RNA editing by adenosine to inosine deamination is a widespread phenomenon, particularly frequent in the human transcriptome, largely due to the presence of inverted Alu repeats and their ability to form double-stranded structures – a requisite for ADAR editing. While several hundred thousand editing sites have been identified within these primate-specific repeats, the function of Alu-editing has yet to be elucidated.cis on sites located several hundred nucleotides from the Alu elements. Furthermore, a computational analysis, based on available RNA-seq data, finds that site-selective editing occurs significantly closer to edited Alu elements than expected. These targets are poorly edited upon deletion of the editing inducers, as well as in homologous transcripts from organisms lacking Alus. Sequences surrounding sites near edited Alus in UTRs, have been subjected to a lesser extent of evolutionary selection than those far from edited Alus, indicating that their editing generally depends on cis-acting Alus. Interestingly, we find an enrichment of primate-specific editing within encoded sequence or the UTRs of zinc finger-containing transcription factors.We show that inverted Alu repeats, expressed in the primate brain, can induce site-selective editing in We propose a model whereby primate-specific editing is induced by adjacent Alu elements that function as recruitment elements for the ADAR editing enzymes. The enrichment of site-selective editing with potentially functional consequences on the expression of transcription factors indicates that editing contributes more profoundly to the transcriptomic regulation and repertoire in primates than previously thought. RNA editing by adenosine deamination is a co- or post-transcriptional alteration of mRNA as well as non-coding RNA, which occurs in metazoans. This adenosine to inosine (A-to-I) editing occurs at single adenosines in transcripts produced by RNA polymerase II. A-to-I RNA editing within double-stranded RNA (dsRNA) is catalyzed by the ADAR family of enzymes (ADAR1 and ADAR2) .A receptor (Gabra-3), where editing (by either ADAR1 or ADAR2) recodes an isoleucine to a methionine (I/M) [cis-acting structural element, downstream of the sequence required for A-to-I catalysis, increases the local concentration of the editing enzyme by attracting ADAR, thus enabling editing in the vicinity.Selective editing of single adenosines is often found in short duplexes interrupted by bulges and internal loops. Since inosine is interpreted as a guanosine by the cellular machinery, editing has the potential to recode an mRNA and thereby increase the protein repertoire. In mammals most transcripts subjected to site-selective A-to-I editing within a coding sequence have been found in genes involved in neurotransmission. ADAR-mediated site-selective editing leads to altered functionality of several ligand- and voltage-gated ion channels as well as G-protein-coupled receptors in the mammalian brain -9. One one (I/M) . We recene (I/M) . AlthougThe repetitive retrotransposable Alu elements, each spanning approximately 300 nucleotides, are abundantly interspersed throughout the primate genome and are present in approximately 75% of all human genes, mostly within introns and untranslated regions (UTRs). Adjacent inverted Alu repeats can pair and form long stable stem-loop structures, which are favorable editing substrates, and are also potentially highly abundant in humans  database). The human transcriptome is therefore exceptionally prone to A-to-I editing in comparison to other mammals and even to other primates, which is mainly attributed to the high frequency of editing within inverted Alu pairs . Even coOur results indicate that inverted Alu repeat elements can act as editing inducers. These elements are often located hundreds of nucleotides away from the specific editing site. We propose that duplexed inverted Alu repeats act as ADAR recruitment elements, which enhance editing efficiency at adjacent sites, ultimately giving rise to new editing events in primate transcriptomes.We have previously shown that a long intronic stem-loop structure, located 150 nucleotides downstream of the I/M editing site in the Gabra-3 transcript, is required for its editing, and is also targeted for editing itself . In the gabra-3 minigene, which includes the I/M site in exon 9 followed by the intron 9 editing inducer stem-loop, was analyzed  d Figure a. The efd Figure a. The reP = 0.001) , although the difference was not statistically significant. A slight decrease in editing efficiency  could be detected when the editing inducer was moved to a double distance downstream of the I/M site (DDS IE). These results suggest that the location of the IE does not affect editing efficiency at the I/M site if it is in the vicinity of the site of editing.As previously shown, no editing was detected by endogenous ADAR in HeLa cells when expressing Gabra-3 lacking the intronic inducer (ΔIE), while the WT transcript was edited to 37% P = 0.00   as the WT intronic IE  Figure a. These E Figure b,c. ThesWe hypothesized that enhancement of site-selective editing by proximal inverted Alu repeats is widespread in the human transcriptome. To examine the relation between inverted Alu repeats and site-selective editing systematically, two datasets were compiled by literature mining . Notably, this difference was due to the high frequency of editing sites located in close proximity (up to 1 to 2 kb) from an edited Alu  of these sites (up to 2 kb from the edited Alu) , we removed editing clusters from the set of non-Alu sites. A cluster was defined as a group of at least three sites that are located up to 40 nucleotides from each other over a minimum total length of 70 nucleotides (from first site to last site). Thus, 1,312 sites were removed in total  and those flanking proximal sites . However, when sites in UTRs were compared, sequences flanking the distal sites (n = 213) were found to be far more conserved than those flanking the sites proximal to inverted Alu repeats (n = 297). See Figure S2 in Additional file Since the editing of Alu-independent sites requires a highly stable and specific stem-loop structure, their flanking sequences have likely been subjected to stringent evolutionary selection. This may not be the case, however, for Alu-dependent editing sites, if their editing efficiency relies on external stem-loop structures (inverted Alu elements). To compare the conservation between these two groups of sequences (near and far from edited Alus), we collected non-clustered editing sites, which are most likely to have functional effects. These were sites leading to non-synonymous recoding of a protein and sites in the UTRs. For each editing site, the average conservation was calculated over the site and 30 flanking nucleotides, 15 from each side, using both PhyloP  and Phasn = 245). To identify annotations for which genes with Alu-dependent editing specifically are enriched, we included all genes containing ‘functional’ sites of editing as a background . In addition, to avoid detecting terms and functions enriched in Alu-containing genes, simply because all the examined genes have Alus, we added all Alu-containing genes to the background list . The results revealed marked enrichment of functions such as transcription regulation (2.2-fold) and the lysosome pathway (46-fold). In agreement with the enrichment found for transcription regulation, high enrichment was observed for genes encoding zinc finger proteins and specific sub-families of these (up to 16-fold), which often function as transcription factors. Table Functional enrichment analysis was performed on genes containing editing sites within 1 kb of an edited Alu (Alu-dependent editing) that are likely to affect functionality. Some of these editing events lead to missense and nonsense recoding and splice signals n = 24. To idenTo unify similar annotation terms, we performed functional annotation clustering analysis with DAVID ,28, whicOne of the genes that harbor site-selective editing in the vicinity of an edited Alu encodes the NEIL1 DNA repair enzyme. NEIL1 has previously been shown to be edited at the second and third positions of the K242 AAA codon within exon 6 ,31. EditNEIL1 minigene including intron 5 (with the inverted Alu repeats) and exon 6, containing the K/R site of editing  in the absence of the inverted Alu repeat  in editing efficiency was also observed in ADAR2 co-transfections and in endogenous editing in HeLa cells  when co-transfected with ADAR1. A twofold increase (P = 0.004) in K/R editing was also observed in co-transfections with ADAR2 and by endogenous editing in HeLa cells  of the transcripts were edited in the editing reporter lacking the Alu inverted repeats  despite the addition of 2.5 μg of transfected ADAR1. To ensure that the different amounts of transfected ADAR1 expression vector were proportional to the amount of produced ADAR1 protein, Western blots were made from extracts prepared after the different transfections. As indicated in Figure Our hypothesis is that a long stem-loop structure forming an almost complete duplex such as the Alu inverted repeats works as a recruitment element of the ADAR proteins. This IE may thereby increase the local concentration of ADAR and facilitate editing at second sites. To determine if this hypothesis holds, we investigated if editing at the K/R site in NEIL1 was less dependent on the concentration of ADAR1 in the presence than in the absence of the Alu IE. We therefore separately transfected a fixed concentration 1.5 μg) of the editing reporters hNEIL1 and hNEIL1 ΔAlu together with titrated concentrations (0 to 2.5 μg) of the ADAR1 expression vector. In theory the substrate containing the Alu IE would require a lower concentration of the editing enzyme to reach a high level of editing than the substrate without the Alu elements. Indeed, already at 0.1 μg of the ADAR1 vector, editing at the K/R site reached a level of 63% in the presence of the Alu inducer, while only 20% ( μg of thP = 0.05) with 0.1 μg of reporter. Editing dramatically decreased when the concentration of the reporter increased to a final editing level of 24% (P = 0.0004). This result indicates that in the presence of the Alu IE, a lower concentration of the ADAR enzyme is required to achieve efficient editing at the selective K/R site. It also suggests that the Alu repeats may help in recruiting the editing enzyme to the transcript.The reciprocal experiment was also performed where the hNEIL1 and hNEIL1 ΔAlu editing reporters were titrated (0.1 to 2.5 μg) with a fixed concentration (1 μg) of the ADAR1 expression vector. This gave high levels of edited hNEIL1 transcripts (60%) at low concentrations of the editing reporter. These continued to be efficiently edited even with high concentrations of the editing reporter and with a peak of editing at 66% with 1 μg of the hNEIL1 reporter . GLI1 act as a transcriptional effector in the Hedgehog (HH) signaling pathway. The transcript of this transcription factor was recently found to be edited in humans, changing an arginine to a glycine (R/G) at position 701 ,32. HoweAnother candidate of the edited transcription factor is the Promyelocytic leukemia zinc finger protein, PLZF . This transcriptional repressor has been shown to regulate major developmental processes, such as spermatogenesis, limb development and hematopoiesis . The traThe Alu sequences aluSz and aluJb are present in other primates, such as the chimpanzee, rhesus and marmoset. However, these Alu sequences are not completely conserved among the primate species. Consequently, a putative stem-loop structure formed by the Alus in rhesuses is disrupted by larger and more frequent bulges compared to humans , this mechanism for regulation will only occur in species with a highly developed brain Figure a. Furthecis-acting Alu elements, thereby contributing to primate and human genome evolution by a process that is absent from other species. The primate-specific enrichment of site-selective editing with functional consequences for transcription factors indicates that editing may contribute profoundly to transcriptomic regulation and the variety in transcript isoforms in the primate brain.The human transcriptome is exceptionally prone to A-to-I RNA editing. This is mainly explained by the frequently edited inverted Alu repeats, which are unique to primates. The function of editing within these repeats has, however, hitherto largely been unknown. In the present work we propose a model where two primate-specific adjacent Alu repeats function as a recruitment element for the ADAR editing enzymes Figure . The incThe Gabra-3 editing reporter construct pGARα3-I/M (Gabra-3 WT) was generated from the mouse genome sequence. The ADAR2 expression vector pcDNA3 FLAG/rADAR2 has previously been described ,36. The •US IE (BamHI): (FW) 5′-aataaggatccaggaagggctgagaagcacttcc-3′, (RW) 5′-aataaggatccaggccagattaccaagaagc-3′•DDS IE (NotI): (FW) 5′-aataagcggccgcaggaagggctgagaagcacacttcc-3′, (RW) 5′-aataagcggccgcaggccagattaccaagaagc-3′•Alu-IE (NheI): (FW) 5′-aatttgctagcgtttcttccatccctataatcc-3′, (RW) 5′-aatttgctagcggtcaagaaccactgttttaatagc-3′NEIL1 gene, and cloned into pcDNA3 FLAG. Primer sequences were as follows:The human NEIL1 and mouse NEIL1 editing reporters were generated by PCR amplification from the genomic •hNEIL1: (FW) 5′-gcccggagctgaccctgagccag-3, (RW) 5′-ggaaccagatggtacggccatgcc-3′•hNeil ΔAlu: (FW) 5′-ggacaaggattcttaatcccactcc-3′, (RW) as above for hNEIL1•mouse NEIL1: (FW) 5′-gcaagtttccactttctacc-3′, (RW) 5′-ccagatggtacggccatgccgg-3'The mouse NEIL1 + Alu was generated by PCR amplification of the human inverted Alus upstream of the K/R site in human NEIL1 using primers with Nhe1 restriction site overhang. The PCR fragment was cloned, using Nhe1, into the mNEIL1 reporter at the position corresponding to the human sequence. Primer sequences were as follows:•mNEIL1 + Alu (Nhe1): (FW) 5′-aatttgctagcggctgggcgcagtggctcatgc-3′, (RW) 5′-atttgctagctggccctgtgcagtggccacac-3′Restriction sites were created and also depleted after insertion of the fragments in the Gabra-3ΔIE reporter and in the mNEIL1 reporter using QuickChange site-directed mutagenesis . All mutants were verified by Sanger sequencing .Reporter constructs (1.5 μg) were co-transfected with ADAR1 or ADAR2 (2.5 μg) expression vectors into HEK293 cells and grown in six-well plates. For endogenous editing, the reporter constructs (4 μg) were transfected into HeLa cells. LIPOFECTAMINE™ 2000  was used in all transfections. The transfection efficiency was comparable between separate experiments. Control transfections using an empty vector co-expressed with or without the substrates were done for each experiment. RNA was isolated 48 hr (HEK293) and 72 hr (HeLa) after transfection using GenElute7™ mammalian total RNA isolation , and treated with DNase-1 Amplification Grade . The cDNA was generated using random hexamer deoxyoligonucleotides and SuperscriptII RT . Negative control reactions without reverse transcriptase were performed in all RT-PCR experiments to exclude genomic DNA contamination. The following PCR was made using Taq . Primers used for the PCR reactions for Gabra-3 WT and Gabra-3 mutant reporters were: (FW) 5′-ggtgtcaccactgttctcacc-3′ and (RE) 5′-gctgtggatgtaataagactcc-3′. Primers used for the PCR reactions of human NEIL1 and mouse NEIL1 reporters were as described above.Experiments were carried out on tissue extracted from an adult NMRI mouse (whole brain) and a human (cerebellum). The experiments on mouse brain tissue were approved by Stockholms Norra Djurförsöksetiska nämnd and have permission number: Dnr N 410/12. The human brain tissue was provided by the Huddinge Brain Bank and the experiments were approved by the Karolinska Institute, Forskningskommité Syd, with the permission number: Dnr 84/02. Total RNA was isolated using the TRIZol reagent protocol  and treated with DNase-1 Amplification Grade . Genomic human and mouse DNA from the same individuals were purified using QIAamp DNA Mini Kit . DNase1-treated total RNA and genomic DNA from the brain of an adult rhesus monkey was purchased . First strand cDNA was synthesized with Superscript II and random hexamer deoxyoligonucleotides . Negative control reactions without reverse transcriptase were performed in all RT-PCR experiments to exclude genomic DNA contamination. A standard PCR protocol was used for NEIL1 and ZFP14 and nested PCR was performed to amplify GLI1 . Primers used for the PCR reactions were as follows:•human NEIL1: (FW) 5′-gcccggagctgaccctgagccag-3′, (RW) 5′- ggaaccagatggtacggccatgcc-3′•mouse NEIL1: (FW) 5′-agcccagagctgaccctgagccag-3′, (RW) 5′- ccagatggtacggccatgccgg-3′•rhesus NEIL1: (FW) 5′-gcctgaagctgaccctcagccag-3′, (RW) 5-′ggaaccagatggtacggccatgcc-3′•human GLI1:  5′-gcagccaatacagacagtggtg-3′, (FW nested) 5′-ccagtgacccagcccaggctg-3′,  5′-ggtggaacctacagccagtgtcc-3′•rhesus GLI1:  5′-tgtcaagacagtgcatggtcctg-3′, (FW nested) 5′-gctccagctagagctcagagg-3′,  5′-ctgtaggctccacctagagcc-3′•mouse GLI1:  5′-acacgtgaagacagtgcatg-3′, (FW nested) 5′-gttcaagagcctgggatgtg-3′,  5′-gacactggctataggcagcac-3′•human ZFP14: (FW) 5′-gaagaagtctaataaatctag-3′, (RW) 5′-ccatcagtggaggatcctggaacc-3′•rhesus ZFP14: (FW) 5′-gaagtctaataaatccagaagg-3′, (RW) 5′-acactgttcatctagtcccc-3′The PCR products were gel-purified using NucleoSpin Gel and PCR Clean-up  and editing was determined by Sanger sequencing .P values were calculated using a two-tailed Student’s t-test. In addition, percentage editing at the I/M site in the Gabra-3 transcript was compared to editing determined by 454 high-throughput sequencing as in [non to full in five stages  according to the mean value of the G peak derived from triplicates of the experiments.To evaluate the level of edited transcripts, RNA from at least three independent experiments was sequenced. Editing was determined by measuring the ratio between the A peak and the G peak heights in individual chromatograms using FinchTV. Percentage editing was calculated as the peak height of G/(A + G) × 100. ng as in , where tWhole-cell extracts of transfected HEK293 cells were prepared using Lysis-M , supplemented with protease inhibitor cocktail . Samples for Western blot were boiled for 10 minutes with a Laemmli sample buffer containing β-mercaptoethanol prior to fractionation by electrophoresis in 10% polyacrylamide gels and transfer to a polyvinyl diflouride (PVDF) membrane. Membranes were probed with α-FLAG (1:1000) or α-actin (1:250), both from Sigma-Aldrich, St. Louis, MO, USA. Horseradish peroxidase-conjugated anti-rabbit IgG  was used as secondary antibodies. Blots were developed using the WesternBright Sirius chemiluminescence detection system .RNA secondary structure predictions were made using Mfold  and Sfolet al. [Editing sites for analysis were compiled from several transcriptome-wide screenings, summarized in Table S1 in Additional file et al. , which iFor each non-clustered analyzed editing site (missense and UTR sites), genomic intervals of 30 flanking nucleotides were obtained, 15 from each side. Non-exonic nucleotides were removed from the intervals, which extended beyond the exon ends, to maintain uniformity in the compared genomic elements. For 46 vertebrates, PhastCons and PhyloP scores were downloaded for the nucleotides in the genomic intervals from the UCSC table browser  and wereTo obtain random adenosines, genomic SNP coordinates, where adenosine is the reference allele, were downloaded from SNPdb  via the Gene ontology and pathway analyses were performed by DAVID ,28. The A-to-I: adenosine to inosine; bp: base pair; DDS IE: downstream inducer element; dsRNA: double-stranded RNA; I/M: isoleucine to methionine; IE: inducer element; K/R: lysine-to-arginine; kb: kilobase; miRNA: microRNA; PCR: polymerase chain reaction; R/G: arginine to glycine; RT-PCR: reverse transcription polymerase chain reaction; SNP: single nucleotide polymorphism; US IE: upstream inducer element; UTR: untranslated region; WT: wild-type; ΔIE: deleted inducer element.The authors declare that they have no competing interests.CD and MÖ designed the experiments. CD performed the molecular genetic studies. MB performed the Western blot analysis. GS designed and performed the computational analyses. CD, GS and MÖ wrote the manuscript. All authors read and approved the final manuscript.Sources of editing sites and edited Alus compiled from literature mining.Click here for fileAll non-Alu editing sites included in the study.Click here for fileFigure S2. Mean PhyloP and PhastCons conservation scores of sequences flanking UTR editing sites proximal to (<=1 kb), and distal to (>1 kb) edited Alu. Figure S3. NEIL1 sequence alignment of selected placental mammals. Figure S4. Editing of the human NEIL1 transcript in the presence and absence of the upstream Alu elements. Figure S5. Titration of hNEIL1 or hNEIL1 ΔAlu reporters co-transfected with a constant concentration of ADAR1 expression vector of 1.5 μg into HEK293 cells. Figure S6. Predicted RNA secondary structures of the inverted Alu repeats in the human and Rhesus ZFP14 transcripts, as presented in Figure Distance distribution between edited sites and nearest edited Alu, after exclusion of clustered sites. Positive/negative distance values indicate that the edited Alu is downstream/upstream of the (non-Alu) editing site, respectively. Green bars: distance from editing sites to the edited Alus. Red bars: distance from random adenosines to edited Alus. After removal of clustered sites, the frequency of sites closest to edited Alus increased, as did the preference for the Alus to be downstream of the edited site (compare with Figure Click here for fileUltra-conserved sequences flanking Alu-independent editing sites.Click here for fileAll significantly enriched terms and genes with editing sites close to inverted Alu repeats.Click here for fileClusters of annotation enriched among genes containing non-Alu editing sites adjacent to edited Alus.Click here for file


This results in cell lysis and release of a circular form of the ICE. Induction of this novel system may allow transfer of an ICE, enhancing its survival potential under conditions not conducive to conjugative transfer.Integrative conjugative elements (ICEs) are a class of bacterial mobile elements that have the ability to mediate their own integration, excision, and transfer from one host genome to another by a mechanism of site-specific recombination, self-circularisation, and conjugative transfer. Members of the SXT/R391 ICE family of enterobacterial mobile genetic elements display an unusual UV-inducible sensitization function which results in stress induced killing of bacterial cells harboring the ICE. This sensitization has been shown to be associated with a stress induced overexpression of a mobile element encoded conjugative transfer gene,  In gene1 .int and xis (int) is a recombinase which catalyzes the recombination reaction between specific recognition sites in a host genome (attB) and the circular ICE element usually called attP  is one of two archetypal members of the SXT/R391 family that was initially isolated from a multidrug resistant clinical isolate of  in 1992 . The sech Africa .Figure 1) and sequences that facilitate regulation of element functions, their integration/excision and their conjugative transfer , which are homologous to transcriptional enhancers, resulted in induction of the SOS response, which promotes autoproteolysis of SetR (Orf96)  and conjugation-associated operons  . SetR/Orpressor.  This in  operons .Escherichia coli AB1157 to UV irradiation, significantly decreasing post-irradiation cell survival rates. It was thought that this sensitization effect was most likely due to interference with one of the RecA∗-induced DNA damage repair pathways of E. coli as the effect was shown to be recA-dependent but lexA-independent  and a conjugative transfer gene orf43 (encoding a TraV homolog) abolished the induced sensitization. This was interesting as orf43 was found to be one of the most conserved of the “core genes” of the SXT/R391 family with all homologs (found to date) being ≥98% similar, indicating that it may be under evolutionary pressure to stay conserved. Orf43 is a TraV homolog which forms part of the mating pore during conjugation . Induction of Jef, which is also known as Xis , might escape in the absence of a functional conjugative mechanism such as might be the case in severely damaged cells. This hypothesis was tested using specific deletion mutants of ICE R391 that were deficient in conjugative transfer  and 1.01 × 10-6 (with UV). Addition of DNase abolished transfer (Transfer rates of <10-10). This indicated that some form of transformation must have occurred.Although, the significance of such a detrimental effect was not immediately obvious when the two phenomenon (increased circular form of the mobile element and cell bursting) were linked, a testable hypothesis emerged. On the one hand DNA damage induces excision of the element  while attransfer . These srestored . EffectiFigure 2) could also provide a back-up escape route to allow conjugation levels discussed above, so that if the cell were so damaged that conjugation cannot take place then a secondary escape mechanism would be available to the element. This appears to be a novel mechanism, which we term a ‘trap-door,’ of mobile element survival and presumably occurs in tandem with formation of circular transfer intermediate, which is subsequently released as a result of pore formation and cell lysis making the element available for transformation allowing for some ICE survival in extreme environments. This proposed mechanism is so far unique to the SXT/R391 family and no comparable escape mechanism has been found in other mobile genetic elements thus far.This mechanism  creating single stranded DNA which stimulates RecA to become its active form RecA∗ .2.∗ cleaves the putative ICE SXT/R391 setCD/orf90-91 repressor protein setR/orf96  in a manner that is cytotoxic to the host cell  and the death of the cell .The cytotoxic effect is the result of damage to the cells outer membrane caused via the action of TraVthe cell , 2015 (F7.The SXT/R391 ICE element is then available to be taken (via ‘apparent transformation’) into other bacterial cells All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Synaptic plasticity is thought to induce memory traces in the brain that are the foundation of learning. To ensure the stability of these traces in the presence of further learning, however, a regulation of plasticity appears beneficial. Here, we take up the recent suggestion that dendritic inhibition can switch plasticity of excitatory synapses on and off by gating backpropagating action potentials (bAPs) and calcium spikes, i.e., by gating the coincidence signals required for Hebbian forms of plasticity. We analyze temporal and spatial constraints of such a gating and investigate whether it is possible to suppress bAPs without a simultaneous annihilation of the forward-directed information flow via excitatory postsynaptic potentials (EPSPs). In a computational analysis of conductance-based multi-compartmental models, we demonstrate that a robust control of bAPs and calcium spikes is possible in an all-or-none manner, enabling a binary switch of coincidence signals and plasticity. The position of inhibitory synapses on the dendritic tree determines the spatial extent of the effect and allows a pathway-specific regulation of plasticity. With appropriate timing, EPSPs can still trigger somatic action potentials, although backpropagating signals are abolished. An annihilation of bAPs requires precisely timed inhibition, while the timing constraints are less stringent for distal calcium spikes. We further show that a wide-spread motif of local circuits—feedforward inhibition—is well suited to provide the temporal precision needed for the control of bAPs. Altogether, our model provides experimentally testable predictions and demonstrates that the inhibitory switch of plasticity can be a robust and attractive mechanism, hence assigning an additional function to the inhibitory elements of neuronal microcircuits beyond modulation of excitability. We must constantly learn in order to meet the demands of a dynamically changing environment. The basis of learning is believed to be synaptic plasticity, i.e. the potential of neuronal connections to change. Depending on context, however, it may be either useful to learn and modify connections or, alternatively, to keep an established network structure stable to maintain what has been already learned . The ability to switch synaptic plasticity on and off in a flexible way hence constitutes an attractive feature of neuronal processing. Here, we analyze a cellular mechanism based on the inhibition-mediated gating of coincidence signals required for Hebbian forms of excitatory synaptic plasticity. While experimental evidence in support of individual steps involved in this mechanism is accumulating, it is as of now unclear whether this mechanism can indeed operate robustly under physiologically realistic parameters of pyramidal cells, in particular, without impairing information flow in these cells altogether. Computational modeling allows us to demonstrate that this is indeed possible if inhibition is well timed (on the order of 1 ms). Moreover, we show that a specific design of the local circuit can ensure the necessary timing. To successfully interact with our environment, we need to adjust to new or changing conditions. It is widely accepted that this ability involves alterations of synaptic connections in the brain, so-called synaptic plasticity . While sIn this context, inhibition has been proposed as a means to regulate plasticity –6. GABAeTo this end, mathematical modeling suggests itself, because it allows to systematically vary both the strength and dendritic location of inhibition and to monitor bAPs and calcium spikes in the whole dendrite with high temporal and spatial resolution. We hence adapted a multi-compartmental model of pyramidal cells to reproduce a number of key electrophysiological characteristics. These included a realistic decrease of the amplitude of bAPs along the dendritic tree , charactWe demonstrate that shunting inhibition gates bAPs and calcium spikes in an all-or-none manner, laying the foundation for a binary switch of plasticity of excitatory synapses. Further, we identify the timing constraints for an efficient inhibitory gating of bAPs and calcium spikes. Implementing an additive spike-timing-dependent plasticity (STDP) rule , we findPyramidal cells form one of the most important classes of projection neurons in the mammalian brain. Their apical and basal dendrites often receive synaptic inputs that originate from different pathways . AccordiWe investigated the feasibility of such inhibition-mediated control of plasticity in a multi-compartmental model neuron with a simplified morphology capturing basic features of the dendritic compartments , as well as the soma and the axon see . ElectroA synapses [Inhibition was assumed to be shunting with rise and decay time constants of 0.5 ms and 5 ms, respectively. This kind of inhibition is commonly observed in pyramidal neurons receiving inhibitory GABAsynapses  and it asynapses , 22. Finsynapses .First, we sought to understand the effect of local dendritic inhibition on bAPs. To this end, we placed a shunting inhibitory input on the proximal apical dendrite, systematically varied its strength, and monitored the amplitude of a somatically-induced bAP along the apical dendrite. For weak inhibition, the bAP amplitude was reduced around the dendritic location of the inhibitory input, but recovered to its full amplitude as the bAP propagated down the dendrite. In contrast, strong inhibition barred the bAP from invading the dendrite . The traA moderate temporal jitter (on the order of 1 ms) in the activation of inhibitory synapses also did not abolish the all-or-none behavior, but when a critical amount of inhibition was reached too early, the neuron was prevented from spiking. Overall, however, timing of inhibition needed to be relatively precise, as we will outline in the following section, where we first present data on the compartment-specificity of the effects.Due to its relatively local effect, shunting inhibition can control dendritic signals in a manner that is distinct between compartments, such as basal and apical dendrites. We hence investigated the effect of inhibition in different compartments , on the bAPs and calcium spikes, in the same and other compartments.First, we consider the general case of bAP modulation in the absence of BAC firing. A systematic illustration of the effects of shunting inhibition on bAPs in different compartments is presented in The first column in To assess whether timing requirements for the modulation of bAPs are realistic, we varied both the strength and the timing of proximal inhibitory inputs and monitored their effect. As discussed above, bAPs were canceled by inhibition in an all-or-none manner. We hence distinguished three cases: (1) inhibition had no effect on the bAP, (2) inhibition canceled the bAP in the dendrite without affecting the somatic spike (the scenario of interest for the modulation of plasticity), and (3) somatic firing was abolished altogether and hence also no bAP was observed. The sensitivity of the bAP to proximal inhibition showed a marked dependence on timing . The intConforming with the local effects of shunting inhibition , 22, disSo far in the analysis, somatic action potentials depended on somatic current injection. The majority of excitatory inputs, however, arrive on the dendrites. For the hypothesized inhibitory regulation of plasticity not to interfere with neuronal processing, it needs to be ensured that shunting inhibition does not impair the forward flow of signals from the dendrites to the soma (while canceling the bAP). We hence also included conductance-based excitatory inputs to the dendrites.In the simplest case, inhibition modulates dendritic signals of postsynaptic spiking that was triggered by an excitatory pathway arriving in a different dendritic compartment. This case proved largely identical to direct somatic stimulation, because inhibitory inputs are not on the electrotonic route from the site of excitatory stimulation to the soma. Functionally, postsynaptic activity triggered by synaptic input in one dendritic compartment A could potentially mediate plasticity in a different compartment B. Our results suggest that in this scenario, synaptic plasticity in dendritic compartment B can be readily modulated by inhibitory inputs, by controling the spread of dendritic signals in this compartment.In a more complex setting, excitatory stimulation and inhibitory control impinge onto the same dendrite. In this case, it is less clear whether inhibition of backpropagating dendritic signals is possible without impairing the ability of forward-directed input integration. A quantitative analysis revealed that an EPSP could reach the soma despite an inhibitory conductance (located 90 μm from the soma) that was capable of suppressing the bAP within a time window of about half a millisecond . The widWhile many neurons exhibit bAPs, some neuron types  can additionally generate distal calcium spikes that trigger BAC firing. Because calcium spikes can also serve as plasticity signals , we analA conductance decayed too fast, and hence could not exert an influence on later bAPs ..Fig 5).To demonstrate that cancelation of the coincidence signals indeed results in the anticipated changes to synaptic plasticity, we next subjected the model cell to a classical plasticity paradigm. Contemporary biophysical and phenomenological models of STDP , 27–29 dAbove, we have shown that the modulation of the bAP requires inhibition to fall into a small time window closely tied to the initiation of the somatic action potential in the pyramidal neuron. The question arises whether such timing is realistic and how it can be achieved. As a proof of principle, we here show that the common local circuit motif of feedforward inhibition  is a gooInterestingly, interneuron dynamics play a role for the timing requirements. Dependent on interneuron type, spike latencies and firing frequencies can vary. The size of the temporal window where inhibition can cancel bAPs (without canceling EPSPs) tends to be smaller for proximal inhibitory synapses than more distal synapses, as outlined above. One may hence speculate that the dynamics of interneurons innervating proximal parts of the dendritic tree should be sufficiently fast. To highlight the role of interneuron dynamics, we probed the circuit with two interneurons differing in their spiking dynamics. We used the interneuron model from the previous paragraph and compared its performance in the circuit to that of a second interneuron with slower dynamics. The latter was implemented by artificially slowing down the opening and closing rates of the interneuron’s sodium channels. In this example, only the interneuron with a short spike latency met the tight timing requirements for bAP modulation at a more proximal synaptic location . The timIn this study, we investigate how dendritic inhibition can serve to regulate Hebbian plasticity of excitatory synapses. Using multicompartmental biophysical neuron models, we show that shunting inhibition can gate the propagation of bAPs as well as dendritic calcium spikes in an all-or-none manner and thus exert direct control over the Hebbian coincidence signals. As a functional consequence, inhibition can provide a binary switch of synaptic plasticity. This switch can be specific for pathways in the local network as well as subsets of synapses within the same neuron. Importantly, forward-directed information flow via orthodromic EPSPs can be maintained when bAPs and calcium spikes are annihilated by shunting inhibition. The trivial scenario, where inhibition cancels not only Hebbian coincidence signals but also the excitatory drive of the neuron, can hence be avoided. Quantitative analyses of the physiologically constrained models reveal that this mechanism imposes strict timing constraints: while for the regulation of bAPs inhibition has to fall into a specific time window of a width of 1 ms, regulation of calcium spikes requires less precise timing. The time scales for proximal and distal inhibitory modulation of plasticity differ by several milliseconds. Finally, we suggest and provide a proof of principle that the precise timing required for bAP modulation can be achieved by a local circuit including the common network motif of feedforward inhibition.The central hypothesis that inhibition can control synaptic plasticity has been discussed in the experimental and theoretical literature –7, 35–37Strong experimental evidence in support of the ability of inhibition to modulate plasticity via gating of bAPs was recently provided by Müllner et al. (2015) . They shOur study makes several testable predictions. In particular, the all-or-none nature of bAP modulation enabling the binary switch as well as the compartment specificity could be tested in experiments. To investigate the latter, classical paired recording paradigms for synaptic plasticity could be extended by optogenetic stimulation of different interneuron classes. Particularly promising candidates are the above mentioned SOM or PV interneurons that target perisomatic and distal dendritic regions of pyramidal cells, respectively , 38.Moreover, perisomatic versus distal modulation of dendritic coincidence signals poses different timing requirements on inhibition. The proximity of inhibition to the soma constrains the modulation window, because the times of passage of the forward-directed EPSP and the backward-directed bAP (at the location of the inhibitory synapse) are very close. Therefore, inhibitory synapses ought to have a certain distance to the soma to be well suited to control the bAP and plasticity without canceling somatic spiking. Our results suggest that at a distance of ∼100 μm, which is relatively proximal for apical dendrites, the timing window becomes wide enough to enable inhibitory control . We predA-mediated inhibition (∼0.5 ms)) is crucial for the mechanism to operate effectively. In contrast, the temporal extent of inhibition  is less important. These variables are likely to be more relevant in setting a lower frequency limit to excitatory signals because they could interfere with a following EPSP if inhibition lasted for too long. For GABAA-typical decay time constants on the order of 5 ms (as used here), such limits to the frequency of EPSPs are sufficiently large (>100 Hz). We note that GABAA-mediated inhibitory postsynaptic potentials (IPSPs) have been described to differ between proximal and distal sites [A-mediated currents.From the perspective of the pyramidal neuron, we find that, a short rise time of synaptic inhibition  plaPer se, when the interneuron is active, plasticity of excitatory pyramidal synapses is switched off. In turn, silencing the interneuron up-regulates pyramidal cell plasticity. This design indicates a disinhibitory regulation of Hebbian plasticity, which is in line with recent findings for behavioral learning [In a feedforward inhibitory circuit, inhibition, by default, comes along with excitation. This means that an additional source is required to switch off the inhibitory influence .Simulations were performed in NEURON , with an2 and 0.01 S/cm2, respectively. The voltage dependence of dendritic sodium channel activation was shifted by +5 mV to allow apical excitatory postsynaptic potentials (EPSPs) to cause somatic spikes without eliciting dendritic spikes. The reversal potentials were: ENa = +60 mV and EK = -80 mV. The A-type potassium conductance increased five-fold up to 500 μm from the soma with an initial value of 0.029 S/cm2.Both the neuron and its dendrites hosted active conductances to allow somatic and dendritic action potentials. The voltage-dependent conductances were limited to those needed for the occurrence of backpropagating action potentials and calcium spikes as experimentally observed. In a first step, inactivating sodium and delayed-rectifying potassium conductances with channel kinetics from  (ModelDB2): gCa,H = 0.00015, gK,Ca = 0.00025 and inside: gCa,H was increased 3-fold, gCa,L = 0.005. In the soma, gCa,H and gK,Ca were increased 2-fold compared to the dendrite. The reversal potential for calcium was ECa = +140 mV.To account for calcium spikes, a calcium spike initiation zone with increased calcium channel densities in the apical tuft between 500 and 750 μm from the soma was incorporated. While high-voltage activated calcium channels , a calcium-activated potassium current  and a calcium decay mechanism were present in all dendritic compartments, we added low-threshold T-type calcium channels  only to the active zone. All channel kinetics were taken from  , matching the order of magnitude of the experimentally measured value  based on modern sodium imaging techniques [The axon initial segment consisted of only a single compartment with increased sodium channel density compared to dendritic compartments (0.6 S/cmchniques ). Voltagchniques –84.If not otherwise indicated, the simplified model was stimulated by somatic step current injection of 0.3 nA for 2 ms. All synapses were modeled as time-dependent conductance changes. The dendritic synapse in A-mediated, and not GABAB-mediated inhibition, because the former is more abundant, and its time constants are better suited for the task. The large time constants of GABAB mediated inhibition are not well-suited, because they lead to (1) slow inhibition, which cannot fulfill the tight timing constraints for bAP inhibition, and (2) long-lasting inhibition, which, next to being a waste of energy, can interfere with subsequent signaling. GABAergic inhibition had a double-exponential time course . The reversal potential of the inhibitory synapses was close to the resting potential, i.e. -73 mV. At rest, inhibition hence did not hyperpolarize the membrane potential, but acted as a local “shunt”, i.e. increased the membrane conductance. The maximum conductance, referred to as inhibition strength, and the onset of inhibition was varied as indicated.Inhibition was modeled to resemble GABAFor control, inhibitory synapses were distributed in space and time according to a Gaussian distribution with varying standard deviation sigma. The spatial distribution was centered around 90 μm and truncated such that synapses were restricted to the apical trunk. The temporal distribution of inhibition onsets was centered around 2 ms after the onset of somatic stimulation.To study the impact of inhibition on dendritic spikes, we recorded the membrane voltage in the basal dendrite (75 μm), the oblique dendrite (370 μm) and the apical tuft (650 μm). Additionally, we monitored the calcium current in the apical tuft at the same spot. Spike amplitudes were measured as the maximum voltage deviation from rest (a membrane voltage reaching 10 mV would be a spike of amplitude 10 mV-(-73 mV) = 83 mV). bAPs were normalized to the non-inhibited amplitude of the first bAP. Calcium spikes were quantified by the integral of the calcium current , normalized to the non-inhibited calcium current. A somatic spike was detected when its amplitude reached a threshold of 80 mV.t is the difference between pre- and postsynaptic spike time: tpost-tpre. A postsynaptic spike was counted when the membrane voltage at the synapse reached a threshold of -20 mV. For apical tuft plasticity, a postsynaptic spike was counted when the calcium concentration reached a threshold of 0.5 mM. We used a different model for distal plasticity, assuming that the calcium spike governs calcium dynamics and thus plasticity in the distal dendrite .The feedforward inhibitory circuit contained the introduced multi-compartment pyramidal neuron model and a model of a fast-spiking interneuron (IN). The latter, a single-compartment model with Hodgkin-Huxley-type sodium and potassium conductances, was taken from  Click here for additional data file.S2 FigModulation of bAPs propagating into the basal dendrite when a calcium spike was triggered by concurrent somatic current injection and dendritic excitation Click here for additional data file.S3 FigResults are in the same format as in (EPS)Click here for additional data file.S4 FigTiming requirements for bAP modulation as a function of the dendritic location of inhibition. The simulation paradigm is identical to that in (EPS)Click here for additional data file.S5 FigBAC-firing and the corresponding distal calcium influx is triggered if pre- and postsynaptic spike times are coincident. Therefore, the amount of calcium, quantified by the time integral of the distal calcium concentration (A) or its maximum amplitude (B), is dependent on the time difference Δt . Because the calcium spike is a nonlinear event, the amount of calcium is bimodally distributed (bottom panel in A and B), such that BAC firing governs calcium dynamics in the distal dendrite. Therefore, a threshold for plasticity based on the calcium concentration could be easily set by locating it in between the peaks of the distribution (dashed line in B bottom). In the presence of  inhibition, the amount of calcium was low, regardless of Δt, because BAC firing was prevented . For both proximal and distal inhibition, the amount of calcium was below the chosen plasticity threshold (bottom in B).(EPS)Click here for additional data file.S6 FigBoth interneuron types were implemented in the feedforward circuit see . The nor(EPS)Click here for additional data file.S1 Tables-soma, d-dendrite, a-apical calcium spike initiation zone, ax-axon. For (PDF)Click here for additional data file.


Transformation of T lymphocytes by HTLV-1 is linked to the capacity of its oncoprotein Tax to interfere with cellular regulatory pathways, including control of cell cycle progression and inactivation of tumor suppressor p53. Tax is modified by a hierarchical sequence of post-translational modifications that controls its intracellular localization and functions via crosstalk. Among these modifications, phosphorylation and acetylation occur at S336 and K346, respectively, in the carboxy-terminal domain of Tax, a domain involved in Tax transforming activity. We determined that the acetylation deficient K346R mutant had a markedly reduced capacity to transform Rat-1 fibroblast, as compared to the acetyl-mimetic K346Q mutant, which transformed Rat-1 cells as wild type Tax. This property correlated with the reduced capacity of mutant K346R to bypass inhibition of CDK4/cyclin D3 complexes by p21, resulting in its reduced capacity to stimulate pRb kinase activity of CDK4. Thus, acetylation at lysine residue 346 by the acetyltransferase p300 likely directly participates in the Tax transforming activity. We also determined that phosphorylation of Tax at the S336P motif involved the proline-directed serine/threonine kinase HIPK2. HIPK2 interacts with Tax and is recruited in the Tax nuclear bodies. Furthermore, the kinase activity of HIPK2 is required for efficient functional inactivation of p53 by Tax. These results indicate that modifications of the Tax carboxy-terminal domain control at least two critical functions in Tax transforming activities: stimulation of cell cycle progression through G1/S via activation of CDK4 kinase activity and functional inactivation of p53 via recruitment of HIPK2 kinase in Tax nuclear bodies.


Clones of labelled cells were induced in adult CAGG-CreER;R26R-LacZ reporter mice and their distributions analysed after different chase periods. Short-lived clones, derived from labelled transient amplifying cells, were shed during the chase period and long-lived clones, derived from stem cells, expanded. At 6 weeks, labelled clones appeared at the periphery, extended centripetally as radial stripes and a few reached the centre by 14 weeks. Stripe numbers depended on the age of tamoxifen treatment. Stripes varied in length, some were discontinuous, few reached the centre and almost half had one end at the limbus. Similar stripes extended across the cornea in CAGG-CreER;R26R-mT/mG reporter mice. The distributions of labelled clones are inconsistent with the CESC hypothesis and support the LESC hypothesis if LESCs cycle between phases of activity and quiescence, each lasting several weeks.The limbal epithelial stem cell (LESC) hypothesis proposes that LESCs in the corneal limbus maintain the corneal epithelium both during normal homeostasis and wound repair. The alternative corneal epithelial stem cell (CESC) hypothesis proposes that LESCs are only involved in wound repair and CESCs in the corneal epithelium itself maintain the corneal epithelium during normal homeostasis. We used tamoxifen-inducible, CreER-  •We used lineage tracing to evaluate alternative corneal epithelial stem cell models.•Clones emerged from the periphery and extended centripetally as radial stripes.•This implies that active stem cells are at the periphery, not throughout the cornea.•Labelling patterns suggested stem cells cycle through quiescent periods.•More labelled clones were induced by treating younger mice. The limbal epithelial stem cell (LESC) hypothesis proposes that all the stem cells are in the basal epithelial layer of the limbus, which lies between the cornea and conjunctiva . The LESThe alternative corneal epithelial stem cell (CESC) hypothesis  accepts 1.1There is ample indirect evidence that stem cells are present in the limbal epithelium. ABCB5 is a promising new candidate LESC marker  and oldeThere is strong experimental evidence from real-time studies in adult mice that, during normal homeostasis, corneal epithelial cells move centripetally, across the whole corneal radius, from the limbus or cornea periphery , as predBoth the LESC and CESC hypotheses accept that LESCs exist but to distinguish between them we need to determine whether there are stem cells in the corneal epithelium itself and if LESCs play a role in corneal epithelial maintenance in the absence of wounding.1.2There is no definitive evidence for or against the existence of CESCs. For example, differences in marker expression, between the corneal and limbal basal epithelia, do not exclude the existence of CESCs that express different markers from LESCs. Likewise, the absence of label-retaining cells from the corneal epithelium does not rule out the existence of CESCs that are not slow cycling. Conversely, the identification of side population cells in the rat corneal epithelium is not good evidence that CESCs exist because, unlike those in the limbal epithelium, they do not express ABCG2 . Some co1.3Compelling evidence for centripetal movement argues against the original CESC hypothesis, because it proposed centrifugal cell movement, but it would not exclude a modified CESC hypothesis with centripetal movement. Other evidence about whether LESCs maintain the corneal epithelium during normal homeostasis is conflicting. The postnatal switch, from a random mosaic pattern to radial stripes, described above, supports the LESC hypothesis. In contrast, when labelled limbal tissue was transplanted surgically to an unlabelled mouse limbus, the transplant failed to colonise the corneal epithelium unless the host corneal epithelium was removed . This re1.4loxP lineage tracing with adult CAGG-CreER;R26R-LacZ reporter mice, without surgical intervention. The ubiquitous CAGG promoter provided unbiased, stochastic labelling of all cell types, including putative stem cells in the limbus or cornea. The observed distribution of long-lived clones, founded by labelled stem cells, was inconsistent with the CESC hypothesis, but supports the LESC hypothesis if LESCs cycle through phases of activity and quiescence.The primary aim of this project was to directly test the LESC and CESC hypotheses and, in particular, determine whether any cells in the corneal epithelium can generate long-lived clones during normal homeostasis. We used tamoxifen-inducible CreER-22.1LacZ mice were bred by crossing homozygous R26R-LacZ (B6.129S4-Gt(ROSA)26Sortm1Sor/J) Cre-reporter mice  in the presence of nuclear-located Cre, so expression is induced when tamoxifen binds to the modified oestrogen receptor, ER, and moves CreER to the nucleus. CAGG-CreER;R26R-mT/mG mice were bred by crossing homozygous R26R-mT/mG (Gt(ROSA)26Sortm4Luo) Cre-reporter mice  was freshly dissolved in corn oil (20–35 mg/ml) with sonication in a 40 °C water bath, prior to injection. Mice were weighed and tamoxifen doses (100 μg/g body weight) were calculated for each mouse. CAGG-CreER;R26R-2.3Eyes were fixed in 0.2% glutaraldehyde for 2 h, stained for β-gal with X-gal, post-fixed in 4% paraformaldehyde (PFA) and stored in 70% ethanol at 4 °C as previously described . This wh2.4Eyes were fixed in 4% PFA overnight and transferred to phosphate buffered saline. Corneas were excised under a dissecting microscope, flattened by making 2–4 radial cuts, mounted in Vectashield (epithelium up) on a glass slide and sealed under a coverslip. They were imaged on a Zeiss LSM 510 confocal inverted microscope using a dry 20 × and oil immersion 40 × and 63 × objectives. Tiled montages, with no overlap between frames, and confocal Z-stacks were acquired.2.5http://vassarstats.net/index.html) was used for chi square goodness of fit tests and 3 × 2 Fisher's exact tests.GraphPad Prism 5 was used for most statistical tests, as described in the text. An online statistical calculator  should be labelled in the reporter mice. Patterns of labelling predicted by the LESC and CESC hypotheses are shown in LacZ mice were used to characterise the β-galactosidase  histochemical staining pattern in the cornea. Mice were injected with tamoxifen three times at 12 weeks, to induce LacZ expression, and left for chase periods of up to 20 weeks before staining for β-gal activity (8–14 corneas from 4–7 mice per group). Another four corneas (from three mice) were analysed after a 30-week chase period to test whether labelled clones were longer-lived, but this small group was not included in the quantitative analysis. The results are illustrated in In the main lineage tracing study, CAGG-CreER;R26R-LacZ mice and 7/8 eyes from 24-week old negative controls showed no β-gal corneal staining. However, one eye had two small, radially aligned, β-gal positive stripes . HoweveAll 12 corneas, which were stained less than one week after tamoxifen injection, had diffuse blue staining over the ocular surface B and hisInitially, the conjunctiva was covered in β-gal positive spots, which enlarged to form circumferentially elongated patches D, F, M. Five eyes from three mice had multiple long stripes . After aSuperficially consistent with both LESC and CESC hypotheses, some stripes had one end at the limbus , intermediate (I) and central (C) regions, shown in KRT5LacZ/− transgenic mice  than our tamoxifen-induced labelling. This suggests that the P:I:C frequency distributions for 14–20 week chase times represent the mature pattern of radial stripe positions. Thus, contrary to the predictions for Time 3 in LacZ corneas includes CC stripes and discontinuous stripes.The published P:I:C distribution for β-gal positive stripes in adult, mosaic, nic mice  is shownThe labelling patterns shown in 3.3The number of active stem cells that maintain the corneal epithelium can be estimated as the number of stripes, induced in adults, which would fit around the limbal circumference if they were evenly distributed. For stripes induced at 12 weeks, the ratio of the modal stripe width at the limbal–corneal boundary (35–40 μm) and the overall mean circumference  implies that 259–296 stripes would fit around the limbal circumference A, B. A s3.4Corneas, from tamoxifen-injected CAGG-CreER;R26R-mT/mG mice, were analysed by confocal microscopy after various chase times. Uninjected negative controls, analysed at 12 and 24 weeks (6 corneas from 3 mice at each age), showed no GFP-positive fluorescence (mG) but the level of red fluorescence (mT) varied, indicating variable expression of the mT/mG reporter transgene A. HoweveStripes close to the limbus were typically 2–5 cells wide I, J. Tog3.5LacZ mice were injected with tamoxifen 3 times at either 4 or 24 weeks and left for chase periods of 6 or 12 weeks. Stripe patterns were compared to those injected at 12 weeks in the main series  is expressed in the basal limbal epithelium but the two studies reported different extents of K14 expression in the corneal epithelium. Thus, our study is the first to demonstrate that CESCs either do not exist or do not contribute significantly to corneal epithelial maintenance under normal homeostatic conditions.Our main aim was to use inducible CreER-reported . Both thIn an elegant real-time experiment, Di Girolamo et al. followed the same clones and demonstrated convincingly that fluorescent stripes emerged from the limbus by about 5 weeks and extended centripetally to the centre of the cornea by 17–21 weeks . The othT2, CAGG-CreER lineage tracing labels all cell types and we found no evidence that any long-lived clones emerged in the central cornea. This allowed us to exclude the possibility that the corneal epithelium is normally maintained by CESCs in the central cornea. Second, our analysis revealed that many stripes were disconnected from the limbus and/or discontinuous and this prompted a new prediction about LESC quiescence (discussed below). Third, we showed that younger mice produced more stripes in response to tamoxifen, which raises further questions about LESC behaviour.All three investigations showed that the corneal epithelium is maintained by LESCs but our study extends this in three ways. First, unlike K14-CreER Minor discrepancies among the three studies may be explained by technical differences. For example, in the other studies there was a shorter lag before stripes emerged (5 weeks vs. 6 weeks in our experiments) but stripes took longer to extend fully (17–21 weeks vs. 14 weeks). The initial lag could be due to delayed production of TACs from LESCs that are labelled during quiescence, retention of some limbal TACs to maintain the limbal epithelium and time required for limbal TACs to reach the cornea and produce an identifiable stripe. Our CAGG-CreER system produced more short-lived, labelled TAC clones, which may have hindered identification of the earliest stripes. In the other studies, mice were injected with tamoxifen at 6–8 weeks. To ensure corneal epithelial homeostasis had stabilised, we labelled most mice at 12 weeks but also compared labelling at 4 and 24 weeks. We found no significant effect of age on the rate of stripe extension so it seems unlikely that the use of younger mice contributed to the slower stripe extension in the other studies. This may simply reflect the difficulty in evaluating when stripe patterns become stabilised because, at least in our study, stripe lengths varied and few stripes reached the centre, even after long chase times.Although CC stripes and discontinuous stripes were not specifically discussed in the other reports, examples of these and multi-coloured stripes were illustrated . This is consistent with both versions of the LESC hypothesis but is strong evidence against both versions of the CESC hypothesis. Stripes extended towards the centre, which is strong evidence against the original CESC-cf hypothesis.For Time 2 in The distance between the limbus and the more peripheral end of the CC stripes did not decrease with longer chase times and the proportion of LC stripes did not increase. This is not consistent with CESC-cf (which predicts stripes should extend towards the limbus and convert some CC stripes to LC stripes) or LESC-cc (which predicts CC stripes should not exist) but is consistent with CESC-cp and LESC-iq.We originally expected that most stripes would extend across the full radius if LESC-cc was correct but not if CESC-cf was correct A, B. HowThe distance between the central end of stripes and the limbus varied widely at all chase times. This is consistent with CESC-cf and CESC-cp if CESCs were scattered throughout the cornea. It is also consistent with LESC-iq because individual LESCs will be labelled at different stages as they cycle through active and quiescent phases, leading to the production of different length stripes. Thus, individual stem cells may only rarely produce full radial stripes if they are usually active for less time than it takes daughter TACs to move across the full radius. In the absence of intermittent quiescence, stripe lengths should increase continuously and would be less variable. Thus, the observed large variations in stripe lengths are not consistent with LESC-cc.Few stripes were induced in most eyes labelled at 12 weeks  and, altThe evidence, summarised in 4.3Our study supports the results of the other two lineage tracing studies , showing4.4Many types of stem cells are quiescent and it has been suggested that, although they have exited the cell cycle, they are not dormant but poised for activation . However4.5Using an unbiased lineage tracing system that labels all cell types, we showed that labelled clones appeared at the periphery and extended centripetally as radial stripes but we found no evidence that long-lived clones also arose in the central corneal epithelium, as proposed by all versions of the CESC hypothesis. These results are consistent with the LESC hypothesis if LESCs cycle through intermittent periods of activity and quiescence, each lasting several weeks. Loss of labelled LESCs or replacement by unlabelled LESCs  may also occur. Detailed cell cycle analysis may be useful for testing whether LESCs show intermittent quiescence, as our study predicts, and for determining whether LESCs exist in different quiescent states.LacZ reporter expression.β-gal staining of corneas and conjunctivas with possible leaky ROSA26R-(A–H) Comparison of β-gal staining in corneas and conjunctivas of five eyes with most extensive radial stripes  and two eyes with more typical labelling patterns (49L and 40R).  Eyes 49L and 49R from the same mouse after a 12-week chase. Eye 49L had two stripes and small patches in the conjunctiva but the contralateral eye, 49R, had several long corneal stripes and medium sized conjunctival patches.  Eyes 39L and 39R from the same mouse, after a 14-week chase, both had long stripes and medium sized conjunctival patches . (E–G) Eyes 161L and 161R from the same mouse, after an 18-week chase. Both eyes had many wide stripes spanning the corneal radius and both medium and large conjunctival patches. The large number of wide corneal stripes and the large conjunctival patches in eyes 161L and 161R, makes it likely that labelling occurred during development. The staining pattern appears similar to that reported for chimeras and X-inactivation mosaics , where lVariation in β-gal positive stripe numbers per cornea after labelling at 12 weeks.s = 0.5311; P < 0.0001 for A and rs = 0.5303; P = 0.0002 for B). (C–F) Regardless of whether eyes 39L, 39R and 49R were included  or excluded , the number of stripes per cornea did not differ significantly among chase times for either left  or right  eyes, which were analysed separately as they were not independent . Data were analysed by the Kruskal–Wallis (KW) test and Dunn's multiple comparison post-test. Males and females are shown in the figures but were not analysed separately.Results were analysed separately with and without data from eyes 39L, 39R and 49R, illustrated in Supplementary Fig. S1, as it was uncertain whether they had any stripes that arose by leaky expression. In each case, data from mouse 161 were excluded, as some stripes were likely to have arisen by leaky expression during development.  The number of stripes per cornea varied and was positively correlated between left and right eyes regardless of whether mice 39 and 49 were included (A) or excluded (B), as shown in the figure. Different chase times and genders are indicated but were not analysed separately. The results shown in A and B were analysed again after omitting eyes with little or no staining, which could have included technical failures and might have increased the correlation but left and right eyes remained correlated  (B) Fisher's exact tests (asterisks) show that a higher percentage of stripes have their central end > 500 μm from the limbus after chase times of 10–20 weeks than for 6 and 8 weeks combined. (C) Fisher's exact tests (asterisks) show that a higher percentage of stripes have their central end > 1000 μm from the limbus after chase times of 14–20 weeks than for 6–12 weeks combined. (D) Comparisons of the distance between the limbus and the central end of the most central 10% of the β-gal stripes. Linear regression showed stripes extended centripetally between 6 and 14 weeks (42–98 days). (E) The percentage of stripes that have one end at the limbus (LC stripes) varied among chase times but did not increase with chase time. (There was a weak trend to decrease with chase time.) (F) Comparisons of the distance between the limbus and the peripheral end of β-gal CC stripes (which do not include the limbus) by Kruskal–Wallis test and Dunn's multiple comparison tests.  Distances increased after 8 weeks. (G) Comparison of the percentage of the central ends of β-gal stripes, in the peripheral (P), intermediate (I) and central (C) regions of the cornea, after different chase times, as described in the text and legend to Fig. 4. For each chase time (6–20 weeks) and the K5 group  Results of the type of analyses shown in Fig. 4 but excluding eyes 49R, 39L and 39R (as well as 161L and 161R), shown in Supplementary Fig. S1. (A) Comparisons of the distance between the limbus and the central end of β-gal stripes by Kruskal–Wallis (KW) test and Dunn's multiple comparison tests.  This also tests for differences in stripe widths among ages because, for this series, age = chase time + 12 weeks. (F) Comparison of circumference/stripe width ratios (for 20–150 μm wide stripes) among chase times of 6–20 weeks after tamoxifen injection at 12 weeks by 1-way analysis of variance (ANOVA) and Tukey's multiple comparison tests.  In the box and whisker plots the whiskers show the minimum and maximum values. The distribution of LC stripes widths  in eyes of mice injected with tamoxifen at 12 weeks and chased for 6–20 weeks  is shown for all LC stripes (A) and 20–150 μm wide stripes (B). In B one outlier that was < 20 μm and six outliers that were > 150 μm wide were excluded, as the wider stripes are likely to be derived from more than one stem cell. The peak stripe width was 35–40 μm and the overall mean circumference was 10,365 μm, so 259–296 stripes would fit around the limbal circumference. This provides an estimate of the number of stem cells that maintain the corneal epithelium but assumes they are evenly distributed around the circumference.  The distribution of circumference / stripe width ratios at the cornea–limbal boundary (using separate circumference measurements for each eye) is shown for all LC stripes (C) and for 20–150 μm wide stripes (D). The peak circumference/stripe width ratio provides another estimate the number of stripes that would fit around the limbal circumference but the peak estimates were rather broad (175–300). (E) Comparison of stripe widths (for 20–150 μm wide stripes) among chase times of 6–20 weeks after tamoxifen injection at 12 weeks by Kruskal–Wallis (KW) tests and Dunn's multiple comparison tests.  The number of stripes per cornea was positively correlated between left and right eyes for mice treated with tamoxifen at 4 weeks (A) but not for the smaller sample treated at 24 weeks (B).  Pearson's correlation coefficients are shown in the figure. Different chase times and genders are indicated but were not analysed separately. (C–F) The distributions of LC stripes widths, measured at the cornea–limbal boundary  and the circumference/stripe width ratios  in eyes of mice injected with tamoxifen at 4 weeks and chased for 6  or 12  weeks. The analysis method is described in the legend to Supplementary Fig. S4.Supplementary material: legends for Supplementary Figs. S1-S5.The following are the supplementary data related to this article.


However, how p31comet's activity is regulated remains unclear. Here we show that the timing of M-phase exit in Xenopus egg extracts (XEEs) depends upon SAC activity, even under conditions that are permissive for spindle assembly. p31comet antagonizes the SAC, promoting XEE progression into anaphase after spindles are fully formed. We further show that mitotic p31comet phosphorylation by Inhibitor of nuclear factor κ-B kinase-β (IKK-β) enhances this role in SAC silencing. Together, our findings implicate IKK-β in the control of anaphase timing in XEE through p31comet activation and SAC downregulation.p31 SACspindle assembly checkpointXEEsXenopus egg extractsIKK-β Inhibitor of nuclear factor κ-B kinase-βKTkinetochoreMTmicrotubuleNEBDnuclear envelope breakdownAPC/Canaphase promoting complex/cyclosomeCSFcytostatic factorDSNdemembranated sperm nucleiTPCA[5-(p-fluorophenyl)-2-ureido] thiophene-3-carboxamideMCCitotic checkpoint complex.1–3). The SAC controls the anaphase promoting complex/cyclosome (APC/C), a ubiquitin ligase that is crucial for mitotic exit. SAC activation causes sequestration of an APC/C activator, Cdc20, within an inhibitory complex called the mitotic checkpoint complex (MCC). The MCC is composed of Cdc20 and SAC proteins Mad2, BubR1, Bub3. The Mad2 protein exists in 2 states, the inactive open state (o-Mad2) and the active closed state (c-Mad2); c-Mad2 is incorporated into the MCC complex. Conversion of o-Mad2 to c-Mad2 occurs at a “catalytic platform” complex on unattached KTs, comprised of c-Mad2 and Mad1.The spindle assembly checkpoint (SAC) ensures faithful separation of mitotic sister chromatids by monitoring interactions between kinetochores (KTs) and microtubules (MTs)  in each cell division. Unattached KTs transiently invoke the SAC during the interval between NEBD and MT attachment, and subsequent SAC silencing helps to determine the timing of anaphase onset.6Mad1 and Mad2 are not essential in budding yeast unless KT MTs are disrupted,1 p31comet is a protein found in higher eukaryotes that plays an important role in SAC silencing.7 p31comet over-expression in tissue culture cells overrides the SAC, while p31comet knockdown delays anaphase onset.8,9 p31comet is structurally related to Mad210 and it binds c-Mad2 in a manner that is essential for its role in SAC silencing.8,11 Two mechanisms have been suggested for p31comet activity: First, p31comet has been proposed to disrupt soluble MCC. This model is supported by the capacity of p31comet to bind c-Mad2 within the MCC in vitro and thereby disrupt that complex.12 Second, p31comet associates with unattached KTs through direct binding to c-Mad2.8,11 p31comet binding to c-Mad2 occludes o-Mad2 binding,10 and a “capping” model has been proposed wherein p31comet blocks MCC generation by inhibition of the Mad1/c-Mad2 catalytic platform.13SAC silencing is not fully understood, although it clearly involves a number of crucial events, including both inactivation of the catalytic platform at KTs and disassembly of existing MCC.ex vivo studies on mitosis. CSF-XEEs preserve the meiotic arrest of the frog egg by cytostatic factor (CSF).14 CSF-XEEs form spindles after addition of demembranated sperm nuclei (DSN). Upon the addition of CaCl2, which mimics fertilization of intact eggs, CSF is lost, spindles are disassembled and CSF-XEEs proceeds into interphase. XEE can recapitulate cell cycle checkpoints, including the SAC. For example, the SAC becomes activated by unattached KTs in CSF-XEEs containing DSN and the MT-depolymerizing agent nocodazole, preventing anaphase onset even after CaCl2 addition and CSF degradation. Cycling XEE are produced through an alternative protocol, so that they mimic the cell cycle of the fertilized egg, spontaneously alternating between interphase and mitosis.14 Like CSF-XEE, cycling XEE activate the SAC in response to unattached KTs. Purified p31comet antagonizes Mad2 inhibition of APC/Ccdc20 in XEE.15Xenopus egg extracts (XEEs) are a convenient system for comet. Here we show that p31comet depletion from XEE caused a SAC-dependent delay in anaphase onset, suggesting that endogenous p31comet is important for mitotic timing in this system. p31comet was mitotically phosphorylated in XEE. While a number of well-established mitotic kinases did not efficiently modify p31cometin vitro, IKK-β (Inhibitor of nuclear factor κ-B kinase-β) was effective for p31comet phosphorylation. Depletion or inhibition of IKK-β delayed mitotic exit of XEEs, and a phosphomimetic p31comet mutant showed increased activity in dissociation of soluble MCC and particularly in disruption of KT association of SAC components. Together, our results suggest that p31comet contributes to the timing of anaphase onset in XEE through antagonism of the SAC and that IKK-β modifies p31comet to enhance its activity.We have used XEEs for investigation of the mitotic role(s) and regulation of p31comet modulates mitotic exit timing in XEE under circumstances permissive for spindle assembly, we incubated control and p31comet-depleted CSF-XEE reactions containing DSN for 15 min. at 23°C, followed by CaCl2 addition to initiate mitotic exit. We examined the progression of each reaction into interphase through Western blotting for Cyclin B. Cyclin B degradation was significantly slower in p31comet-depleted samples , arguing that p31comet depletion did not delay mitotic exit by disrupting spindle assembly, and p31comet-depleted XEEs could activate their SAC in response to fully unattached KTs. .We postulated that transiently unattached KTs temporarily activate the SAC in XEEs, and that p31comet promotes anaphase by antagonizing the SAC. This observation is consistent with reports that p31comet-depleted HeLa cells show mitotic exit delays because they are unable to mediate SAC silencing.8,11,16Together, these observations suggest that the SAC slows mitotic exit in CSF-XEE containing DSN, and that p31comet is phosphorylated during mitosis,17,18 and that phosphorylation of human p31comet on Serine-102 weakens its interaction with Mad2 and its capacity to silence the SAC.19 We wished to determine whether p31comet might be phosphorylated in XEE, and, if so, what role this modification might play in its activity. To address this question, we took samples from a cycling XEE reaction at different times, and examined the activity of kinases that can phosphorylate p31comet by adding GST-p31comet and [γ−32P]ATP to each sample. After 30 min., we re-isolated GST-p31comet and subjected it to SDS-PAGE and autoradiography  and declining abruptly as the cycling XEE returned to interphase.It has been reported that mammalian p31comet phosphorylation, we examined the capacity of purified Mps1, Aurora B, Cyclin B/Cdk1 and IKK-β to phosphorylate GST-p31comet . Mps1, Aurora B and Cyclin B/Cdk1 are well-studied mitotic kinases. IKK-β has been implicated in spindle assembly20 and SAC regulation.21 The only kinase that efficiently phosphorylated GST-p31cometin vitro was IKK-β. To test whether IKK-β has a major role in p31comet phosphorylation, we performed kinase assays using CSF-XEE treated with IKK-β inhibitor TPCA ([5-(p-fluorophenyl)-2-ureido] thiophene-3-carboxamide).22 TPCA both inhibited phosphorylation of GST-p31comet in CSF-XEE . TPCA did not inhibit phosphorylation of the Cyclin B/Cdk1 substrate Histone H1 within CSF-XEE , Threonine-6 (T6) and Threonine-179 (T179) of p31comet as candidate IKK-β sites . Mutation of either S4 and T6 to alanine or S179 to alanine reduced in vitro p31comet phosphorylation, while mutation of all 3 residues to alanines nearly abolished p31comet modification . We raised rabbit antibodies that specifically recognized p31comet phosphorylated on S4 and T6 : Anti-p31comet-S4p,T6p antibodies did not recognize bacterially expressed GST-p31comet on Western blots, but showed a prominent band of the appropriate molecular weight after GST-p31comet was treated with IKK-β . The appearance of this band was suppressed by the addition of TPCA to the reaction. In a similar fashion, the anti-p31comet-S4p,T6p antibodies recognized GST-p31comet that had been incubated in XEE alone or in XEE plus DMSO, but not GST-p31comet incubated in XEE reactions containing TPCA . We also raised rabbit antibodies against Xenopus IKK-β (anti-xIKK-β), which were used to immunodeplete IKK-β from XEE. We examined the capacity of these depleted XEE to phosphorylate GST-p31comet by Western blotting with anti-p31comet-S4p,T6p antibodies . We observed that phosphorylation of p31comet was abolished after IKK-β depletion, but could be restored through the addition of purified human IKK-β at a similar concentration. These experiments collectively suggest that IKK-β phosphorylates p31comet on S4 and T6, and that its kinase activity is essential for the modification of these residues in CSF-XEE.We performed mass spectrometry to identify the sites on p31comet in cycling XEE by Western blotting with anti-p31comet-S4p,T6p antibodies . Interestingly, the peak of anti-p31comet-S4p,T6p kinase activity was somewhat sharper than overall kinase activity against p31comet  of recombinant wild type p31comet (p31comet-wt), a phosphomimetic form of p31comet, in which S4, T6 and T179 were mutated to glutamic acid (p31comet-EEE), or a non-phosphoryated form, in which those residues were mutated to alanine (p31comet-AAA). We added DSN to each reaction and incubated them at 23°C. After 15 min., we added CaCl2 and took periodic samples for Western blotting analysis of Cyclin B levels  staining , p31comet-EEE was similarly more efficient than p31comet-wt or p31comet-AAA. Together, these results suggest that p31comet phosphorylation by IKK-β enhances its capacity for MCC dissociation, thus improving its capacity for SAC silencing.We compared the capacity of recombinant p31comet activity at unattached KTs, we isolated chromatin from XEE incubated with nocodazole and p31comet-wt, p31comet-AAA and p31comet-EEE. We analyzed the levels of KT-bound SAC components , KT structural components (Spc24) and chromosome-associated inner centromeric proteins (Shugoshin1 and Aurora B).23 p31comet-wt and p31comet-AAA had little effect on the chromosome-bound levels of these components . By contrast, chromosomes showed a progressive loss of MCC components  and non-MCC SAC proteins (Bub1) with increasing p31comet-EEE concentrations . p31comet-EEE was not effective in releasing SAC components from KT in Mad2-depleted XEEs , consistent with the idea that p31comet-EEE must associate with KT8,11 in order to act in this context. These data suggest Mad2-dependent p31comet recruitment to KT allows it to play a positive role in dispersion of SAC proteins from KT, and that this function is enhanced by IKK-β phosphorylation. We did not observe appreciably higher levels of SAC proteins on KTs in p31comet-depleted XEEs than in control samples, so we do not believe that p31comet normally determines the maximal amount of their recruitment.To determine if IKK-β phosphorylation controls p31comet plays an important role in anaphase onset in XEE, and that its activity is significantly enhanced through phosphorylation by IKK-β. Together, these observations predicted that IKK-β should promote anaphase in XEE. To directly test this prediction, we examined the consequences of IKK-β inhibition or depletion upon mitotic exit in CSF-XEE. First, we added TPCA to CSF-XEE containing DSN. After 20 min. at 23°C, we added CaCl2 and followed mitotic exit . However, mitotic exit was significantly delayed after CaCl2 addition, with longer persistence of both high Cyclin B levels . This delay was reversed through the addition of purified human IKK-β . As with the delay caused by p31comet depletion . Notably, while p31comet-EEE was somewhat more efficient than p31comet-wt at mediating the dissociation of Mad2 from Cdc20 within the soluble faction (comet-wt (comet phosphorylation by IKK-β particularly enhances its capacity to act at KT. We estimate that the concentration of p31comet (15 nM) is much lower than the concentration of Mad2 (300 nM) within XEE . Because of this difference, endogenous p31comet may only be sufficient to neutralize a small fraction of the soluble Mad2 through stoichiometric binding.13 It is interesting to speculate that p31comet phosphorylation may promote its activity specifically against the smaller pool of Mad2 associated to KT, and that this might be its primary site of action within XEE.p3124 IKK-β depletion causes mitotic delays in mammalian cells, accompanied by spindle assembly defects and stabilization of the Aurora A kinase.20 On the other hand, the IKK-β inhibitor BMS-345541 causes precocious APC/C activation in tissue culture cells, resulting in early mitotic exit, defective chromosome separation and improper cytokinesis, and BMS-345541 can override the SAC in nocodazole-arrested cells.21 It is notable in this context that the transcription factor NF-κB, a critical IKK-β target, controls the expression of numerous cell cycle regulators.24 NF-κB's activity is stimulated by drugs that act on the MT cytoskeleton, including taxol or nocodazole,25,26 suggesting the interesting possibility that the SAC may regulate IKK-β.Our findings are interesting in light of the underappreciated role of IKK-β within mitosis, which is distinct from its function in cellular stress and inflammatory signaling pathways.20 and its chemical inhibition21 have opposite effects on mitotic exit in mammalian cells, with the former causing mitotic arrest while the latter promotes SAC release. These distinct phenotypes may reflect the activity of IKK-β against multiple downstream targets. Within XEEs, a simpler system that does not require gene expression,14 IKK-β depletion and its inhibition by TPCA both caused mitotic exit delays . Nevertheless, alignment of their sequences shows that mouse p31comet has residues that may correspond to the S4 and T6 phosphorylation sites, while human p31comet shares the site corresponding to T6. This relationship suggests that p31comet phosphorylation by IKK-β could be important for mitotic control in mammals as well as amphibians, a possibility that we are currently testing.It is not clear why IKK-β depletioncomet as a SAC release factor. While previous reports have indicated a role of IKK-β within mitosis, our findings are among the first mechanistic insights into the nature of this role. These observations point toward an interesting possible convergence of two disparate fields through the action of a classical signaling kinase upon the mitotic machinery.In summary, our results show that the SAC can modulate anaphase timing in XEE without external perturbation of spindle assembly, and suggest a model in which IKK-β enhances mitotic exit in XEE at least partially through phosphorylation and activation of p31comet protein sequence against the X. laevis and X. tropicalis mRNA plus EST cluster database (protein query to DNA database) to identify the Xenopus homolog of p31comet. The cDNA sequence that we retrieved (Genbank: CA973841.1) encodes a protein that is 52% identical and 64% similar to the human p31comet protein .We performed a blast search using the human p31Xenopus p31comet was amplified from a X. laevis cDNA library using forward (ATGGCGCAGAGTGGCACAGATCTACC) and reverse (TCAGTTGTGAAACCCCTTTACTAT) primers. It was subcloned into a pGEX4T1 vector for protein expression in E. Coli, or into pFastBac HT for expression in Baculovirus infected insect cells. p31comet phosphomimetic and phosphodeficient mutants were generated using PCR-based site-directed mutagenesis. Xenopus Mad2 was cloned into pGEX4T1 using forward (ATGTCGTCCATCTTGCCTTTCACCCCGCCAGTAG) and reverse (TTAGGACATGCTTGAGCAGCGGACTGAAGGGGATCCCATCTGTGT) primers. Xenopus Mad2L12A was generated by site direct mutagenesis.A cDNA encoding wild type comet variants and Mad2 were purified from E. Coli using glutathione Sepharose beads (Amersham). The GST tag of GST-Mad2L12A was subsequently removed by thrombin digestion. His6-tagged p31comet variants were purified from Baculovirus infected insect cell lysates using Ni2+-NTA resin (Qiagen).GST-tagged p31Xenopus p31comet, anti-Xenopus Mad2 and anti-Xenopus IKK-β were raised by Pacific Immunology Corporation  against p31comet (full length), Mad2 (full length) and IKK-β (aa 100–300) proteins. Anti-peptide antibodies that specifically recognize p31comet phosphorylated on residues S4 and T6 were generated in rabbits by the Pacific Immunology Corporation using peptide MAQ-Sp-G-Tp-DLPLRRAH.Rabbit anti-comet , anti- IKKβ , anti-Xenopus cyclin B , anti-CENP-A , anti-Mad1  and anti-GST , anti-xMad2 , anti-BubR1 , anti-Cdc20 , anti-Spc24 , anti-Bub1 (1:1000 rabbit), anti-Aurora B , Histone H3 , anti-Shugoshin , anti-Nup93 .Antibodies were used at the following concentrations: anti-p31Figure S5A, images were captured with Gel Logic 6000 Pro system (Carestream) and quantified using Carestream Imaging software.For quantification of Western blots in Kinases  were purchased from SignalChem.32P]ATP and 2.5 µg GST-p31comet in a final volume of 25 µl of reaction buffer . Reactions were incubated for 1 hr at 23°C. GST-p31comet was precipitated on glutathione sepharose beads, and washed 3 times with buffer A , 0.1 µM Okadic acid, 40 mM β–glycerolphosphate) for 4 times (5 min each). The samples were subjected to SDS-PAGE and32P labeled GST-p31comet was visualized using a phosphoimager and captured by phospho-scanner. GST-p31comet panel shows the kinase reaction blotted with anti-GST antibodies (third panel).In comet and 4 ng/ul . The reactions were incubated for 1 hour at 23°C, and terminated though addition of 20 mM EDTA. 2.5 mM p-nitrobenzyl mesylate (PNBM) was added to each sample, followed by an additional hour of incubation at 23°C. The samples were subjected to SDS-PAGE and Western blotting using the thiophosphate ester specific rabbit (Abcam) antibodies to identify phosphorylated proteins.In 27,14 Unless otherwise indicated, DSN were added at 10,000 DSN/µl, and CaCl2 was added to a concentration of 0.6 mM to promote mitotic exit of CSF-XEEs. Where indicated, nocodazole was added at 65 µM. Immunodepletions of p31comet, IKKβ and Mad2 were made using antibodies coupled to protein-A Dynal magnetic beads (Life Technologies) through 2 round of depletion. The p31comet variants were added to depleted XEEs when indicated. In 27CSF-XEEs, cycling XEEs and DSN were prepared as described.6-tagged p31comet-wt, p31comet-AAA or p31comet-EEE were added at the indicated concentrations to CSF-XEE containing DSN and nocodazole, and incubated at 23°C for 30 min. MCC was immunoprecipitated from each sample using anti-Xenopus Cdc20 antibodies coupled to protein-A Dynal magnetic beads. The beads were washed 3 times with buffer A, eluted with SDS sample buffer and subjected to Western blotting.To assay MCC disruption in 6-tagged p31comet-wt, p31comet-AAA or p31comet-EEE were incubated in 1 ml of TBS with 0.05% Tween20 (TBST) buffer at 23°C for 1 hour. Mad2 was Immunoprecipitated using anti-Mad2 antibodies coupled to Protein A-agarose beads. The beads were washed 3 times using Buffer A and the associated proteins were visualized by SDS-PAGE and CBB staining.In