had more significant tooth movement than those in the other groups. Normal salivary VitDBP was between 2.75 and 6.48 ng/ml, which showed a tight interval. Normal VitDBP levels in saliva significantly correlated with a higher resolution of tooth irregularity than "high' or "low" groups. This finding is striking as it demonstrates that an optimal range of VitD is necessary for predictive orthodontic treatment. Therefore, for individuals with suboptimal (low or high) VitD levels at the start of the orthodontic therapy, a customized treatment strategy can be planned, and completion of the orthodontic treatment would be predictably longer.Another important finding in our work was that VitDBP did not demonstrate significant variation between race, age, and gender, further supporting the value of salivary VitDBP as a measure of VitD. There has been controversy in whether VitD levels vary according to race, age, and gender. African Americans and Asians were thought to have higher VitD deficiency than Caucasians with stronger overall bone health , 39. Mosp = 0.007 with no significant differences in VitDBP levels [p < 0.05) difference in salivary VitDBP levels when considering a two-season year model. During the "winter" season defined as October to February, salivary VitDBP levels were statistically higher than in the "summer" season defined as March to September, correlating with changes noted in 25-OHD and 1,25-OHD. Further studies are needed to determine the seasonal effects on VitDBP levels and its activity on 25-OHD and 1,25-OHD levels.Previous studies have shown that there was a significant seasonal fluctuation in 25-OHD and 1,25-OHD levels . During P levels . Our stuPrevious research has focused on the ALP as a biomarker of osteoblastic activity that could reflect tissue responses during orthodontic tooth movement . There iClinical outcomes of orthodontic treatment are associated with the level of VitDBP in saliva. Normal levels of VitDBP are shown to have higher orthodontic tooth movement levels than higher or lower levels. Given the strong trend that is independent of the confounding factors , the predictive value or salivary VitDBP for tooth movement should be studied in larger cohorts in future studies.