Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

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.     

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.     

Nuclear lamin antigen and messenger RNA expression in bovine in vitro produced and nuclear transfer embryos

The nuclear lamina is a complex meshwork of nuclear lamin filaments that lies on the interface of the nuclear envelope and chromatin and is important for cell maintenance, nucleoskeleton support, chromatin remodeling, and protein recruitment to the inner nucleolus. Protein and mRNA patterns for the major nuclear lamins were investigated in bovine in vitro fertilized (IVF) and nuclear transfer embryos. Expression of lamins A/C and B were examined in IVF bovine germinal vesicle (GV) oocytes, metaphase II oocytes, zygotes, 2-cell, 8-cell, 16-32-cell embryos, morulae, and blastocysts (n = 10). Lamin A/C was detected in 9/10 immature oocytes, 10/10 zygotes, 8/10 2-cell embryos, 4/10 morulae, 10/10 blastocysts but absent during the maternal embryonic transition. Lamin B was ubiquitously expressed during IVF preimplantation development but was only detected in 4/10 GV oocytes. Messenger RNA expression confirms that the major lamins, A/C and B1 are expressed throughout preimplantation development and transcribed by the embryo proper. Lamin A/C and B expression were observed (15 min, 30 min, 60 min, 120 min) following somatic cell nuclear transfer using adult fibroblasts and at the 2-cell, 8-cell, 16-32-cell, morula and blastocyst stage (n = 5). Altered expression levels and localization of nuclear lamins A/C and B was determined in nuclear transfer embryos during the first 2 hr post fusion, coincidental with only partial nuclear envelope breakdown as well as during the initial cleavage divisions, but was restored by the morula stage. This mechanical and molecular disruption of the nuclear lamina provides key evidence for incomplete nuclear remodeling and reprogramming following somatic cell nuclear transfer.     

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.      

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.