Cellular & Molecular Imaging

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.

Imaging dataset for PMID: 39314434

This submission includes 3D images of fixed Arabidopsis thaliana ovules at all developmental stages acquired by confocal laser scanning microscopy. It also contains the corresponding segmentation masks for cell boundaries.

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.

A hallmark of microbial biofilms is the self-production of extracellular matrix that encases the cells resident within the community. The matrix provides protection from the environment, while spatial heterogeneity of expression influences the structural morphology and colony spreading dynamics. Bacillus subtilis is a model bacterial system used to uncover the regulatory pathways and key building blocks required for biofilm growth and development. Previous reports have suggested that poly-?-glutamic acid (PGA) production is suppressed during biofilm formation and does not play a major role in biofilm morphology of the undomesticated isolate NCIB 3610. In this work we report on the observation of multiple travelling fronts that develop during the early stage of B. subtilis colony biofilm formation. We find the emergence of a highly motile population of bacteria that is facilitated by the extraction of fluid from the underlying agar substrate. Motility develops behind a moving front of fluid that propagates from the boundary of the biofilm towards the interior. The extent of proliferation is strongly modulated by the presence of extracellular polysaccharides (EPS). We trace the origin of this moving front of fluid to the production of PGA. We find that PGA production is correlated with higher temperatures, resulting in a mature biofilm morphology that is distinct from the biofilm architecture typically associated with B. subtilis. Our results suggest that B. subtilis NCIB 3610 produces distinct biofilm matrices in response to environmental conditions.

All images used to generate the figures of this manuscript

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.

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.

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.