Plasmodium ARK2 and EB1 drive unconventional spindle dynamics, during chromosome segregation in sexual transmission stages

The Aurora family of kinases orchestrates chromosome segregation and cytokinesis during cell division, with precise spatiotemporal regulation of its catalytic activities by distinct protein scaffolds. Plasmodium spp., the causative agents of malaria, are unicellular eukaryotes with three unique and highly divergent aurora-related kinases (ARK1-3) that are essential for asexual cellular proliferation but lack most canonical scaffolds/activators. Here we investigate the role of ARK2 during sexual proliferation of the rodent malaria Plasmodium berghei, using a combination of super-resolution microscopy, mass spectrometry, and live-cell fluorescence imaging. We find that ARK2 is primarily located at spindle microtubules in the vicinity of kinetochores during both mitosis and meiosis. Interactomic and co-localisation studies reveal several putative ARK2-associated interactors including the microtubule-interacting protein EB1, together with MISFIT and Myosin-K, but no conserved eukaryotic scaffold proteins. Gene function studies indicate that ARK2 and EB1 are complementary in driving endomitotic division and thereby parasite transmission through the mosquito. This discovery underlines the flexibility of molecular networks to rewire and drive unconventional mechanisms of chromosome segregation in the malaria parasite.</p

Genome-wide functional analysis reveals key roles for kinesins in the mammalian and mosquito stages of the malaria parasite life cycle

Kinesins are microtubule (MT)-based motors important in cell division, motility, polarity, and intracellular transport in many eukaryotes. However, they are poorly studied in the divergent eukaryotic pathogens Plasmodium spp., the causative agents of malaria, which manifest atypical aspects of cell division and plasticity of morphology throughout the life cycle in both mammalian and mosquito hosts. Here, we describe a genome-wide screen of Plasmodium kinesins, revealing diverse subcellular locations and functions in spindle assembly, axoneme formation, and cell morphology. Surprisingly, only kinesin-13 is essential for growth in the mammalian host while the other 8 kinesins are required during the proliferative and invasive stages of parasite transmission through the mosquito vector. In-depth analyses of kinesin-13 and kinesin-20 revealed functions in MT dynamics during apical cell polarity formation, spindle assembly, and axoneme biogenesis. These findings help us to understand the importance of MT motors and may be exploited to discover new therapeutic interventions against malaria

Protein phosphatase 1 regulates atypical mitotic and meiotic division in Plasmodium sexual stages

PP1 is a conserved eukaryotic serine/threonine phosphatase that regulates many aspects of mitosis and meiosis, often working in concert with other phosphatases, such as CDC14 and CDC25. The proliferative stages of the malaria parasite life cycle include sexual development within the mosquito vector, with male gamete formation characterized by an atypical rapid mitosis, consisting of three rounds of DNA synthesis, successive spindle formation with clustered kinetochores, and a meiotic stage during zygote to ookinete development following fertilization. It is unclear how PP1 is involved in these unusual processes. Using real-time livecell and ultrastructural imaging, conditional gene knockdown, RNA-seq and proteomic approaches, we show that Plasmodium PP1 is implicated in both mitotic exit and, potentially, establishing cell polarity during zygote development in the mosquito midgut, suggesting that small molecule inhibitors of PP1 should be explored for blocking parasite transmission

Expansion microscopy of Plasmodium gametocytes reveals the molecular architecture of a bipartite microtubule organisation centre coordinating mitosis with axoneme assembly

Transmission of malaria-causing parasites to mosquitoes relies on the production of gametocyte stages and their development into gametes. These stages display various microtubule cytoskeletons and the architecture of the corresponding microtubule organisation centres (MTOC) remains elusive. Combining ultrastructure expansion microscopy (U-ExM) with bulk proteome labelling, we first reconstructed in 3D the subpellicular microtubule network which confers cell rigidity to Plasmodium falciparum gametocytes. Upon activation, as the microgametocyte undergoes three rounds of endomitosis, it also assembles axonemes to form eight flagellated microgametes. U-ExM combined with Pan-ExM further revealed the molecular architecture of the bipartite MTOC coordinating mitosis with axoneme formation. This MTOC spans the nuclear membrane linking cytoplasmic basal bodies to intranuclear bodies by proteinaceous filaments. In P. berghei, the eight basal bodies are concomitantly de novo assembled in a SAS6- and SAS4-dependent manner from a deuterosome-like structure, where centrin, γ-tubulin, SAS4 and SAS6 form distinct subdomains. Basal bodies display a fusion of the proximal and central cores where centrin and SAS6 are surrounded by a SAS4-toroid in the lumen of the microtubule wall. Sequential nucleation of axonemes and mitotic spindles is associated with a dynamic movement of γ-tubulin from the basal bodies to the intranuclear bodies. This dynamic architecture relies on two non-canonical regulators, the calcium-dependent protein kinase 4 and the serine/arginine-protein kinase 1. Altogether, these results provide insights into the molecular organisation of a bipartite MTOC that may reflect a functional transition of a basal body to coordinate axoneme assembly with mitosis

The Skp1-Cullin1-FBXO1 complex is a pleiotropic regulator required for the formation of gametes and motile forms in Plasmodium berghei

Malaria-causing parasites of the Plasmodium genus undergo multiple developmental phases in the human and the mosquito hosts, regulated by various post-translational modifications. While ubiquitination by multi-component E3 ligases is key to regulate a wide range of cellular processes in eukaryotes, little is known about its role in Plasmodium . Here we show that Plasmodium berghei expresses a conserved SKP1/Cullin1/FBXO1 (SCF FBXO1 ) complex showing tightly regulated expression and localisation across multiple developmental stages. It is key to cell division for nuclear segregation during schizogony and centrosome partitioning during microgametogenesis. It is additionally required for parasite-specific processes including gamete egress from the host erythrocyte, as well as integrity of the apical and the inner membrane complexes (IMC) in merozoite and ookinete, two structures essential for the dissemination of these motile stages. Ubiquitinomic surveys reveal a large set of proteins ubiquitinated in a FBXO1-dependent manner including proteins important for egress and IMC organisation. We additionally demonstrate an interplay between FBXO1-dependent ubiquitination and phosphorylation via calcium-dependent protein kinase 1. Altogether we show that Plasmodium SCF FBXO1 plays conserved roles in cell division and is also important for parasite-specific processes in the mammalian and mosquito hosts

<i>Cbp80</i> is needed for the expression of piRNA components and piRNAs

<div><p>Cap binding protein 80 (Cbp80) is the larger subunit of the nuclear cap-binding complex (nCBC), which is known to play important roles in nuclear mRNA processing, export, stability and quality control events. Reducing <i>Cbp80</i> mRNA levels in the female germline revealed that <i>Cbp80</i> is also involved in defending the germline against transposable elements. Combining such knockdown experiments with large scale sequencing of small RNAs further showed that <i>Cbp80</i> is involved in the initial biogenesis of piRNAs as well as in the secondary biogenesis pathway, the ping-pong amplification cycle. We further found that <i>Cbp80</i> knockdown not only led to the upregulation of transposons, but also to delocalization of Piwi, Aub and Ago3, key factors in the piRNA biosynthesis pathway. Furthermore, compared to controls, levels of Piwi and Aub were also reduced upon knock down of <i>Cbp80</i>. On the other hand, with the same treatment we could not detect significant changes in levels or subcellular distribution (nuage localization) of piRNA precursor transcripts. This shows that <i>Cbp80</i> plays an important role in the production and localization of the protein components of the piRNA pathway and it seems to be less important for the production and export of the piRNA precursor transcripts.</p></div

Germline <i>Cbp80</i> is involved in producing piRNAs.

<p><b>(A-C)</b> Genotypes of ovaries analyzed are depicted on top. Ovaries showing the "d" phenotype (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181743#pone.0181743.g001" target="_blank">Fig 1A</a>) upon <i>Cbp80</i> knockdown were used. <b>(A-C, F)</b> Scale and axis depicted in the left pannel also apply to the corresponding panels on the right. <b>(A-B)</b> Histogram showing small RNAs (23–29 nucleotides) mapping to the soma-specific <i>flamenco</i> (<i>flam</i>) cluster <b>(A)</b> and to the germline-specific cluster <i>42AB</i> <b>(B)</b>. Ovaries expressed specifically in the germline shRNAs against <i>Cbp80</i> or <i>mCherry</i> (as control). <b>(C)</b> Plots showing the size distribution of small RNAs derived from each strand of the <i>42AB</i> and the <i>flam</i> clusters. Small RNA reads derived form the <i>42AB</i> cluster were normalized to small RNAs mapping to the somatic <i>flam</i>, which is unaffected in this germline-specific knockdown. Number of small RNA reads of the characteristic size for piRNAs (23–29 nt) mapping to the germline-specific, dual-strand <i>42AB</i> cluster were reduced upon <i>Cbp80</i> knockdown. The same results were obtained when normalizing to the total number of small RNA reads. Small RNA reads derived form the <i>flam</i> cluster were normalized to the total number of small RNA reads. <b>(D)</b> A histogram showing the relative levels of <i>42AB</i> derived piRNAs upon <i>Cbp80</i> knockdown compared to the control knockdown. The data is normalized to the number of reads from the <i>flam</i> locus. Differences between control (<i>shmCherry</i>) and <i>shCbp80</i> treatment are highly significant (p-value is < 2x10^-16 using a chi-square test). <b>(E)</b> Relative abundance of sense-antisense piRNA pairs overlapping by 10 nt (compared to the total number of sense-antisense pairs mapping to the <i>flam</i> or the <i>42AB</i> clusters, in the small RNA libraries of the <i>Cbp80</i> and the control knockdowns. Differences between <i>42AB</i> control (<i>shmCherry</i>) and <i>42AB</i> levels upon <i>shCbp80</i> treatment are highly significant (p-value is < 2x10^-16 using a chi-square test). <b>(F)</b> Histograms showing the relative enrichment of RNAs overlapping by the indicated number of nucleotides, plotted by Z-score, for the <i>42AB</i> and <i>flam</i> clusters. Knockdown targets in the female germline are indicated on top of the figure. The peak at position 10 (arrow) is indicative of a ping-pong signature.</p

Expression of piRNA pathway components requires <i>Cbp80</i>.

<p><b>(A, D)</b> Knock down of <i>Cbp80</i> in egg chambers expressing a <i>dsRNA</i> against <i>Cbp80 (dsCbp80</i>) driven by the GAL4-nos.NGT40 driver and the UAS-Dcr2. Control flies expressed a <i>dsRNA</i> against <i>GFP</i> (<i>dsGFP</i>) <b>(B, C)</b> Ovaries expressing specifically in the germline (<i>pCog-Gal4</i> driver) shRNAs against <i>Cbp80</i> or <i>mCherry</i> (as control). Control flies used for RT-PCR experiments expressed also a Jupiter-mCherry fusion protein. <b>(A-B)</b> mRNA levels of piRNA pathway factors (<i>piwi</i>, <i>aub</i>, <i>Rhi</i>, <i>zuc</i> and <i>Ago3</i>) and <i>Cbp80</i> were measured by qRT-PCR. Fold expression levels of each mRNA in the knockdown samples relative to its expression in the corresponding control are shown for each mRNA. Total starting RNA amounts were the same in both samples. Error bars represent ⁺/- SD of 2 controls in B and 3 control samples in A and 3 biological knockdown replicates. *p<0.05; **p<0.01; ***p<0.001. <b>(C-D)</b> Levels of Cbp80, Piwi, Aub, BicD, Cdk7, Clc and Tub (as loading control) were assessed by Western blotting. Ponceau staining is also shown to reveal total proteins as loading control. Levels Piwi, Aub and Cdk7 were strongly reduced upon <i>Cbp80</i> reduction. On the other hand, levels of Tub, Clc and BicD were less affected. For Cbp80 knockdown samples were extracted from egg chambers showing the phenotype “d” (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181743#pone.0181743.g001" target="_blank">Fig 1A</a>).</p

<i>Cbp80</i> phenotypes in the female germline and its role in silencing transposable elements.

<p>(<b>A</b>) Germline-specific knock down of <i>Cbp80</i> was performed either with the driver <i>pCog-GAL4</i> and an shRNA against <i>Cbp80 (shCbp80)</i> or by the GAL4-nos.NGT40 driver combined with UAS-Dcr2 and a dsRNA against <i>Cbp80 (dsCbp80)</i>. The knockdown leads to 3 phenotypic classes with the frequencies shown in (<b>A’</b>): normal wild-type phenotype [n], ovaries that appear to lack germ cells [gc^-], and partially developing ovaries that mostly degenerate after stages 7–9 [d]. <b>(B-B’)</b> Ovaries showing the "d" phenotype upon <i>Cbp80</i> knockdown were tested for Cbp80 levels by Western blotting. The sample contained only a minor fraction of the other phentypes. In <b>(B)</b> wild-type ovaries and wild-type ovaries expressing a Myc-tagged Cbp80 were used as controls, and knock down was performed with the shRNA. 2 different amounts of the <i>shCbp80</i> samples were loaded. In <b>(B’)</b> ovaries expressing a dsRNA against <i>Cbp80</i> or <i>GFP</i> were used. Tubulin was used as a loading control. (<b>C</b>) Fold increases in RNA levels of indicated TEs upon germline-specific RNAi-mediated knock down of <i>piwi</i> and <i>Cbp80 (shRNA</i> against Cbp80). The germline GAL4 driver alone was used as control. Fold-changes in RNA levels relative to control were normalized to <i>rp49</i> levels. Error bars indicate SD; n = 3, with 2 biological replicates. (D) Fold increases in RNA levels relative to <i>rp49</i> of the same TEs upon knock down of <i>Cbp80</i> using the <i>dsRNA</i>. Control ovaries expressed the <i>dsGFP</i> RNAi construct. The upregulation of the TE was also observed when <i>Tub</i> and <i>BicD</i> were used to normalize the reads (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181743#pone.0181743.s003" target="_blank">S3 Fig</a>). *p<0.05; **p<0.01; ***p<0.001.</p