AID and caspase 8 shape the germinal center response through apoptosis.

Germinal centers (GCs) are clusters of activated B cells that form in secondary lymphoid organs during a T-dependent immune response. B cells enter GCs and become rapidly proliferating centroblasts that express the enzyme activation-induced deaminase (AID) to undergo somatic hypermutation and class-switch recombination. Centroblasts then mature into centrocytes to undergo clonal selection. Within the GC, the highest affinity B cell clones are selected to mature into memory or plasma cells while lower affinity clones undergo apoptosis. We reported previously that murine Aicda(-/-) GC B cells have enhanced viability and accumulate in GCs. We now show that murine Aicda(-/-) GC B cells accumulate as centrocytes and inefficiently generate plasma cells. The reduced rate of plasma cell formation was not due to an absence of AID-induced DNA lesions. In addition, we show that the deletion of caspase 8 specifically in murine GC-B cells results in larger GCs and a delay in affinity maturation, demonstrating the importance of apoptosis in GC homeostasis and clonal selection.

Negative supercoiling creates single-stranded patches of DNA that are substrates for AID-mediated mutagenesis.

Antibody diversification necessitates targeted mutation of regions within the immunoglobulin locus by activation-induced cytidine deaminase (AID). While AID is known to act on single-stranded DNA (ssDNA), the source, structure, and distribution of these substrates in vivo remain unclear. Using the technique of in situ bisulfite treatment, we characterized these substrates-which we found to be unique to actively transcribed genes-as short ssDNA regions, that are equally distributed on both DNA strands. We found that the frequencies of these ssDNA patches act as accurate predictors of AID activity at reporter genes in hypermutating and class switching B cells as well as in Escherichia coli. Importantly, these ssDNA patches rely on transcription, and we report that transcription-induced negative supercoiling enhances both ssDNA tract formation and AID mutagenesis. In addition, RNaseH1 expression does not impact the formation of these ssDNA tracts indicating that these structures are distinct from R-loops. These data emphasize the notion that these transcription-generated ssDNA tracts are one of many in vivo substrates for AID.

Tissue remodeling: a mating-induced differentiation program for the Drosophila oviduct.

BACKGROUND: In both vertebrates and invertebrates, the oviduct is an epithelial tube surrounded by visceral muscles that serves as a conduit for gamete transport between the ovary and uterus. While Drosophila is a model system for tubular organ development, few studies have addressed the development of the fly's oviduct. Recent studies in Drosophila have identified mating-responsive genes and proteins whose levels in the oviduct are altered by mating. Since many of these molecules (e.g. Muscle LIM protein 84B, Coracle, Neuroglian) have known roles in the differentiation of muscle and epithelia of other organs, mating may trigger similar differentiation events in the oviduct. This led us to hypothesize that mating mediates the last stages of oviduct differentiation in which organ-specific specializations arise. RESULTS: Using electron- and confocal-microscopy we identified tissue-wide post-mating changes in the oviduct including differentiation of cellular junctions, remodeling of extracellular matrix, increased myofibril formation, and increased innervation. Analysis of once- and twice-mated females reveals that some mating-responsive proteins respond only to the first mating, while others respond to both matings. CONCLUSION: We uncovered ultrastructural changes in the mated oviduct that are consistent with the roles that mating-responsive proteins play in muscle and epithelial differentiation elsewhere. This suggests that mating triggers the late differentiation of the oviduct. Furthermore, we suggest that mating-responsive proteins that respond only to the first mating are involved in the final maturation of the oviduct while proteins that remain responsive to later matings are also involved in maintenance and ongoing function of the oviduct. Taken together, our results establish the oviduct as an attractive system to address mechanisms that regulate the late stages of differentiation and maintenance of a tubular organ.

Mating induces an immune response and developmental switch in the Drosophila oviduct.

Mating triggers physiological and behavioral changes in females. To understand how females effect these changes, we used microarray, proteomic, and comparative analyses to characterize gene expression in oviducts of mated and unmated Drosophila females. The transition from non-egg laying to egg laying elicits a distinct molecular profile in the oviduct. Immune-related transcripts and proteins involved in muscle and polarized epithelial function increase, whereas cell growth and differentiation-related genes are down-regulated. Our combined results indicate that mating triggers molecular and biochemical changes that mediate progression from a "poised" state to a mature, functional stage.

Mating-responsive genes in reproductive tissues of female Drosophila melanogaster.

Male-derived accessory gland proteins that are transferred to females during mating have profound effects on female reproductive physiology including increased ovulation, mating inhibition, and effects on sperm utilization and storage. The extreme rates of evolution seen in accessory gland proteins may be driven by sperm competition and sexual conflict, processes that may ultimately drive complex interactions between female- and male-derived molecules and sperm. However, little is known of how gene expression in female reproductive tissues changes in response to the presence of male molecules and sperm. To characterize this response, we conducted parallel genomic and proteomic analyses of gene expression in the reproductive tract of 3-day-old unmated and mated female Drosophila melanogaster. Using DNA microarrays, we identified 539 transcripts that are differentially expressed in unmated vs. mated females and revealed a striking peak in differential expression at 6 h postmating and a marked shift from primarily down-regulated to primarily up-regulated transcripts within 3 h after mating. Combining two-dimensional gel electrophoresis and liquid chromatography mass spectrometry analyses, we identified 84 differentially expressed proteins at 3 h postmating, including proteins that appeared to undergo posttranslational modification. Together, our observations define transcriptional and translational response to mating within the female reproductive tract and suggest a bimodal model of postmating gene expression initially correlated with mating and the final stages of female reproductive tract maturation and later with the declining presence of male reproductive molecules and with sperm maintenance and utilization.

Positively charged peptides can interact with each other, as revealed by solid phase binding assays.

Solid phase assay systems such as enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), and overlay gels are used to study processes of protein-protein interactions. The common principle of all these methods is that they monitor the binding between soluble and surface-immobilized molecules. Following the use of bovine serum albumin (BSA)-peptide conjugates or isolated synthetic peptides and the above-mentioned solid phase assay systems, the results of the current work demonstrate that positively charged peptides can interact with each other. Both the ELISA and SPR methods demonstrated that the binding process reached saturation with K(d) values ranging between 1 and 14 nM. No interaction was observed between BSA conjugates bearing positively charged peptides and conjugates bearing negatively charged peptides or with pure BSA molecules, strengthening the view that interaction occurs only between positively charged peptides. However, interactions between peptides in solution were not observed by nuclear magnetic resonance (NMR) or by native gel electrophoresis. It appears that for positively charged molecules to interact, one of the binding partners must be immobilized to a surface, a process that may lead to the exposure of otherwise masked groups or atoms. We discuss the relevance of our findings for the use of solid phase assay systems to study interactions between biomolecules.

Protein interactions involved in nuclear import of the Agrobacterium VirE2 protein in vivo and in vitro.

Agrobacterium, the only known organism capable of trans-kingdom DNA transfer, genetically transforms plants by transferring a segment of its DNA, T-DNA, into the nucleus of the host cell where it integrates into the plant genome. One of the central events in this genetic transformation process is nuclear import of the T-DNA molecule, which to a large degree is mediated by the bacterial virulence protein VirE2. VirE2 is distinguished by its nuclear targeting, which occurs only in plant but not in animal cells and is facilitated by the cellular VIP1 protein. The molecular mechanism of the VIP1 function is still unclear. Here, we used in vitro assays for nuclear import and quantification of protein-protein interactions to directly demonstrate formation of ternary complexes between VirE2, VIP1, and a component of the cellular nuclear import machinery, karyopherin alpha. Our results indicate that VIP1 functions as a molecular bridge between VirE2 and karyopherin alpha, allowing VirE2 to utilize the host cell nuclear import machinery even without being directly recognized by its components.