IL13, and the HLA-DPA1 and HLA-DQA1 were, respectively, the most significantly associated with the IgE production at newborn and 6 years of age. Analyses of gene-gene interactions indentified that the combination of NPSR1, rs324981 TT with FGF1, rs2282797 CC had the highest risk (85.7%) of IgE elevation at 1.5 years of age (P = 1.46×10−4). The combination of IL13, CYFIP2 and PDE2A was significantly associated with IgE elevation at 3 years of age (P = 5.98×10−7), and the combination of CLEC2D, COLEC11 and CCL2 was significantly associated with IgE elevation at 6 years of age (P = 6.65×10−7). Our study showed that the genetic association profiles of the IgE production among fetus, infancy and childhood are different. Genetic markers for early prediction and prevention of allergic sensitization may rely on age-based genetic association profiles.Elevation of serum IgE levels has long been associated with allergic diseases. Many genes have been linked to IgE production, but few have been linked to the developmental aspects of genetic association with IgE production. To clarify developmental genetic association, we investigated what genes and gene-gene interactions affect IgE levels among fetus, infancy and childhood in Taiwan individuals. A birth cohort of 571 children with completion of IgE measurements from newborn to 1.5, 3, and 6 years of age was subject to genetic association analysis on the 384-customized SNPs of 159 allergy candidate genes. Fifty-three SNPs in 37 genes on innate and adaptive immunity, and stress and response were associated with IgE production. Polymorphisms of the  The prevalence of allergic diseases has dramatically increased over the past decades, especially in children A large body of evidence shows that regulation of serum IgE levels is under strong genetic control with heritability estimates of 50–80% IgE production and allergic diseases have a complex background, affected by innate and adaptive immunity genes To study the association between genetic polymorphisms and blood IgE levels, we collected blood samples from neonatal umbilical cord blood in a birth cohort study after informed consent as previously described Total IgE levels were measured using a Pharmacia CAP system . We used Pharmacia Cap System to detect allergic sensitization as reflected on allergen specific IgE levels. The allergen specific IgE levels detected by Pharmacia Cap System have been shown to closely correlate to skin prick tests ADAM33, CAT, NOS1, NOS2, NOS3, GSTP1 and MSRA have been shown to be responsible for modulation of allergy reaction in prenatal or postnatal environment In an attempt to select 384 customized SNPs in and around allergy candidate genes for this study, we searched allergy candidate genes and its SNPs associated with IgE production or allergic diseases reported in literature IL13, 2044 A/G and CTLA-4, 49 A/G was performed as described in our previous report IL13, 2044 A/G polymorphism between these two methods was 99%, and that for the CTLA-4, 49 A/G polymorphism between these two methods was 98%.In brief, DNA samples were quantified with a PicoGreen dsDNA Quantitation Kit . After DNA quantification, the DNA samples were adjusted to 50 ng/ul in TE buffer  for PCR amplification in an Oligo Pool All (OPA) containing a set of all the primers for the 384 SNPs. The PCR products were hybridized and analyzed using BeadStudio software with genotyping analysis module version 2.3.43 . The overall call rate of the 384 SNPs was 97.1%. After excluding 14 SNPs with a call rate less than 90%, the average call rate of the SNPs subjected to the analysis was 99.0%. Verification of the accuracy of the 384 SNP microarray data by repeat measurements and parallel experiments of restriction fragment length polymorphisms of The associations of elevated IgE in subjects with different genotypes or alleles were analyzed by univariate analyses using SPSS 17.0 . Genes associated with IgE elevation were arbitrarily classified into three categories: innate immunity, adaptive immunity, and stress and response genes. Total IgE levels are not normally distributed. We followed previous articles to use quartile classification http://sourceforge.net/projects/mdr/. The MDR approach of the gene-gene interactions has been described previously P value derived from the permutation test was 0.05 or less. Because IgE elevation was defined by a binary cut-off at >75% vs. <25% rather than continuous variable in this study, we used a binary logistic regression to confirm the high and low risk classifications of the significant gene-gene interactions revealed by MDR analyses. Moreover, we also used rate displays of the IgE elevation for Chi-square tests to validate the differences between high and low risk genotypes using the whole dataset at the ages of 1.5, 3 and 6 years.Gene-gene interactions were analyzed by multifactor dimensionality reduction software (MDR version 1.1.0), which is an open source project available at P>0.05). The intra-correlations of IgE levels among newborn, 1.5, 3 and 6 years were highly significant correlated one another  IL-13, rs1800925 was the most significantly associated with CBIgE elevation, and HLA-DPA1, rs1431399 was the most significantly associated with the IgE elevation at 6 years of age. Five genes associated with CBIgE elevation persisted into childhood associated with infant IgE elevation at 3 years of age. The CD209 polymorphisms were associated with infant IgE elevation both at 3 and 6 years of age. The CYFIP2 polymorphisms were associated with IgE elevation at newborn, 3 and 6 years of age (We have previously shown that twenty-one SNPs in 14 genes are associated with CBIgE elevation (≧ 0.5 kU/L) s of age ; Fig. 1BCCR5, TNFA, IL6, CLEC2D, CLEC7A, CLEC9A, CCL17, CX3CL1 and CD209, were significantly associated with higher or lower IgE levels. These genes were mainly clustered in chromosomes 3, 6, 7, 12, 16 and 19  and false negative rate (FNR) of these putative combinations were 0.385, 0.258 and 0.350 (FPR); 0.299, 0.341 and 0.225 (FNR), respectively, at 1.5, 3, and 6 years of age.Given the important role of genes related to higher or lower IgE production, we sought to study gene-gene interactions on IgE production at newborn, 1.5, 3 and 6 years of age. Multifactor dimensionality reduction analysis was used to investigate whether certain SNP combinations of the 350 SNPs studied were significantly associated with IgE production . In MDR,NPSR1, rs324981 and FGF1, rs2282797 was significantly associated with IgE production at 1.5 years of age (P = 1.46×10−4). The interaction among IL13, rs1800925, CYFIP2, rs767007 and PDE2A, rs755933 was significantly associated with IgE production at 3 years of age (P = 5.98×10−7). The interaction among CLEC2D, rs1560011, COLEC11, rs10210631 and CCL2, rs2857656 was significantly associated with IgE levels at 6 years of age (P = 6.65×10−7). We also validated the risk classifications by rate displays of the Chi-square test. As shown in NPSR1, rs324981 and FGF1, rs2282797 and then validated the significantly different (P<0.001) high and low risk classifications by the Chi-square test  of IgE production at 1.5 years of age, and the combination with NPSR1, rs324981 TT and FGF1, rs2282797 TC had the lowest risk (19.4%) of infant IgE production at 1.5 years of age.To validate the gene-gene interactions revealed by MDR analyses, we controlled for the significant SNPs of allergy candidate genes at the ages of 1.5, 3 and 6 years and for the perinatal factors including maternal atopy, paternal atopy, parental smoking, gender, and maturity in the multivariate analysis. As shown in are test . In the IL13, rs1800925, CYFIP2, rs767007 and PDE2A, rs755933 had the highest average testing balance accuracy at 63.1%, classifying high and low risk classifications to the IgE elevation at 3 years of age  at 6 years of age. In contrast, the SNPs of HLA-DPA1 and HLA-DQA1 revealed a significant association with IgE production when the children grew up to 6 years of age. IgE is produced by B cells, which interact with TH2 cells and undergo isotype class-switching after the induction of TH2 cell-derived cytokines. It is well known that an imbalance between TH1 and TH2 immune response is critical for IgE production and to the subsequent development of allergic diseases. In addition, evidence has suggested that inappropriate TH1 and TH2 responses can be suppressed by regulatory T (Treg) cells H2 cytokine, which increased in the sera of individuals with atopic diseases and also in the bronchoalveolar lavage fluids of asthmatic patients. The overexpression of IL-13 in the airway leads to an increase in mucus production, bronchial hyperresponsiveness, and goblet cell hyperplasia IL13 with CBIgE production has been reported in our birth cohort IL13, CYFIP2, and PDE2A genotypes caused different high and low risk classifications for IgE production at 3 years of age, showing the highest risk in the combinations of IL13, rs1800925 CC, CYFIP2, rs767007 CC and PDE2A, rs755933 AA or IL13, rs1800925 TC, CYFIP2, rs767007 CC and PDE2A, rs755933 AG or IL13, rs1800925 TC, CYFIP2, rs767007 GG and PDE2A, rs755933 AA. Different gene-gene interactions associated with IgE production among newborn, infancy and childhood suggests prediction of allergic sensitization in perinatal stage may rely on age-based genetic association profiles, and environmental factors in different ages.Interestingly, we also found that the SNPs of In this study, we initially analyzed associations of the 350 SNPs with IgE elevation and arbitrarily summarized the risk SNPs into different compartments of the immune genes studied. We then traced kinetic changes of the genetic associations from newborn, 1.5, 3 to 6 years of age. For gene-gene interactions are difficult to be assessed by multivariate logistic regression analyses, we used MDR analysis, which could identify high order gene-gene interactions without previous information of disease-associated genes The CBIgE levels detected in this cohort are similar to other cohorts in Germany and Canada The strengths of this study are its larger sample size and prospective follow-up to 6 years of age, which enabled us to evaluate the kinetic genetic associations of IgE production during early childhood. The limitations of the study are its analysis with dichotomical dataset to do permutation; the dichotomical cut-off values of IgE levels at 75th vs. 25th percentile may not completely represent the high and low risk groups. This study only analyzed serum IgE pool because this study did not measure cell bound IgE levels that are age-dependent Figure S1(TIFF)Click here for additional data file.Figure S2(TIFF)Click here for additional data file.Figure S3(TIF)Click here for additional data file.Figure S4(TIF)Click here for additional data file.Table S1(DOC)Click here for additional data file.


Euphausia superba with the under-ice habitat was investigated in the Lazarev Sea (Southern Ocean) during austral summer, autumn and winter. Data were obtained using novel Surface and Under Ice Trawls (SUIT), which sampled the 0–2 m surface layer both under sea ice and in open water. Average surface layer densities ranged between 0.8 individuals m−2 in summer and autumn, and 2.7 individuals m−2 in winter. In summer, under-ice densities of Antarctic krill were significantly higher than in open waters. In autumn, the opposite pattern was observed. Under winter sea ice, densities were often low, but repeatedly far exceeded summer and autumn maxima. Statistical models showed that during summer high densities of Antarctic krill in the 0–2 m layer were associated with high ice coverage and shallow mixed layer depths, among other factors. In autumn and winter, density was related to hydrographical parameters. Average under-ice densities from the 0–2 m layer were higher than corresponding values from the 0–200 m layer collected with Rectangular Midwater Trawls (RMT) in summer. In winter, under-ice densities far surpassed maximum 0–200 m densities on several occasions. This indicates that the importance of the ice-water interface layer may be under-estimated by the pelagic nets and sonars commonly used to estimate the population size of Antarctic krill for management purposes, due to their limited ability to sample this habitat. Our results provide evidence for an almost year-round association of Antarctic krill with the under-ice habitat, hundreds of kilometres into the ice-covered area of the Lazarev Sea. Local concentrations of postlarval Antarctic krill under winter sea ice suggest that sea ice biota are important for their winter survival. These findings emphasise the susceptibility of an ecological key species to changing sea ice habitats, suggesting potential ramifications on Antarctic ecosystems induced by climate change.The association of Antarctic krill  Euphausia superba often dominates the zooplankton community in numbers and biomass south of the Antarctic Polar Front (APF). It is a globally important fisheries resource Antarctic krill The life cycle and winter survival of Antarctic krill is closely linked with sea ice In winter, ice algae accessible from the underside of ice floes constitute an important resource for larval In order to investigate the importance of the ice-water interface layer for euphausiids and other macrofauna, a new sampling device was developed for the quantitative sampling of this environment, the Surface and Under Ice Trawl SUIT . Three eThis study aims toQuantify the density and the population structure of Antarctic krill in the immediate (0–2 m) surface layer, both under sea ice and in open water in summer, autumn and winter;relate these data to environmental parameters, such as sea ice properties, light regime and hydrography;assess the importance of the ice-water interface layer in relation to conventional standardised sampling of the 0–200 m depth layer.Wet bescherming Antarctica (WBA) were issued by the Netherlands Ministry of Agriculture, Nature and Food Quality .All necessary permits were obtained for the described field studies. Permits as required by the Dutch Polarstern” in the Lazarev Sea (Southern Ocean) in austral summer , autumn , and winter . The expeditions were part of a multi-year field experiment embedded in the interdisciplinary LAzarev Sea KRIll Study (LAKRIS). The surveys sampled a regular station grid covering the seasonal sea ice zone, from 6°W to 3°E and from 60°S to the continental ice shelf at approximately 70°S (Sampling was performed during three research missions of RV “ely 70°S . A detaiSurface and Under Ice Trawls SUIT  were useDiscovery” method). Thysanoessa macrura were measured from the tip of the rostrum to the tip of the telson. Euphausiid furcilia larvae from the mesozooplankton fraction were identified to species level and counted.The catch was immediately sorted on board. After the collection of all macrofauna >0.5 cm from either the entire sample or a representative subsample, the mesozooplankton fraction was preserved on 4% hexamine-buffered formaldehyde-seawater solution. In autumn 2004, the mesozooplankton fraction was obtained from the separate plankton net. Animals >0.5 cm collected from this net were combined with the shrimp net catch in subsequent analyses. Euphausiids were separated by species. Displacement volume and number of individuals of each species were noted. Euphausiids for length-frequency analysis were fixed in formaldehyde solution for 48 to 96 hours before sex determination and length measurement. Antarctic krill were measured from the front edge of the eye to the tip of telson  mounted outside the net opening allowed the volume of water passing through the net to be estimated. The average trawling speed was 2.5 knots (1.3 m s−1).Standardized double-oblique hauls to a depth of 200 m were conducted with a rectangular midwater trawl RMT  on all LImmediately after catch retrieval, euphausiids were removed from the RMT 8 sample. If the sample size was larger than 1 litre, a representative subsample was analysed. Euphausiids were stored in 4% formalin-seawater solution for length measurements and maturity stage analyses. RMT 1 samples were immediately preserved in a 4% formaldehyde-seawater solution. A detailed description of the RMT sampling procedure was provided by Hunt et al. (2011) plus) was supplemented by an altimeter (type Benthos PSA-916) to measure the distance to the sea floor, a transmissometer (type Wet Labs C-Star) to measure the attenuation of light, and a chlorophyll-sensitive fluorometer . The temperature-salinity profiles were used to calculate the mixed layer depth (MLD [m]) for each station −2] was measured by the ship's meteorological system. Bottom depth [m] was estimated for each station position using modeled global bathymetry from a publicly available database Vertical profiles of temperature, salinity and density were obtained by lowering a CTD  probe to depths varying between 1,000 m and the sea floor. The CTD (type SBE 911−2) were excluded from the autumn and winter datasets because of the known diel vertical migration behaviour of Antarctic euphausiids in these seasons −2].Of the 56 quantitative SUIT hauls, 18 were conducted in summer, 16 in autumn, and 22 in winter . Data frInitial data exploration showed that the distribution of animal densities was highly skewed. Densities were therefore log (x+0.001)-transformed to conform with the model assumptions of subsequent statistical analyses. Variance analysis (ANOVA) was conducted to assess the significance of the effect of season and the presence of sea ice during SUIT hauls, and the interaction of the two factors. Test results for ice-open water comparisons within each sampling season were confirmed with the non-parametric Mann-Whitney U-test, which is robust against non-normal distribution of the data. Overall densities of animals for different years and ice conditions were expressed both as averages (i.e. arithmetic mean) and geometric means (i.e. the exponent of the mean of the log(x+0.001)-transformed densities).Non-linear relationships between environmental variables and densities of postlarval Antarctic krill were investigated with Generalized Additive Models GAM, . The folMLD;water temperature (0 m – MLD);water temperature (0–200 m);salinity (0 m – MLD);salinity (0–200 m);attenuation (0 m – MLD);attenuation (0–200 m); - potential temperature at depth of temperature maximum;chlorophyll a conc. ;ocean depth;solar radiation;proportional ice coverage during SUIT hauls;average ice thickness during SUIT hauls.P-value, until the Akaike information criterion (AIC) reached a minimum. Sometimes the estimated degrees of freedom of smooth terms were so low that a linear relationship may have been sufficient. In that case the model was tested with parametric terms, and preferred if these were significant and the AIC was lower. Regression assumptions were assessed visually by plotting the response variable against fitted values and residuals against variables.Due to multiple interactions of environmental variables with sampling season, separate models were computed for each season. Collinearity of variables was assessed by calculating correlation coefficients and variance inflation factors  was situated . Much co−2), followed by autumn (2004) (0.82 ind. m−2), and summer (2007/2008) (0.79 ind. m−2) (−2), one haul in autumn (9.60 ind. m−2), and two hauls in winter (17.86 and 23.11 ind. m−2).Antarctic krill was clearly the most abundant krill species and the only euphausiid caught in the 0–2 m surface layer in all three seasons. The average surface density was highest in winter (2006) (2.70 ind. mnd. m−2) . In summnd. m−2) . Elevatend. m−2) , and at nd. m−2) . A few eThe overall size range of postlarval Antarctic krill (13–54 mm) was largely similar in all three sampling seasons . Modes, A diel pattern in the surface density of postlarval Antarctic krill was apparent from the five day/night comparative locations that yielded a sufficient number of animals in summer and winter. In summer, densities of postlarval Antarctic krill were higher at day than at night, both under ice and in open water . In wintFurcilia larvae of Antarctic krill were the dominant euphausiid larvae in the 0–2 m surface layer in autumn and winter, but had not yet developed from earlier stages in summer, because sampling was performed early in the season. In contrast to postlarval Antarctic krill, they were significantly more abundant under ice than in open water in autumn . In wintThe relation between the under-ice and open water 0–2 m surface layer densities of postlarval Antarctic krill significantly differed among sampling seasons (ANOVA: p<0.01). In summer, Antarctic krill density at the surface was significantly higher under ice than in open water. In autumn, the density of postlarval Antarctic krill was significantly lower under ice than in open waters. The integrated under-ice density, however, was based on only three of the 16 quantitative stations sampled on the LAKRIS grid in that season . The higThe density of postlarval Antarctic krill in the 0–200 m stratum significantly differed among the sampling seasons (ANOVA: p<0.01), but was not significantly related to the presence of sea ice (ANOVA: p>0.1). In summer, both the average and geometric mean densities in the 0–200 m stratum were lower than in the 0–2 m layer in ice-covered waters, but above the values of the 0–2 m surface layer in open waters . In autuThe density of postlarval Antarctic krill in the surface layer was significantly related to different combinations of environmental variables in each sampling season. In summer, parametric terms for average ice thickness and attenuation in the mixed layer, combined with smooth functions of proportional ice coverage during SUIT hauls and MLD obtained a very good model fit, explaining 94.6% of the deviance. The modelled krill density was positively affected by both decreasing ice thickness and attenuation in the mixed layer, an approximate proportional ice coverage >12%, and a MLD <12 m or between 20 and 30 m ; Table 3Thysanoessa macrura were caught in the 0–2 m surface layer. Occurring at 16 of the 18 quantitative hauls, average surface layer density was below that of Antarctic krill. (0.24 ind. m−2), although they locally reached higher densities , postlarval ensities . Overall sea ice ; Table 4T. macrura occurred in all three seasons. During summer, they were on the average tenfold more abundant in the 0–200 m layer than at the surface (T. macrura was not related to the presence of sea ice (ANOVA: p>0.1). In autumn 2004 and winter 2006, very low average densities (<1.0 ind. m−2) were recorded in the 0–200 m stratum compared to summer values. In summer, the overall size distributions of the 0–2 m surface and 0–200 m stratum were not significantly different from each other .In the 0–200 m layer, postlarval  surface . The denThe present study is the first multi-seasonal investigation of the distribution of Antarctic krill in the top 2 m layer of the ocean, using a micronekton net capable of sampling under closed pack-ice. Covering an area of about 1,000 km in latitudinal, and 200–300 km in longitudinal extent, our results show that significant parts of the Antarctic krill population in the Lazarev Sea can be found in the ice-water interface layer almost year-round, both near the ice edge and hundreds of kilometres deep in the pack-ice . This coth century The potential importance of sea ice habitats for the recruitment and survival of Antarctic krill was highlighted for the south-west Atlantic sector of the Southern Ocean, where populations have been declining in parallel with a decrease in the duration and extent of the winter sea ice coverage during the last 3 decades of the 20Thysanoessa macrura is more omnivorous, and not associated with sea ice T. macrura were similar under ice and in open water . (PDF)Click here for additional data file.


The cerebellar cortex comprises a stereotyped array of transverse zones and parasagittal stripes, built around multiple Purkinje cell subtypes, which is highly conserved across birds and mammals. This architecture is revealed in the restricted expression patterns of numerous molecules, in the terminal fields of the afferent projections, in the distribution of interneurons, and in the functional organization. This review provides an overview of cerebellar architecture with an emphasis on attempts to relate molecular architecture to the expression of long-term depression (LTD) at the parallel fiber-Purkinje cell (pf-PC) synapse. The general hypothesis explored in this review is that the elaborate molecular architecture of the cerebellar cortex has its counterpart in the compartmentation of function. In particular, many forms of synaptic plasticity have been identified in the cerebellar cortex  synapse. A brief consideration of its opposite –LTP—is also included. Other forms of Purkinje synaptic plasticity in the cerebellum are not included since so little is known of their relationship to the stripe architecture. Therefore, to set the stage the review begins with a brief overview of the patterning of the main players—Purkinje cells, climbing and mossy fiber afferents, and granule cells.Several recent reviews have described the architecture of the adult cerebellar cortex . On the other hand—and central to what follows—different stripes display distinctly different intrinsic properties, notably a variety of different forms of synaptic plasticity  results in a long-lasting depression of transmission at the pf-PC synapse . The downstream consequence is the internalization of AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)-sensitive glutamate receptors (AMPAR), and consequent synaptic desensitization. NO and CRF play supporting roles. It is instructive to examine the expression patterns of the different players in this pathway.An influential model of the molecular basis of LTD at the pf-PC synapse, due to Ito /Ca2+ signaling pathway and plays a key role in the induction of pf-PC LTD , GluRδ2 and AMPAR. mGluRs are G protein-coupled receptors with 7 transmembrane segments that do not form ion channels but rather signal via intracellular chemical messenger systems. Eight genes coding for different subtypes of mGluRs have been identified, seven of which are expressed in the cerebellum. In particular, Purkinje cell mGluR1 is localized in the peri- and extra-synaptic membranes. It is functionally coupled to PLC through which it modulates the IPThere are at least 4 mGluR1 splice variants with differing subcellular and cellular distributions  results in striped Purkinje cell degeneration  results in the selective Purkinje cell death of the zebrin II- Purkinje cell subset, but again the evidence suggests that this is due to differential sensitivity to an abnormal Ca2+ influx rather than restricted expression of the α1a channel; Fletcher et al., One consequence of mGluR1 activation is Ca2+ influx is modulated by calpacitins, notably the 2 Purkinje cell proteins PEP-19 and neurogranin/RC3. Sutcliffe and colleague have proposed that calpacitins regulate calmodulin availability in dendritic spines and thus regulates their ability to amplify the mobilization of Ca2+ in response to metabotropic glutamate receptor stimulation, releasing calmodulin rapidly in response to large influxes of Ca2+ and slowly in response to small increases. This action is inhibited by PKC-mediated phosphorylation , reveals stripes of granule cells similar to those revealed by PLCβ4 expression —in particular, the PLA2 isoform: e.g., Linden, 3 release from intracellular stores to bind to its receptor on the endoplasmic reticulum , is questionable. Immunocytochemistry with antibodies against IP3R do not show Purkinje cell stripes  Ca2+-independent , if any, of the four CaBRs are predominantly located perisynaptically at the dendritic spines of Purkinje cells  receptor signaling. In the adult cerebellum GABA3R, is the phosphorylation  and PLCβ isoforms  are expressed in stripes. How this expression of LTP relates to that described above, is unclear.In contrast, another apparent manifestation of pf-PC LTP has a close relationship to cerebellar stripes. This instance comes from the flavoprotein autofluorescence imaging of cerebellar activity by Ebner and colleagues  to intriguing , to being of marginal significance at best (e.g., IP3R; IGF-1; PKCδ: Table In this review, LTD at the pf-PC synapse has been used as an example of a correlation between the molecular architecture of the cerebellar cortex and the specialization of cerebellar function. To recapitulate, the data show two things: first, LTD is manifested differently in different stripes; and secondly, some of the molecules implicated in the LTD signaling pathways also show expression patterns restricted to stripes, ranging from convincing (e.g., mGluR1b, EAAT4, PLCβ3, PLCβ3/4, GABAThe author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Dicistroviridae family adopts an overlapping triple pseudoknot structure to directly recruit the 80S ribosome in the absence of initiation factors. The pseudoknot I (PKI) domain of the IRES mimics a tRNA-like codon:anticodon interaction in the ribosomal P site to direct translation initiation from a non-AUG initiation codon in the A site. In this study, we have performed a comprehensive mutational analysis of this region to delineate the molecular parameters that drive IRES translation. We demonstrate that IRES-mediated translation can initiate at an alternate adjacent and overlapping start site, provided that basepairing interactions within PKI remain intact. Consistent with this, IGR IRES translation tolerates increases in the variable loop region that connects the anticodon- and codon-like elements within the PKI domain, as IRES activity remains relatively robust up to a 4-nucleotide insertion in this region. Finally, elements from an authentic tRNA anticodon stem-loop can functionally supplant corresponding regions within PKI. These results verify the importance of the codon:anticodon interaction of the PKI domain and further define the specific elements within the tRNA-like domain that contribute to optimal initiator Met-tRNAi-independent IRES translation.The intergenic region internal ribosome entry site (IGR IRES) of the  For most eukaryotic mRNAs, through the concerted action of several eukaryotic initiation factors (eIFs), the 40S ribosomal subunit is recruited to the 5′-end of the transcript and subsequently scans for an AUG initiation codon cis-acting RNA elements that can directly recruit and position the ribosome, usually utilizing only a subset of canonical initiation factors and trans-acting factors trans-acting and initiation factor requirements. Moreover, a subset of IRES mechanisms do not involve scanning but instead these IRESs directly recruit and position the 40S subunit near or at the initiation codon. For example, the Hepatitis C virus (HCV) IRES directly binds to 40S subunits and only requires eIF3 and the ternary complex of eIF2-GTP-Met-tRNAi to position the initiation Met-tRNAi at the AUG start codon Although the majority of cellular mRNAs initiate translation via a cap-dependent mechanism, a subset of transcripts and viral RNAs can be expressed through non-canonical, cap-independent means using an internal ribosome entry site (IRES) Dicistroviridae family also contains a pseudoknot structure (PKI) to direct ribosome positioning at the initiation codon. However, unlike the HCV IRES, the IGR IRES has the unique ability to recruit the ribosome in the absence of all canonical initiation factors and establishes the translational reading frame by occupying the ribosomal P site such that the initiating non-AUG codon is positioned in the A site Ala, to the A site and eEF2 catalyzes the initial pseudo-translocation step which occurs in the absence of peptide bond formation The intergenic region (IGR) IRES of the The secondary and tertiary structures of the IGR IRES are intrinsic to its function. Although there is a lack of conservation at the primary sequence, the secondary structures of the IGR IRESs are well conserved. The IGR IRES adopts an overlapping triple pseudoknot structure  which contains two independently folded domains Several biochemical approaches have obtained insight into the structure-derived function of the IRES and have demonstrated that the IGR IRES undergoes conformational rearrangements in the progression from the unbound state, to the 40S subunit- and 80S-bound states A main role of the PKI tRNA mimicry domain is to position the ribosome on the IRES such that translation initiates within a specific reading frame and at a specific non-AUG codon in the ribosomal A site. To gain insight into this process, we performed a comprehensive mutagenesis study targeting three constituents of the PKI tRNA mimicry domain, including the PKI helical stem, the codon:anticodon-like basepairing interactions, and the variable loop region (VLR) and assessed their functional contribution to a key step of IRES-mediated translation initiation – ribosome positioning. Alterations in the helical stem adjoined to the anticodon-like element by introducing nucleotide bulges or basepair deletions tend to reduce IRES translation, suggesting a functional importance of this region to IRES translation. Furthermore, the defect in translation and ribosome positioning observed with the VLR insertion/deletion mutations suggests that there is a stringent requirement in the length of the VLR for IRES activity and that this region may be involved in optimal docking of the IRES within the ribosome to drive translation. Here, we have elucidated the molecular parameters of the PKI tRNA mimicry domain that contribute to IGR IRES-mediated translation and reading frame selection.The dicistronic luciferase plasmid containing the CrPV IGR IRES has been previously described in vitro and subsequently purified using an RNeasy kit (Qiagen). The purity and integrity of the RNAs were confirmed by gel electrophoresis. For in vitro translation assays, 0.15 µg of uncapped, pre-folded dicistronic RNAs were incubated in RRL containing potassium acetate to a final concentration of 154 mM. Following incubation for 1 hour at 30°C, the reactions were analyzed by SDS-PAGE and luciferase expression was assessed by [35S]-methionine incorporation. The gels were dried and subjected to PhosphorImager analysis .The dicistronic DNA construct containing the wild-type or mutant IRES was linearized using XbaI. RNAs were transcribed 2, 1 mg/ml heparin) and the supernatant was subjected to brief centrifugation to remove cellular debris. The supernatant was applied to a 30% (w/w) sucrose cushion containing 0.5M KCl and centrifuged at 100,000 g to pellet ribosomes. Ribosomes were resuspended in buffer B (20 mM Tris-HCl (pH 7.5), 6 mM magnesium acetate, 150 mM KCl, 6.8% (w/w) sucrose, 2 mM DTT) and subsequently treated with puromycin to dissociate ribosomes from the mRNAs. KCl was added to obtain a final concentration of 0.5 M. The dissociated ribosomes were resolved on a 10–30% (w/w) sucrose gradient where the peaks corresponding to the free 40S and 60S subunits were detected by measuring the absorbance at 260 nm. The corresponding fractions were collected, pooled, and concentrated in buffer C (20 mM Tris-HCl (pH 7.5), 0.2 mM EDTA, 10 mM KCl, 1 mM MgCl2, 6.8% (w/w) sucrose) using Amicon Ultra spin concentrators (Millipore). The resultant concentrations of the ribosomal subunits were determined by spectrophotometry using the conversions 1 A260 nm = 50 nM and 1 A260 nm = 25 nM for the 40S and 60S subunits, respectively.Ribosomal subunits were purified from HeLa cell pellets  as previously described 5′- GTAAAAGCAATTGTTCCAGGAACCAG - 3′ in 40 mM Tris (pH 7.5) and 0.2 mM EDTA by slow cooling from 65°C to 30°C. Annealed RNAs were added to RRL pre-incubated with either 15 µM edeine or 0.68 mg/mL cycloheximide and containing 20 µM amino acid mix, 8 units of Ribolock (Fermentas) and 154 nM final concentration of potassium acetate (pH 7.5). The reaction was incubated at 30°C for 20 minutes. Following incubation, ribosome positioning was determined by primer extension/reverse transcription using 10 units of AMV reverse transcriptase (Promega) in the presence of 125 µM of each of dTTP, dGTP, dCTP, 25 µM dATP, 0.5 µL of α- [32P] dATP , 8 mM MgOAc, 10 units of Ribolock, 1X buffer E , 100 mM KCl, 2.5 mM MgOAc, 0.25 mM spermidine, 2 mM DTT) in the final reaction volume. The reverse transcription reaction was incubated at 30°C for 1 hour, after which it was quenched by the addition of STOP solution . Toeprinting/primer extension analysis using purified 80S was performed in a similar manner using 150 µg dicistronic RNA and 100 nM and 150 nM final concentrations of 40S and 60S, respectively, at 37°C. Following the reverse transcription reaction, the samples were extracted by phenol/chloroform (twice), chloroform alone (once), and ethanol-precipitated. The cDNA was analyzed under denaturing conditions on 6% (w/v) polyacrylamide/8M urea gels, which were dried and subjected to phosphorimager analysis. In each figure, representative gels are shown from at least 2 independent experiments.Toeprinting analysis of ribosomal complexes in RRL was performed as previously described in vitro transcribed dicistronic reporter RNAs containing the wild-type or chimeric IRESs in the intercistronic space . Although both mutations reduced IRES translation, the activities fall within a range that was consistent with the chimeric IRESs containing the ProtRNA anticodon loop . To determine if other nucleotide bulges can be tolerated, one or two adjacent nucleotides were deleted from the 5′ proximal region of the helical stem and the resultant effect on IRES activity was assessed (6176 and ΔUU6176-7). Deletion of U6176 or UU6176-7 reduced IRES translation to ∼50% and 25% of wild-type, respectively (6176/A6200 and UU6176-7/AA6199-200). While deletion of one complete basepair rendered the IRES moderately active (∼50%), removal of two complete basepairs was sufficient to abrogate IRES activity (6176/A6200 and UU6176-7/AA6199-200). These results therefore demonstrate that the PKI helical region can tolerate additional bulges, consistent with the lack of conservation in the bulged nucleotides within the helical stem. However, the position of the bulge may be important for optimal IRES activity, as deletion of A6182 adversely affected IRES-mediated translation  and at A6182  eliminated Toeprint A and the translocated toeprint, but not Toeprint B or at A6182 , which effectively disrupted basepairing at the authentic site to potentially promote basepairing exclusively at the inserted sequence. Interestingly, the CC6214-15GG mutation resulted in negligible FLuc expression , suggesting that the authentic translational start site may be preferentially selected in IRES-mediated translation. When the second codon-like element was introduced adjacent to the authentic site, translation from the inserted site was observed (9–14% of the wild-type IGR IRES activity) (6214-15GG mutation did not significantly affect translation from the adjacent inserted site (6214-15GG). Taken together, these findings indicate that IRES-mediated translation initiation is permissible at an alternate start site, but only when the inserted site is located proximally to the authentic site.For the construct where the second codon-like element was inserted two nucleotides downstream of the authentic site, negligible reporter expression from the inserted site was observed (6226-7, indicating a properly positioned ribosome at the authentic translational start site (6232-3, six nucleotides downstream, which was consistent with a ribosome positioned after two cycles of translocation (6233-4 and AU6239-40 under edeine and cycloheximide treatment, respectively (6226-7, which is inconsistent with the observation that both sites supported translation (6232-3 and a lower intensity toeprint at AC6236-7 which corresponded to translocation from the authentic and inserted sites, respectively (6214-5GG construct, which supported translation initiation exclusively from the inserted site, generated an observable toeprint at AC6236-7 only upon cycloheximide, but not edeine treatment (To confirm that ribosomes are in fact positioning at both sites of initiation, toeprinting analysis was performed in RRL . For theart site , lane 2.location , lane 3.ectively , bottom.nslation , lane 2.ectively , lane 3.The recent acquisition of a high-resolution structure of the CrPV IGR IRES PKI domain has yielded significant insights into the remarkable ability of structured RNAs to manipulate the translational machinery Plautia stali intestine virus and suggests that there may be functional implications in the flexible nature of this region Ala to the ribosomal A site and subsequent translocation steps further stabilize the IRES-80S complex (The disordered nature of the VLR in the ribosome-bound state is a conserved feature for both the CrPV and the related  complex . AlthougWe have recently demonstrated that a subset of IGR IRESs can initiate translation in the +1 reading frame through a non-canonical basepair adjacent to PKI 6205, and A6208 and U6211 to specific nucleotides resulted in a decrease in IRES activity (6205 or other nucleotides within the VLR affect the conformation of PKI or may interact, possibly transiently, with the ribosome, IRES and/or the incoming tRNA to mediate IRES-dependent translation.Our results indicate that the nucleotide identity within the VLR has a role in IRES activity. Translation initiation at the adjacent or downstream alternate translational start sites effectively increases the length of the VLR by 4 or 7 nucleotides, respectively . While tactivity . It rema6190-1 or UA6212-3 to their Watson-Crick complements such that the basepairing more closely resembles a true codon:anticodon pairing – suggests that three basepairing interactions can confer IRES translation, albeit weakly (6191 results in an enhancement in IRES translation, suggesting that IRES activity is not maximal but is optimized in the context of the viral genome Structural comparison between the anticodon mimic of the IRES and an authentic tRNA anticodon:codon pair reveals remarkable similarities, although some deviations occur in the vicinity of the PKI basepairs t weakly . Additioicis the anticodon mimicry stem-loop with the initiation codon, occupies the ribosomal P site to engage the ribosome to initiate translation from the A site. For both IRESs, the molecular framework of the pseudoknot domains has been optimized for efficient IRES translation . The current study provides the basis for future work into understanding how different regions of the PKI domain, especially specific elements of the VLR, coordinately function and direct IRES-mediated translation. As we gain more structural and biochemical insights into these viral tRNA-like elements, it will be interesting to see whether other viral and cellular IRESs use pseudoknot structures as a common strategy for ribosome positioning and reading frame selection. Finally, it is interesting to note that some plant RNA viruses also utilize a tRNA-like element within their 3'UTR to mediate viral translation and replication, suggesting that tRNA mimicry may be a more general strategy for viral translation Viruses have evolved unique mechanisms for translating their genomes as a strategy to hijack the ribosome during infection when cap-dependent translation is compromised. Both HCV and CrPV IGR IRESs have acquired a pseudoknot to directly manipulate the ribosome and position it properly at the initiation codon. However, each IRES utilizes the pseudoknot distinctly. The HCV pseudoknot acts as a connector region to link the ribosome binding domain and the initiation codon-containing domain and orients the HCV initiation codon in the mRNA binding cleft of the 40S subunit to presumably mediate binding of the ternary complex eIF2-GTP-Met-tRNA


Interleukin-33 (IL-33) is a new member of the IL-1 cytokine family. The objectives of present study are to assess whether IL-33 can protect cardiomyocytes from anoxia/reoxygenation (A/R)-induced injury and the mechanism involved in the protection.−/− mice were challenged with an A/R with or without IL-33. Myocyte apoptosis was assessed by measuring caspase 3 activity, fragmented DNA and TUNEL staining. In addition, cardiomyocyte oxidative stress was assessed by measuring DHR123 oxidation; PKCβII and JNK phosphorylation were assessed with Western blot.Cardiomyocytes derived from either wild type or JNK1Challenge of cardiomyocytes with an A/R resulted in cardiomyocyte oxidative stress, PKCβII and JNK phosphorylation, and myocyte apoptosis. Treatment of the cardiomyocytes with IL-33 attenuated the A/R-induced myocyte oxidative stress, prevented PKCβII and JNK phosphorylation and attenuated the A/R-induced myocyte apoptosis. The protective effect of the IL-33 did not show in cardiac myocytes with siRNA specific to PKCβII or myocytes deficient in JNK1. Inhibition of PKCβII prevented the A/R-induced JNK phosphorylation, but inhibition of JNK1 showed no effect on A/R-induced PKCβII phosphorylation.Our results indicate that IL-33 prevents the A/R-induced myocyte apoptosis through inhibition of PKCβ/JNK pathway. Cardiovascular diseases remain the number one killer in Western society. Coronary artery disease which is associated with myocardial ischemia accounts for 1 in 6 of deaths in the United States Reperfusion of ischemic myocardium can activate apoptotic pathways leading to the death of cardiomyocytes that were viable just before reperfusion c-Jun N-terminal kinase (JNK) is an important member of the mitogen-activated protein kinase (MAPK) family. It plays a pivotal roles in regulation of cell function and is readily to be activated by many environmental stimuli including myocardial reperfusion Protein kinase C (PKC) is another important kinase by which the cell functions are regulated. It is a serine/threonine protein kinase family which has at least 11 isoforms that function in different biological systems. PKCβ is one of conventional (c-PKC) isoforms in vitroin vivoInterleukin-33 (IL-33) is a 30 kD protein and is a new member of the IL-1 cytokine family. It plays its biological roles by interacting with ST-2L receptor. IL-33 is broadly expressed in many tissues including the heart in vitro count part to I/R in vivo, induced myocyte apoptosis and studied the mechanism involved in the protective action of IL-33 by using cultured cardiomyocytes. We demonstrated that 1) A/R-induced myocyte apoptosis is attenuated by exogenous IL-33; 2) A/R challenge to myocytes resulted in increase in PKCβII phosphorylation which is prevented by the IL-33 and inhibition of PKCβII with siRNA specific to PKCβII attenuated the A/R-induced myocyte apoptosis; 3) A/R challenge to myocytes led to increase in JNK phosphorylation which was also prevented by the IL-33 and genetically inhibition of JNK1 diminished the A/R-induced myocyte apoptosis; 4) IL-33 attenuated myocyte oxidative stress after A/R; inhibition of PKCβII prevented the A/R-induced JNK phosphorylation, while inhibition of JNK1 showed no effect on A/R-induced PKCβII phosphorylation.In the present study, we further confirmed the beneficial effect of IL-33 in preventing anoxia/reoxygenation (A/R), an −/− mice (C57BL/6 background) were obtained from Jackson laboratories . The mice were housed in Victoria Research Laboratory Vivarium Service with a 12-hour light/dark cycle and free access to rodent chow and tap water. Breeding pairs were set up to generate the neonatal mice as a source for isolation of cardiac myocytes. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health . The experimental protocols were approved by the University of Western Ontario Animal Care and Use Committee (Protocol No. 2008-053).C57BL/6 mice were obtained from Charles River Canada  and JNK1Cardiomyocytes were isolated from neonatal mouse hearts as previously described in vitro correlate to ischemia/reperfusion in vivo, cardiomyocytes were exposed to an A/R as described previously As Cardiomyocyte oxidative stress was assessed by measuring the oxidation of intracellular dihydrorhodamine 123 , an oxidant-sensitive fluorochrome, as described previously Lysates of cardiomyocytes were resolved on 10% SDS-PAGE and transferred to PVDF membranes. Blocked (5% non-fat milk) membranes were used for detection of phosphorylated and total JNK or PKCβII with specific antibodies , respectively. Myocyte JNK or PKCβII phosphorylation status was expressed as a ratio of phosphorylated to total JNK or PKCβII protein Small-interference RNA (siRNA) specific for PKCβII was purchased from Santa Cruz Biotechnology. Cardiac myocytes were transfected the siRNA using Lipofectamine 2000 reagent (Invitrogen Canada) according to the manufacturer’s instructions g for 10 min at 4°C. The supernatants were incubated with a caspase-3 fluorometric substrate DEVA-AFC (BioVision). The caspase 3 activity was determined by measuring the fluorescence intensity with a Victor 3 multi-label counter (Perkin Elmer).Cardiomyocyte apoptosis was assessed by determination of caspase 3 activity and fragmented DNA g for 10 min at 4°C, the supernatants were collected for detection of histone-associated DNA fragments with ELISA.Apoptotic myocyte death was determined with a quantitative Cell Death Detection ELISA Kit (Roche) to detect fragmented DNA according to manufacturer’s instructions For TUNEL assay, cardiomyocytes were grown on coverslips, and were washed with PBS and fixed with 4% paraformaldehyde 24 hours post A/R or N/R. Cardiomyocyte apoptosis was detected with TUNEL labeling using an In Situ Cell Death Detection Kit, Fluorescein (Roche) following the manufacturer’s instructions. After TUNEL labeling, nuclei were stained with Hochest 33258, and the TUNEL positive cells were observed using microscope . The percentage of apoptotic cells per total cells was determined in five randomly chosen fields p value of less than 0.05 is considered to be statistically significant.Data are expressed as mean ± SEM. Statistical analysis was performed with two-way ANOVA followed by a Bonferroni correction for multiple comparisons. Graph Pad Software program was used for statistical analysis. A To examine the effect of IL-33 on cardiomyocyte apoptosis after A/R, the myocyte caspase-3 activity, DNA fragments and TUNEL staining were measured. As shown in , open bar). Inhibition of PKCβII with siRNA attenuated the A/R-induced myocyte apoptosis (PKCβ activation has been reported contributes to reperfusion-induced lung injury(8). To determine the role of PKCβ on A/R-induced myocyte apoptosis, PKCβII phosphorylation and siRNA knock-down PKCβII were used in the study. As shown in  (open bar), A/R challenge to myocytes increase in JNK phosphorylation. Genetically inhibition of JNK1 attenuated the A/R-induced myocyte apoptosis  belongs to mitogen-activated protein kinase family It is believed that PKCβ and JNK are sensitive to intracellular oxidant stress. We and others have previously reported that IL-33 plays protective effect on myocardium in the setting of I/R It has been demonstrated that IL-33 interacts with its receptor ST-2L and plays antihypertropic effect in the pressure overload-induced myocardial hypertrophy through modulating the NFκB It has been reported that JNKs are regulated by a multiple-tier module of protein kinase Taken together, in the present study we have demonstrated: 1) PKCβ/JNK, a novel cell signaling pathway is involved in the A/R-induced myocyte apoptosis; 2) IL-33 protects the cardiomyocytes from A/R-induced myocyte apoptosis through inhibition of the PKCβ/JNK pathway. Our study indicates that IL-33 may have therapeutic potential for ischemia/reperfusion-related myocardial injury.


Natural killer (NK) cells contribute to the defense against infected and transformed cells through the engagement of multiple germline-encoded activation receptors. Stimulation of the Fc receptor CD16 alone is sufficient for NK cell activation, whereas other receptors, such as 2B4 (CD244) and DNAM-1 (CD226), act synergistically. After receptor engagement, protein kinases play a major role in signaling networks controlling NK cell effector functions. However, it has not been characterized systematically which of all kinases encoded by the human genome (kinome) are involved in NK cell activation.A kinase-selective phosphoproteome approach enabled the determination of 188 kinases expressed in human NK cells. Crosslinking of CD16 as well as 2B4 and DNAM-1 revealed a total of 313 distinct kinase phosphorylation sites on 109 different kinases. Phosphorylation sites on 21 kinases were similarly regulated after engagement of either CD16 or co-engagement of 2B4 and DNAM-1. Among those, increased phosphorylation of FYN, KCC2G (CAMK2), FES, and AAK1, as well as the reduced phosphorylation of MARK2, were reproducibly observed both after engagement of CD16 and co-engagement of 2B4 and DNAM-1. Notably, only one phosphorylation on PAK4 was differentally regulated.The present study has identified a significant portion of the NK cell kinome and defined novel phosphorylation sites in primary lymphocytes. Regulated phosphorylations observed in the early phase of NK cell activation imply these kinases are involved in NK cell signaling. Taken together, this study suggests a largely shared signaling pathway downstream of distinct activation receptors and constitutes a valuable resource for further elucidating the regulation of NK cell effector responses. Natural killer (NK) cells are lymphocytes belonging to the innate immune system. They can eliminate infected or transformed cells through direct killing of target cells 2+ mobilization as well as degranulation and cytokine production SH2D1A whereupon engagement of 2B4 results in inhibitory signals In terms of signaling by NK cell receptors, structurally distinct inhibitory receptors all contain immunoreceptor tyrosine-based inhibition motifs (ITIMs). The signaling by such motifs has been extensively studied and is mediated by activation of tyrosine phosphatases as well as the tyrosine kinase c-Abl In conclusion, further studies focusing at signaling downstream of co-activating receptors and the prototypical NK cell activation receptor CD16 are needed to identify common nodes as well as pathway specificities of receptors with distinct cytoplasmic domains, helping to define the core-signal network regulating NK cell activation.Mass spectrometry (MS) can provide insights into protein phosphorylation events that coordinate signaling networks. In particular, Fourier transform mass spectrometry (FTMS) routinely achieves a very high data accuracy, facilitating unambiguous protein sequencing and identification of amino acid site-specific post-translational modifications 2+-sensitive fluorescent dyes and incubated either with IgG isotype control, anti-CD56 (both served as negative controls), anti-CD16, anti-2B4, or anti-DNAM-1, as well as co-incubated with anti-2B4 and anti-DNAM-1 monoclonal antibodies (mAbs) on ice. Thereafter, cells were pre-warmed for 5 minutes at 37°C and analyzed on a flow cytometer. The strength and dynamics of intracellular Ca2+-mobilization were assessed in NK cells following crosslinking with a secondary goat anti-mouse Fc antibody . Our data indicate a CD16-induced dephosphorylation of LCK on the inhibitory tyrosine Y192, increased phosphorylation of LCK on the activating region Y394/T395, phosphorylation of LYN on S38, dephosphorylation of LYN on serine S164 and phosphorylation of KPCT on S685. The dephosphorylation of LCK on Y192 and phosphorylation of LYN on S38 were also observed in NK cells co-activated by 2B4 and DNAM-1. Furthermore, kinases implicated in NK cell function, but not specifically assigned to CD16 signaling, i.e. FAK2, FGR, FYN, ITK, and KCC2G/D, were phosphorylated upon CD16 stimulation. FAK2 was dephosphorylated on the amino acids T842 and Y579, which is part of the kinase domain, upon CD16 engagement or 2B4 and DNAM-1 co-engagement. Engagement of CD16 and co-stimulation of 2B4 and DNAM-1 induced phosphorylation of FGR on its supposed auto-phosphorylation site Y412. Triggering of CD16 led to the phosphorylation of FYN on the N-terminal serines S21, S25, and S26, whereas the adjacent Y28 showed a trend toward dephosphorylation . PhosphoThe majority of kinases identified by our study have not been characterized in NK cells before. Phosphorylation on the activating Y714 of FER was reduced, as was FES phosphorylation decreased on the activating Y713 by engagement of CD16 or co-engagement of 2B4 and DNAM-1. In contrast, phosphorylation of the proximate S716 residue of FES was significantly induced by engagement of CD16 or co-engagement of 2B4 and DNAM-1. Further, MARK2 phosphorylation was significantly reduced on S456 in NK cells following engagement of CD16 and co-engagement of 2B4 and DNAM-1. Remarkably, PAK4 phosphorylation on S181 was diminished after CD16 engagement, but significantly up-regulated upon co-engagement of 2B4 and DNAM-1. Moreover, NK cell receptor activation led to the phosphorylation of AAK1 and GAK, both of which are involved in clathrin-mediated endocytosis. AAK1 phosphorylation on T620 was significantly enhanced following engagement of CD16 and co-engagement of 2B4 and DNAM-1, whereas the region S672–S678 was reversely regulated. Our data also suggested the phosphorylation of GAK on S829 following CD16 activation. NK cell activation via CD16 and or 2B4 and DNAM-1 co-engagement also led to the phosphorylation of AAPK1 on its predicted auto-phosphorylation site S496, enhanced phosphorylation of GSK3A on the inhibitory site S21, phosphorylation of KPCD2 on S214, phosphorylation of NEK9 on S331-T333 and PCTK2 phosphorylation on S137 and S165. Furthermore, PRPK was phosphorylated on T8 in response to CD16 engagement. In summary, proteome analysis of receptor-induced kinase phosphorylation implicated several new kinases in the regulation of NK cell activation.The potential of kinase-selective phosphoproteomics (phosphokinomics) to provide comprehensive access to the human kinome has previously been demonstrated 9 NK cells) for phosphokinome analyses. Importantly, as compared to freshly isolated NK cells, IL-2–cultured NK cells were not significantly altered with respect to the expression of activation receptors, kinetics of intracellular calcium mobilization, or magnitude of degranulation following receptor stimulation. The non-specific kinase inhibitor VI16743 in combination with the commercially available ATP-competitor Purvalanol-B were used to capture the kinome and phosphorylation status of stimulated NK cells. The number of kinases identified by our workflow equaled the number of kinases detected by chemical proteome approaches using a pool of 5 different kinase inhibitors Phosphokinomics is useful for the systematic characterization of kinase-based signaling on a minute time scale Many of the regulated kinases presented in this study have not been previously described in NK cells . For mosFYN was consistently phosphorylated upon engagement of all receptors investigated on its N-terminal serines S21, S25 and S26, but phosphorylation of the adjacent tyrosine Y28 was unchanged or even reduced. FYN is known to play an important role in proximal signaling by 2B4 The calcium/calmodulin-dependent kinase 2  subunits gamma and delta were phosphorylated on S381–S384 and S330 following CD16 engagement or 2B4 and DNAM-1 co-engagement. Furthermore, our data suggests simultaneous phosphorylation of the S287 activation site on KCC2G. Poggi and colleagues have demonstrated that pharmacological inhibition of KCC2 blocks IL-2–cultured NK cell killing of autologous dendritic cells, but not of K562 cells Results revealed that AAK1, the ubiquitously expressed AP2 associated kinase 1 implicated in clathrin-mediated endocytosis Stimulation of CD16 or 2B4 and DNAM-1 induced FES phosphorylation of S716 and dephosphorylation of the postulated auto-phosphorylation site Y173 MARK2 was consistently dephosphorylated on S456 following engagement of CD16 or co-engagement of 2B4 and DNAM-1. MARK family kinases phosphorylate microtubule-associated proteins, regulate microtubule-based intracellular transport, and are implicated in cellular polarity This study characterizes a significant portion of the NK cell kinome and provides a first unbiased and systematic view into kinase signaling in primary NK cells. Proteomics detected receptor-dependent kinase phosphorylations already after 2 minutes of receptor engagement and allowed the discrimination of differentially phosphorylated kinase populations as demonstrated by FYN. Several kinases were consistently regulated following the engagement of activating receptors. Co-engagement of DNAM-1 and 2B4 generates a phosphorylation pattern at kinases, which was rather similar to that of CD16-stimulated cells, supporting the idea of a core signal network for NK cell activation. Kinases and phosphorylations, which have not previously been implicated in NK cell signaling were regulated and may thus contribute to the process of NK cell activation. Regulation of kinases such as MARK2 and AAK1 are interesting in terms of understanding the molecular pathways for cellular polarization and endocytosis, processes that are induced upon NK cell activation. Thus, contemplating data presented here will direct future studies focused on elucidating the molecular regulation of NK cell effector functions.5 per well) and irradiated, allogeneic PBMCs (105 per well) were resuspended in complete medium supplemented with 500 U/ml recombinant IL-2  and 10 µg/ml PHA-L (Sigma). Cells were co-cultured in 96-well plates for one week. NK cells were transferred to culture flasks and cultured further for 2 weeks in complete medium supplemented with 50 U/ml IL-2. The purity and viability of NK cells were assessed by flow cytometry prior to analysis. The percentage of CD3−CD56+ NK cells was greater than 95% in all experiments.This study was approved by The Regional Ethics Review Board in Stockholm. Peripheral blood was obtained with informed consent from healthy volunteers. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation . NK cells were subsequently purified by negative magnetic selection . Freshly isolated NK cells were resuspended in complete medium  and analyzed within two days after isolation. For polyclonal expansion, purified NK cells (10The human erythroleukemia cell line K562 and murine mastocytoma cell line P815 (both American Type Culture Collection) were maintained in complete medium.For flow cytometry, NK cell activation, calcium flux analyses, and CD107a degranulation assays, the following mouse monoclonal antibodies (mAbs) were used: anti-CD107a , anti-CD16 , anti-CD56 , anti-DNAM-1 , anti-NKG2D , and anti-CD3 , anti-2B4 , and isotype control mAb . For determination of intracellular calcium mobilization, the calcium-sensitive dyes Fluo-4 and FuraRed (both Molecular Probes) were used.7 NK cells) for 30 min on ice. 300 µl staining solution  were added, and cells were incubated for an additional 30 minutes at 4°C. Dye-loaded NK cells were washed, resuspended in HBSS, and kept on ice. Prior to analysis NK cells were resuspended in 300 µl HBSS supplemented with 1% FBS, pre-warmed at 37°C in a water bath, and analyzed by flow cytometry. After 30 seconds, samples were removed from the flow cytometer, and F(ab′)2-anti-mouse IgG crosslinking antibodies  were added. Thereafter, samples were measured for 4 minutes by flow cytometry. Acquired flow cytometric data were analyzed using the FlowJo software (Treestar). NK cells were gated on forward/side scatter, and the ratios of FL-1 and FL-3 intensities were displayed as a function of time.NK cells were resuspended in 50 µl Hanks buffered salt solution  and incubated with the primary mAbs indicated  containing fluorochrome-conjugated anti-CD56 and anti-CD107a mAbs, and analyzed by flow cytometry. Data were analyzed with FlowJo software.For assessment of NK cell degranulation, 2×109 IL-2-cultured NK cells were used (5×108 NK cells per stimulation). IL-2-cultured NK cells were starved overnight in complete medium without IL-2. Prior to stimulation, cells were resuspended in complete medium at 2×107 cells/ml. NK cells were incubated with primary mAbs directed against CD16, DNAM-1 and 2B4 for 30 minutes on ice (1 µg mAb per 107 NK cells). MAb-coated NK cells were pre-warmed at 37°C in a water bath, thereafter added F(ab′)2-anti-mouse-IgG crosslinking antibody (Jackson ImmunoResearch), and stimulated for the indicated time.For kinome experiments, up to 1,5×10g for 30 minutes at 4°C and filtered using a 0.45 µm syringe membrane (Millipore). The protein concentrations of lysates were determined using a Bradford protein assay (BioRad) to ensure equal loading of kinase affinity columns. For small molecule affinity chromatography (SMAC), the kinase inhibitors VI16743 and Purvalanol-B (Tocris) were used. Synthesis of V16743 and generation of both kinase affinity materials were conducted as previously described Following stimulation, NK cells were lysed in ice-cold lysis buffer containing 50 mM HEPES pH 7.5, 1 M NaCl, 1 mM EGTA, protease inhibitor cocktail (Roche), and 1% Triton-X100. Lysates were centrifuged at 70000× Proteins were extracted from concentrated SMAC eluate fractions by chloroform/methanol precipitation 2O containing 2% acetic acid, pH 2.8) and incubated with two Ga3+-chelated gel disks (Pierce) for one hour at room temperature or overnight at 4°C. The Ga3+-matrix was washed 3 times with 100 µl IMAC-binding buffer to remove unmodified peptides. Retained phosphopeptides were eluted with 600 µl IMAC elution buffer . The phosphopeptide-enriched fraction was vacuum-dried, desalted and analyzed by LC-MS/MS. IMAC flow-through and washing fractions were collected and constituted the phosphopeptide-depleted fraction. The phosphopeptide-depleted fraction was further sub-fractioned by SCX chromatography. Thereafter, desalted peptides were resolved and separated on a MonoS PC1.6/5 column  using an Ettan micro LC system . SCX-separated peptide fractions were vacuum-dried, desalted, and analyzed separately by LC-MS/MS.Phosphorylated peptides (phosphopeptides) were enriched from complex peptide samples using the following immobilized metal affinity chromatography (IMAC) protocol: iTRAQ-labeled and desalted peptide mixtures were resolved in IMAC binding buffer . In this study, proteins were only considered if they were identified at least with one unique peptide having an individual Mascot peptide score above 20, which indicated identity or extensive homology (p<0.05) using the following Mascot search parameters: enzyme, trypsin (specificity: K/R); maximum missed cleavages, 1; fixed modifications, iTRAQ 4-plex (K), iTRAQ (N terminus), Methylthio (C); variable modifications, phosphorylation , oxidation (M); peptide tolerance, 10 ppm; MS/MS tolerance, 0.1 Da. Proteins identified by one peptide and Mascot-aided assignment of tyrosine (Y) phosphorylation (n = 21) were checked by manual inspection of corresponding MS/MS spectra, whereas annotation of threonine  and serine  phosphorylation was statistically validated by the AScore algorithms www.ebi.ac.uk/pride/).LC-MS/MS analyses of phosphopeptide-enriched and -depleted fractions were performed on an Acquity Ultra Performance LC system (Waters Corp.) connected to an LTQ Orbitrap XL Fourier transform mass spectrometer (Thermo Scientific). Raw MS data were converted to data formats compatible with Mascot search engine (Matrix Science). Data from all peptide fractions were merged using Mascot Daemon (version 2.1.6). The UniProtKB/Swiss-Prot primary sequence database was used for protein identification  PBMC obtained from human peripheral blood by Ficoll gradient centrifugation and (B) IL-2–cultured NK cell were stained with fluorochrome-conjugated anti-CD3 and anti-CD56 mAbs and analyzed by flow cytometry. IL-2–cultured NK cell were 95 to 99% CD3−CD56+ as determined by flow cytometry. Data represent IL-2–cultured NK cell populations used in the phosphoproteome studies.(PDF)Click here for additional data file.Figure S2VI16743/Purvalanol-B-based affinity chromatography permits comprehensive enrichment of human protein kinases. (A) Kinases were significantly enriched by VI16743/Purvalanol-B-based affinity chromatography. Percentages of kinases and non-kinases (others: proteins without kinase activity) after VI16743/Purvalanol-B-mediated affinity purification are shown. (B) Kinases are identified based on a significantly higher number of MS spectra reflecting a better peptide identification and coverage of kinases achieved by MS. Assigned MS spectra per kinase and non-kinases (others) are depicted as mean values ± standard deviation (SD) of all conducted kinase-selective phosphoproteome experiments. Differences between two groups were examined using the Student's t-test .(PDF)Click here for additional data file.Figure S3Statistical validation of phosphorylation site regulation at protein kinases by iTRAQassist. Statistical evaluation of quantitative peptide data was conducted by iTRAQassist, a MS device specific noise algorithm, as previously described (PDF)Click here for additional data file.Figure S4Number of kinase phosphorylation sites identified in respective experiments.(PDF)Click here for additional data file.Figure S5Distribution of iTRAQ-based protein and phosphorylation site regulation factors. This figure provides a general overview of all protein (black curve) and phosphorylation site (red curve) regulation factors calculated for two representative proteome experiments Click here for additional data file.Figure S6ITRAQ-MS-based quantification of N-terminal FYN phosphorylation induced by the engagement of CD16 or 2B4 and DNAM-1. Distinct phosphopeptide populations were resolved by nano-UPLC (2O). Relative iTRAQ intensities of the corresponding phosphopeptides are shown at the right. FYN was differentially phosphorylated at S21, S25, S28 and Y28 following engagement of CD16 or co-engagement of 2B4 and DNAM-1.ano-UPLC  and sepa(PDF)Click here for additional data file.Table S1Performed phosphokinome experiments. Footnote: In experiment I and II cIgG-, CD16- and 2B4 and DNAM-1 co-activated NK cells were analyzed. In experiment III cIgG- and 2B4 and DNAM-1 co-activated NK cells were analyzed. §Mascot-based annotation of serine and threonine phosphorylation was checked by the AScore algorithm. 47 phosphorylation sites could not be assigned unambiguously.(PDF)Click here for additional data file.Table S2Kinases expressed in human NK cells. Footnote: *Kinase grouping according to Manning et al., 2002.(PDF)Click here for additional data file.Table S3Kinase phosphorylation in human NK cells. Footnote: 1Serine/threonine annotations by Mascot were validated using AScore algorithm apredicted phosphorylation site (UniProt); *not listed in UniProt database; when phosphorylation sites could not be assigned unambiguously, alternative sequences are given. Note: Only for peptides containing more than one single serine/threonine residue AScore is required and annotation of tyrosine phosphorylation is not evaluated by AScore algorithm, but was inspected manually.(PDF)Click here for additional data file.


Sprattus sprattus) throughout an entire winter in a 150-m-deep Norwegian fjord. During ice-free conditions, the sprat surfaced and released gas bubbles at night with an estimated surfacing rate of 3.5 times per fish day−1. The vertical swimming speeds during surfacing were considerably higher (~10 times) than during diel vertical migrations, especially when returning from the surface, and particularly when the fjord was not ice covered. The sprat released gas a few hours after surfacing, suggesting that the sprat gulped atmospheric air during its excursions to the surface. While the surface activity increased after the fjord became ice covered, the records of gas release decreased sharply. The under-ice fish then displayed a behavior interpreted as “searching for the surface” by repeatedly ascending toward the ice, apparently with limited success of filling the swim bladder. This interpretation was supported by lower acoustic target strength in ice-covered waters. The frequent surfacing behavior demonstrated in this study indicates that gulping of atmospheric air is an important element in the life of sprat. While at least part of the population endured overwintering in the ice-covered habitat, ice covering may constrain those physostome fishes that lack a gas-generating gland in ways that remain to be established.Upward-facing echosounders that provided continuous, long-term measurements were applied to address the surfacing behavior and gas release of the physostome sprat ( Sprattus sprattus) is a physostome fish with an open swim bladder. Clupeids have one pneumatic duct connecting the swim bladder to the stomach and one anal duct leading from the posterior swim bladder to the vent  with artificially emptied swim bladders could refill them by swallowing air at the surface; meanwhile, the individuals without access to the surface suffered a high mortality.The clupeid sprat  is the innermost part of the Oslofjord, and due to restricted water exchange, the deep water of the fjord is normally hypoxic. Bunnefjorden often becomes ice covered during winter. A map depicting the study area and the location of the echosounders  is given in Klevjer and Kaartvedt .2 and was towed at two knots. The mesh size is 20 cm near the opening, declining to around 1 cm at the rear part and 3 mm in the cod-end. A Scanmar depth sensor was connected to the trawl, measuring the trawl depth during sampling. The trawl was equipped with a multisampler codend  carried out during two sampling days in December 2009 and one sampling day in April 2010. The pelagic trawl has an aperture of about 100 m−1 (38 kHz), 2 ping s−1 (120 kHz), and 2.5–6 ping s−1 (200 kHz). The echosounders were connected to land with cables that provided electricity and transmitted data to a pc on shore. All raw data were stored for later analyses.Three submerged echosounders kept in pressure proof casings were deployed from November 11, 2009 until April 12, 2010. A Simrad EK60 38 kHz echosounder (7° beam width) was deployed at the bottom of the fjord (150 m), and adjacent buoys with echosounders floating at 80 m  and 30 m  were deployed for enhanced resolution in shallow waters. The upward-facing transducers were mounted in steel frames with gimbal couplings to ensure horizontal orientation of their surfaces. The temporal resolution of data (the ping rate) was set to 1–2 ping sWe here use data from the bottom-mounted echosounder for abundance estimates (echointegration) and also present echograms from this echosounder to illustrate the population distribution and behavior. Individual surfacing and gas release behavior, and vertical swimming speed are analyzed based on data from the shallowest echosounder that provided superior resolution of near-surface waters. Data from the echosounder at 80 m (120 kHz) were used as a supplement to assess the amount and depth of gas releases that may have occurred below the shallowest echosounder.Acoustic data from 200 kHz  were processed in the Sonar_5 Pro software program  where echo traces from single organisms are selected from the echogram and combined into tracks by the researcher. The data were tracked in the single echo detection (SED) echogram. This only displays a subset of the data that fulfill criteria for a well-defined single target, in contrast to the ordinary amplitude (Amp) echogram that shows all backscatter. The SED threshold was set to −70 dB. Any tracks with TS stronger than −45 dB were removed during post-processing (to sort out fish larger than sprat). The tracking was done between the surface and the upper 15–20 m of the water column. The surface area of the beam was 10.5 mReleased gas could be seen on the echogram as a thin line ascending from the fish up to the surface  was determined by integration of 38 kHz acoustic data from 0 to 100 m depth, using the LSSS software  . The average number of sprat per m2 per day was calculated by dividing the backscatter value with the TS value of sprat (−48.2 dB) based on manual tracking of 38 kHz acoustic data . Average cloud cover per day, measured in octas, was used to map the occurrence of clear sky and overcast weather during the study period. A Web camera providing images every hour was used to monitor the ice conditions of the fjord. The fjord became ice covered in January this winter.Weather data from a nearby station in Oslo (Blindern) were obtained from the climate database of the Norwegian Meteorological Institute available at , 58 gobiids (Gobiidae spp.), 12 whitings (Merlangius merlangus), 4 anchovies (Engraulis encrasicolus), 1 pollack (Pollachius pollachius), and some gelatinous plankton. Catches of other invertebrates like krill and shrimps were negligible this winter.A total of 7,345 sprat were caught during the sampling in December 2009 and April 2010, making sprat the most abundant species in the trawl catches. In December, the highest catches of sprat were obtained in the range of 60–80 m during the day (~720 sprat per 10 min of trawling) and 40–70 m at night (~85 sprat per 10 min of trawling). There were no catches of sprat below 80 m in the beginning of the winter. During daytime in April, most of the sprat were collected from 70 to 95 m (~520 sprat per 10 min) with nearly none distributed deeper than 100 m or shallower than 60 m (<5 sprat per 10 min). The catches contained furthermore a total of 162 herring . Most fish were distributed in a layer ranging from around 30–60 m at night Fig. a.Fig. 3AThe nocturnal layer became more dispersed and extended to shallower depths by the end of the month, and by December, a part of the population carried out normal DVM and schooled in deep waters during the day. A bimodal distribution was displayed at night Fig. b. A simia (interpreted as acoustic biomass), were highest in the period before the fjord froze over with a monthly average of >300 in November and ~200 in December  with daily values fluctuating between 100 and 400  and number of surfacing fish per day . There were no records of surfacing sprat during daytime.Surfacing sprat was detected every date during ice-free conditions  and 35 times fish−1 day−1 in the period after the ice had melted, i.e., the rate of gas release was an order of magnitude higher than the rate of surfacing . The low relationship may to some extent be explained by fish releasing air also below the depths of our records with the 200 kHz echosounder as data from the 120 kHz echosounder showed that the fish released air between 0 and 40 m depths. However, the majority of gas release appeared to take place in the 30 m interval covered by the 200 kHz echosounder.Sprat surfaced after sunset, particularly early at night, and released gas in the following hours during nighttime Fig. b, d. Reling Fig. . The rel−1 for that period. The high number of surfacing fish was only apparent for around 1 week followed by a period with a few detections until the second half of February  and number of surfacing fish during the months with ice cover , but the coefficient of determination was very low.The fjord became ice covered between 06 January and 08 January. There were few surfacing fish during the first 2 weeks of January (<10). This was followed by a conspicuous increase in recorded surface events during the third week of January ranging from 39 to 101 per day Fig. a giving ary Fig. b. In Marary Fig. c. Combin−1 day−1  (not shown). No consistent pattern between frequency of surfacing events and cloud index or moon phase was evident.−1, respectively . A post hoc Tukey’s test  revealed that among fifteen possible comparisons, only three were not significantly affected by interaction: downward swimming before ice versus downward swimming after ice (P = 0.18), upward swimming before ice versus upward swimming after ice (P = 0.99), and downward swimming during ice versus upward swimming after ice (P = 0.86). The two factors are difficult to interpret because of the significant interaction; however, the results show that the swimming speed depended on environmental conditions and that the difference between upward and downward swimming speed was greater in ice-free waters , and a significant interaction between the factors was also present here . TS of upward swimming sprat in ice-free waters versus TS of downward swimming sprat in ice-covered waters was the one comparison (among six) that was not affected by interaction .The TS values were weaker in ice-covered waters than in ice-free waters. The average TS values of ascending and descending sprat during surfacing in ice-free waters were −54.2 and −53.5 dB, respectively, and −57.8 and −54.3 dB in ice-covered waters, respectively. The TS values differed significantly by both swimming direction and ice conditions (ice-free waters versus ice-covered waters) , and the TS values of descending sprat in ice-free waters were significantly stronger than the TS of descending sprat in ice-covered waters. This was the case in spite of much faster diving , which might have caused a steeper tilt angle, and thus rather expectedly reduced TS  (facultative air-breather and physostome fish) used oxygen directly from gas bubbles found under the ice to prolong its survival in a hypoxic lake. Similar to the mud-minnow, the sprat may have searched for trapped gas underneath the ice. As we found a significant relation between surfacing and gas release in ice-free waters, the latter might be used as indication of how “successful” under-ice surfacing (inferred as under-ice gulping) was (see below).As air bubbles can be trapped in pockets during ice formation, and gas may accumulate under the ice from photosynthesis and anaerobic decomposition, there might have been an alternate source of gas underneath the ice cover. In relation to aerial respiration, Klinger  and KlinIn ice-free waters, release of gas was observed some hours after surfacing. Gas release in herring has been documented in previous studies . Noticeably, during some days in January when the fjord was ice covered, the average rate of surfacing increased to ~26 times fish−1 day−1, and combining all months with ice cover, the surfacing rate was ~12.5 times fish−1 day−1. Negligible amount of observed gas bubbles during this period indicates an unsuccessful outcome in terms of obtaining air. Yet, the consistent pattern of repetitive trials underneath the ice suggests that atmospheric air is an important element in the life strategy of sprat. Fish with negative buoyancy may compensate by tilted swimming (Huse and Ona Coregonus hoyi) when exposed to increased pressure (four atmospheres) due to enhanced swimming activity. Some fish may use oxygen in the swim bladder for respiration during hypoxic conditions, but it is unclear to what extent this also relates to sprat  have shown that denial of surface access leads to negative buoyancy and may cause increased mortality, reduced welfare  because of their migrations to the surface (Thorne and Thomas


Many infectious agents utilize CD46 for infection of human cells, and therapeutic applications of CD46-binding viruses are now being explored. Besides mediating internalization to enable infection, binding to CD46 can directly alter immune function. In particular, ligation of CD46 by antibodies or by measles virus can prevent activation of T cells by altering T-cell polarity and consequently preventing the formation of an immunological synapse. Here, we define a mechanism by which CD46 reorients T-cell polarity to prevent T-cell receptor signaling in response to antigen presentation. We show that CD46 associates with lipid rafts upon ligation, and that this reduces recruitment of both lipid rafts and the microtubule organizing centre to the site of receptor cross-linking. These data combined indicate that polarization of T cells towards the site of CD46 ligation prevents formation of an immunological synapse, and this is associated with the ability of CD46 to recruit lipid rafts away from the site of TCR ligation. CD46 is a human receptor for complement and many pathogens, including Neisseria, Group A Streptococcus, Species B adenoviruses, vaccine strains of the measles virus, and Human Herpes Virus 6 (HHV6) –3. The bHowever, recent work has identified cellular processes that are directly affected by CD46 ligation, and that provide an opportunity to dissect the molecular interactions through which CD46 exerts its effects. Mounting evidence suggests that CD46 signaling affects cell morphology and polarity , 26, andCD46Cyt1.C-A and CD46Cyt1.C-A,L-R were generated by site-directed mutagenesis as described , and wit4) expressing CD46 constructs were ligated with anti-CD46  antibody or anti-Tfr antibody for 30 min at 37°C, then incubated with antiCD3/CD28 coated beads and stained as described [μM between slices. Approximately 20–30 slices were acquired per image, with the pin hole set to 1 AU, using a 60 × oil immersion objective (NA 1.42). Digital images were processed with Image J  and MetaMorph . For MTOC recruitment, slides were de-identified and scored for concentration of tubulin staining at the bead interface.MD45 cells  and ligated and stimulated as above were excited with a 2-photon laser at 800 nm and emission intensities simultaneously recorded in the range of 400–460 nm and 470–530 nm , 32. IntNA = 1.4 was used. GP histograms were fitted to two Gaussian populations using the nonlinear fitting algorithm Solver in Microsoft Excel [Final GP images were pseudocolored in Adobe Photoshop. For all images a 100 × oil objective, ft Excel . The meaft Excel , 33. Sta4, 5 mM EGTA and protease inhibitors) containing 0.5% Triton X-100 (Sigma) for 25 min at 4°C and fractionated on a bottom-loaded discontinuous sucrose gradient as described [μg/mL, 4°C) [μg/mL, 37°C).Cells were scraped, lysed at 3 mg proteins/ml in ice-cold TNE buffer   and with6) were cultured with 5 mCi  3H palmitoic acid (Amersham Biosciences UK) for 4 hours at 37°C and 5% CO2, washed once in PBS, lysed in 0.5% Nonidet-P40, TBS (pH7.8), 5 mM EDTA, and Complete protease inhibitors  on ice, and immunoprecipitated with anti-CD46 (1840) antibody for PAGE.Cells , heterogeneous, highly dynamic, sterol- and sphingolipid-enriched domains that compartmentalize cellular processes” , have be0–200 nm,To test whether the transmembrane cysteine residue of CD46 provides a site for palmitoylation, cells were incubated with radiolabeled palmitoic acid, and immunoprecipitated for CD46. Radiolabeled bands of appropriate size for CD46 were detected from HeLa cells expressing endogenous CD46, and from CHO cells transfected with CD46Cyt1, but not from nontransfected CHO cells, indicating that CD46 is constitutively palmitoylated  upon ligation of CD46Cyt1. In contrast, transfection and ligation of CD46Cyt1.C-A had no significant effect on global membrane order . These data indicate that expression of CD46 influences the organisation of the membrane fluidity, and that ligation of CD46 further enhanced the formation of ordered domains. Notably, CD46-mediated membrane condensation required the transmembrane cysteine. Detergent extraction alone is not always a reliable indicator of raft recruitment , 39, 40,id rafts , 43, 44.id rafts . Firstlyon, “Pf” . Note thP < .05), indicating that palmitoylation of CD46 indeed targets the protein to more ordered domains. Ligated CD46Cyt1 was recruited to regions of average GP values 0.437 ± 0.077 , but not to the levels indicative of ordered domains . These studies together demonstrate that (i) CD46 constitutively localizes to a domain that, although not a classic raft as defined by detergent insolubility or GP values, is somewhat more ordered than the general cell membranes, (ii) CD46 is recruited to stable lipid rafts upon ligation, thus increasing the abundance of ordered domains, and (iii) recruitment of CD46 into ordered domains (both constitutive and ligated) is dependent upon a transmembrane cysteine.We next determined whether CD46 was preferentially recruited into ordered domains. To correlate CD46 with ordered domains, we used positive CD46 staining to mask the GP images . Ligation of Tfr prior to stimulation with CD3/CD28 beads did not reduce membrane condensation at CD3/CD28 contact sites . In contrast, ligation of CD46 prior to TCR stimulation significantly reduced raft formation at T cell activation sites to 0.305 ± 0.84 (P < .001). Thus, as well as inhibiting recruitment of MTOC and cytotoxic granules to the immunological synapse [Raft-like domains are concentrated at the immunological synapse during antigen presentation , 45–47. P < .05), and ligation abrogated (0.207 ± 0.084) this recruitment. However, expression (0.388 ± 0.077) and ligation (0.403 ± 0.057) of CD46Cyt1.C-A had no effect. Ligated CD46Cyt1 colocalized with ordered domains (P < .05) reduction in raft recruitment to CD3/CD28 beads when CD46Cyt1 was expressed and not ligated, compared to parental cells that do not express CD46 or cells expressing the CD46 palmitoylation mutant. This is consistent with the effect of CD46Cyt1 expression on global membrane organization and the location of CD46Cyt1 in ordered domains, suggesting that CD46-mediated raft domains are a separate entity to pre-existing or TCR-induced raft domains. These data together indicate that CD46 must associate with lipid rafts to prevent the polarization of rafts to the IS and that CD46 ligation competes with TCR signalling to control the localisation of higher-ordered membranes.Secondly, we tested whether association of CD46 with rafts was required for the inhibitory effect on raft recruitment to the IS, utilizing MD45 cells Figures . As in tP < .001). This effect was specific for the CD46 antibody, because antibodies to Tfr had no effect, and was mediated by CD46Cyt1 but not by CD46Cyt2 (P < .001 for mutated CD46 compared with wild-type). The observation that reorientation was not completely abolished by mutating the raft-association site, and that CD46Cyt2 was not able to mediate reorientation, indicates that entities other than lipid rafts also play a role. Given that CD46 associates with the polarity regulator, Discs large (Dlg) via its C-terminus  is not sufficient to prevent polarisation of either rafts or the MTOC to the site of TCR stimulation. This, combined with our observation that abrogation of raft accumulation only partially prevents re-orientation, indicates that the dominant effect of CD46 on T-cell polarity is mediated by a combined contribution from lipid rafts and other entities. We have previously found that CD46 interacts with Dlg, a member of a network that regulates polarity of epithelial cells and T cells , 28, 48,These experiments indicate that CD46 associates with lipid rafts, and that this association facilitates the repolarisation of T cells triggered by CD46 ligation. CD46 ligation prevents the recruitment of rafts (this study) and the MTOC (this study and ) to the K. Gaus and S. M. Russell contributed equally to the paper.


Saccharomyces cerevisiae. In a candidate screen for checkpoint genes, we identified bud2Δ as deficient for the SPC. Bud2 is a GTPase activating protein (GAP), and the only known substrate of Bud2 was Rsr1/Bud1, a Ras-like GTPase and a central component of the bud-site-selection pathway. Mutants lacking Rsr1/Bud1 had no checkpoint defect, as did strains lacking and overexpressing Bud5, a guanine-nucleotide exchange factor (GEF) for Rsr1/Bud1. Thus, the checkpoint function of Bud2 is distinct from its role in bud site selection. The catalytic activity of the Bud2 GAP domain was required for the checkpoint, based on the failure of the known catalytic point mutant Bud2R682A to function in the checkpoint. Based on assays of heterozygous diploids, R682Abud2, was dominant for loss of checkpoint but recessive for bud-site-selection failure, further indicating a separation of function. Tem1 is a Ras-like protein and is the critical regulator of mitotic exit, sitting atop the mitotic exit network (MEN). Tem1 is a likely target for Bud2, supported by genetic analyses that exclude other Ras-like proteins.The spindle position checkpoint (SPC) ensures correct mitotic spindle position before allowing mitotic exit in the budding yeast  Eukaryotic cells partition their chromosomes between mother and daughter cells on cell division. In budding yeast, the plane of cell division is determined at the start of the cell cycle by selection of a site of polarized growth - the bud site. As the bud grows, the bud site becomes the position of the mother-bud neck. At anaphase, the mitotic spindle must be perpendicular to and intersect to the plane of cell division, and this requires that one spindle pole body (SPB) move into the bud while the other remains in the mother. To position the mitotic spindle, cytoplasmic  microtubules interact with the bud cortex and pull the spindle into the mother/bud neck, via the independent actions of the dynein and Kar9 pathways arp1Δ mutant). In such a strain, a fraction of the cells in the population fail to move the spindle into the neck, and the cell cycle halts. When the checkpoint mechanism is defective, these cells proceed to exit from mitosis with the spindle in the mother. In our screen, a bud2Δ null mutant gave a relatively strong phenotype, so we have investigated the role of Bud2 in the spindle position checkpoint.To investigate the molecular mechanisms of the spindle position checkpoint, with a focus on the molecular basis by which the cell senses spindle position, we screened candidate null mutants lacking proteins with structural or functional connections to the mother-bud neck. To activate the checkpoint, we performed the screen in a strain lacking dynein function  select a bud site adjacent to the previous budding site a/α cells have a bipolar budding pattern, in which the newly born daughter places her first bud site at the distal pole, away from its mother lte1 null mutation prevents the premature mitotic exit. This pathway also involves a BUD gene, BUD6.Exit from mitosis appears to be controlled by the activity of another Ras-like GTPase, Tem1 bud2. However, checkpoint failure in the bud2 mutant was independent of Lte1, providing a contrast to the pathway that involves Bud6 Here, we show that loss of Bud2 also causes failure of the checkpoint in about half of cells with mis-positioned spindles. The checkpoint defects caused by point mutations affecting the Bud2 GAP domain were similar to the defects caused by null mutations of arp1Δ bud2Δ mutants had many cells with greater than three nuclei, consistent with but not specific for a defect in the SPC. To assay the integrity of the checkpoint, we performed time-lapse movie analysis of single cells with mitotic spindles in the mother. Using GFP-tubulin as a marker for mitotic spindle breakdown, we calculated the percentage of cells in which the checkpoint was intact  cell. The bud2Δ mutants underwent inappropriate spindle breakdown in the mother with a MT in the mother-bud neck, eliminating the possibility that the bud2Δ mutant accumulates multinucleate cells as a result of aberrant MT dynamics. An example is shown in bud2 lte1 double mutant had a checkpoint defect similar to that of a bud2 single mutant, indicating that the checkpoint defect was independent of Lte1.In our screen for candidate checkpoint genes, dynactin/s intact . About 4BUD6 had a checkpoint defect with magnitude similar to the one observed here for bud2, and the bud6 checkpoint defect did depend completely on LTE1bud2Δ bud6Δ double mutant showed an exacerbated checkpoint defect , suggesting that these bud2 mutants are defective in the GAP activity.The Bud2 GAP domain contains the ‘FLR motif’, which is highly conserved in Ras-GAPs from other organisms 2 strain . The budbud2 mutant alleles on a low-copy-number CEN plasmid. The R682Abud2 allele did not rescue checkpoint function at all  or Sigma-Aldrich , except as indicated. Yeast were cultured and manipulated by standard techniques BUD2 were produced by PCR-mediate mutagenesis using a YCp50 plasmid carrying wt BUD2, pHP519, as template F680Abud2 and R682Abud2, respectively.Point mutations of Activation and failure of the spindle position checkpoint were assayed with time-lapse video microscopy observing the behavior of the mitotic spindle in individual living cells expressing GFP-tubulin (GFP-Tub1), as described BUD2 plasmid.To assay bud site selection, haploid cells carrying each plasmid were grown to mid-log phase in rich liquid medium lacking uracil, and then stained with Calcofluor, as described γ -32P]GTP, and then incubated with GST or GST-Bud2 at 23°C for 20 min, followed by a filter binding assay to measure the protein-bound radioactivity. We calculated and plotted the percent of GTP remaining, compared to the amount at time zero.For GAP assays, the proteins were a kind gift of Drs. Paul Polakis and Frank McCormick  except for GST-Bud2, which was purified from yeast in our lab. The assay conditions were as previously described, except that only one time point was measured after 20 min incubation see . BrieflyFigure S1Bud2 acts as a GAP for Rsr1/Bud1 but not for Ras2. Rsr1/Bud1 or Ras2 preloaded with [γ -32P]GTP was incubated with GST-Bud2 or GST, and the percentage of radiolabelled GTP remaining bound to each GTPase is plotted. This plot represents an average of two experiments with similar results.(TIF)Click here for additional data file.Movie S1Mitotic exit, marked by breakdown of the mitotic spindle, occurs in a bud2 mutant cell in which a cytoplasmic microtubule is present in the mother-bud neck.(MOV)Click here for additional data file.