Protein kinase Cα (PKCα) is one of the predominant PKC isoforms that phosphorylate cardiac troponin. PKCα is implicated in heart failure and serves as a potential therapeutic target, however, the exact consequences for contractile function in human myocardium are unclear. This study aimed to investigate the effects of PKCα phosphorylation of cardiac troponin (cTn) on myofilament function in human failing cardiomyocytes and to resolve the potential targets involved.in vitro cross-phosphorylation of the PKA sites by PKCα. Isometric force was measured at various [Ca2+] after exchange. The maximal force (Fmax) in the cTn (DD+PKCα) group (17.1±1.9 kN/m2) was significantly reduced compared to the cTn (DD) group (26.1±1.9 kN/m2). Exchange of endogenous cTn with cTn (DD+PKCα) increased Ca2+-sensitivity of force (pCa50 = 5.59±0.02) compared to cTn (DD) (pCa50 = 5.51±0.02). In contrast, subsequent PKCα treatment of the cells exchanged with cTn (DD+PKCα) reduced pCa50 to 5.45±0.02. Two PKCα-phosphorylated residues were identified with mass spectrometry: Ser198 on cTnI and Ser179 on cTnT, although phosphorylation of Ser198 is very low. Using mass spectrometry based-multiple reaction monitoring, the extent of phosphorylation of the cTnI sites was quantified before and after treatment with PKCα and showed the highest phosphorylation increase on Thr143.Endogenous cTn from permeabilized cardiomyocytes from patients with end-stage idiopathic dilated cardiomyopathy was exchanged (∼69%) with PKCα-treated recombinant human cTn (cTn (DD+PKCα)). This complex has Ser23/24 on cTnI mutated into aspartic acids (D) to rule out max and increases myofilament Ca2+-sensitivity, while subsequent treatment with PKCα in situ decreased myofilament Ca2+-sensitivity. The known PKC sites as well as two sites which have not been previously linked to PKCα are phosphorylated in human cTn complex treated with PKCα with a high degree of specificity for Thr143.PKCα-mediated phosphorylation of the cTn complex decreases F Protein kinase C (PKC) is a member of the serine/threonine protein kinase family and is expressed in most tissues, including the heart. PKC is able to modify cardiac function via phosphorylation of proteins involved in calcium handling and in the regulation of contractile proteins. The thin filament proteins troponin T (cTnT) and I (cTnI) 2+-sensitivity of force upon incubation of single permeabilized human cardiomyocytes with the catalytic subunit of PKC, as well as with the PKCε and PKCα isoforms, while no changes in isometric force at saturating Ca2+-concentration were observed The troponin complex is an important substrate of PKCα and both positive and negative inotropic and lusitropic effects have been reported  2+-sensitivity 2+2+-desensitization of force. So far, the site-specific effects in human tissue remain illusive. Therefore, current study aimed to investigate the specific effects of human cTn phosphorylation by PKCα on contractility, and to explore which phosphorylation targets might be involved.Known PKC phosphorylation targets include on cTnI: Ser42, Ser44, Ser76 (or Thr77) and Thr143 and on cTnT: Ser1, Thr194, Ser198, Thr203 and Thr284  in vitro cross-phosphorylation of these PKA sites by PKCα. To identify the origin of the changes in contractile function observed, we investigated the targets of PKCα in human cTn using site-specific phospho-antibodies, liquid chromatography (LC) MS/MS and a targeted MS-based method, multiple reaction monitoring (MRM), which allows quantitation of site-specific phosphorylation.The specific role of PKCα-mediated phosphorylation of cTn in cardiomyocytes was analyzed in myocardium from end-stage heart failure patients using our previously described cTn exchange method 2+ but a depression of the maximal force generating capacity (Fmax) of cardiomyocytes. The overall effects of PKCα-mediated phosphorylation of the cTn complex on cardiomyocyte force development were negative. In contrast to the Ca2+-sensitizing effect of the PKCα-treated cTn (DD) complex, subsequent PKCα-incubation of the cardiomyocytes after exchange resulted in a desensitization of the myofilaments to Ca2+. This indicates that PKCα-mediated phosphorylation of sarcomeric proteins other than cTn exerts opposing effects on Ca2+-sensitivity. LC MS/MS analysis of human recombinant cTn complex treated with PKCα revealed two sites that, for the first time, are identified as PKCα substrates: Ser198 located on cTnI and Ser179 on cTnT. In addition, MRM analysis revealed target-specificity in the in vitro PKCα-mediated phosphorylation of Ser42, Ser44, Thr143, and Ser198 on cTnI.Our results revealed that exchange using PKCα-treated cTn (DD) resulted in a sensitization of the myofilaments to CaAn expanded methods section is available in in vitro but not in vivo (2ATP (sodium adenosine-5′-triphosphate), 4 mmol/L MgCl2, 6 mmol/L DTT, 10 μmol/L PMA , 10 μl/ml phosphatase inhibitor cocktail , and 5 μl/ml protease inhibitor cocktail  for 180 minutes at 30°C. The phosphorylated cTn (DD) complex (cTn (DD+PKCα)) was dialyzed overnight in order to remove ATP. Samples were taken at different time–points to assess the time course of phosphorylation.Human recombinant cTn complex was prepared as described before  in vivo  [1]. CarAnalysis of PKCα-mediated phosphorylation of recombinant cTn (DD) complex proteins was determined using ProQ Diamond stained (Molecular Probes) 1D gradient gels and Western blotting using antibodies against cTnI Ser42 and Thr143 (Abcam) as described previously n = 6) from end-stage failing idiopathic dilated myocardium  and permeabilized by addition of 0.5% Triton X-100 for 5 minutes 2, 3 mmol/L CaCl2, 2.5 mmol/L EGTA, pH 6.9). Single cardiomyocytes were exchanged with recombinant cTn complex as described before In the exchange experiments, left ventricular samples . During this slack test, force first dropped to zero and after the restretch quickly redeveloped to the original steady state level. A single exponential was fitted to estimate the rate constant of force redevelopment at maximal activation (Ktr-max).Force measurements on permeabilized cardiomyocytes were performed as described previously Coomassie-stained protein bands were excised and processed for trypsin in-gel digestion according to the protocol of Gundry et al. n = 4 per group; technical replicates  = 3).Tissue from end-stage heart failure was incubated with PKCα as described before Human recombinant cTn was maximally phosphorylated by PKCα (based on the phospho specific Pro-Q Diamond staining) and MRM was used to determine PKCα induced phosphorylation levels of recombinant cTnI . Analysit-test or ANOVA, where appropriate. Values are given as means ± S.E.M. of n myocytes.Myofilament data analysis was performed using the modified Hill equation to fit force-pCa relations. Comparisons between the cTn (DD) and cTn (DD+PKCα) groups were made using an unpaired Student MRM data acquisition, processing and analysis were performed using Applied Biosystems/MDS Sciex Analyst software and Multiquant 1.0. The average peak areas for each phosphorylated tryptic peptide were expressed in absolute quantities (fmol) using standard curves constructed using known amounts of synthetic peptides and the values obtained were corrected for loading differences by densitometric analysis of the bands excised from the SDS-PAGE gel .Incubation of recombinant cTn (DD) complex with PKCα resulted in phosphorylation of both cTnT and cTnI. The ProQ Diamond stained gel shown in Exchange experiments were conducted in tissue samples from 6 different end-stage failing hearts. The choice to use tissue from patients with end-stage heart failure instead of control tissue was based on our previous results where an effect of PKCα phosphorylation was only observed in the failing and not in the control group n = 3)  , i.e. ve2+-sensitivity of force measurements were performed at various [Ca2+] in single cardiomyocytes (n = 41). Exchange using 1.0 mg/ml unphosphorylated cTn (DD) complex (n = 34 myocytes) resulted in a significant decrease of the Ca2+-sensitivity compared to the time control cells (ΔpCa50 = 0.09±0.02), which were kept in exchange solution without cTn complex  complex, which showed no functional differences compared to control cardiomyocytes . This la2+-sensitivity compared to exchange with cTn (DD) (ΔpCa50 = 0.08±0.02). The cardiomyocytes exchanged with cTn (DD+PKCα) were subsequently incubated for 60 minutes with PKCα to determine the effects of additional PKCα-mediated protein phosphorylation as performed in a previous study decrease in myofilament Ca2+-sensitivity upon incubation of the cells with PKCα (ΔpCa50 = 0.14±0.03)  . This obmax) at saturating Ca2+ concentration (pCa 4.5)  group compared to the cTn (DD) group. Thus, the cumulative effect of PKCα-mediated phosphorylation of cTnI and cTnT on Fmax and pCa50 is negative.Exchange of endogenous cTn with cTn (DD) did not change the maximal isometric force (FpCa 4.5) . Howeverol cells  and cellol cells . The commax after incubation with PKCα (Fmax = 22.4±2.2 kN/m2) compared to the Fmax before incubation (Fmax = 25.2±3.1 kN/m2)   additional force measurements were performed before and after (1 hour) incubation with PKCα. These results did not show a decrease in F n = 13) . Myocytepas) measured at pCa 9 compared to untreated time control cells (pas did not significantly differ between cTn (DD) and cTn (DD+PKCα) (pas.Exchange of endogenous cTn with cTn (DD) did not change passive force (Fol cells . In addiDD+PKCα) . Recentltr-max) measured at pCa 4.5 after a slack test (tr-max upon exchange with cTn (DD+PKCα) compared to cTn (DD)  exchange was seen on the steepness of the force-pCa relation (nH) or the rate of force redevelopment (Kack test . FurthercTn (DD) . PKCα tr  comprising residues 193–203 were observed in the PKC treated sample , and that of the unphosphorylated peptide  comprised residues 177–188 identified Ser179 as a target for PKCα  and after 180 minutes of incubation. The amino acid sequence coverage of cTnT and cTnI was on average 47% and 40%, respectively. Both the phosphorylated and unphosphorylated cTnI peptides of NIDALsGMEGR  and failing tissue a fold increase of 4.26±0.68  after PKCα treatment. This indicates that cTnI Ser198 is a substrate for PKCα in both donor and failing tissue.Site-specific quantification of PKCα-treated human recombinant cTnI was performed using MRM analysis against Ser42, Ser44, Thr143 and Ser198. Synthetic peptides corresponding to unmodified and modified forms of each cTnI tryptic fragment containing the PKC phosphorylatable residues  were usein vitro resulted in an increase in Ca2+-sensitivity and a reduction in the force generating capacity. Conversely, PKCα treatment after exchange resulted in a decrease in Ca2+-sensitivity, most likely via phosphorylation of other targets within the myofilament lattice. Moreover we identified two PKCα phosphorylation substrates on human cTn: Ser198 located on cTnI and Ser179 on cTnT, which have not previously been linked to PKC and provided evidence of target specificity in the phosphorylation of cTnI.This study aimed to investigate the functional effects of PKCα-mediated phosphorylation of the troponin complex in human cardiomyocytes and to resolve the targets involved. The main finding from the cTn exchange experiments was that specific PKCα-mediated phosphorylation of the troponin complex 2+-sensitivity in human cardiomyocytes exchanged with cTn (DD+PKCα) when compared to the cTn (DD) group. This indicates that there are specific sites on cTnT and/or cTnI phosphorylated by PKCα that enhance myofilament Ca2+-sensitivity. Previously, Wang et al. 2+-sensitivity after PKC-βII application by direct phosphorylation of cTnI at Thr143, a unique residue in the inhibitory region of cTnI. Our results  have been shown to reduce the Ca2+-sensitivity, which is in agreement with our earlier findings in failing cardiomyocytes incubated with PKCα in vitro. Apart from cTnI and cTnT also cMyBP-C and titin can be phosphorylated by PKCα. On cMyBP-C are sites Ser275 and Ser304 (human sequence) identified as PKC substrates 2+-sensitivity of force in human cardiomyocytes remains to be established.PKCα-treatment after exchange resulted in a decrease in Cain vivo situation. It can be noted that the extent of phosphorylation of the different phosphorylation sites as observed by Zhang et al. 2+-sensitivity of force and a reduction in the maximal force generating capacity (Fmax) and that the combined effect on force development is negative. Subsequent incubation of the cardiomyocytes with PKCα resulted in a decrease in Ca2+-sensitivity but did not affect Fmax. While the increase in Ca2+-sensitivity is due to phosphorylation of sites on cTn, we attribute the decrease in Ca2+-sensitivity after subsequent incubation with PKCα to phosphorylation of one or more myofilament protein (s) other than cTn.We show that the newly identified site on cTnI, Ser198, is phosphorylated to some extent in failing myocardium where it is a substrate of PKCα and, in particular, that Thr143 is the preferred substrate for PKCα on cTnI. Therefore, phosphorylation of Thr143 is likely to play a major role in the observed effects but caution should be exerted when extrapolating these data to the 2+-sensitivity is greater in failing than in healthy control cardiomyocytes even though it has been shown that the expression and activity is upregulated in many models of cardiac injury, hypertrophy and failure Van der Velden et al. showed that the effect of PKC phosphorylation on Camax was observed upon treatment with recombinant PKCα max was only observed in healthy tissue and not in failing rat myocardium max in failing tissue in the study of Belin et al. and in our study could imply that the relevant site in their study in rats was already saturated, whereas this was not the case in our failing human samples.Exchange of endogenous cTn in failing cardiomyocytes with cTn (DD+PKCα) complex decreased the maximal force generating capacity of the failing cardiomyocytes. These findings resemble the results of Belin et al. in rat skinned myocytes in which a marked depression of the Fmax in the cTn (DD+PKCα) group could not be reduced further nor could it be corrected by subsequent incubation with PKCα. One explanation for the lack of a decrease in maximal force in the human failing myocytes directly incubated with PKCα might be that certain PKC sites (eg. Ser42/44 on cTnI and Thr203 on cTnT) are only exposed and sufficiently phosphorylated in recombinant cTn protein but not when the cTn complex is part of the intact filaments. Alternatively, coincident phosphorylation of other myofilament proteins (eg. cMyBP-C) may exert an opposing effect on the maximal force generating capacity in intact myofilaments and thereby obscure an effect of PKCα on maximal force The reduction in maximal force was not observed in failing cardiomyocytes that were incubated with PKCα. Moreover, the decreased FPhosphorylation of the known PKC sites Ser42/44 and Thr143 is evidently fast as can be judged from the relatively high phosphorylation at these sites in cTn complex incubated with PKCα for less than 1 minute . This im2+-sensitivity of force might be caused by in vitro phosphorylation of Thr143 on cTnI or from phosphorylation of our newly identified sites on cTnI and cTnT. Subsequent incubation of the exchanged cardiomyocytes from failing hearts with PKCα decreased the Ca2+-sensitivity of force, which is in agreement with our previous study In conclusion, the PKCα-induced increase in CaFigure S1Cross-phosphorylation of the PKA sites Ser23/Ser24 by PKCα incubation.(TIF)Click here for additional data file.Figure S2Exchange of cTn (DD) complex in donor cardiomyocytes.(TIF)Click here for additional data file.Figure S3Exchange of endogenous cTn with cTn (DD) complex in non-failing donor tissue.(TIF)Click here for additional data file.Figure S4Coomassie stained SDS-PAGE gel of cTn complex.(TIF)Click here for additional data file.Figure S5MRM MS traces of PKCα phosphorylated cTnI peptides.(TIF)Click here for additional data file.Figure S6Calibration curves of two standard peptides by MRM.(TIF)Click here for additional data file.Table S1List of the (synthetic) phosphorylated peptides used for the MRM assay and the corresponding transitions.(DOCX)Click here for additional data file.Table S2Data overview of site-specific quantification of PKCα-treated human recombinant cTnI.(DOCX)Click here for additional data file.File S1Online Supplement.(DOCX)Click here for additional data file.


Single-copy integration vectors based upon the site-specific recombination systems of bacteriophage are invaluable tools in the study of bacterial pathogenesis. The utility of such vectors is often limited, however, by the fact that integration often results in the inactivation of bacterial genes or has undesirable effects on gene transcription. The aim of this study is to develop an integration vector that does not have a detectable effect on gene transcription upon integration.Staphylococcus aureus. This system is based on the lysogenic phage L54a site-specific recombination system in which the L54a phage (attP) and chromosome (attB) attachment sites, which share an 18-bp identical core sequence, were modified with identical mutations. The integration vector, pLL102, was constructed to contain the modified L54a attP site (attP2) that was altered at 5 nucleotide positions within the core sequence. In the recipient strain, the similarly modified attB site (attB2) was inserted in an intergenic region devoid of detectable transcription read-through. Integration of the vector, which is unable to replicate in S. aureus extrachromosomally, was achieved by providing the L54a integrase gene in a plasmid in the recipient. We showed that pLL102 integrated specifically at the engineered site rather than at the native L54a attB site and that integration did not have a significant effect on transcription of genes immediately upstream or downstream of the integration site.We have developed a single-copy integration system that enables the cloning vector to integrate at a specific engineered site, within an untranscribed intergenic region, in the chromosome of E. coli-S. aureus shuttle vector that can be used to introduce any cloned gene into the S. aureus chromosome at a select site without affecting gene expression. The vector should be useful for genetic manipulation of S. aureus and for marking strains for in vivo studies.In this work, we describe an  Staphylococcus aureus, an important human pathogen that has become one of the most serious infectious agents worldwide [S. aureus, most plasmid vectors are multi-copy in nature which could result in overexpression of the complementing gene or could place a heavy metabolic burden on the host bacterium. In order to circumvent these and other problems, we previously developed S. aureus vectors that allow cloning of genes in single copy [S. aureus, but because they each carry a bacteriophage attP site, they can integrate into the bacterial chromosome at the attB site for L54a or Ø11, respectively, in the presence of the bacteriophage integrase (Int) [The ability to genetically manipulate bacterial pathogens is essential for the advancement of research on the mechanism of pathogenesis. This holds true for orldwide . One of gle copy ,3. Thesese (Int) ,3.attP, which is much larger than attB, provides binding sites for Int and accessory factors whereas the attB site only has a binding site for Int [attP, the arm-type and core-type sites. In the recombination reaction, the binding of accessory proteins to attP causes DNA bending which allows an Int monomer to bind both the arm site and the half core site simultaneously. During the recombination reaction, a synapsis is formed in which 4 Int monomers bind to 4 attP arm sites, 2 attP half core sites and 2 attB half core sites. The resulting higher order nucleoprotein complex allows the integrase to catalyze the strand exchange between short 7 bp sequences, termed overlap sequences, within the 15 bp core sequence [The L54a site-specific recombination system is similar to that of coliphage λ. λ site-specific recombination is mediated by a tyrosine recombinase known as integrase, or Int, protein [ for Int . λ Int battP site and the bacterial attB site share an identical 18 bp core sequence [attB site is located near the 5' end of the geh gene (SAOUHSC0030), which encodes a glycerol ester hydrolase, and therefore lysogenization by L54a results in a lipase negative phenotype [attP site is between 228-235 bp in length but the L54a attB site is less than 27 bp [int-encoded integrase of L54a, like λ Int, has a tyrosine residue that is required for site-specific recombination [In the L54a phage site-specific recombination system, the phage henotype . Inactivhenotype -12. We han 27 bp . The intbination . Thus, tattB site of the respective phage or pathogenicity island. One drawback of using the S. aureus phage site-specific recombination systems for construction of single copy vectors is that the attB site of most staphylococcal phage is located within an open reading frame (ORF) of a gene [attB site would interrupt an S. aureus gene. This is often undesirable because it could inactivate a gene with an important biological function. In addition, a number of sRNAs have been found in the intergenic regions of the S. aureus chromosome which may be affected even if a phage integrates within an intergenic region [attB site is within the geh gene, whereas the Ø11 attB site is within a hypothetical gene that was previously misannotated to be in an intergenic region [attB site used for these vectors in some strains may already have an integrated prophage or pathogenicity island. This will interfere with the subsequent transduction from the readily transformable S aureus strain RN4220 used as the initial recipient of these vectors. In addition to the site-specific recombination systems mentioned above, a Cre/lox recombination system has also been used as a genetic tool for maker removal in strain construction in S. aureus [In addition to the single copy vectors developed by us, other researchers have developed vectors based on the site-specific recombination systems of Ø13 , and thef a gene -12. As ac region ,17. For c region . Further. aureus .S. aureus based on the L54a site-specific recombination system in which the same mutation was introduced into both attB and attP. The vector can replicate in Escherichia coli, which allows for easy DNA manipulations. In S. aureus, the vector integrates with high fidelity into an engineered attB site that is devoid of transcription activity.In this report, we describe the construction of a single-copy integration vector for attP or attB, drastically reduces the efficiency of recombination. However, the identical change in the partner site restores recombination efficiency [® cloning system developed by Invitrogen Life Science Technologies).In the λ site-specific recombination system, a single bp alteration within the 7 bp overlap sequence, in either ficiency ,20. BaseS. aureus strain NCTC8325 annotation) was initially selected for insertion of a mutated attB site. We detected very little transcriptional activity in this region in strain 8325-4 cultures grown for 2 h, 4 h or 16 h (data not shown), although two sRNAs are known to be encoded within this region [Since the staphylococcal phage L54a Int-mediated recombination system closely parallels that of λ, we hypothesized that a similar alteration in integration specificity could be achieved in the L54a system . This wos region .attP and attB affect L54a integration specificity. As shown in Figure attP and attB sites [attP and attB pair by overlapping PCR. The altered attP was named attP1  of the bacterial chromosome. This is within the intergenic region between SAOUHSC00937 and SAOUHSC00938 of strain RN4220, 29 bp downstream of sRNA RsaF [The altered 1 system  as previ1 system  using RNRNA RsaF ,23. The attB1 or the wild type attB site. Out of 4 integrants that we tested, 3 were integrated at the correct location, attB1 (results not shown). However, in one of the integrants the plasmid had integrated at the wild type L54a attB site in the geh gene. These results indicate that the altered att sites can be recognized by L54a Int but that the specificity of integration is less than perfect. These results also indicate that the size of the L54a attB site is less than 21 bp in length, which is shorter than the 27 bp that we previously determined [PCR was used in order to determine whether pLL3961 had integrated at termined .attP and attB, but 2-bp alteration is not enough to ensure high fidelity. Therefore, to increase the specificity, we replaced 5 bp of the att core sequence as shown in Figure att site was named attP2. The attP2 fragment was then cloned into pCL25 [attP fragment, to form pLL102  by RT-PCR (data not shown). The 21 bp attB2 site was then inserted into the intergenic region between G and C at coordinates 14208 and 14209 (NCTC8325 genome) of strain RN4220  and CYL12376 ) (data not shown) indicating that the insertion of attB2 or the integration of pLL102 at the attB2 site does not affect expression of the surrounding genes.SAOUHSC00009 (NCTC8325 genome) is annotated as s region ,17. To aS. aureus chromosome at a location devoid of detectable transcription activity. Once a plasmid is inserted into the attB2 site, it can then be moved to any other strain by phage transduction. Since there is no detectable transcription activity at the attB2 site, the insertion is not likely to alter biological function of the clone. Furthermore, the region flanking the attB2 site is highly conserved among all sequenced strains suggesting that moving the integrated plasmid from CYL12349 to other strains is not likely to be hindered by a lack of sequence homology. The system reported here uses a novel, artificial attB site, attB2, thereby avoiding the disadvantages associated with other bacteriophage derived integration vectors. Additionally, the attB2 site is portable and could be inserted into other locations of the chromosome. Thus, our results indicate that plasmid pLL102 is a suitable vector for cloning genes in single copy. In addition, our system can be used to mark a strain with a readily detectable reporter gene, which could be useful for studying pathogenesis in vivo, without affecting gene expression of the marked strains.We have developed a single copy integration system that allows insertion of a plasmid vector into the E. coli was grown in Luria broth or Luria agar (Difco). S. aureus was grown in tryptic soy broth (TSB) or TSB agar (Difco). E. coli media were supplemented with 34 μg/ml chloramphenicol, 100 μg/ml penicillin, or 50 μg/ml spectinomycin, as appropriate. S. aureus media were supplemented with 10 μg/ml chloramphenicol, or 3 μg/ml tetracycline as appropriate. Recombinant plasmids were initially constructed in E. coli then electroporated into S. aureus as described [S. arueus strains was carried out by phage 52A.The strains and plasmids used in this study are listed in Table escribed . TransduRNA for RT-PCR and qRT-PCR was isolated as described . RT-PCR attB2 site and the subsequent integration of pLL102 affected transcription of the flanking genes, we performed qRT-PCR as described [To determine whether the insertion of the escribed  of the aattP1 and attB1 sites were created by altering two bp within the core of attP and attB, respectively, by overlapping PCR using primers L54aInt1, L54aAttP1, L54aAttP2 and L54aAttP3  of the bacterial chromosome. This is within the intergenic region between SAOUHSC00937 and SAOUHSC00938 of strain RN4220, 29 bp downstream of sRNA RsaF [attB site.The altered escribed ,22 usingRNA RsaF ,23. The attP and attB sites with 5 bp substitutions were constructed by overlapping PCR using primers L54aAttP4, L54aAttP5, L54aAttP6 and L54aAttP7  of strain RN4220 (Figure The P7 Table . The amp2 Figure . The 289The authors declare that they have no competing interests.MGL, DC and CYL designed the research and wrote the manuscript. MGL carried out chromosome site selection experiments. DC carried out gene expression experiments. JA performed technical experiments in plasmid and strain construction. JJ and JWG carried out strain construction and integration experiments. All authors read and approved the final manuscript.


Ischemic and traumatic brain injury is associated with increased risk for death and disability. The inhibition of penumbral tissue damage has been recognized as a target for therapeutic intervention, because cellular injury evolves progressively upon ATP-depletion and loss of ion homeostasis. In patients, thiopental is used to treat refractory intracranial hypertension by reducing intracranial pressure and cerebral metabolic demands; however, therapeutic benefits of thiopental-treatment are controversially discussed. In the present study we identified fundamental neuroprotective molecular mechanisms mediated by thiopental. Here we show that thiopental inhibits global protein synthesis, which preserves the intracellular energy metabolite content in oxygen-deprived human neuronal SK-N-SH cells or primary mouse cortical neurons and thus ameliorates hypoxic cell damage. Sensitivity to hypoxic damage was restored by pharmacologic repression of eukaryotic elongation factor 2 kinase. Translational inhibition was mediated by calcium influx, activation of the AMP-activated protein kinase, and inhibitory phosphorylation of eukaryotic elongation factor 2. Our results explain the reduction of cerebral metabolic demands during thiopental treatment. Cycloheximide also protected neurons from hypoxic cell death, indicating that translational inhibitors may generally reduce secondary brain injury. In conclusion our study demonstrates that therapeutic inhibition of global protein synthesis protects neurons from hypoxic damage by preserving energy balance in oxygen-deprived cells. Molecular evidence for thiopental-mediated neuroprotection favours a positive clinical evaluation of barbiturate treatment. The chemical structure of thiopental could represent a pharmacologically relevant scaffold for the development of new organ-protective compounds to ameliorate tissue damage when oxygen availability is limited. Traumatic brain injury and cerebral infarction initiate deleterious events in the penumbra that exacerbate the initial injury Maintenance of ion homeostasis by ion-motive ATPases and protein synthesis are dominant energy-consuming processes of the cells Protein synthesis depends on initiation and elongation factors whose activity is tightly regulated by posttranslational modification The Brain Trauma Foundation Guidelines recommend high-dose thiopental treatment of patients with severe brain injury who present with refractory intracranial hypertension. This practice is the only second-level measure with class II evidence, demonstrating the ability of thiopental to reduce intracranial pressure Because cerebral metabolism and translation are closely intertwined, the aim of the present study was to examine thiopental-mediated effects on global protein synthesis, high-energy phosphate metabolism, and its impact on neuronal damage following oxygen deprivation.4, 0.025% DNase I, and 0.4 mg/ml trypsin inhibitor from soybean  and triturated for 10–15 times using sterile glass Pasteur pipettes. After centrifugation at 100 g for 5 min, dissociated cells were collected from the supernatant. Neurons were enriched by a second centrifugation at 800 g for 5 min and seeded on Poly-d-Lysine coated culture plates in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum and 2 mM Glutamax (Life Technologies) at 37°C, 8% CO2. After 3 h, the medium was changed to Neurobasal-A containing 2% B27-supplement and 1 mM Glutamax (Life Technologies).The human neuronal cell line SK-N-SH was purchased from the American Tissue Culture Collection  and maintained in Eagle’s minimum essential medium, supplemented with 1 mM sodium pyruvate, 2 mM Glutamax, 1500 mg/l sodium bicarbonate, 100 IU streptomycin, 100 IU penicillin, and 10% fetal calf serum . Primary cortical neurons were dissected from C57/BL6 mouse neonates (P0–P2) as described Cells were treated with thiopental , thapsigargin , forskolin , cycloheximide , and actinomycin D (Sigma-Aldrich) as indicated. The eEF2K inhibitor A484954, the calmodulin inhibitor camstatin, and the cAMP antagonist cAMPS-Rp were obtained from Tocris Bioscience . The AMPK inhibitor compound c (dorsomorphin dihydrochloride) was purchased from Calbiochem.Immunoblotting was performed using whole cell lysates as described previously 35S]methionine  for 2 h and lysed in SDS-sample buffer . Proteins were denatured by boiling for 5 min and separated by 10% SDS-PAGE. To demonstrate loading of equal amounts of protein, gels were fixed in 25% isopropanol and 10% acetic acid for 30 min, stained in 10% acetic acid containing 0.006% Coomassie G250 for 2 h, destained in 10% acetic acid for 6 h and dried on a Whatman 3 MM paper for 2 h at 80°C. [35S]methionine incorporation was visualized by exposure to x-ray films .Cultures were incubated two times for 20 min in methionine free Eagle’s minimum essential medium  to deplete internal methionine pools before cells were pulse labeled with 200 µCi/ml of methionine, and the extend of synthesized nascent polypeptides was visualized by SDS-PAGE and autoradiography. To ensure that incorporation of [35S]methionine was directly proportional to the length of labeling time, SK-N-SH cells were labeled for 20 min to 6 h in preliminary experiments. [35S]methionine incorporation into proteins was linear within this time period (data not shown). Consequently, cells were pulse-labeled with [35S]methionine for 2 h in the following experiments. Our results depicted in Phosphorylation of eEF2 at Thr56 is sufficient to inhibit global protein synthesis eEF2 phosphorylation is regulated by the calcium and calmodulin-dependent kinase eEF2K Several protein kinases regulate calcium-independent eEF2K activity The significance of eEF2K, calcium-dependent calmodulin, and cAMP in eEF2 phosphorylation were additionally analyzed by the use of the eEF2K inhibitor A484954, the calmodulin antagonist camstatin, and the cAMP analog cAMPS-Rp. As shown in Barbiturates inhibit mitochondrial respiration Next we investigated the importance of AMPK in eEF2K-dependent eEF2 phosphorylation. Because the eEF2K-Ser398 antibody is commercially not available, we directly analyzed eEF2 phosphorylation in the presence of the AMPK inhibitor compound c. Surprisingly, thiopental induced eEF2 phosphorylation was only partially reduced by 10 µM of compound c , suggestThe penumbra, surrounding the primary necrotic core in brain injured patients, is composed of a rim of ischemic tissue potentially destined for infarction but not yet irreversibly injured The observed difference in susceptibility to hypoxic neuronal cell death would imply that metabolically active cells are particularly vulnerable to oxygen deprivation, whereas metabolic inactive, resting cells are protected under these conditions. Therefore we speculated that inhibitors of protein synthesis might significantly reduce hypoxic cell damage. To examine whether a reduction in global protein synthesis prevents ischemic damage, we first used the translational inhibitor cycloheximide to test the viability of oxygen deprived neuronal cells in the presence of serum . In agreIn the following experiments we examined whether thiopental, that has previously been shown to inhibit global protein translation also mediates cytoprotection during hypoxia. As shown in Next we examined the execution of hypoxic neuronal cell death. Surprisingly we could neither demonstrate caspase-3 activity in oxygen deprived SK-N-SH cells , nor werIn mRNA translation is an energy-consuming process that depletes up to 60% of the ATP synthesized by the cells 35S]methionine (The metabolism of differentiated primary cortical neurons and the established SK-N-SH cell line might significantly vary. To analyze the effect of thiopental on translational regulation in terminally differentiated cells, primary neurons were isolated from the cortex of mice and cell cultures were tested for phosphorylation of eEF2 and AMPK. As shown in thionine .In analogy to SK-N-SH cells, the inactivating modification of eEF2 and translational repression in differentiated neurons by thiopental might result in the preservation of intracellular energy content and consequently in protection from hypoxic damage. As shown in Deprivation of high energy phosphate metabolites during ischemia results in the disruption of ion homeostasis, membrane depolarization, and neuronal cell death Neuroprotection by thiopental has been described in the clinic and the associated reduction of metabolic demands is well established In our experiments thiopental elevated cytoplasmatic calcium levels and camstatin, a peptide antagonist of calmodulin, prevented eEF2 phosphorylation indicating that this mechanism is essential for thiopental-mediated translational arrest. Calcium in resting cells is about 0.1 µM and the observed 4 to 6 fold increase may be close to optimum for induction of eEF2K or other proteins Barbiturates attenuate NMDA receptor mediated intracellular calcium elevations and calcium induced membrane depolarisation, suggesting a reduction of excitotoxic neuronal injury A mechanism which may synergistically lead to thiopental-dependent eEF2 phosphorylation is the induction of AMPK during energy starvation. Barbiturates depress mitochondrial respiration AMPK activation is involved in numerous cytoprotective molecular mechanisms such as stress resistance, autophagy, inhibition of plaque formation in Alzheimer’s disease, and energy intake and expenditure de novo protein synthesis including iNOS, COX-2, MMPs or proinflammatory cytokines Previously we reported that thiopental-mediated modulation of intracellular signal transduction depends on the sulfur substitution at the C2 and thus is a characteristic of thiobarbiturates Furthermore, inhibition of protein synthesis might be useful in the treatment of diseases associated with excessive or deregulated protein expression. AMPK-dependent pathways are crucial in metabolic and structural remodeling, inflammation, cell growth, and tumor suppression Previously we described that thiopental inhibits calcium-dependent caspase activation and apoptosis in neuronal cells A crucial question is how translational inhibitors reduce ischemic neuronal damage. There is only limiting data available demonstrating the reduction of deleterious effects in ischemic neurons by translational inhibitors and participating cytoprotective mechanisms are inadequately resolved In patients, a therapeutic benefit of thiopental therapy is controversially discussed, mainly because of severe medical complications and the lack of clinical trials providing clear evidence for neuroprotection by this drug. Our study shows molecular evidence that thiopental and other translational inhibitors modify intracellular energy expenditure, thus supporting cellular adaptation to hypoxia and improving survival. These findings raise the possibility that thiobarbiturates may indeed improve clinical outcome in selected patients. Lack of clinical evidence for thiopental-mediated neuroprotection might be due to insufficient control of medical complications or the time frame barbiturates are employed after traumatic brain injury - the opportunity for the drugs to prevent secondary injury may have past due to the rapid kinetics of energy depletion. Our findings therefore suggest the initiation of new trials to revaluate neuroprotection by thiopental under these aspects. Furthermore, our results indicate that these studies shouldn’t be limited to patients with refractory intracranial pressure but should also include other hypoxic insults, such as stroke.In summary, the presented findings indicate that inhibition of global protein synthesis by thiopental or other general translational inhibitors effectively protects from hypoxic neuronal cell damage by stabilizing high energy metabolite content under oxygen-limited conditions. Based on our results, we suggest that translational inhibition and preservation of intracellular ATP content by thiopental is mediated by a combined activation of AMPK and calcium/calmodulin complexes, resulting in eEF2 phosphorylation and inactivation. Compounds, based on the structure of heterocyclic thiourylene derivates might therefore reveal a pharmacologically relevant scaffold for the development of novel organ-protective compounds. The identification and modification of chemically active sites within the molecule might improve molecular or metabolic defence mechanisms against limited oxygen supply and allow for efficient screening of compound banks for new organ protective agents containing these biologically relevant structures. Furthermore, our findings suggest examining other general translational inhibitors, agents that maintain mitochondrial respiratory activity during hypoxia, and the therapeutic application of high energy molecules to ameliorate ischemic tissue damage.


IC-2 is a subunit of cytoplasmic dynein, a minus-end directed motor protein necessary for transport of diverse cargos along microtubules. Emerging data support the hypothesis that post-translational modification regulates dynein but the signaling mechanisms used are currently unknown. We find that ERK phosphorylates IC-2 on a novel, highly conserved Serine residue proximal to the binding site for the p150Glued subunit of the cargo adapter dynactin. Surprisingly, neither constitutive phosphorylation nor a phosphomimetic substitution of this Serine influences binding of p150Glued to IC-2. These data suggest that ERK phosphorylation of IC-2 regulates dynein function through mechanisms other than its interaction with dynactin.Extracellular-signal regulated kinase (ERK) signaling is required for a multitude of physiological and patho-physiological processes. However, the identities of the proteins that ERK phosphorylates to elicit these responses are incompletely known. Using an affinity purification methodology of general utility, here we identify cytoplasmic dynein intermediate chain  The ERK cascade regulates a variety of processes including proliferation, survival, motility and differentiated cell function downstream of receptor tyrosine kinase stimulation. Many receptor tyrosine kinases, including the EGF receptor, are internalized by endocytosis following ligand-binding . The oriDiverse observations suggest that ERK regulates movement of membrane bounded organelles along microtubules. A fraction of ERK has long been known to associate with microtubules –15 and iGlued subunit of dynactin was unaffected by Serine 81 phosphorylation. We hypothesize that ERK phosphorylation regulates dynein through mechanisms other than its interaction with dynactin.Here we identify cytoplasmic dynein intermediate chain 2  aERK often phosphorylates substrate proteins on Serine and Threonine residues in PXSP and PXTP sequence motifs (where X is any amino acid) . We firsTo identify new ERK substrates we treated cells with EGF for 30 min. with or without prior treatment with UO126. Anti-phospho PXSP immunoprecipitates were made from each lysate, labeled with Cy5 and Cy3 dye respectively, combined and resolved on a single 2D gel. By comparing fluorescence intensities between Cy3 and Cy5 channels a number of proteins whose apparent abundance was substantially decreased in immunoprecipitates from UO126-treated cells were identified. One spot much less abundant in the UO126-treated sample  was pickThere are several isoforms of the vertebrate cytoplasmic dynein intermediate chain, the product of two genes and alternative splicing –31. HoweThe migration of the IC-2C band shifted to a slightly higher apparent molecular weight in response to EGF stimulation, and this mobility shift was blocked by pre-treatment with UO126 . While nIC-2C contains a single PXSP motif  MEK1 in which the sites of activating phosphorylation, Serines 218 and 222, are mutated to phosphomimetic aspartate residues . Cells wntiserum .Glued subunit of the dynactin cargo adapter complex [Glued binding. First we asked if mimicking S81 phosphorylation modulates binding of p150Glued to recombinant IC-2C protein. Bacterially expressed (non-phosphorylated) wild type and S81D IC-2C proteins were equally able to bind p150Glued in these pull-down assays . The dyelopment , and indelopment  and redielopment . Furtherelopment . From thelopment , and traelopment . These eN-terminal domain that encodes the cargo binding function of the complex. Most of the dynein cargo binding subunits bind to the base of the molecule. Thus, the intermediate and light intermediate chains bind as dimers to the base of the heavy chain, while the light chain dimers bind the intermediate chains. While it is clear that the dynein heavy chain functions as an ATP-dependent microtubule motor [Each cytoplasmic dynein complex has two heavy chains that contain a motor domain encoding ATPase units and microtubule binding elements, and a smaller le motor ,49, the le motor  that arele motor ,53. Thisle motor  and specle motor . Howeverle motor ,30,33,37le motor ,37, and le motor ,54. RelaTo identify new targets for ERK phosphorylation in the context of EGF stimulation we used Cell Signaling Technology, Inc.’s PTMScan technology. This approach uses antibodies to phosphorylated kinase consensus motifs as a means to identify substrates. Using antisera that recognize phosphorylated Serine or Threonine residues in consensus motifs for ERK phosphorylation , initiaInspection of the IC-2C sequence reveals a single PXSP motif (PMSP) encompassing Serine 81  that is N- and C-terminal by a number of negatively charged residues making it a candidate for phosphorylation by casein kinase II [in vitro phosphorylate dynein intermediate chain on an unknown site(s) [Xenopus dynein intermediate chain and stimulate minus-end directed movement of melanophores [2+/calmodulin-dependent kinase II in some proteins [In addition to the predominant site of phosphorylation at Serine 81, we identified five additional minor (low +P/−P ratio) phosphorylation sites in mouse IC-2C: Serine 51, Threonine 89, Serine 95, Serine 98 and Threonine 154 . The weainase II . Casein  site(s)  but to o site(s)  and Casenophores . Serinesproteins ,58,59. Fproteins  which waproteins .In vitro reconstitution assays verified that ERK directly phosphorylates IC-2C on Serine 81 [Using MEK inhibitor and a constitutively active form of MEK1 we determined that MEK activity is necessary and sufficient for phosphorylation of IC-2C in fibroblasts . Similarerine 81 . AlthougSecond, it should be noted that neither serum-starvation nor UO126 completely eliminate reactivity with the phospho Serine 81 antibody in fibroblasts, while mutation of Serine 81 to Alanine does. Similarly, MEK inhibitors do not entirely eliminate S81 phosphorylation of endogenous intermediate chain(s) in A375 melanoma cells. These observations indicate that some intermediate chain is likely phosphorylated on Serine 81 by kinases distinct from ERK and/or resides in compartments not accessed by the MEK inhibitors.We postulated that IC S81 phosphorylation triggered by EGF stimulation of ERK might be important for the recruitment of dynein to EGF receptor signaling endosomes. This is consistent with the observations that MEK1 is required for perinuclear localization of late endosomes containing EGFR  and thatGlued subunit of dynactin which binds directly to the N-terminus of dynein intermediate chain. Previously reported sites of dynein phosphorylation (Serine 84 and Threonine 89) proximal to the p150Glued binding site are reported to inhibit the IC-2/p150Glued interaction in gel overlay assays [Glued binding site, we reasoned that Serine 81 phosphorylation might also modulate p150Glued binding. Since gel overlay and solution binding assays have yielded contradictory results [Glued from the high speed pellet material of malignant melanoma cells exhibiting constitutive ERK-dependent phosphorylation of IC-2C. Similarly, recombinant (dephospho) wild type and phosphomimetic S81D IC-2C did not detectably differ in their binding to p150Glued in pull-down assays. These observations are in agreement with those of Vaughan and colleagues who, before our formal demonstration of phosphorylation of Serine 81, also found an S81D mutant to be without effect on p150Glued binding in gel overlay assays [Glued binding to IC-2C. We hypothesize that Serine 81 phosphorylation might recruit dynein to receptor signaling endosomes by engaging a novel cargo adapter, much like Threonine 89 phosphorylation promotes dynein binding to kinetochores via zw10 [Dynactin is thought to be important in dynein binding to many cargos (reviewed in ). This iy assays ,33 and iy assays ,61. Intey assays ,61 indic results ,61, we cy assays . While iGlued, implying that Serine 81 phosphorylated intermediate chains bind to a distinct cargo adapter(s) on receptor signaling organelles destined for retrograde transport.The retrograde movement of Trk-containing endosomes is important for axonal and neuronal survival . Trk recvs. NGF in PC12 cells [in situ ERK activation and dynein phosphorylation could determine the journey time and/or destination of such receptor signaling endosomes.It will be important to determine if other receptor tyrosine kinases, and other classes of cell surface receptor that trigger ERK signaling, also utilize Serine 81 phosphorylation as a means to modify dynein. While EGF, BDNF and NGF are currently the only extracellular stimuli known to cause Serine 81 phosphorylation, our data are consistent with the idea that these receptors utilize Serine 81 phosphorylation as a means to target dynein intermediate chains to receptor tyrosine kinase-containing organelles, perhaps pointing to a universal mechanism for engaging receptor signaling organelles destined for retrograde transport by the dynein motor complex. Moreover, since these receptor tyrosine kinases continue to signal from endosomes on their intracellular journey, and it is known that the amplitude and kinetics of ERK activity can trigger different cell fate outcomes , it is While additional studies are clearly necessary to further discern the role of dynamic Serine 81 phosphorylation in regulation of dynein function, the methodology described here is of general utility in identifying novel substrates for ERK in diverse contexts.MEK1-null fibroblasts  reconstimRFP-tagged IC-2C constructs are described elsewhere . PolyhisGeneration of the phospho Serine 81 IC-2C antiserum is described elsewhere . We founrd April 2007 [th November 2007 [2, 1% Trition X-100, 1 mM PMSF, 3 mM benzamidine, 10 μg/mL each of leupeptin and pepstatin, 10 nM microcystin LR, 1 mM sodium orthovanadate, 5 mM sodium pyrophosphate, 50 mM sodium fluoride), and centrifuged at 13,000g for 5 min. Cell lysates were precleared with 40 μL of rProtein-A Sepharose CL4B  for 1 h at 4 °C, and centrifuged at 600g for 5 min. Cleared supernatant was assayed for protein concentration with BCA kit . Two micrograms protein (in 1 mL volume) was incubated with anti-PXSP (15 μL) or anti-PXTP  overnight at 4 °C. Immune complexes were sedimented with 40 μL of rProtein-A Sepharose CL4B for 2 h at 4 °C followed by two washes in lysis buffer (1 mL each time). Beads were suspended in 100 μL 2X SDS-Sample buffer, boiled, centrifuged and the supernatant was assayed by western blotting with anti-dynein intermediate chain mAb . WT celone 74.1 ; Covancend dimension gels using an overlay consisting of 0.5% agarose in SDS running buffer . Second-dimension SDS-10% PAGE was performed using a DALTsix system  at 5 W/gel for 30 min. followed by 13 W/gel for 4 h at 25 °C. The Cy3 and Cy5 signals for each gel were individually imaged on a Typhoon 9400 scanner  using mutually exclusive excitation/emission wavelengths of 532nm (ex) and 580nm (em) for Cy3, and 633nm (ex) and 670 nm (em) for Cy5. After imaging, the gels were fixed , rinsed in water three times and then incubated overnight in SYPRO Ruby in the dark. The SYPRO Ruby post-stain allows for the correction of the unlabeled proteins’ migration in relation to the cognate Cy-Dye labeled proteins, and ensures accurate spot excision. SYPRO Ruby images were acquired on the Typhoon using 450 nm (ex) and 610 nm (em) filters, and re-imaged post-excision to ensure accurate spot picking. Proteins whose Cy5 fluorescence was decreased  were identified using DeCyder software  and picked using a robotic Ettan Spot Handling Workstation . Spots were de-stained by successive changes of 20 mM ammonium bicarbonate and 50% acetonitrile, followed by dehydration with 100% acetonitrile (20 min.). Dehydrated gel plugs were digested in-gel with 8 μL 20 μg/mL porcine modified trypsin protease (Promega) in 20 mM ammonium bicarbonate for 6 h at 37 °C. Tryptic peptides were then extracted from the gel plugs in two cycles of 50% acetonitrile/0.1% trifluoroacetic acid and dried by evaporation. Peptides were reconstituted in 5 μL 2% acetonitrile/0.1% formic acid prior to LC-MS analysis. A Thermo LTQ-XL (CA) was coupled with an Eskigent nanoLC system . Peptides were loaded on a Dionex C18 trap column and separated on a PicoFrit C18 column/emitter at 200 nL/min with gradient 2% acetonitrile/0.1% formic acid to 40% acetonitrile/0.1% formic acid in 20 min., to 60% acteonitrile in 5 min, to 90% acetonitrile in 15 min. before returning to starting solvent. The peptides are eluted directly into the LTQ-XL mass spectrometer. One survey scan was acquired first. The 5 most abundant peptide ions (precursor ions) were selected for MS/MS for partial sequencing. In-house MASCOT 2.2  was used to search against SWISS-PROT and NCBI non-redundant rodent databases (updated 6/2009) without constraining protein molecular weight or isoelectric point and allowing for carbamidomethylation of cysteine, partial oxidation of methionine residues, and one missed trypsin cleavage. The significance threshold of 5% probability (Mascot default) was used. Proteins with protein scores above 55 and two or more peptides with ion scores greater than 20 are considered matches.Anti-phospho PXSP immunoprecipitates from control and UO126-treated cells were suspended in 7 M urea, 2 M thiourea, 4% CHAPS, 20% glycerol, 30 mM Tris, pH 8.5, prior to labeling with 400 pmol either Cy3 (DMSO/EGF) or Cy5 (UO126/EGF) for 30 min. on ice in the dark. Reactions were quenched by the addition of 10 mM lysine for 10 min. on ice in the dark. This protocol results in minimal labeling (~1%) to minimize molecular weight shifting and results in sensitivities on the order ~1 ng. The Cy3- and Cy5-labeled samples were combined, mixed with an equal volume of Destreak Rehydration Buffer  and actively rehydrated into 24 cm 3–10 nL immobilized pH gradient (IPG) strips  for 15 h, followed by isoelectric focusing using an Ettan IPGphor II  for a total of 90,000 Vhrs . Cysteines were reduced and carbamidomethylated while the proteins were equilibrated into the second-dimensional loading buffer by incubating the focused strips in equilibration buffer  supplemented with 20 mg/mL DTT for 15 min. at room temperature with shaking, followed by 25 mg/mL iodoacetamide in equilibration buffer for an additional 15 min. room temperature incubation. IPG strips are then cemented onto 2Confluent 15 cm plates of WT cells (2 per time point) were serum deprived overnight in DMEM/0.1% FBS before incubation with or without 10 ng/mL EGF for 30 min. at 37 °C. Lysates were prepared in RIPA buffer containing protease and phosphatases inhibitors, clarified by centrifugation and triplicate 1.7 mg aliquots of lysate protein were immunoprecipitated with 10 μg anti-dynein intermediate chain antibody 74.1  for 3 h 18 reversed-phase capillary column. 7.5 μL volumes of the extracts were injected and the peptides eluted from the column by an acetonitrile/0.1 M acetic acid gradient at a flow rate of 0.5 μL/min over 1 h. The nanospray ion source was operated at 2.5 kV. The digest was analyzed by acquiring 1 full scan mass spectra  to determine peptide molecular weights followed by 10 product ion spectra (MS/MS-ion trap) to determine amino acid sequences. The data were analyzed by using the Sequest search algorithm (Bioworks 3.3.1) against mouse IC-2C. Any potentially phosphorylated peptides that passed minimal cutoff scores and 10ppm parent mass error were manually verified for identity and modification site. Once the sequence and modification site were verified, the data was further examined to determine all possible forms of each peptide that could be identified in each time point digest—nonphosphorylated, phosphorylated, missed cleavages, oxidized Met, acrylamide Cys, etc. Areas (phosphorylated and nonphosphorylated) for all forms of a peptide were determined using selected ion chromatograms from QualBrowser  using the most abundant isotope with a mass window of +/− 0.02Da. A ratio of phosphorylated divided by nonphosphorylated was determined for each sample. Semi-quantitative changes in extent of phosphorylation were determined by comparing this ratio in the EGF-stimulated sample to the unstimulated sample.The LC-MS system consisted of a Thermo Electron LTQFT (Surveyor HPLC) mass spectrometer system with a Protana nanospray ion source interfaced to a self-packed 8 cm × 75 um i.d. Phenomenex Jupiter 10 μm Cg for 10 min. to yield low speed supernatant (S1) and pellet (P1) fractions. The S1 fraction was subsequently centrifuged at 100,000g for 60 min. to yield the high speed supernatant (S2) and pellet (P2) fractions. Portions equivalent to a fixed number of cells were resolved on 8%–15% gradient gels.Isolation of membrane-bound organelles was accomplished essentially as described ,45. BrieE. coli Rosetta (DE3) pLysS  harboring rat dynein intermediate chain 2C in pET 21a were grown to OD600 = 0.6 in 3L LB media at 37 °C. Cultures were cooled to 30 °C and induced with 0.3 mM IPTG for 2 h. The following steps were carried out at 0–4 °C unless noted. Cells were lysed in TN buffer  containing 15 mM imidazole and protease inhibitors by two passes through an Emulsiflex C5 . Lysates were clarified by centrifugation at 100,000g for 60 min, and loaded onto a 1 mL HiTrap HP Nickel column . The column was washed extensively with TN/15 mM imidazole before elution with a linear gradient of 15–500 mM imidazole in TN. Fractions containing IC-2C (200–300 mM imidazole) were pooled and applied to a Q2 anion exchange column  equilibrated in TN before elution with a linear gradient of 0–450 mM NaCl in TN. Fractions containing IC-2C were pooled, frozen as aliquots in liquid nitrogen and stored at −80 °C. Yield was approximately 0.5–1 mg per 3 L culture, of which approximately 50%–75% was full length IC-2C protein. To determine if phosphorylation of Serine 81 modulates binding of IC-2C to p150Glued 0, 0.5, 2.5 and 12.5 ug of recombinant polyhistidine-tagged wild type or S81D IC-2C were incubated with rat brain lysate overnight, subsequently captured with Nickel agarose and extensively washed with FLAG buffer [Glued antiserum.G buffer . Bound pGlued antisera .P2 fractions were prepared from A375 melanoma cells transfected with mRFP-tagged IC-2C constructs. Pellets were resuspended in cold CB buffer and immunoprecipitated with monoclonal anti-RFP antibody  for 2.5 h at 4 °C. Immune complexes were bound to protein G-coupled Dynabeads  and collected with a magnetic tube rack according to manufacturer’s instructions. After washing 3 times with cold CB buffer, bound proteins were eluted with 200 mM glycine, pH 2 for 30 min. on ice and blotted with anti-RFP  and anti-p150


RhoGDI proteins are important regulators of the small GTPase Rac, because they shuttle Rac from the cytoplasm to membranes and also protect Rac from activation, deactivation and degradation. How the binding and release of Rac from RhoGDI is regulated is not precisely understood.We report that the non-receptor tyrosine kinase Fer is able to phosphorylate RhoGDIα and form a direct protein complex with it. This interaction is mediated by the C-terminal end of RhoGDIα. Activation of Fer by reactive oxygen species caused increased phosphorylation of RhoGDIα and pervanadate treatment further augmented this. Tyrosine phosphorylation of RhoGDIα by Fer prevented subsequent binding of Rac to RhoGDIα, but once a RhoGDIα-Rac complex was formed, the Fer kinase was not able to cause Rac release through tyrosine phosphorylation of preformed RhoGDIα-Rac complexes.These results identify tyrosine phosphorylation of RhoGDIα by Fer as a mechanism to regulate binding of RhoGDIα to Rac. Rac proteins belong to the larger family of Rho GTPases that act as molecular switches. When bound to GTP, they are in an active conformation. Hydrolysis of GTP to GDP causes an inactivating conformational change. Racs are post-translationally modified by the removal of C-terminal amino acid residues, followed by the attachment of a lipid moiety in the form of a geranyl-geranyl group .Because of this modification, mature Rac proteins are lipophilic and are only present in the cytosol when bound to the chaperone and transport protein RhoGDI. RhoGDI regulates the movement of Rac between the cytosol and membranes. Moreover, a recent study showed that Rho proteins compete for binding to a limited pool of RhoGDI, which, once it has bound them, provides stabilization and protection from degradation .There are only three distinct RhoGDI proteins, of which RhoGDIα is the most ubiquitously expressed. The structure of RhoGDI bound to small GTPases has been elucidated. Rac makes contact with both N-and C-terminal regions in RhoGDI, with its hydrophobic tail fitting into a pocket in the C-terminal part of RhoGDI. However, the exact mechanism by which binding and release of Racs from RhoGDI is mediated is not well known ,4.RhoGDI can be phosphorylated on serine S101 and S174 by the serine/threonine kinase Pak  and PKCαBCR/ABL oncogene encodes a chimeric protein that contains most of the Abl tyrosine kinase. Proteomic analysis of cells transfected with Bcr/Abl ATP (5000 Ci/mmol) (Amersham). After 40 min on ice, reactions were terminated by the addition of 2× SDS-sample buffer and boiling for 5 min. Samples were run on 8% SDS-PAGE gels and the radioactively labeled proteins visualized by autoradiography.The kinase activity of Fer or Bcr/Abl was measured as described previously . Briefly2, 10 mM MgCl2, 1 mM DTT). 20 μM of ATP was added to the mixture, which was incubated for 30 min at 4°C for the kinase reaction. Next, 400 μl of binding buffer  and 50 pmol Rac1 protein were added, followed by incubation for 1 h at 4°C. Incubation with antibody against RhoGDIα for 1 h at 4°C was followed by Protein A agarose addition and a second incubation 1 h at 4°C. To investigate if phosphorylation of RhoGDIα by Fer releases Rac1, GST-RhoGDIα protein was incubated with Rac1 in binding buffer for 1 h at 4°C; GST-Fer or GST alone was added, and a pull down with glutathione agarose was done, followed by washing of the precipitate. Kinase reactions were initiated by addition of kinase buffer and ATP for 30 min at 4°C.To investigate possible binding of phosphorylated-RhoGDIα to Rac1, GST-RhoGDIα protein (50 pmol) was first combined with GST-Fer (25 pmol) or GST alone (25 pmol) in 100 μl of kinase buffer (20 mM HEPES, pH 7.5, 50 mM NaCl, 25 mM MnClRhoGDI: Rho GDP-dissociation inhibitor; ROS: reactive oxygen speciesFF performed experiments, brought in new ideas and wrote part of the manuscript; S-M K performed experiments and wrote part of the manuscript; LH made the baculovirus Fer protein; PS provided critical reagents; JG designed experiments and provided critical input; NH provided ideas and wrote the manuscript. All authors read and approved the final manuscript.


Anisakis-sensitization-associated chronic urticaria (CU+) differ with respect to specific IgE levels. We hypothesised different immunoglobulin avidities in both entities as well as their dependence on TI and fish consumption. 16 patients with GAA and 17 patients with CU+ were included, and immunoglobulin levels were analysed by CAP (Phadia). IgE and IgG avidity indexes  were also determined. IgG avidity was higher in GAA than in CU+ (P = 0.035), whereas there was a tendency to lower IgE avidity in GAA (P = 0.095). When analysing all patients, AvIgG was positively correlated with specific IgE, IgG, and IgG4 as well as total IgE , but AvIgE was negatively correlated with specific IgE , specific IgG4 , and total IgE . In GAA, weekly fish consumption was positively associated with AvIgE . A multivariate regression showed that time interval was the main explaining factor for AvIgE in GAA. We could show a differential behaviour of immunoglobulin isotype avidities in both entities and their dependence on fish-eating habits as well as on the time elapsed to the last parasitic episode.Gastroallergic anisakiasis (GAA) and  Anisakis simplex is a fish parasite of worldwide distribution, whose third stage larva can be present in a huge number of marine teleost fishes. Humans can therefore be accidental hosts by consuming crude or undercooked fish [A. simplex produces also a clinically relevant IgE-mediated hypersensitivity reaction with appearance of acute urticaria, angioedema, or anaphylaxis [ked fish . Whereasked fish , 3. Gastphylaxis .It is well known that, in GAA, specific as well as total IgE and other specific immunoglobulin isotype levels depend upon the time interval elapsed (TI) between the acute parasitic episode and the obtaining of the serum sample . The parAnisakis sensitization-associated chronic urticaria (CU+) and has been described as a different phenotype of urticaria with distinct clinical as well as immunologic features with respect to other chronic urticaria forms, but also compared to GAA [A. simplex hypersensitivity is now reported from different geographic regions [Another clinical entity, where a previous acute parasitic episode is suspected to play at least a necessary factor in a disease of multifactorial genesis, has been proposed to be an d to GAA –9. Furth regions . Therefo regions , the sea regions , 12. The experience of immune features in GAA teaches us that the allergic reaction is elicited after a secondary contact with the parasite , 13. The4 [Acute allergic hypersensitivity reactions have mainly been studied in the fields of food or insect allergy. Besides the necessary production of IgE induced by proteins with specific allergenic properties, several other factors are necessary to produce allergic reactions, as IgE presence is not equivalent to clinical allergy. Further measurable amounts of detectable IgE do not properly predict allergic reactions. Further relevant factors could include the production of high-affinity IgE or the ratio of other blocking immunoglobulin isotypes, such as IgG4 , 15.Affinity and avidity studies have shown that continuous or repeated contact with antigen leads to an antibody affinity maturation . This knA. simplex-associated allergic diseases is to assess hypothesized differences in avidity in GAA and CU+ and to associate them with the time interval elapsed since the parasitic episode and also with fish-eating habits as a possible correlation to the probability of previous subclinical parasitic episodes. Therefore, one approach to get more insight into the clinical relevance of specific IgE and IgG in Patients were recruited at our allergy service and included prospectively if they matched the inclusion criteria. The study protocol was approved by the University Hospital La Princesa Ethical Committee.A. simplex at ≥0.7 kU/L.All patients had to display a positive SPT and detectable IgE antibodies against GAA patients had an acute episode of acute urticaria, angioedema, or anaphylaxis of less than 48-hour duration within 24 hours after eating raw or clearly undercooked fish, and other eliciting causes have been discarded by history and allergological workup.CU+ patients were included if they had at least twice per week recurring wheals for at least 6 weeks. Patients were not included in this study if physical stimuli were the main eliciting agents of the urticarial reaction. Other factors associated with CU were not an exclusion factor, as CU is mainly multifactorial.Thus 16 patients with GAA and 17 patients with CU+ were included and questioned about weekly fish and monthly anchovies in vinegar sauce consumption. Further time interval was defined as the interval between serum sample and GAA episode or onset of CU symptoms.Anisakis specific IgE, IgG, and IgG4 and total IgE were analysed by CAP-FEIA  [A. simplex whole body extract were further assessed by ELISA [Anti- Sweden) . Specifiby ELISA . A. simplex whole body extract at 10 μg/mL overnight and incubated for 30 min with 6 M urea in PBS at room temperature. Microplates were washed and blocked with BSA for 1 h. Each serum was studied in quadruplicate (2 with urea treatment and 2 without), diluted in PBS-Tween 20, BSA 0.1%, and incubated for 2 h at 37°C. After incubating and washing, two of the quadruplicate sera were treated with PBS 6 M urea for 30 min at room temperature and the remaining sera were incubated with PBS for 30 min. After washing, goat anti-human IgG and horseradish peroxidase (HRP) conjugate  were used. For the IgE determination, a murine monoclonal antibody against an ε-human IgE chain  was added and incubated, followed by a goat anti-mouse IgG (γ1), HRP conjugate . The reactions were developed with o-phenylenediamine with hydrogen peroxide and read at 490 nm. The avidity index  was defined as the mean optical density (OD) of urea-treated wells/mean OD urea-untreated wells × 100.Avidity ELISA was based on the dissociative method using urea as a denaturing agent . Plates Statistical analysis was performed using SPSS ver. 15.0 for Windows. 4 as well as for avidity indexes and compared by Mann-Whitney U test.Median values and interquartile ranges (IQR) were calculated for total and specific IgE or IgGWe performed nonparametric correlation (Spearman Rho) studies between AvIgE, AvIgG, immunoglobulins, time interval, and fish consumption.4, fish intake, and time interval as possible explaining variables with stepwise exclusion of nonsignificant variables.Furthermore, a linear regression was performed separately for GAA and CU+ for IgE avidity as dependent variable. We analysed specific IgE, total IgE, IgG, IgGMean age of the studied patients was 52.5 ± 14.3 years. 18 patients were females and 15 were males. These data did not differ in both groups.Mean time from the acute parasitic episode to the serum sample in GAA was 5.0 (±5.2) months and mean duration of urticaria in CU+ was 27.0 (±20.8) months.Mean number of fish meals per week was 2.7 (±1.5) in all patients without differences in both studied groups.4 was higher in GAA than in CU+ , whereas there was a tendency to lower IgE avidity in GAA (P = 0.095). Median AvIgE in all patients was 80.3 (interquartile range 73.2–91.8), while median AvIgG was 53.9 (37.4–91.8). 4 as well as total IgE , but AvIgE was negatively correlated with specific IgE , specific IgG4 , and total IgE .When analysing all patients, AvIgG was positively correlated with specific IgE, IgG, and IgGP = 0.02). Also, in GAA weekly fish consumption was positively associated with AvIgE .This divergent correlation depending on AvIgE or AvIgG was mainly maintained when patients with GAA were analysed Figures  and 2. HP = 0.015) was the main explaining factor for AvIgE in GAA  with possibly different avidities. In this respect, a previous study in peanut allergic subjects showed IgE : IgG avidity ratios to be higher than 1 for a total antigenic source, whereas the opposite ratio was found when analysing avidity to a major allergen Ara h2 [Whereas avidity describes the overall strength of the interaction between antibodies and their antigens, it would therefore be interesting to perform a similar study analysing affinities to already known n Ara h2 . In our Whereas all immunoglobulin isotypes are known to be lower in CU+ than in GAA, a fact we could also confirm for specific IgG in our study, avidities could also be interpreted when analysing the time interval between parasitism and obtaining of the serum sample. Likewise, regression analysis showed that, in GAA, time interval was the main explaining factor for IgE avidity.Anisakis proteins [Affinity is overall expected to rise in time after infection, but this probably applies to only a few relevant allergens or epitopes . A possiproteins , 13. Eveproteins . Otherwiproteins .Anisakis proteins of other specificities than those leading to the allergic episode, leads to maintaining lower affinity antibodies. This would be an explanation to the fact that patients sensitized to Anisakis do not react with acute type 1 hypersensitivity reactions in provocation tests with non viable Anisakis larvae [4 included in the IgG compartment, with confirmed higher avidities in GAA could, by competing with the same epitopes recognized by IgE, prevent mast cell activation and the subsequent allergic episode, when Anisakis contaminated fish (nonviable or inactivated larvae) is well tolerated by patients with previous GAA. Again, the time interval seems to be the main explaining factor. Also in this case we could speculate that, once a sensitization process has occurred, following continuous contact with s larvae , 29. Furwithin an antibody isotype, but also the outcome of a mechanism, in which probably outcompeting of an immunoglobulin isotype over another could have been resulted by high-affinity antibodies in the maturation process [Interestingly, if we look at the diverging correlation of AvIgG and AvIgE with specific IgE, we can also postulate that avidity is not only the outcome of an affinity maturation process of antibodies  process .A possible drawback of these results is of methodological concern, as there could be some controversy about the influence of IgG antibodies on an ELISA assay in avidity studies. However, previous studies with using thiocyanate as denaturing agent showed independence of immunoglobulin levels and avidity and lack of independence between avidity indexes and immunoglobulin concentrations , 31. TheAnisakis-associated allergic disease has not been studied so far, but our results could lead to a testable hypothesis, where clinically different CU+ and GAA after parasitism could be motivated by distinct class switching of specific antibodies.We did not show a direct negative correlation between AvIgE and AvIgG, but this could be due to the fact that we did not measure differential epitope affinity and the high variation of binding capacities and maturation process of antibodies between individuals . In thisA. simplex parasitism, we could thus show a differential behaviour of immunoglobulin isotype avidities in GAA and CU+ and their dependence on fish-eating habits as well as on the time elapsed to the last parasitic episode. Further studies should address avidity studies against excretory-secretory proteins as well as affinity studies against known allergens.In this first study on immunoglobulin avidities in humans affected by


Free space is necessary for larval recruitment in all marine benthic communities. Settling corals, with limited energy to invest in competitive interactions, are particularly vulnerable during settlement into well-developed coral reef communities. This situation may be exacerbated for corals settling into coral-depauperate reefs where succession in nursery microhabitats moves rapidly toward heterotrophic organisms inhospitable to settling corals. To study effects of benthic organisms  on newly settled corals and their survivorship we deployed terra-cotta coral settlement plates at 10 m depth on the Mesoamerican Barrier Reef in Belize and monitored them for 38 mo. During the second and third years, annual recruitment rates declined by over 50% from the previous year. Invertebrate crusts (primarily sponges) were absent at the start of the experiment but increased in abundance annually from 39, 60, to 73% of the plate undersides by year three. Subsequently, substrates hospitable to coral recruitment, including crustose coralline algae, biofilmed terra-cotta and polychaete tubes, declined. With succession, substrates upon which spat settled shifted toward organisms inimical to survivorship. Over 50% of spat mortality was due to overgrowth by sponges alone. This result suggests that when a disturbance creates primary substrate a “recruitment window” for settling corals exists from approximately 9 to 14 mo following the disturbance. During the window, early-succession, facilitating species are most abundant. The window closes as organisms hostile to coral settlement and survivorship overgrow nursery microhabitats. Larval recruitment is one critical process leading to the recovery of community structure following disturbances in the marine realm. Yet, Caribbean coral reefs are suffering from increasing rates of disturbances and decreasing rates of recruitment Free space is necessary for settling larvae, and thus, is important in all marine benthic communities In shallow reef environments, corals recruit to nursery microhabitats where rates of predation and competition are lower than on exposed surfaces and post-settlement survivorship is higher Titanoderma prototypum, a preferred settlement substratum for certain species of corals T. prototypum, are present and inhibitory organisms, such as sponges and bryozoans, have yet to colonize nursery microhabitats for corals. While this window is maximally open, newly settled corals may be more likely to successfully “run the gauntlet” to recruitment Since the 1970s, many Caribbean coral reefs have shifted from coral to macroalgal-dominated systems The fate of coral larvae settling into an increasingly hostile world is addressed in this study. We sought to observe the succession of benthic organisms and their interactions in coral nursery microhabitats with newly settled corals on a typical, contemporary, macroalgae-dominated Caribbean reef. Is there an identifiable period following a perturbation on a reef when substrate conditions are optimal for successful coral recruitment? The emerging view regarding recruitment patterns is that benthic interactions, such as competition, drive patterns when space is limiting, but larval supply or very early post-settlement mortality drives spatial variability in recruitment when free space is abundant We studied recruitment and survivorship of corals over three years as succession altered the species composition of nursery microhabitats. By observing interactions between corals and other sessile organisms on coral settlement plates in Belize, we sought to provide new insights into the important ecological interactions that occur within typical epibenthic Caribbean reef communities.To assess patterns in coral recruitment and early post-settlement survivorship in relation to other benthic organisms, we conducted studies on the Mesoamerican Barrier Reef in Belize (16°48.18 N 88°04.93 W) from June 2005 to August 2008. We selected two sites on the forereef adjacent to Carrie Bow Cay and two sites on the forereef adjacent to South Water Cay, just to the north. Sites were all at 10 m water depth and separated by approximately 0.5 km. The coordinates for the individual sites are as follows: Site 1- 16°48.098 N 88°04.725 W, Site 2- 16°48.363 N 88°04.472 W, Site 3- 16°48.769 N 88°04.686 W, and Site 4- 16°49.168 N 88°04.677 W.Peyssonnelia spp.), macroalgae (primarily Dictyota spp.), articulated algae , turf algae, sponges/gorgonians, and sand) falling directly under the tape.At each site we established 5 10-m line transects along which we addressed benthic cover. Using the line intercept transect (LIT) method To study the effect of benthic organisms  on coral recruitment and survivorship, we used 10 cm×10 cm×1 cm terra-cotta settlement plates to mimic primary substrate on a reef. We define ‘recruitment’ in an operational sense, referring to those newly settled corals that have survived metamorphosis and have recognizable skeletons, dead or alive, at the time of the retrieval of the plates. In June 2005, we deployed 25 tiles at each of the 4 sites. Five plates per transect were affixed to dead coral and separated from the bottom by a 1-cm PVC spacer. Plate deployment was modified from Mundy Plates were monitored in August 2005, March 2006, August 2006, August 2007 and August 2008 for newly recruited corals, survivorship of previously identified corals and succession of benthic organisms. One full day was required to sample each site. Thus the monitoring occurred over four days. In the results, we refer to the monitoring periods mentioned above as “approximate days following deployment”, or day 64 (for August 2005), day 271 (for March 2006), day 427 (for August 2006), day 802 (August 2007), and day 1157 (for August 2008). At each of the 5 monitoring intervals, all plates were transported from the reef to the lab in seawater, analyzed under a dissecting microscope (25×) while immersed in seawater, and returned to the reef within 6 h. In the Caribbean at 10 m depth, corals settle primarily on undersides of settlement plates Agaricia spp. and Porites spp. Shearer and Coffroth Agaricia spp. and Porites spp. recruits as Agaricia agaricites  and Porites astreoides. Porites spp. are characterized by a porous coenosteum, septa with prominent teeth, often an epitheca, and they can form multiple polyps within a short time (wks-mos). Agaricia spp. do not develop an epitheca and have prominent, laminar septa, and can remain in single polyp form for over a year. Thus, newly settled corals were identified to genus where possible, measured at maximum diameter, and mapped relative to an x-y coordinate scale around the plate. We recorded the substrate upon which the coral recruited and the percent cover of all other encrusting biota on the plate underside . To determine percent cover, we used visual estimates because this method was shown to be more accurate and have less within-observer variation than random-point-quadrats (RPQ), particularly when estimating cover of rare sessile organisms Optimally, when identifying coral recruits, the tissue is removed, often with bleach, to reveal the skeletal elements helpful for identification While plates were searched several times at each observation period for coral recruits, carefully looking under foliose algae, it is possible that recruitment rates would have been higher if plates were bleached and dried before searching. This methodological difference should be taken into consideration if recruitment rates reported in this study are compared to other recruitment studies in the region.i, which is the proportional use divided by the proportional availability of each substrate.Special attention was paid to the “subcryptic settlement microhabitat” To test whether pooling data from the four sites was justified, we first used non-metric multidimensional scaling ordination . Furthermore, there was no significant difference in total recruitment among sites . At the end of the 3.17-yr experiment, mean coral recruits (± SE) per entire plate underside were: Site 1 = 2.43±0.09, Site 2 = 2.59±0.10, Site 3 = 2.79±0.14, Site 4 = 3.0±0.26. Thus, we pooled recruitment data from the four sites for all subsequent analyses.The surrounding biota that might influence coral recruitment varied little among sites . Site weakly influenced adult live coral cover, abundance of CCA, macroalgae, articulated algae, turf algae, and sponges  of all substrates, ranging from high to low, were polychaete tubes = 8.30, crustose coralline algae (not including T. prototypum) = 3.45, T. prototypum = 2.50, biofilmed terra-cotta = 1.90, Peyssonnelia = 1.40, invertebrate crusts = 0.11, turf algae = 0.03, and macroalgae = 0.00. A wi value greater than 1 indicates a positive selection for the substrate and a value less than 1 indicates avoidance of the substrate. A value near 1 indicates that the resource was used proportionally to its availability and no resource selection was observed. Thus, we deduce coral recruitment success to vary along the spectrum of substrates ranging from those that facilitate to those that inhibit this process  continued to increase in abundance through day 1157, peaking at 69%. By the end of the study (day 1157), all inhibitor species combined accounted for 87% of the nursery microhabitat , whereas days deployed had a strong influence . Similarity percentage (SIMPER) analysis Changes in substrate composition of nursery microhabitats were examined   . These lsponges) .No increased survivorship of earlier cohorts was obvious . For exaAgaricia and Porites revealed Porites spp.'s inability to compete with the increasingly hostile environment, with no survivorship beyond the first cohort  algae, and invertebrates . Among encrusting marine organisms, thicker margins overgrow and thus outcompete thinner margins Titanoderma prototypum (crust thickness 40 mm) lose space competition to thicker, overgrowing organisms. Replacement of thin, early-succession facilitating species by thick late successional species often ends with the thickest sponges covering everything and sometimes completely filling the nursery habitat . We have monitored one sponge-filled nursery habitat in Belize for eight years with most of the last five years remaining in a sponge-filled state.Succession in coral nursery habitats, as it affects coral recruitment, is largely unstudied. We found functional group (growth form) changes conforming to patterns described by Jackson Recruitment rates fell as the nursery microhabitat shifted towards encrusting invertebrates and away from biofilms, CCA, and polychaete tubes , 5. SurpPorites spp. recruits, all but those in the earliest cohort (March 2006) that faced relatively hospitably fouled plates had zero survivorship, indicating that they may be highly vulnerable to overgrowth in well-developed settlement microhabitats  to inhibiting and deadly heterotrophs (sponges) Compared to IndoPacific reefs, Caribbean reefs have distinct disadvantages when it comes to recovering following disturbances. The Caribbean has naturally lower biodiversity, particularly of the reef-building, branching corals that never readily recruited even when they were abundant