Cellular & Molecular Imaging

Raw data corresponding to the publication, "Cell surface localisation of GPI-anchored receptors in Trypanosoma brucei", published in eLife (https://doi.org/10.7554/eLife.107191.1)

Trypanosoma brucei bloodstream form tagging protein localisation data. Widefield epifluorescence microscope images of protein subcellular localisation in the bloodstream form life cycle stage of the unicellular eukaryotic pathogen Trypanosoma brucei by endogenous tagging with mNeonGreen (mNG). This deposition includes a summary of the localisations, primer sequences, provided in a directory structure analogous to the TrypTag genome-wide procyclic form project: https://doi.org/10.6019/S-BIAD1866 .

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.

Raw data corresponding to the publication "Continuous endosomes form functional subdomains and orchestrate rapid membrane trafficking in trypanosomes" published in eLife (https://doi.org/10.7554/eLife.91194.2).

Imaging dataset for PMID: 39314434

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

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.

Morphological modifications and shifts in organelle relationships are hallmarks of dormancy in eukaryotic cells. Communications between altered mitochondria and nuclei are associated with metabolic quiescence of cancer cells that can survive chemotherapy. In plants, changes in the pathways between nuclei, mitochondria, and chloroplasts are associated with cold stress and bud dormancy. Plasmodium falciparum parasites, the deadliest agent of malaria in humans, contain a chloroplast-like organelle (apicoplast) derived from an ancient photosynthetic symbiont. Antimalarial treatments can fail because a small fraction of the blood stage parasites enter dormancy and recrudesce after drug exposure. Altered mitochondrial-nuclear interactions in these persisters have been described for P. falciparum, but interactions of the apicoplast remained to be characterized. In the present study, we examined the apicoplasts of dormant persisters obtained after exposure to dihydroartemisinin (a first-line antimalarial drug) followed by sorbitol treatment, or after exposure to sorbitol treatment alone. As previously observed, the mitochondrion of persisters was consistently enlarged and in close association with the nucleus. In contrast, the apicoplast varied from compact and oblate, like those of active ring stage parasites, to enlarged and irregularly shaped. Enlarged apicoplasts became more prevalent later in dormancy, but regular size apicoplasts subsequently predominated when actively replicating parasites recrudesced. All three organelles, nucleus, mitochondrion, and apicoplast, became closer during dormancy. Understanding their relationships in erythrocytic-stage persisters may lead to new strategies to prevent recrudescences and protect the future of malaria chemotherapy.

Targeted protein degradation using conditional degron tags (CDTs) technologies is a powerful method for rapidly degrading a protein of interest (POI) upon the addition of a degrader drug. A prerequisite for the controlled degradation of an endogenous POI is the generation of homozygous knock-in cells that have the degron tag either integrated at the N- or C-terminus of their gene loci. However, obtaining those homozygous knock-in cells often requires selecting many single-cell clones, as human cells typically exhibit low homology-directed repair (HDR) activities. Additionally, tagging a degron to an endogenous protein may inadvertently impair the protein expression level, potentially affecting the protein function even before the drug is administered. Here, we develop a method for generating degron-tagged knock-in cells that allows us to skip the laborious single-cell cloning. The method was developed from our observation that most knock-in cells contain the degron tag only in one allele (heterozygous), while the other allele has a frame-shift insertion/deletion. This allowed us to bypass the need for single-cell cloning. We validated our method by knocking in degron tags at the N-terminus of cytoplasmic dynein1 subunits or Adaptor Protein 2 (AP2) subunit. Our experiments confirmed the rapid degradation of these proteins and their functional inhibition in bulk cell populations. To mitigate the reduced expression often associated with the degron tagging, we established a method to control expression levels by inserting a mini-promoter immediately upstream of the knock-in cassette. Our method simplifies the workflow for degron tag knock-in and enhances the versatility of these valuable technologies.