Tyrosine phosphorylation of Munc18‐1 inhibits synaptic transmission by preventing SNARE assembly

Tyrosine kinases are important regulators of synaptic strength. Here, we describe a key component of the synaptic vesicle release machinery, Munc18‐1, as a phosphorylation target for neuronal Src family kinases (SFKs). Phosphomimetic Y473D mutation of a SFK phosphorylation site previously identified by brain phospho‐proteomics abolished the stimulatory effect of Munc18‐1 on SNARE complex formation (“SNARE‐templating”) and membrane fusion in vitro. Furthermore, priming but not docking of synaptic vesicles was disrupted in hippocampal munc18‐1‐null neurons expressing Munc18‐1_Y473D. Synaptic transmission was temporarily restored by high‐frequency stimulation, as well as by a Munc18‐1 mutation that results in helix 12 extension, a critical conformational step in vesicle priming. On the other hand, expression of non‐phosphorylatable Munc18‐1 supported normal synaptic transmission. We propose that SFK‐dependent Munc18‐1 phosphorylation may constitute a potent, previously unknown mechanism to shut down synaptic transmission, via direct occlusion of a Synaptobrevin/VAMP2 binding groove and subsequent hindrance of conformational changes in domain 3a responsible for vesicle priming. This would strongly interfere with the essential post‐docking SNARE‐templating role of Munc18‐1, resulting in a largely abolished pool of releasable synaptic vesicles.     

Quantum-dye labeled proteins for glycobiology: a viable nonradioactive alternative tracer

Quantum dye (QD), a macrocyclic europium-chelate, developed as a cytological marker, has never been used for quantitative applications. It would be ideal, however, if the same tracer can be used for both qualitative and quantitative purposes. We have labeled some lectins and neoglycoproteins with QD for the purpose of quantitative analyses in glycobiology, and tested its suitability in three different areas in glycobiology: (1) glycosyltransferase, (2) an animal lectin - mannose- binding protein, and (3) the Gal/GalNAc receptor of rat liver membrane. Usefulness of QD-labeled lectins was amply demonstrated by the quantification of galactosyltransferase activity using QD-soybean agglutinin and QD-RCA120 ( Ricinus communis agglutinin). We also showed that QD-labeled neoglycoproteins, QD-Man-BSA and QD-Gal-BSA, can replace radioiodinated counterparts in the binding assays of animal lectins (serum mannose binding protein and hepatic Gal/GalNAc receptor.) The advantage of QD and other europium labels is that it does not decay as radioiodides do. The long shelf-life results in more consistent results from repeated experiments.      

Construction of a bacterial artificial chromosome (BAC) library of Lycopersicon esculentum cv. Stevens and its application to physically map the Sw-5 locus

The Sw-5 gene is a dominantly inherited resistance gene in tomato and functional against a number of tospovirus species. The gene has been mapped on chromosome 9, tightly linked to RFLP markers CT220 and SCAR421. To analyse the Sw-5 locus, a BAC genomic library was constructed of tomato cv. Stevens, homozygous for the Sw-5 gene. The library comprised 18 816 clones with an average insert size of 100 kb, corresponding to two genome equivalents. The library was screened by PCR using primers designed for the CT220 and SCAR421 sequences, resulting in a 250 kb contig of known orientation on the long arm of chromosome 9. Using degenerate primers based on homologous sequences in the nucleotide binding site of resistance gene sequences, three discrete PCR fragments obtained from this contig were cloned and sequenced. Analysis of these fragments revealed a high similarity with numerous resistance genes or resistance gene like sequences. The present data indicate that at least three different resistance gene candidate (RGC) sequences are present in the vicinity of marker CT220, supporting the view that a resistance gene family may be responsible for the unusually broad resistance to tospoviruses conferred by the Sw-5 locus. This revised version was published online in June 2006 with corrections to the Cover Date.     

Bill redness is positively associated with reproduction and survival in male and female zebra finches

Sexual traits can serve as honest indicators of phenotypic quality when they are costly. Brightly coloured yellow to red traits, which are pigmented by carotenoids, are relatively common in birds, and feature in sexual selection. Carotenoids have been linked to immune and antioxidant function, and the trade-off between ornamentation and these physiological functions provides a potential mechanism rendering carotenoid based signals costly. Mutual ornamentation is also common in birds and can be maintained by mutual mate choice for this ornament or by a correlated response in one sex to selection on the other sex. When selection pressures differ between the sexes this can cause intralocus sexual conflict. Sexually antagonistic selection pressures have been demonstrated for few sexual traits, and for carotenoid-dependent traits there is a single example: bill redness was found to be positively associated with survival and reproductive output in male zebra finches, but negatively so in females. We retested these associations in our captive zebra finch population without two possible limitations of this earlier study. Contrary to the earlier findings, we found no evidence for sexually antagonistic selection. In both sexes, individuals with redder bills showed higher survival. This association disappeared among the females with the reddest bills. Furthermore, females with redder bills achieved higher reproductive output. We conclude that bill redness of male and female zebra finches honestly signals phenotypic quality, and discuss the possible causes of the differences between our results and earlier findings.     

A quantitative analysis of the interplay of environment,neighborhood, and cell state in 3D spheroids

Cells react to their microenvironment by integrating external stimuli into phenotypic decisions via an intracellular signaling network. To analyze the interplay of environment, local neighborhood, and internal cell state effects on phenotypic variability, we developed an experimental approach that enables multiplexed mass cytometric imaging analysis of up to 240 pooled spheroid microtissues. We quantified the contributions of environment, neighborhood, and intracellular state to marker variability in single cells of the spheroids. A linear model explained on average more than half of the variability of 34 markers across four cell lines and six growth conditions. The contributions of cell‐intrinsic and environmental factors to marker variability are hierarchically interdependent, a finding that we propose has general implications for systems‐level studies of single‐cell phenotypic variability. By the overexpression of 51 signaling protein constructs in subsets of cells, we also identified proteins that have cell‐intrinsic and cell‐extrinsic effects. Our study deconvolves factors influencing cellular phenotype in a 3D tissue and provides a scalable experimental system, analytical principles, and rich multiplexed imaging datasets for future studies.     

The cost of incubation in relation to clutch-size in the Collared Flycatcher Ficedula albicollis

This study examines the importance of avian incubation costs as determinants of clutch-size variation by performing clutch-size and brood-size manipulations in the same population of Collared Flycatchers Ficedula albicollis during the same breeding season. In 2 5 cases when three or more clutches of the same size were completed on the same day, we moved two eggs on the day after the last egg had been laid from one randomly selected clutch (C) to another (C) and moved two other eggs from this to a third clutch (C⁺). In 20 other cases of simultaneously completed clutches of the same size, we moved two randomly selected young from one brood to a second and from that moved two other young to a third (B, B and B⁺groups). Most females were weighed the day after completion of the clutch and 1–4 days before hatching of the young, and some of them also 10–14 days after hatching of the young. We measured the daily energy expenditure of females incubating manipulated clutches of 4, 6 and 8 eggs by means of the doubly-labelled water (D₂¹⁸O) technique and also recorded their nest attendance. Hatching success of fertilized eggs was reduced in the enlarged clutches compared with control and reduced clutches. Females expired on average 3142.6 ml CO₂ and expended 78.6 kJ per day while incubating, which corresponds to a metabolic intensity of 3.3 times BMR. Daily energy expenditure increased with clutch-size due to higher costs while incubating, and not because of changed activity patterns. There were no significant differences in length of incubation, female mass or mass changes between phases for the C, C and C⁺groups. In both the C and B groups, enlarged broods produced significantly more fledged young than control broods, and those significantly more than reduced broods. Fledgling tarsus-length and mass did not differ significantly between treatments in either the C or B groups. There was no significant difference in breeding success between clutch and brood manipulations. In this season, incubation costs did not entail significant fitness losses, expressed either as fledgling production or female condition. Also, control females could have raised more young to fledging age than they did with no apparent costs.     

The use of in vitro peptide binding profiles and in silico ligand-receptor interaction profiles to describe ligand-induced conformations of the retinoid X receptor alpha ligand-binding domain

It is hypothesized that different ligand-induced conformational changes can explain the different interactions of nuclear receptors with regulatory proteins, resulting in specific biological activities. Understanding the mechanism of how ligands regulate cofactor interaction facilitates drug design. To investigate these ligand-induced conformational changes at the surface of proteins, we performed a time-resolved fluorescence resonance energy transfer assay with 52 different cofactor peptides measuring the ligand-induced cofactor recruitment to the retinoid X receptor-alpha (RXRalpha) in the presence of 11 compounds. Simultaneously we analyzed the binding modes of these compounds by molecular docking. An automated method converted the complex three-dimensional data of ligand-protein interactions into two-dimensional fingerprints, the so-called ligand-receptor interaction profiles. For a subset of compounds the conformational changes at the surface, as measured by peptide recruitment, correlate well with the calculated binding modes, suggesting that clustering of ligand-receptor interaction profiles is a very useful tool to discriminate compounds that may induce different conformations and possibly different effects in a cellular environment. In addition, we successfully combined ligand-receptor interaction profiles and peptide recruitment data to reveal structural elements that are possibly involved in the ligand-induced conformations. Interestingly, we could predict a possible binding mode of LG100754, a homodimer antagonist that showed no effect on peptide recruitment. Finally, the extensive analysis of the peptide recruitment profiles provided novel insight in the potential cellular effect of the compound; for the first time, we showed that in addition to the induction of coactivator peptide binding, all well-known RXRalpha agonists also induce binding of corepressor peptides to RXRalpha.     

Adaptive and nonadaptive plasticity in changing environments: Implications for sexual species with different life history strategies

Populations adapt to novel environmental conditions by genetic changes or phenotypic plasticity. Plastic responses are generally faster and can buffer fitness losses under variable conditions. Plasticity is typically modeled as random noise and linear reaction norms that assume simple one‐to‐one genotype–phenotype maps and no limits to the phenotypic response. Most studies on plasticity have focused on its effect on population viability. However, it is not clear, whether the advantage of plasticity depends solely on environmental fluctuations or also on the genetic and demographic properties (life histories) of populations. Here we present an individual‐based model and study the relative importance of adaptive and nonadaptive plasticity for populations of sexual species with different life histories experiencing directional stochastic climate change. Environmental fluctuations were simulated using differentially autocorrelated climatic stochasticity or noise color, and scenarios of directional climate change. Nonadaptive plasticity was simulated as a random environmental effect on trait development, while adaptive plasticity as a linear, saturating, or sinusoidal reaction norm. The last two imposed limits to the plastic response and emphasized flexible interactions of the genotype with the environment. Interestingly, this assumption led to (a) smaller phenotypic than genotypic variance in the population (many‐to‐one genotype–phenotype map) and the coexistence of polymorphisms, and (b) the maintenance of higher genetic variation—compared to linear reaction norms and genetic determinism—even when the population was exposed to a constant environment for several generations. Limits to plasticity led to genetic accommodation, when costs were negligible, and to the appearance of cryptic variation when limits were exceeded. We found that adaptive plasticity promoted population persistence under red environmental noise and was particularly important for life histories with low fecundity. Populations producing more offspring could cope with environmental fluctuations solely by genetic changes or random plasticity, unless environmental change was too fast.