Cellular & Molecular Imaging

Phytoplankton is a minor fraction of the global biomass playing a major role in primary production and climate. Despite improved understanding of phytoplankton diversity and genomics, we lack nanoscale subcellular imaging approaches to understand their physiology and cell biology. Here, we present a complete Focused Ion Beam - Scanning Electron Microscopy (FIB-SEM) workflow (from sample preparation to image processing) to generate nanometric 3D phytoplankton models. Tomograms of entire cells, representatives of six ecologically-successful phytoplankton unicellular eukaryotes, were used for quantitative morphometric analysis. Besides lineage-specific cellular architectures, we observed common features related to cellular energy management: i) conserved cell-volume fractions occupied by the different organelles; ii) consistent plastid-mitochondria interactions, iii) constant volumetric ratios in these energy-producing organelles. We revealed detailed subcellular features related to chromatin organization and to biomineralization. Overall, this approach opens new perspectives to study phytoplankton acclimation responses to abiotic and biotic factors at a relevant biological scale.

Multiplexed immunofluorescence images (4 stain, nuclear and cytoplasmic/membrane markers), single-cell segmentation, and tissue annotations for whole slide, developmental human manual digits.

All symptoms of malaria disease are associated with the asexual blood stages of development, involving cycles of red blood cell (RBC) invasion and egress by the Plasmodium spp. merozoite. Merozoite invasion is rapid and is actively powered by a parasite actomyosin motor. The current accepted model for actomyosin force generation envisages arrays of parasite myosins, pushing against short actin filaments connected to the external milieu that drive the merozoite forwards into the RBC. In Plasmodium falciparum, the most virulent human malaria species, Myosin A (PfMyoA) is critical for parasite replication. However, the precise function of PfMyoA in invasion, its regulation, the role of other myosins and overall energetics of invasion remain unclear. Here, we developed a conditional mutagenesis strategy combined with live video microscopy to probe PfMyoA function and that of the auxiliary motor PfMyoB in invasion. By imaging conditional mutants with increasing defects in force production, based on disruption to a key PfMyoA phospho-regulation site, the absence of the PfMyoA essential light chain, or complete motor absence, we define three distinct stages of incomplete RBC invasion. These three defects reveal three energetic barriers to successful entry: RBC deformation (pre-entry), mid-invasion initiation, and completion of internalisation, each requiring an active parasite motor. In defining distinct energetic barriers to invasion, these data illuminate the mechanical challenges faced in this remarkable process of protozoan parasitism, highlighting distinct myosin functions and identifying potential targets for preventing malaria pathogenesis.

This dataset comprises transmission electron microscopy (TEM) images of villus enterocytes from the distal ileum and jejunal-ileal junction of adult mice with conditional inactivation of caudal-family transcription factors. It includes control, intestine-specific Cdx2 knockout, Cdx1 knockout, and Cdx1/Cdx2 double-knockout samples, acquired to examine enterocyte ultrastructure. One-centimetre segments of distal ileum were fixed, embedded in Taab 812 resin, thin-sectioned (95 nm), stained with lead citrate, and imaged on a JEOL 1200 transmission electron microscope at 80 kV. Images are organised by experiment (Cdx2 KO; Cdx1/Cdx2 double KO) and by sample/genotype; scale bars are present in the images. The dataset is associated with Verzi et al., Molecular and Cellular Biology 2011 (PMID 21402776).

In multicellular organisms, tissue architecture reflects not only dedicated patterning genes but the integrated state of core cellular physiology. Whether the same holds for the communities that bacteria build has been difficult to test at scale. To this end, we developed µPULLI, a high-throughput platform that pairs low-magnification, label-free brightfield timelapse microscopy with computer vision. Using µPULLI, we screened a genome-wide Vibrio cholerae transposon library. Our screen revealed that community architecture is governed as much by core physiology as by dedicated biofilm genes: perturbations to central metabolism, cofactor biosynthesis, and cell-envelope architecture each leave distinctive, pathway-specific fingerprints on how a community develops. These fingerprints arise through both transcriptional and non-transcriptional mechanisms, including cell-surface changes invisible to RNA sequencing. Small molecules drive communities predictably through this phenotype space, and the principle holds across taxonomically diverse pathogens. Emergent community architecture thus encodes the integrated physiological state of its constituent cells, revealing a rich, largely untapped layer of biological information that can be read from simple, low-magnification brightfield videos of growing cultures.

Imaging dataset for PMID: 39314434

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.

Plasmodium oocysts within the mosquito midgut epithelium produce sporozoites, the stage which is infective for the vertebrate host. The formation process of these sporozoites is termed sporogony and is still poorly understood. During sporogony a 30 nm thick longitudinal fiber, the rootlet fiber, connects the centriolar plaque of the nucleus to the apical polar ring at the tip of the forming sporozoite. It is hypothesized to play a role in trafficking secretory organelles and nuclear uptake into the newly forming sporozoite bud. A similar fiber was previously observed in Toxoplasma gondii which is formed by two striated fiber assemblins (SFAs). Here we characterize the two SFA homologs in P. berghei (PbSFA1/PBANKA_1227000, PbSFA2/PBANKA_1012000) via fluorescent tagging, confirming a fiber-like localization at the tips of nascent sporozoites in oocysts. Knockout of either SFA gene lead to significant reduction in oocyst numbers and a complete failure to produce any sporozoites. Upon investigation by electron microscopy and electron tomography, initiation of sporozoite formation could be observed but the rootlet fiber was absent in sporozoite buds. Strikingly, nuclear uptake was strongly affected, as no nuclei could be observed in elongated sporozoite buds. Further, elongated sporozoite buds failed to bud off, ultimately creating a network of incompletely formed ‘sporozoites’. These findings emphasize the role of the rootlet fiber in nuclear uptake und organization of sporozoite formation, making them essential for the progression through the mosquito.

Multiple myeloma involves early dissemination of malignant plasma cells across the bone marrow; however, the initial steps of dissemination remain unclear. Human bone marrow-derived mesenchymal stromal cells (hMSCs) stimulate myeloma cell expansion (e.g., IL-6) and simultaneously retain myeloma cells via chemokines (e.g., CXCL12) and adhesion factors. Hence, we hypothesized that the imbalance between cell division and retention drives dissemination. We present an in vitro model using primary hMSCs co-cultured with INA-6 myeloma cells. Time-lapse microscopy revealed proliferation and attachment/detachment dynamics. Separation techniques (V-well adhesion assay and well plate sandwich centrifugation) were established to isolate MSC-interacting myeloma subpopulations that were characterized by RNAseq, cell viability and apoptosis. Results were correlated with gene expression data (n=837) and survival of myeloma patients (n=536). On dispersed hMSCs, INA-6 saturate hMSC-surface before proliferating into large homotypic aggregates, from which single cells detached completely. On confluent hMSCs, aggregates were replaced by strong heterotypic hMSC-INA-6 interactions, which modulated apoptosis time-dependently. Only INA-6 daughter cells (nMA-INA6) detached from hMSCs by cell division but sustained adherence to hMSC-adhering mother cells (MA-INA6). Isolated nMA-INA6 indicated hMSC-autonomy through superior viability after IL­6 withdrawal and upregulation of proliferation-related genes. MA-INA6 upregulated adhesion and retention factors (CXCL12), that, intriguingly, were highly expressed in myeloma samples from patients with longer overall and progression-free survival, but their expression decreased in relapsed myeloma samples. Altogether, in vitro dissemination of INA-6 is driven by detaching daughter cells after a cycle of hMSC-(re)attachment and proliferation, involving adhesion factors that represent a bone marrow-retentive phenotype with potential clinical relevance.

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.