Cellular & Molecular Imaging

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.

Gene regulation by RNA interference (RNAi) is a conserved process driven by double-stranded RNA (dsRNA). It responds to exogenous cues and drives endogenous gene regulation. In Caenorhabditis elegans, RNAi can be inherited from parents to offspring. While a number of factors have been implicated in this inheritance process, we do not understand how and when they function. Using a new inheritance assay, we establish a hierarchy amongst previously identified inheritance factors. The nuclear Argonaute protein HRDE-1 was required for RNAi establishment in parents and offspring, but not for the inheritance process. In contrast, the cytoplasmic Argonaute protein WAGO-3 was the only factor essential for inheritance, via sperm and oocyte, while not affecting establishment in either parent or offspring. We propose a cycle between nuclear and cytoplasmic Argonaute proteins, where nuclear activity drives most of the silencing and cytoplasmic activity ensures inheritance. Finally, we implicate the RNA helicase ZNFX-1 as a factor that links the inherited WAGO-3 protein to nuclear RNAi in the offspring.

Plasmodium parasites, the causative agents of malaria, undergo complex replication within vertebrate and insect hosts, presenting unique opportunities for therapeutic intervention. A key challenge during these replication events, i.e., schizogony in vertebrate red blood cells and sporogony in oocysts within mosquitos, is ensuring the faithful partitioning of nuclei and organelles into the numerous daughter cells that form at once from a single parent. While nuclear microtubule-organizing centers, or centriolar plaques (CPs), have been hypothesized to play a central role in this process, the molecular mediators linking the CPs and organelles remain incompletely defined. Here, we characterize two striated fiber assemblin (SFA) homologs, SFA1 and SFA2, in Plasmodium falciparum and Plasmodium berghei across two hosts. We show that these SFAs form a physical bridge between the CP and the nascent apical poles of daughter cells, facilitating high-fidelity progeny formation during schizogony and sporogony. Loss of SFA function disrupts merozoite and sporozoite formation, with profound consequences for transmission. These findings establish SFAs as essential organizers of parasite morphogenesis and highlight them as potential targets for antimalarial therapies. This submission includes the source microscopy image data for experiments performed in P. falciparum.

OME-NGFF converted study from idr0090. Drug resistance threatens the effective prevention and treatment of an ever-increasing range of human infections. This highlights an urgent need for new and improved drugs with novel mechanisms of action to avoid cross-resistance. Current cell-based drug screens are, however, restricted to binary live/dead readouts with no provision for mechanism of action prediction. Machine learning methods are increasingly being used to improve information extraction from imaging data. Such methods, however, work poorly with heterogeneous cellular phenotypes and generally require time-consuming human-led training. We have developed a semi-supervised machine learning approach, combining human- and machine-labelled training data from mixed human malaria parasite cultures. Designed for high-throughput and high-resolution screening, our semi-supervised approach is robust to natural parasite morphological heterogeneity and correctly orders parasite developmental stages. Our approach also reproducibly detects and clusters drug-induced morphological outliers by mechanism of action, demonstrating the potential power of machine learning for accelerating cell-based drug discovery.

to be described

Immunofluorescence microscopy images supporting expression of HA-tagged PfMD3 in specific stages of P. falciparum development

Protein synthesis is a core cellular process, necessary throughout the complex lifecycle of Plasmodium parasites, thus specific translation inhibitors would be a valuable class of antimalarial drugs, capable of both treating symptomatic infections in the blood and providing chemoprotection by targeting the initial parasite population in the liver, preventing both human disease and parasite transmission back to the mosquito host. As increasing numbers of antiplasmodial compounds are identified that converge mechanistically at inhibition of cytoplasmic translation, regardless of molecular target or mechanism, it would be useful to gain deeper understanding of how their effectiveness as liver stage translation inhibitors relates to their chemoprotective potential. Here, we probed that relationship using the P. berghei-HepG2 liver stage infection model. Using o-propargyl puromycin-based labeling of the nascent proteome in P. berghei-infected HepG2 monolayers coupled with automated confocal feedback microscopy to generate unbiased, single parasite image sets of P. berghei liver stage translation, we determined translation inhibition EC50s for five compounds, encompassing parasite-specific aminoacyl tRNA synthetase inhibitors, compounds targeting the ribosome in both host and parasite, as well as DDD107498, which targets Plasmodium eEF2, and is a leading antimalarial candidate compound being clinically developed as cabamiquine. Compounds were then tested at equivalent effective concentrations to compare the parasite response to, and recovery from, a brief period of translation inhibition in early schizogony.

This dataset includes microscopy and sequence data related to the study and used in the figures. It includes light and electron microscopy images. The sample preparation, image acquisition and analysis protocols are described in the methods.

to be described

Fluorescence imaging across the near-infrared (NIR, 700–1000 nm) and shortwave infrared (SWIR, 1000–2000 nm) regions offers significant advantages for biomedical applications, yet photophysical enhancements achieved with NIR and SWIR chromophores observed in solution often fail to translate to complex biological environments. Fluorous-soluble fluorophores, fluorofluorophores, face additional challenges, exhibiting poor brightness and photostability when dissolved in perfluorocarbons (PFCs) due to unfavorable interactions with the fluorous phase. Here, we report counterion exchange as a strategy to enhance the photophysical properties of two heptamethine cyanine fluorofluorophore for NIR and SWIR imaging. Exchanging the small chloride counterion with a large, fluorinated aryl borate counterions significantly improved the brightness (10-fold) and photostability (57-fold) in PFCs. These enhancements were successfully translated across multiple biological systems from macrophage cells to NIR imaging zebrafish retinal tissue and finally to SWIR imaging in mice. These results demonstrate that strategic counterion modification provides a straightforward approach to optimize fluorofluorophores, with solution-phase improvements that translate to in vivo NIR and SWIR imaging.