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Abstract The copackaging of mRNAs into biomolecular condensates called germ granules is a conserved strategy to posttranscriptionally regulate germline mRNAs. In Drosophila melanogaster, mRNAs accumulate in germ granules by forming homotypic clusters, aggregates containing multiple transcripts from the same gene. Nucleated by Oskar (Osk), homotypic clusters are generated through a stochastic seeding and self-recruitment process that requires the 3′ untranslated region (UTR) of germ granule mRNAs. Interestingly, the 3′ UTR belonging to germ granule mRNAs, such as nanos (nos), have considerable sequence variations among Drosophila species and we hypothesized that this diversity influences homotypic clustering. To test our hypothesis, we investigated the homotypic clustering of nos and polar granule component (pgc) in four Drosophila species and concluded that clustering is a conserved process used to enrich germ granule mRNAs. However, we discovered germ granule phenotypes that included significant changes in the abundance of transcripts present in species’ homotypic clusters, which also reflected diversity in the number of coalesced primordial germ cells within their embryonic gonads. By integrating biological data with computational modeling, we found that multiple mechanisms underlie naturally occurring germ granule diversity, including changes in nos, pgc, osk levels and/or homotypic clustering efficacy. Furthermore, we demonstrated how the nos 3′ UTR from different species influences nos clustering, causing granules to have ∼70% less nos and increasing the presence of defective primordial germ cells. Our results highlight the impact that evolution has on germ granules, which should provide broader insight into processes that modify compositions and activities of other classes of biomolecular condensate.

Monocyte-derived dendritic cells (moDC) are an important scientific and clinical source of functional dendritic cells (DC). However, the optimization of the generation process has to date mainly been limited to the variation of soluble factors. In this study, we investigated the impact of the cell culture dish surface on phenotype and cytokine profile. We compared a standard cell culture dish to a non-adherent culture dish for two immunogenic maturation conditions, two tolerogenic conditions, and an unstimulated control. Phenotype, cytokine profile and T cell stimulatory capacity were determined after a 3-day culture. Light microscopy revealed an increase in homotypic cluster formation correlated with the use of non-adherent surfaces, which could be reduced by using blocking antibodies against CD18. All surface markers analyzed showed moderate to strong differences depending on the culture dish surface, including significantly decreased expression of key maturation markers such as CD80, CD86, and CCR7 as well as PD-L1 on cells stimulated with the Jonuleit cytokine cocktail cultured on a non-adherent surface. Significant differences in the secretion of many cytokines were observed, especially for cells stimulated with LPS, with over 10-fold decreased secretion of IL-10, IL12-p40, and TNF-α from the cells cultured on the non-adherent surface. All immunogenic moDC populations showed similar capacity to induce antigen-specific T cells. These results provide evidence that the DC phenotype depends on the surface used during moDC generation. This has important implications for the optimization of DC-based immunotherapy development and underlines that the local surrounding can interfere with the final DC population beyond the soluble factors.

The periodicity of nucleotide arrangement was studied for conserved regions of the regulatory modules of 16 early development genes in two Drosophila species. The relative disposition of the conserved segments was found to be a multiple of the length of one coil of the nucleosomal DNA superhelix. Homotypic and heterotypic clusters of transcription-factor binding sites were observed in the conserved regions. The sites are usually at a distance that corresponds to the coil length of superhelical DNA of the nucleosome. The transcription-factor binding sites were assumed to play a role in regulating DNA condensation according to the nucleosome type.