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Annotation of distinct elements in the apical complex of Toxoplasma tachyzoites using a mixed-scale dense neural network. (A) A representative slice from a tomogram that was not used in the training data and that is shown annotated in (B) and (C). Scale bar, 200 nm. (B) Annotation produced by the neural network after different iterative rounds of training (see results section for details) overlaid on the tomographic slice from (A) showing the AVs (yellow), rhoptries and micronemes (red), and microtubules (blue). Note the small but marked improvement in the annotation accuracy of the AVs, rhoptry, and IMT with each additional training as pointed by the arrows. (C) Manual correction (cyan) of the NN annotation after training #3. A zoomed-in view of the square in the left panel is shown in the right panel. Scale bar for the zoomed-in view, 50 nm.
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Host cell invasion by intracellular, eukaryotic parasites within the phylum Apicomplexa, is a remarkable and active process involving the coordinated action of apical organelles and other structures. To date, capturing how these structures interact during invasion has been difficult to observe in detail. Here, we used cryogenic electron tomography...
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Intracellular infectious agents, like the malaria parasite, Plasmodium falciparum , face the daunting challenge of how to invade a host cell. This problem may be even harder when the host cell in question is the enucleated red blood cell. Evolution has provided P. falciparum and related single-celled parasites within the phylum Apicomplexa with a c...
Citations
... The second one is associated with three to five aligned vesicles interconnected by linker densities [6,8,54]. These MT-associated vesicles (MVs) resemble the AV in terms of size, shape, density, and coating [8,68]. Remarkably, T. gondii tachyzoites contain eight to 12 rhoptries and are able to undergo multiple rounds of rhoptry secretion, delivering their contents into uninfected host cells [69]. ...
... Interestingly, in the absence of ICMAP2, an MV was found in contact with the parasite plasma membrane and containing a rosette-like structure. This supports the notion that MVs may function in reloading the subsequent AV, enabling the sequential discharge of rhoptries [8,68]. The gene encoding ICMAP3 undergoes alternative splicing to generate two isoforms: ICMAP3 I/II . ...
Microtubules (MTs) play a vital role as key components of the eukaryotic cytoskeleton.
The phylum Apicomplexa comprises eukaryotic unicellular parasitic organisms
defined by the presence of an apical complex which consists of specialized
secretory organelles and tubulin-based cytoskeletal elements. One apicomplexan
parasite, Toxoplasma gondii, is an omnipresent opportunistic pathogen with
significant medical and veterinary implications. To ensure successful infection
and widespread dissemination, T. gondii heavily relies on the tubulin structures
present in the apical complex. Recent advances in high-resolution imaging,
coupled with reverse genetics, have offered deeper insights into the composition,
functionality, and dynamics of these tubulin-based structures. The apicomplexan
tubulins differ from those of their mammalian hosts, endowing them with unique
attributes and susceptibility to specific classes of inhibitory compounds.
... The organization and composition of the conoid tubulin fibers and SPMTs were described at an unprecedented level of resolution [27][28][29][30] . Specifically, cryo-ET unraveled details of the machinery involved in rhoptry discharge 20,23,31,32 . The necks of the two most apical rhoptries were seen near the ICMTs, inside the conoid, and docking to an apical vesicle (AV), which in turn was docked to the parasite plasma membrane. ...
In Apicomplexa, rhoptry discharge is essential for invasion and involves an apical vesicle (AV) docking one or two rhoptries to a macromolecular secretory apparatus. Toxoplasma gondii is armed with 10–12 rhoptries and 5-6 microtubule-associated vesicles (MVs) presumably for iterative rhoptry discharge. Here, we have addressed the localization and functional significance of two intraconoidal microtubule (ICMT)-associated proteins instrumental for invasion. Mechanistically, depletion of ICMAP2 leads to a dissociation of the ICMTs, their detachment from the conoid and dispersion of MVs and rhoptries. ICMAP3 exists in two isoforms that contribute to the control of the ICMTs length and the docking of the two rhoptries at the AV, respectively. This study illuminates the central role ICMTs play in scaffolding the discharge of multiple rhoptries. This process is instrumental for virulence in the mouse model of infection and in addition promotes sterile protection against T. gondii via the release of key effectors inducing immunity.
... TgFER2, a conoid-associated Ca 2+binding protein, localizes to the cytosolic surface of the rhoptries and is essential for their discharge (Coleman et al, 2018). Moreover, treatment of parasites with Ca 2+ ionophores leads to apparent fusion between the apical vesicle and the docked rhoptries (Segev-Zarko et al, 2022). The common reliance of both micronemes and rhoptries on Ca 2+ signaling complicates deconvolution of the series of events that mediate invasion. ...
Apicomplexan parasites discharge specialized organelles called rhoptries upon host cell contact to mediate invasion. The events that drive rhoptry discharge are poorly understood, yet essential to sustain the apicomplexan parasitic life cycle. Rhoptry discharge appears to depend on proteins secreted from another set of organelles called micronemes, which vary in function from allowing host cell binding to facilitation of gliding motility. Here we examine the function of the microneme protein CLAMP, which we previously found to be necessary for Toxoplasma gondii host cell invasion, and demonstrate its essential role in rhoptry discharge. CLAMP forms a distinct complex with two other microneme proteins, the invasion‐associated SPATR, and a previously uncharacterized protein we name CLAMP‐linked invasion protein (CLIP). CLAMP deficiency does not impact parasite adhesion or microneme protein secretion; however, knockdown of any member of the CLAMP complex affects rhoptry discharge. Phylogenetic analysis suggests orthologs of the essential complex components, CLAMP and CLIP, are ubiquitous across apicomplexans. SPATR appears to act as an accessory factor in Toxoplasma , but despite incomplete conservation is also essential for invasion during Plasmodium falciparum blood stages. Together, our results reveal a new protein complex that mediates rhoptry discharge following host‐cell contact.
... Intracellular Ca 2+ release is necessary and sufficient to trigger the rapid trafficking and exocytosis of micronemes (Carruthers et al., 1999a(Carruthers et al., , 1999bEndo et al., 1982 ;Sidik et al., 2016b ). While Ca 2+ is also necessary for rhoptry discharge, discharge relies on additional cellular processes such as microneme exocytosis (Coleman et al., 2018 ;Segev-Zarko et al., 2022 ). While the exocytosis of micronemes and rhoptries is known to be critical for parasite motility, the mechanisms linking Ca 2+ signaling to their trafficking and fusion to the plasma membrane are still unclear. ...
... However, formally demonstrating the relationship between Ca 2+ /CDPK1 and rhoptry discharge is complicated by the dependency of the latter on the secretion of certain microneme proteins (Ben Chaabene et al., 2021 ;Carruthers and Sibley, 1997 ). T. gondii tachyzoites have several rhoptries, yet only two are docked for exocytosis at a given time (Aquilini et al., 2021 ;Mageswaran et al., 2021 ;Segev-Zarko et al., 2022 ). Regulating the activity of ARO during motile stages may influence the ability to mobilize and re-dock rhoptries in preparation for invasion. ...
Apicomplexan parasites use Ca2+-regulated exocytosis to secrete essential virulence factors from specialized organelles called micronemes. Ca2+-dependent protein kinases (CDPKs) are required for microneme exocytosis; however, the molecular events that regulate trafficking and fusion of micronemes with the plasma membrane remain unresolved. Here, we combine sub-minute resolution phosphoproteomics and bio-orthogonal labeling of kinase substrates in Toxoplasma gondii to identify 163 proteins phosphorylated in a CDPK1-dependent manner. In addition to known regulators of secretion, we identify uncharacterized targets with predicted functions across signaling, gene expression, trafficking, metabolism, and ion homeostasis. One of the CDPK1 targets is a putative HOOK activating adaptor. In other eukaryotes, HOOK homologs form the FHF complex with FTS and FHIP to activate dynein-mediated trafficking of endosomes along microtubules. We show the FHF complex is partially conserved in T. gondii, consisting of HOOK, an FTS homolog, and two parasite-specific proteins (TGGT1_306920 and TGGT1_316650). CDPK1 kinase activity and HOOK are required for the rapid apical trafficking of micronemes as parasites initiate motility. Moreover, parasites lacking HOOK or FTS display impaired microneme protein secretion, leading to a block in the invasion of host cells. Taken together, our work provides a comprehensive catalog of CDPK1 targets and reveals how vesicular trafficking has been tuned to support a parasitic lifestyle.
... Defining the components and function of the Toxoplasma conoid The past few years have seen tremendous efforts to define the apical end of Toxoplasma tachyzoites in incredible detail, using a variety of imaging modalities(Li et al., 2023;Mageswaran et al., 2021;Segev-Zarko et al., 2022;Sun et al., 2022). At the apical end of a tachyzoite is the conoid, a barrel-shaped structure comprised angled tubulin fibres and containing a further two intraconoidal microtubules (Dos Santos Pacheco et al., 2020). ...
Apicomplexan parasites comprise significant pathogens of humans, livestock and wildlife, but also represent a diverse group of eukaryotes with interesting and unique cell biology. The study of cell biology in apicomplexan parasites is complicated by their small size, and historically this has required the application of cutting‐edge microscopy techniques to investigate fundamental processes like mitosis or cell division in these organisms. Recently, a technique called expansion microscopy has been developed, which rather than increasing instrument resolution like most imaging modalities, physically expands a biological sample. In only a few years since its development, a derivative of expansion microscopy known as ultrastructure‐expansion microscopy (U‐ExM) has been widely adopted and proven extremely useful for studying cell biology of Apicomplexa. Here, we review the insights into apicomplexan cell biology that have been enabled through the use of U‐ExM, with a specific focus on Plasmodium, Toxoplasma and Cryptosporidium. Further, we summarize emerging expansion microscopy modifications and modalities and forecast how these may influence the field of parasite cell biology in future.
... The invasive stages of most apicomplexans, including T. gondii and C. parvum, are highly polarized and contain an ensemble of apically localized structures known as the apical complex that participates in gliding motility [18][19][20][21] . Traditional electron microscopy (EM) 22,23 and cryo-electron tomography (cryo-ET) 19,20,[24][25][26] studies have revealed the apical complex to contain a unique structure called the conoid, consisting of a cone-like arrangement of tubulin fibers and two associated rings above called the preconoidal rings (PCRs). Interestingly, when the parasite moves, the conoid extrudes through the apical polar ring (APR) 22,[27][28][29] at the collar of the inner membrane complex (IMC; a network of flattened alveolar sacs underneath the parasite plasma membrane). ...
The phylum Apicomplexa comprises important eukaryotic parasites that invade host tissues and cells using a unique mechanism of gliding motility. Gliding is powered by actomyosin motors that translocate host-attached surface adhesins along the parasite cell body. Actin filaments (F-actin) generated by Formin1 play a central role in this critical parasitic activity. However, their subcellular origin, path and ultrastructural arrangement are poorly understood. Here we used cryo-electron tomography to image motile Cryptosporidium parvum sporozoites and reveal the cellular architecture of F-actin at nanometer-scale resolution. We demonstrate that F-actin nucleates at the apically positioned preconoidal rings and is channeled into the pellicular space between the parasite plasma membrane and the inner membrane complex in a conoid extrusion-dependent manner. Within the pellicular space, filaments on the inner membrane complex surface appear to guide the apico-basal flux of F-actin. F-actin concordantly accumulates at the basal end of the parasite. Finally, analyzing a Formin1-depleted Toxoplasma gondii mutant pinpoints the upper preconoidal ring as the conserved nucleation hub for F-actin in Cryptosporidium and Toxoplasma. Together, we provide an ultrastructural model for the life cycle of F-actin for apicomplexan gliding motility.
... Intracellular Ca 2+ release is necessary and sufficient to trigger the rapid trafficking and exocytosis of micronemes (Carruthers et al., 1999a;Endo et al., 1982;. While Ca 2+ is also necessary for rhoptry discharge, exocytosis relies on additional cellular processes such as microneme exocytosis (Coleman et al., 2018;Segev-Zarko et al., 2022). While the exocytosis of micronemes and rhoptries is known to be critical for parasite motility, the mechanisms linking Ca 2+ signaling to their trafficking and fusion to the plasma membrane are still unclear. ...
... Phosphorylation on ARO was observed near the N-terminal acylation sites required for rhoptry targeting and may regulate the bundling and positioning of mature rhoptries during motile stages (Mueller et al., 2016). However, formally demonstrating the relationship between Ca 2+ /CDPK1 and rhoptry discharge is complicated only two are docked for exocytosis at a given time (Aquilini et al., 2021;Mageswaran et al., 2021;Segev-Zarko et al., 2022). Regulating the activity of ARO during motile stages may influence the ability to mobilize and re-dock rhoptries in preparation for invasion. ...
Apicomplexan parasites use Ca2+-regulated exocytosis to secrete essential virulence factors from specialized organelles called micronemes. Ca2+-dependent protein kinases (CDPKs) are required for microneme exocytosis; however, the molecular events that regulate trafficking and fusion of micronemes with the plasma membrane remain unresolved. Here, we combine sub-minute resolution phosphoproteomics and bio-orthogonal labeling of kinase substrates in Toxoplasma gondii to identify 163 proteins phosphorylated in a CDPK1-dependent manner. In addition to known regulators of secretion, we identify uncharacterized targets with predicted functions across signaling, gene expression, trafficking, metabolism, and ion homeostasis. One of the CDPK1 targets is a putative HOOK activating adaptor. In other eukaryotes, HOOK homologs form the FHF complex with FTS and FHIP to activate dynein-mediated trafficking of endosomes along microtubules. We show the FHF complex is partially conserved in T. gondii, consisting of HOOK, an FTS homolog, and two parasite-specific proteins (TGGT1_306920 and TGGT1_316650). CDPK1 kinase activity and HOOK are required for the rapid apical trafficking of micronemes as parasites initiate motility. Moreover, parasites lacking HOOK or FTS display impaired microneme protein secretion, leading to a block in the invasion of host cells. Taken together, our work provides a comprehensive catalog of CDPK1 targets and reveals how vesicular trafficking has been tuned to support a parasitic lifestyle.
... The mixed-scale dense network (MSDNet) [7] was developed as a deep learning framework for image classification and pixel-by-pixel segmentation tasks with a relatively simple architecture containing roughly two to three orders of magnitude fewer trainable parameters than U-Nets [8] and other typical encoder-decoder convolutional neural networks [9,10]. MSDNets have proven effective and been tested in several use cases for tomographic reconstruction [11,12,13,14], nano-CT denoising [15], segmentation of sub-nuclear structures in focused-ion beam scanning electron microscopy (FIB-SEM) [16], X-ray scattering imaging inpainting [17], and X-ray in-line phase contrast imaging [18]. ...
Scientific user facilities present a unique set of challenges for image processing due to the large volume of data generated from experiments and simulations. Furthermore, developing and implementing algorithms for real-time processing and analysis while correcting for any artifacts or distortions in images remains a complex task, given the computational requirements of the processing algorithms. In a collaborative effort across multiple Department of Energy national laboratories, the "MLExchange" project is focused on addressing these challenges. MLExchange is a Machine Learning framework deploying interactive web interfaces to enhance and accelerate data analysis. The platform allows users to easily upload, visualize, label, and train networks. The resulting models can be deployed on real data while both results and models could be shared with the scientists. The MLExchange web-based application for image segmentation allows for training, testing, and evaluating multiple machine learning models on hand-labeled tomography data. This environment provides users with an intuitive interface for segmenting images using a variety of machine learning algorithms and deep-learning neural networks. Additionally, these tools have the potential to overcome limitations in traditional image segmentation techniques, particularly for complex and low contrast images.
... TgFER2, a conoid-associated Ca 2+ -binding protein, localizes to the cytosolic surface of the rhoptries and is essential for their discharge (Coleman et al, 2018). Moreover, treatment of parasites with Ca 2+ ionophores leads to apparent fusion between the apical vesicle and the docked rhoptries (Segev-Zarko et al, 2022). The common reliance of both micronemes and rhoptries on Ca 2+ signaling complicates deconvolution of the series of events that mediate invasion. ...
Apicomplexan parasites discharge specialized organelles called rhoptries upon host cell contact to mediate invasion. The events that drive rhoptry discharge are poorly understood, yet essential to sustain the apicomplexan parasitic life cycle. Rhoptry discharge appears to depend on proteins secreted from another set of organelles called micronemes, which in Toxoplasma gondii includes MIC8 and the microneme-associated CRMP complex. Here, we examine the function of the microneme protein CLAMP, uncovering its essential role in rhoptry discharge. CLAMP forms a distinct complex with two other microneme proteins, the invasion-associated SPATR, and a previously uncharacterized protein we name CLAMP-linked invasion protein (CLIP). CLAMP-deficiency does not impact parasite adhesion or microneme protein secretion; however, knockdown of any member of the CLAMP complex affects rhoptry discharge. Phylogenetic analysis suggests orthologs of the essential complex components, CLAMP and CLIP, are ubiquitous across apicomplexans. Nevertheless, SPATR, which appears to act as an accessory factor in Toxoplasma, is essential during Plasmodium falciparum blood stages. Our results reveal a new protein complex that mediates rhoptry discharge following host-cell contact.
... A 100-layer mixed-scale dense neural network 39 was trained to detect phage ϕKp24 tail fibers in reconstructed tomograms. Here, we used a similar training setup as in other studies that use mixed-scale dense networks 56,73,74 . For more information about this training setup, we refer to these earlier studies. ...
The Klebsiella jumbo myophage ϕKp24 displays an unusually complex arrangement of tail fibers interacting with a host cell. In this study, we combine cryo-electron microscopy methods, protein structure prediction methods, molecular simulations, microbiological and machine learning approaches to explore the capsid, tail, and tail fibers of ϕKp24. We determine the structure of the capsid and tail at 4.1 Å and 3.0 Å resolution. We observe the tail fibers are branched and rearranged dramatically upon cell surface attachment. This complex configuration involves fourteen putative tail fibers with depolymerase activity that provide ϕKp24 with the ability to infect a broad panel of capsular polysaccharide (CPS) types of Klebsiella pneumoniae. Our study provides structural and functional insight into how ϕKp24 adapts to the variable surfaces of capsulated bacterial pathogens, which is useful for the development of phage therapy approaches against pan-drug resistant K. pneumoniae strains. The jumbo contractile bacteriophage, Kp24, has been isolated from clinical strains of Klebsiella pneumonia. Here, the authors present structural and functional insight into the capsid, tail and tail fibres and how this impacts infectivity.
