Biology subjects

Bottrill, A. R.

Publications and source records attributed to Bottrill, A. R..

5 recordsLinked to original sources

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↗

bioRxiv2024

Divergent Plasmodium protein kinases drive MTOC, kinetochore and axoneme organisation in male gametogenesis

Sexual development and male gamete formation of the malaria parasite in the mosquito midgut is governed by rapid endomitosis in the activated male gametocyte. This process is highly regulated by protein phosphorylation, specifically by three divergent male-specific protein kinases (PKs): CDPK4, SRPK1 and MAP2. Here, we localise each PK during rapid male gamete formation using live-cell imaging, identify their putative substrates by immunoprecipitation, and determine the morphological consequences of their absence using ultrastructure expansion and transmission electron microscopy. Each PK has a distinct location in either the nuclear or cytoplasmic compartment. Protein interaction studies revealed that CDPK4 and MAP2 interact with key drivers of rapid DNA replication, while SRPK1 is involved in RNA translation. The absence of each PK results in severe defects in either microtubule organising centre (MTOC) organisation, kinetochore segregation or axoneme formation. This study reveals the crucial role of these PKs during endomitosis in formation of the flagellated male gamete and uncovers some of their potential substrates that drive this process. Summary blurbThis study reveals how Plasmodium kinases regulate MTOC, axoneme and kinetochore organisation in male gametogenesis, providing key insights into potential targets for malaria transmission control.

microbiology↗

bioRxiv2024

Plasmodium NEK1 coordinates MTOC organisation and kinetochore attachment during rapid mitosis in male gamete formation

Mitosis is an important process in the cell cycle required for cells to divide. Never in mitosis (NIMA)-like kinases (NEKs) are regulators of mitotic functions in diverse organisms. Plasmodium spp, the causative agent of malaria is a divergent unicellular haploid eukaryote with some unusual features in terms of its mitotic and nuclear division cycle that presumably facilitate proliferation in varied environments. For example, during the sexual stage of male gametogenesis that occurs within the mosquito host, an atypical rapid closed endomitosis is observed. Three rounds of genome replication from 1N to 8N and successive cycles of multiple spindle formation and chromosome segregation occur within eight minutes followed by karyokinesis to generate haploid gametes. Our previous Plasmodium berghei kinome screen identified four Nek genes, of which two, NEK2 and NEK4, are required for meiosis. NEK1 is likely to be essential for mitosis in asexual blood stage schizogony in the vertebrate host, but its function during male gametogenesis is unknown. Here, we study NEK1 location and function, using live cell imaging, ultrastructure expansion microscopy (U-ExM) and electron microscopy, together with conditional gene knockdown and proteomic approaches. We report spatiotemporal NEK1 location in real-time, coordinated with microtubule organising centre (MTOC) dynamics during the unusual mitoses at various stages of the Plasmodium spp. life cycle. Knockdown studies reveal NEK1 to be an essential component of the MTOC in male cell differentiation, associated with rapid mitosis, spindle formation and kinetochore attachment. These data suggest that Plasmodium berghei NEK1 kinase is an important component of MTOC organisation and essential regulator of chromosome segregation during male gamete formation.

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

Expanding the Zebrafish Genetic Code through Site-Specific Introduction of Diazirine-lysine, Bicyclononyne-lysine and Azido-lysine

Site-specific incorporation of un-natural amino acids (UNAA) is a powerful approach to engineer and understand protein function [1-4]. Site-specific incorporation of UNAAs is achieved through repurposing the amber codon (UAG) as a sense codon for the UNAA, a tRNACUA that base pairs with an UAG codon in the mRNA and an orthogonal amino-acyl tRNA synthetase (aaRS) that charges the tRNACUA with the UNAA [5, 6]. Here, we report expansion of the zebrafish genetic code to incorporate the UNAAs, Azido-lysine (AzK), bicyclononyne-lysine (BCNK), and Diazirine-lysine (AbK) into green fluorescent protein (GFP) and Glutathione-S-transferase (GST). We also present proteomic evidence for UNAA incorporation into GFP. Our work sets the stage for the use of UNAA mutagenesis to investigate and engineer protein function in zebrafish.

synthetic biology↗