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Plasmodium parasites, the causative agents of malaria, undergo closed mitosis without breakdown of the nuclear envelope. Unlike closed mitosis in yeast, Plasmodium berghei parasites undergo multiple rounds of asynchronous nuclear divisions in a shared cytoplasm. This results in a multinucleated organism prior to the formation of daughter cells within an infected red blood cell. During this replication process, intact nuclear pore complexes (NPCs) and their component nucleoporins play critical roles in parasite growth, facilitating selective bi-directional nucleocytoplasmic transport and genome organization. Here, we utilize ultrastructure expansion microscopy to investigate P. berghei nucleoporins at the single nucleus level throughout the 24-hour blood-stage replication cycle. Our findings reveal that these nucleoporins are distributed around the nuclei and organized in a rosette structure previously undescribed around the centriolar plaque, responsible for intranuclear microtubule nucleation during mitosis. By adapting the recombination-induced tag exchange system to P. berghei through a single plasmid tagging system, which includes the tagging plasmid as well as the Cre recombinase, we provide evidence of NPC formation dynamics, demonstrating Nup221 turnover during parasite asexual replication. Our data shed light on the distribution of NPCs and their homeostasis during the blood-stage replication of P. berghei parasites., Importance: Malaria, caused by Plasmodium species, remains a critical global health challenge, with an estimated 249 million cases and over 600,000 deaths in 2022, primarily affecting children under five. Understanding the nuclear dynamics of Plasmodium parasites, particularly during their unique mitotic processes, is crucial for developing novel therapeutic strategies. Our study leverages advanced microscopy techniques, such as ultrastructure expansion microscopy, to reveal the organization and turnover of nuclear pore complexes (NPCs) during the parasite's asexual replication. By elucidating these previously unknown aspects of NPC distribution and homeostasis, we provide valuable insights into the molecular mechanisms governing parasite mitosis. These findings deepen our understanding of parasite biology and may inform future research aimed at identifying new targets for anti-malarial drug development.

The precise mode of action of ganaplacide (KAF156), a phase III antimalarial candidate, remains elusive. Here we employ omics-based methods with the closely related chemical analog, GNF179, to search for potential Plasmodium targets. Ranking potential targets derived from chemical genetics and proteomic affinity chromatography methodologies identifies SEY1 , or Synthetic Enhancement of YOP1, which is predicted to encode an essential dynamin-like GTPase implicated in homotypic fusion of endoplasmic reticulum (ER) membranes. We demonstrate that GNF179 decreases Plasmodium SEY1 melting temperature. We further show that GNF179 binds to recombinant Plasmodium SEY1 and subsequently inhibits its GTPase activity, which is required for maintaining ER architecture. Using ultrastructure expansion microscopy, we find GNF179 treatment changes parasite ER and Golgi morphology. We also confirm that SEY1 is an essential gene in P. falciparum . These data suggest that SEY1 may contribute to the mechanism of action of imidazolopiperazines and is a new and attractive druggable target.

Malaria is a global and deadly human disease caused by the apicomplexan parasites of the genus Plasmodium. Parasite proliferation within human red blood cells (RBCs) is associated with the clinical manifestations of the disease. This asexual expansion within human RBCs begins with the invasion of RBCs by P. falciparum, which is mediated by the secretion of effectors from 2 specialized club-shaped secretory organelles in merozoite-stage parasites known as rhoptries. We investigated the function of the Rhoptry Neck Protein 11 (RON11), which contains 7 transmembrane domains and calcium-binding EF-hand domains. We generated conditional mutants of the P. falciparum RON11. Knockdown of RON11 inhibits parasite growth by preventing merozoite invasion. The loss of RON11 did not lead to any defects in processing of rhoptry proteins but instead led to a decrease in the amount of rhoptry proteins. We utilized ultrastructure expansion microscopy (U-ExM) to determine the effect of RON11 knockdown on rhoptry biogenesis. Surprisingly, in the absence of RON11, fully developed merozoites had only 1 rhoptry each. The single rhoptry in RON11-deficient merozoites were morphologically typical with a bulb and a neck oriented into the apical polar ring. Moreover, rhoptry proteins are trafficked accurately to the single rhoptry in RON11-deficient parasites. These data show that in the absence of RON11, the first rhoptry is generated during schizogony but upon the start of cytokinesis, the second rhoptry never forms. Interestingly, these single-rhoptry merozoites were able to attach to host RBCs but are unable to invade RBCs. Instead, RON11-deficient merozoites continue to engage with RBC for prolonged periods eventually resulting in echinocytosis, a result of secreting the contents from the single rhoptry into the RBC. Together, our data show that RON11 triggers the de novo biogenesis of the second rhoptry and functions in RBC invasion., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Anaguano et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Apicomplexan parasites exhibit tremendous diversity in much of their fundamental cell biology, but study of these organisms using light microscopy is often hindered by their small size. Ultrastructural expansion microscopy (U-ExM) is a microscopy preparation method that physically expands the sample by ~4.5×. Here, we apply U-ExM to the human malaria parasite Plasmodium falciparum during the asexual blood stage of its lifecycle to understand how this parasite is organized in three dimensions. Using a combination of dye-conjugated reagents and immunostaining, we have cataloged 13 different P. falciparum structures or organelles across the intraerythrocytic development of this parasite and made multiple observations about fundamental parasite cell biology. We describe that the outer centriolar plaque and its associated proteins anchor the nucleus to the parasite plasma membrane during mitosis. Furthermore, the rhoptries, Golgi, basal complex, and inner membrane complex, which form around this anchoring site while nuclei are still dividing, are concurrently segregated and maintain an association to the outer centriolar plaque until the start of segmentation. We also show that the mitochondrion and apicoplast undergo sequential fission events while maintaining an association with the outer centriolar plaque during cytokinesis. Collectively, this study represents the most detailed ultrastructural analysis of P. falciparum during its intraerythrocytic development to date and sheds light on multiple poorly understood aspects of its organelle biogenesis and fundamental cell biology., Competing Interests: BL, AC, JB, DA, SF, VM, DW, JD, SA No competing interests declared, (© 2023, Liffner, Cepeda Diaz et al.)

Recent reports have indicated that a manganese oxide spinel component, when embedded in a relatively small concentration in layered xLi 2 MnO 3 ·(1-x)LiMO 2 (M = Ni, Mn, or Co) electrode systems, can act as a stabilizer that increases their capacity, rate capability, cycle life, and first-cycle efficiency. These findings prompted us to explore the possibility of exploiting lithiated cobalt oxide spinel stabilizers by taking advantage of (1) the low mobility of cobalt ions relative to that of manganese and nickel ions in close-packed oxides and (2) their higher potential (∼3.6 V vs Li 0 ) relative to manganese oxide spinels (∼2.9 V vs Li 0 ) for the spinel-to-lithiated spinel electrochemical reaction. In particular, we revisited the structural and electrochemical properties of lithiated spinels in the LiCo 1-x Ni x O 2 (0 ≤ x ≤ 0.2) system, first reported almost 25 years ago, by means of high-resolution (synchrotron) X-ray diffraction, transmission electron microscopy, nuclear magnetic resonance spectroscopy, electrochemical cell tests, and theoretical calculations. The results provide a deeper understanding of the complexity of intergrown layered/lithiated spinel LiCo 1-x Ni x O 2 structures when prepared in air between 400 and 800 °C and the impact of structural variations on their electrochemical behavior. These structures, when used in low concentrations, offer the possibility of improving the cycling stability, energy, and power of high energy (≥3.5 V) lithium-ion cells.