Biology subjects

Rea, E.

Publications and source records attributed to Rea, E..

10 recordsLinked to original sources

bioRxiv2026

Single-shot light-field microscopy captures delivery-dependent dye and autofluorescence patterns in Caenorhabditis elegans

Three-dimensional imaging of live Caenorhabditis elegans commonly relies on sequential z-stack acquisition, which can be slow and susceptible to movement and photobleaching. Here, we used light-field microscopy to capture dye and delivery-dependent fluorescence patterns across intact adult worms in a single exposure per channel. With a 40x/1.20 NA water-immersion objective, each reconstructed dataset contained 121 axial slices spanning 109 {micro}m at 0.91 {micro}m spacing. In a representative adult, nematode signal extended from 40.88 to 85.4 {micro}m, corresponding to approximately 44.5 {micro}m of captured axial depth without sequential z scanning. Three fluorescence channels were acquired with a summed exposure time of 112 ms across a 685 x 1010 {micro}m field of view. FM1-43 labelled intestinal, cuticular and vesicular structures, FM4-64 highlighted ingested bacteria and intestinal compartments, and Nile Red revealed vesicular and broader whole-organism fluorescence. Intrinsic blue and green autofluorescence signals were also resolved. These observations demonstrate rapid single-shot acquisition of whole-organism three-dimensional fluorescence information and show how delivery route influences the spatial distribution of commonly used fluorescent probes in C. elegans.

biophysics↗

bioRxiv2025

Plasmodium ARK1 regulates spindle formation during atypical mitosis and forms a divergent chromosomal passenger complex

Mitosis in Plasmodium spp., the causative agent of malaria, is fundamentally different from model eukaryotes, proceeding via a bipartite microtubule organising centre (MTOC) and lacking canonical regulators such as Polo kinases. During schizogony, asynchronous nuclear replication produces a multinucleate schizont, while rapid male gametogony generates an octaploid nucleus before gamete formation. Here, we identify Aurora-related kinase 1 (ARK1) as a key component of inner MTOC and spindle formation, controlling kinetochore dynamics and driving mitotic progression. Conditional ARK1 depletion disrupts spindle biogenesis, kinetochore segregation, karyokinesis and cytokinesis in both stages, and affects parasite transmission. Interactome analysis shows that ARK1 forms the catalytic core of a non-canonical chromosomal passenger complex (CPC) containing two highly divergent inner centromere proteins (INCENPs), which we term INCENP-A and INCENP-B, and lacking the canonical chromatin-targeting subunits Survivin and Borealin. Comparative genomics suggests that apicomplexan INCENPs arose through recurrent lineage-specific duplications, reflecting an evolutionary rewiring of CPC architecture in this eukaryotic lineage. Together, these findings reveal key adaptations in Plasmodium mitosis involving ARK1 and its INCENP scaffolds, and identify the ARK1-INCENP interface as a potential multistage target for antimalarial intervention.

microbiology↗

bioRxiv2023

Discovery of essential kinetoplastid-insect adhesion proteins and their function in Leishmania-sand fly interactions

Leishmania species, members of the kinetoplastid parasites, cause leishmaniasis, a neglected tropical disease, in millions of people worldwide1. Leishmania has a complex life cycle with multiple developmental forms, as it cycles between a sand fly vector and a mammalian host; understanding their life cycle is critical to understanding disease spread2. One of the key life cycle stages is the haptomonad form, which is attached to the insect through its flagellum. This adhesion, which is conserved across kinetoplastid parasites, is implicated to have an important function within their life cycles and hence on disease transmission3-5. Here, we discovered kinetoplastid-insect adhesion proteins (KIAPs), which are localised in the attached haptomonad flagellum. Deletion of these KIAPs impaired cell adhesion in vitro and prevented Leishmania from colonising the stomodeal valve in the sand fly, without affecting cell growth. This result will provide important insights for a comprehensive understanding of the Leishmania life cycle.

microbiology↗

bioRxiv2023

Plasmodium ARK2-EB1 axis drives the unconventional spindle dynamics, scaffold formation, and chromosome segregation of sexual transmission stages

Mechanisms of cell division are remarkably diverse, suggesting the underlying molecular networks among eukaryotes differ extensively. The Aurora family of kinases orchestrates the process of chromosome segregation and cytokinesis during cell division through precise spatiotemporal regulation of their catalytic activities by distinct scaffolds. Plasmodium spp., the causative agents of malaria, are unicellular eukaryotes that have three divergent aurora-related kinases (ARKs) and lack most canonical scaffolds/activators. The parasite uses unconventional modes of chromosome segregation during endomitosis and meiosis in sexual transmission stages within mosquito host. This includes a rapid threefold genome replication from 1N to 8N with successive cycles of closed mitosis, spindle formation and chromosome segregation within eight minutes (termed male gametogony). Kinome studies had previously suggested likely essential functions for all three Plasmodium ARKs during asexual mitotic cycles; however, little is known about their location, function, or their scaffolding molecules during unconventional sexual proliferative stages. Using a combination of super-resolution microscopy, mass spectrometry, and live-cell fluorescence imaging, we set out to investigate the role of the atypical Aurora paralog ARK2 to proliferative sexual stages using rodent malaria model Plasmodium berghei. We find that ARK2 primarily localises to the spindle apparatus in the vicinity of kinetochores during both mitosis and meiosis. Interactomics and co-localisation studies reveal a unique ARK2 scaffold at the spindle including the microtubule plus end-binding protein EB1, lacking conserved Aurora scaffold proteins. Gene function studies indicate complementary functions of ARK2 and EB1 in driving endomitotic divisions and thereby parasite transmission. Our discovery of a novel Aurora kinase spindle scaffold underlines the emerging flexibility of molecular networks to rewire and drive unconventional mechanisms of chromosome segregation in the malaria parasite Plasmodium.

cell biology↗

bioRxiv2022

Formation and three-dimensional architecture of Leishmania adhesion in the sand fly vector

Attachment to a substrate to maintain position in a specific ecological niche is a common strategy across biology, especially for eukaryotic parasites. During development in the sand fly vector, the eukaryotic parasite Leishmania adheres to the stomodeal valve, as the specialised haptomonad form. Dissection of haptomonad adhesion is a critical step for understanding parasite transmission. Nevertheless, haptomonad studies are limited, as this is a technically challenging life cycle form to investigate. Here, we have combined three-dimensional electron microscopy approaches, including serial block face scanning electron microscopy (SBFSEM) and serial tomography to dissect the organisation and architecture of haptomonads in the sand fly. We showed that the attachment plaque contains distinct structural elements. Using time-lapse light microscopy, we identified five stages of haptomonad differentiation, and showed that calcium is necessary for haptomonad adhesion to the surface. This study provides the structural and regulatory foundations of the haptomonad form, which are critical for a holistic understanding of Leishmania transmission.

microbiology↗

bioRxiv2020

A divergent cyclin/cyclin-dependent kinase complex controls the atypical replication of Plasmodium berghei during gametogony and parasite transmission.

Cell cycle transitions are generally triggered by variation in the activity of cyclin-dependent kinases (CDKs) bound to cyclins. Malaria-causing parasites have a life cycle with unique cell-division cycles, and a repertoire of divergent CDKs and cyclins of poorly understood function and interdependency. We show that Plasmodium berghei CDK-related kinase 5 (CRK5), is a critical regulator of atypical mitosis in the gametogony and is required for mosquito transmission. It phosphorylates canonical CDK motifs of components in the pre-replicative complex and is essential for DNA replication. During a replicative cycle, CRK5 stably interacts with a single Plasmodium-specific cyclin (SOC2), although we obtained no evidence of SOC2 cycling by transcription, translation or degradation. Our results provide evidence that during Plasmodium male gametogony, this divergent cyclin/CDK pair fills the functional space of other eukaryotic cell-cycle kinases controlling DNA replication.

microbiology↗

bioRxiv2019

Kinesin-8B controls basal body function and flagellum formation and is key to malaria parasite transmission

Eukaryotic flagella are conserved microtubule-based organelles that drive cell motility. Plasmodium, the causative agent of malaria, has a single flagellate stage: the male gamete in the mosquito. Three rounds of endomitotic division together with an unusual mode of flagellum assembly rapidly produce eight motile gametes. These processes are tightly coordinated but their regulation is poorly understood. To understand this important developmental stage, we studied the function and location of the microtubule-based motor kinesin-8B, using gene-targeting, electron microscopy and live cell imaging. Deletion of the kinesin-8B gene showed no effect on mitosis but disrupted 9+2 axoneme assembly and flagellum formation during male gamete development and also completely ablated parasite transmission. Live cell imaging showed that kinesin-8B-GFP did not colocalise with kinetochores in the nucleus but instead revealed dynamic, cytoplasmic localisation with the basal bodies and the assembling axoneme during flagellum formation. We thus uncovered an unexpected role for kinesin-8B in parasite flagellum formation that is vital for the parasite life cycle.

cell biology↗

bioRxiv2019

Plasmodium condensin core subunits (SMC2/SMC4) mediate atypical mitosis and are essential for parasite proliferation and transmission

Condensin is a multi-subunit protein complex regulating chromosome condensation and segregation during cell division. In Plasmodium spp., the causative agent of malaria, cell division is atypical and the role of condensin is unclear. Here we examine the role of SMC2 and SMC4, the core subunits of condensin, during endomitosis in schizogony and endoreduplication in male gametogenesis. During early schizogony SMC2/SMC4 localize to a distinct focus, identified as the centromeres by NDC80 fluorescence and ChIP-seq analyses, but do not form condensin I or II complexes. In mature schizonts and during male gametogenesis, there is a diffuse SMC2/SMC4 distribution on chromosomes and in the nucleus, and both condensin I and II complexes form at these stages. Knockdown of smc2 and smc4 gene expression revealed essential roles in parasite proliferation and transmission. The condensin core subunits (SMC2/SMC4) form different complexes and may have distinct functions at various stages of the parasite life cycle.

cell biology↗

bioRxiv2019

Systematic analysis of Plasmodium myosins reveals differential expression, localization and function in invasive and proliferative parasite stages

The myosin superfamily comprises of actin-dependent eukaryotic molecular motors important in a variety of cellular functions. Although well studied in many systems, knowledge of their functions in Plasmodium, the causative agent of malaria, is restricted. Previously, six myosins were identified in this genus, including three Class XIV myosins found only in Apicomplexa and some Ciliates. The well characterised MyoA, is a class XIV myosin essential for gliding motility and invasion. Here, we characterize all other Plasmodium myosins throughout the parasite life cycle and show that they have very diverse patterns of expression and cellular location. MyoB and MyoE, the other two Class XIV myosins, are expressed in all invasive stages, with apical and basal locations, respectively. Gene deletion revealed that MyoE is involved in sporozoite motility, MyoF and MyoK are likely essential in the asexual blood stages, and MyoJ and MyoB are not essential. Both MyoB and its essential light chain (MCL-B) are localised at the apical end of ookinetes but expressed at completely different time points. This work provides a better understanding of the role of actomyosin motors in Apicomplexan parasites, particularly in the motile and invasive stages of Plasmodium during sexual and asexual development within the mosquito.

cell biology↗

bioRxiv2019

Plasmodium Kinesin-8X associates with mitotic spindles and is essential for oocyst development during parasite proliferation and transmission

Kinesin-8 proteins are microtubule motors that are often involved in regulation of mitotic spindle length and chromosome alignment. They move towards the ends of spindle microtubules and regulate the dynamics of these ends due, at least in some species, to their microtubule depolymerization activity. Plasmodium spp. exhibit an atypical endomitotic cell division in which chromosome condensation and spindle dynamics are not well understood in the different proliferative stages. Genome-wide homology analysis of Plasmodium spp. revealed the presence of two Kinesin-8 motor proteins (Kinesin-8X and Kinesin-8B). Here we have studied the biochemical properties of Kinesin-8X and its role in parasite proliferation. In vitro, Kinesin-8X showed motile and depolymerization activities like other Kinesin-8 motors. To understand its role in cell division, we have used protein tagging and live cell imaging to define the location of Plasmodium Kinesin-8X during all proliferative stages of the P berghei life cycle. Furthermore, we have used gene targeting to analyse the function of Kinesin-8X. The results reveal a spatio-temporal involvement of Kinesin-8X in spindle dynamics and its association with both mitotic and meiotic spindles and the putative microtubule organising centre (MTOC). Deletion of the Kinesin-8X gene showed that this protein is required for endomitotic division during oocyst development and is therefore necessary for parasite replication within the mosquito gut, and for transmission to the vertebrate host. Consistently, transcriptome analysis of{Delta} kinesin-8X parasites reveals modulated expression of genes involved mainly in microtubule-based processes, chromosome organisation and the regulation of gene expression supporting a role in cell division.\n\nAuthor SummaryKinesins are microtubule-based motors that play key roles in intracellular transport, cell division and motility. Members of the Kinesin-8 family contribute to chromosome alignment during cell division in many eukaryotes. However, the roles of kinesins in the atypical cell division in Plasmodium, the causative agent of malaria, is not known. In contrast to many other eukaryotes, Plasmodium proliferates by endomitosis, in which genome replication and division occur within a nucleus bounded by a persistent nuclear envelope. We show that the Plasmodium genome encodes only nine kinesins and we further investigate the role of Kinesin-8X throughout the Plasmodium life cycle using biochemical and gene targeting approaches. We show that Plasmodium Kinesin-8X has microtubule-based motility and depolymerization activity. We also show that Kinesin-8X is probably localized on putative MTOCs and spindles during cell division in most of the stages of P. berghei life cycle. By gene deletion we demonstrate that Kinesin-8X is essential for normal oocyst development and sporozoite formation. Genome-wide RNA analysis of{Delta} kinesin-8X parasites reveals modulated expression of genes involved in microtubule-based processes. Overall, the data suggest that Kinesin-8X is a molecular motor that plays essential roles during endomitosis in oocyst development in the mosquito, contributing to parasite transmission.

cell biology↗