Cellular & Molecular Imaging

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.

The strains LEGE 06306, LEGE 06307, LEGE 11436, and LEGE 10375, isolated from northern Portugal, were characterized using a polyphasic taxonomic approach that included 16S rRNA gene phylogenetic analyses (ML and BI), 16S-23S ITS secondary structures, p-distance calculations, phylogenomics, morphological and ultrastructural (TEM) observations, MALDI-TOF MS profiling, as well as ecological and biochemical characterization. Although LEGE 06306, LEGE 06307, and LEGE 11436 share high 16S rRNA gene sequence similarity and display very similar morphology, the three ML and BI phylogenetic analyses revealed that these strains form three distinct lineages within the family Prochlorococcaceae, without consistent affiliation to any previously described genera. The phylogenomic analysis, ITS comparisons, and the biochemical statistical comparisons further supported the separation of these lineages. MALDI-TOF analysis revealed unique spectral profiles for each strain, while their pigment compositions were similar but varied in pigment concentrations, supporting their distinction. Based on these combined results, the three strains are described as representatives of three new cyanobacterial genera. Strain LEGE 10375 showed an uncertain placement in both the 16S rRNA and whole-genome phylogenies and therefore could not be taxonomically assigned. The high content of bioactive metabolites of the studied strains, including pigments, phenolic compounds, and sugars, positions them as sustainable sources for functional ingredients with potential applications in the food, feed, and pharmaceutical industries.

This dataset contains comprehensive image-based morphological profiling data from a systematic drug repurposing screen for host-directed antivirals. The repository includes three major study components. The primary screen employed high-content Cell Painting analysis combined with viral immunostaining of 5,275 repurposable compounds screened against SARS-CoV-2 infection in Vero E6 monkey kidney cells. This initial morphological profiling identified compounds that reversed the infected cell phenotype towards non-infected controls. Hit validation was performed through dose-response morphological profiling experiments by Cell Painting and antibody staining in human A549 lung epithelial cells engineered to express the ACE2 receptor, confirming antiviral activity in a human-relevant cell model. A counter screen assessed compound antiviral activity that could be attributed to drug-induced phospholipidosis (DIPL) instead of specific engagement of viral or host targets. This component includes high-content data from human A549-ACE2 cells stained with HCS LipidTOX Green Phospholipidosis Detection Reagent, CellTracker Deep Red Dye, and Hoechst 33342. The dataset includes raw images and extracted morphological features. This resource enables identification of host-targeting antivirals with potential broad-spectrum activity and supports future pandemic preparedness efforts.

Indocyanine Green (ICG) has seen widespread use in the operating room as a fluorescent vascular imaging agent. However, its rapid vascular clearance often necessitates redosing, resulting in procedural delays, greater background, and potential adverse physiological effects. Here, we present Chromenylium Green (ChromG), a novel fluorescent tracer exhibiting excellent visualization of vasculature in mice with greater contrast and seven times the vascular half-life relative to ICG. ChromG allows for high-resolution imaging of vasculature at greater depths, enabling whole-body 3D vascular reconstruction with up to 74% connectivity. As a proof of concept of ChromG’s utility in disease contexts, we demonstrate the ability to visualize saphenous artery stenoses above the clinically accepted contrast limit for eight times longer than ICG. With its excellent biocompatibility profile and compatibility with current clinical imaging technologies, ChromG is a promising fluorescent probe for prolonged, high-resolution intraoperative vascular imaging.

All images used to generate the figures of this manuscript

Bacteria occupy heterogeneous environments, attaching and growing within pores in materials, living hosts, and matrices like soil. Systems that permit high-resolution visualization of dynamic bacterial processes within the physical confines of a realistic and tractable porous media environment are rare. Here we use microfluidics to replicate the grain shape and packing density of natural sands in a 2D platform to study the flow-induced spatial evolution of bacterial biofilms underground. We introduce a wildtype strain (Pantoea sp. YR343, n=3) or an EPS-defective strain (Pantoea sp. YR343 ΔUDP, n=3) to the porous media platform and then simulate a rainfall event using gravity-driven flow of bacterial growth media.

Cell state transitions underlie the emergence of diverse cell types and are traditionally defined by changes in gene expression. Yet these transitions also involve coordinated shifts in cell morphology and behaviour, which remain poorly characterized in densely packed epithelia. We developed a quantitative live-imaging and computational framework to track thousands of individual cells over time in the rapidly differentiating Xenopus mucociliary epithelium (MCE). From segmentations and trajectories, we extracted dynamic features—cell and nuclear shape, movement, and position—to create a time-resolved morphodynamic dataset spanning the full course of differentiation. While single features showed high noise and low separability of ground-truth cell types, supervised machine learning revealed that integrating time-resolved features robustly predicts final cell fate. Gradient-boosted trees and multinomial logistic regression achieved moderate but consistent accuracy, especially for abundant epithelial lineages. Key discriminants included normalized Z position, membrane–nucleus offset, and absolute experimental time, whereas movement contributed minimally to the results. Our data show that morphodynamic signatures encode predictive information about cell identity and provide a framework linking cellular dynamics with molecular state.

Preventing parasite transmission from humans to mosquitoes is recognised to be critical for achieving elimination and eradication of malaria. Consequently developing new antimalarial drugs with transmission-blocking properties is a priority. Large screening campaigns have identified many new transmission-blocking molecules, however little is known about how they target transmissible Plasmodium falciparum stage V gametocytes, or how they affect their underlying cell biology. To respond to this knowledge gap, we have developed a machine learning image analysis pipeline to characterise and compare the cellular phenotypes generated by transmission-blocking molecules during male gametogenesis. Using this approach, we studied 40 molecules, categorising their activity based upon timing of action and visual effects on the organisation of tubulin and DNA in the cell. Our data both proposes new modes of action and corroborates existing modes of action of identified transmission-blocking molecules. Furthermore, the characterised molecules provide a new armoury of tool compounds to probe gametocyte cell biology and the generated imaging dataset provides a new reference for researchers to correlate molecular target or gene deletion to specific cellular phenotype. Our analysis pipeline is not optimised for a specific organism and could be applied to any fluorescence microscopy dataset containing cells delineated by bounding boxes, and so is extendible to any disease model.

The ability of a single protein to trigger different functions is an assumed key feature of cell signaling, yet there are very few examples demonstrating it. Here, using an optogenetic tool to control membrane localization of RhoA nucleotide exchange factors (GEFs), we present a case where the same protein can trigger both protrusion and retraction when recruited to the plasma membrane, polarizing the cell in two opposite directions. We show that the basal concentration of the GEF prior to activation predicts the resulting phenotype. A low concentration leads to retraction, whereas a high concentration triggers protrusion. This unexpected protruding behavior arises from the simultaneous activation of Cdc42 by the GEF and sequestration of active RhoA by the GEF PH domain at high concentrations. We propose a minimal model that recapitulates the phenotypic switch, and we use its predictions to control the two phenotypes within selected cells by adjusting the frequency of light pulses. Our work exemplifies a unique case of control of antagonist phenotypes by a single protein that switches its function based on its concentration or dynamics of activity. It raises numerous open questions about the link between signaling protein and function, particularly in contexts where proteins are highly overexpressed, as often observed in cancer.

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).