Cellular & Molecular Imaging

Alpha-toxin is a major virulence factor of Staphylococcus aureus. Monomer binding to host cell membranes result in the formation of heptameric transmembrane pores. Among human model airway epithelial cell lines, A549 cells were most sensitive toward the toxin followed by 16HBE14o- and S9 cells. In this study we investigated the processes of internalization of pore-containing plasma membrane areas as well as potential pathways for heptamer degradation (lysosomal, proteasomal) or disposal (formation of exosomes/microvesicles). The abundance of toxin hep-tamers upon applying an alpha-toxin pulse to the cells declined both in extracts of whole cells and of cellular membranes of S9 cells, but not in those of 16HBE14o- or A549 cells. Comparisons of heptamer degradation rates under inhibition of lysosomal or proteasomal degradation revealed that an important route of heptamer degradation, at least in S9 cells, seems to be the lysosomal pathway while proteasomal degradation appears to be irrelevant. Exosomes prepared from cul-ture supernatants of toxin-exposed S9 cells contained alpha-toxin as well as low amounts of ex-osome and microvesicle markers. These results indicate that the ability of lysosomal degradation of internalized toxin heptamers may be the most important determinant of toxin-resistance of some types of airway epithelial cells.

The Darwin Tree of Life project has the goal to sequence the genomes of 70,000 species of eukaryotic organisms in Britain and Ireland. This is a collection of photographs of the samples included in the study, provided by the National History Museum (NHM).

Background Microsporidia sp. MB, a microsporidian symbiont found naturally in Anopheles mosquitoes, has potential as a novel malaria control tool since it can inhibit Plasmodium development and transmission. The most feasible MB-based Plasmodium control strategy would involve dissemination through live mosquito releases, or release of spores infective to mosquito larvae. To implement either strategy, establishment of stable mosquito colonies carrying MB at a high frequency is likely to be essential. The progeny of field caught An. gambiae s.l from Burkina Faso were isolated for individual egg laying and tested for MB. The progeny of the MB positive females were pooled and this process was repeated for multiple generations. The relative density of MB in different life stages and tissues of the An. coluzzii host was examined using a novel duplex qPCR assay. We also examined the impact of MB on fecundity through individualization for egg laying and counting of eggs. Finally, we examined laid eggs for presence of MB spores. Results Three An. coluzzii colonies and one An. gambiae s.l hybrid colony were established with high prevalence and density of MB and were maintained for more than two years with minimal intervention. MB prevalence and density was highest in eggs and adult females and lowest in L4 larvae; in adults density was highest in the gonads. Additionally, MB density increased in ovary samples following blood feeding which was likely due to the activation of sporogony. The production of spores is the reason why MB-carrying females lay more white non-hatching eggs and show a small reduction in fecundity. Conclusions Establishment of several stable MB carrying An. gambiae s.l colonies and understanding the impact of spores on fecundity are significant steps forward in developing MB as a malaria control tool.

Proper chromosome segregation is crucial for cell division. In eukaryotes, this is achieved by the kinetochore, an evolutionarily conserved multi-protein complex that physically links the DNA to spindle microtubules and takes an active role in monitoring and correcting erroneous spindle-chromosome attachments. Our mechanistic understanding of these functions and how they ensure an error-free outcome of mitosis is still limited, partly because we lack a complete understanding of the kinetochore structure in the cell. In this study, we use single-molecule localization microscopy to visualize individual kinetochore complexes in situ in budding yeast. For major kinetochore proteins, we measured their abundance and position within the metaphase kinetochore. Based on this comprehensive dataset, we propose a quantitative model of the budding yeast kinetochore. While confirming many aspects of previous reports based on bulk imaging, our results present a unifying nanoscale model of the kinetochore in budding yeast.

Polyphasic studies on cyanobacterial biodiversity are scarce in the Caribbean and have never been conducted in the Dominican Republic. In this study we analysed sixteen cyanobacterial isolates sampled from biofilms growing on rocks and related to freshwater, saline and thermal environments across the country. A polyphasic approach was employed, incorporating 16S rRNA gene phylogenetic analysis, molecular identity assessments (p-distance), 16S–23S ITS sequence comparison and secondary structure analysis, alongside morphological characterization and habitat comparison. The strains were distributed across the orders Nodosilineales, Oculatellales, Oscillatoriales and Nostocales. Eleven strains were identified as belonging to Almyronema, Euryhalinema, Salileptolyngbya, Nodosilinea, Reticulonema, Vacuolonema, Capilliphycus, Purpureonostoc, Violetonostoc, Desmonostoc and Hapalosiphon. Additionally, four strains formed three distinct clades and presented low 16S rRNA gene identities with their phylogenetically closest genera, suggesting the existence of three undescribed lineages pending comprehensive polyphasic study. At the species level, four strains were considered undescribed lineages pending comprehensive polyphasic study and one strain (LEGE 171504) was described as a new species of Salileptolyngbya. This study represents the first investigation into the cyanobacterial diversity of Dominican Republic expanding the distribution of the identified taxa. Moreover, this is the first report of Salileptolyngbya in the Caribbean region and in brackish waters, as well as the first newly described cyanobacterial species from the Dominican Republic.

Dysregulated kinase activity drives oncogenic signalling, perturbs cellular homeostasis, and promotes tumour progression. Despite major success in targeting kinases therapeutically, the downstream consequences of kinase inhibition and the mechanisms underlying drug resistance remain incompletely understood. One of the most frequent oncogenic kinase mutations, BRAFV600E, constitutively activates the MAPK pathway and represents a major therapeutic target in melanoma and other cancers. However, the functional relevance of most phosphorylation events downstream of BRAF signalling is unknown, limiting mechanistic interpretation and rational therapeutic design. Here, we established a global, multi-omic model of BRAF inhibition response in BRAFV600E-mutant melanoma cells by integrating time-resolved phosphoproteomics, biophysical PTM-proteomics, transcriptomics, and thermal proteome profiling. Our ultradeep phosphoproteomic analysis revealed widespread phosphorylation changes upon Dabrafenib treatment, while biophysical phosphoproteomics uncovered phosphorylation events associated with altered solubility and subcellular localisation, indicative of biomolecular condensation and nuclear reorganisation. Integration of these modalities into a network-based mechanistic model enabled the prioritisation of functionally relevant phosphorylation sites and kinases. Experimental validation confirmed CDK9, CLK3, and TNIK as key regulators of BRAFV600E signalling and as candidate targets for combinatorial inhibition strategies capable of re-sensitising resistant melanoma cells in a synthetic lethal manner. The transcription factor ETV3 emerged from the network as a previously unrecognised effector of oncogenic BRAF signalling. Using phosphosite-specific biophysical data, imaging, and FRAP experiments, we demonstrated that ETV3 phosphorylation controls its DNA-binding kinetics. Functional assays combining ETV3 knockdown, metabolomics, and drug screening revealed that ETV3 modulates transcriptional and metabolic responses to BRAF inhibition, linking oncogenic signalling to metabolic rewiring. Together, this study provides a comprehensive systems-level framework that connects phosphorylation dynamics to protein function and cellular phenotype, highlights ETV3 as a novel signalling node, and illustrates how multi-omic, site-resolved network models can reveal actionable mechanisms of kinase-driven oncogenesis.

The use of CRISPR-associated enzymes in iPSC-derived neurons for precise gene targeting and high-throughput gene perturbation screens offers great potential but presents unique challenges compared to dividing cell lines. CRISPRi screens in iPSC-derived neurons and glia have already been successful in relating gene function to neurological phenotypes; however, loss of dCas9-KRAB expression after differentiation has been observed by many labs and has been largely ascribed to transgene silencing after differentiation. Here, we investigated the expression levels of different CRISPR enzymes in iPSC and Ngn2-derived neurons using piggybac delivery. We found that the commonly used dCas9-KRAB (using the KOX1 domain) displayed dramatic reduction in protein expression levels following neuronal differentiation, yet surprisingly, nCas9 constructs retained comparable protein expression between iPSCs and neurons. We further found that CRISPR constructs, primarily relying on the SV40 Nuclear Localization Signal (NLS), fail to efficiently localize to the nuclei of neurons, despite having robust nuclear levels in iPSCs, leading to KRAB(KOX1)-specific cytoplasmic degradation. By testing other NLSs, we were able to correct neuronal nuclear localization and protein expression, confirming the contribution of mislocalization to the instability of dCas9-KRAB(KOX1) in neurons. As the lack of nuclear localization can have a profound impact on editing and gene perturbation efficiency, we suggest further investigation across both cultured and in-vivo post-mitotic cell models.

The Darwin Tree of Life project has the goal to sequence the genomes of 70,000 species of eukaryotic organisms in Britain and Ireland. This is a collection of photographs of the samples included in the study, provided by the National History Museum (NHM).

This dataset contains raw 3D whole-mount multiplexed immunofluorescence imaging data from early zebrafish embryos used in the study “Multiplexed embryo profiling links cellular state to zygotic genome activation in single cells.” The dataset comprises 3D iterative in toto immunofluorescence imaging (3D-4i) of 212 embryos collected between 2 and 4 hours post-fertilization, spanning division cycles 7 to 12 during the major wave of zygotic genome activation. Embryos were imaged by automated spinning-disk confocal microscopy across four sequential staining rounds, including DAPI and markers of cell cycle progression, transcriptional activity, chromatin state, and pluripotency-associated factors (including PCNA, pH3, Pol-II-S2p, Pol-II-S5p, H3K27ac, H2B, Nanog, Alyref, β-catenin, and Pou5f3/FLAG).

Tara Expeditions are global scientific voyages that probe morphological and molecular diversity, evolution and ecology of marine plankton to explore how they are impacted by changes in the Earth's climate. The first expeditions collected samples of marine plankton containing viruses, bacteria, archaea, protists and planktonic metazoans living in the photic layer of the world's oceans. These expeditions, the first taking place between 2009 and 2013, include Tara Oceans: a global view, and Tara Oceans Polar Circle, both of which followed the same sampling protocol. This dataset includes 1074 pictures of 284 planktonic taxa (mainly microalgae and other Ciliate and Radiolarian protists) collected from the vicinity of the Marquesas Islands in the Southern Pacific Ocean during the Tara Oceans expedition. Multiple samples particularly of the size fractions 5-20 and 20 180 um from four sites and two depths were processed with different methods and studied in detail using scanning electron microscopy.