New Results

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

Rajan Pandey, Steven Abel, Matthew Boucher, Richard J Wall, Mohammad Zeeshan, Edward Rea, Aline Freville, Xueqing Maggie Lu, Declan Brady, Emilie Daniel, Rebecca R. Stanway, Sally Wheatley, Gayani Batugedara, Thomas Hollin, Andrew R. Bottrill, Dinesh Gupta, Anthony A. Holder, Karine G. Le Roch, Rita Tewari
doi: https://doi.org/10.1101/674028

Summary

Condensin is a multi-subunit protein complex that regulates chromosome organization, segregation and condensation during cell division in eukaryotes. 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. SMC2 and SMC4 localize at discrete foci during schizogony, and with a diffuse nuclear distribution during male gametogenesis. ChIP-seq analyses suggest a centromeric location of SMC2/SMC4 only during schizogony. Co-immunoprecipitation data reveal the presence of both condensin complex I and II during male gametogenesis, but only the SMC2/SMC4 heterodimer during schizogony. Finally, knockdown of smc2 and smc4 gene expression revealed their essential roles in parasite proliferation and transmission. This study shows that condensin core subunits (SMC2/SMC4) have differential complex and distinct functions at different stages of the parasite life cycle.

Introduction

Cellular proliferation in eukaryotes requires chromosome replication and segregation, followed by cell division, to ensure that daughter cells have identical copies of the genome. During classical open mitosis in many eukaryotes, chromosome condensation, centrosome migration and formation of the mitotic spindle are followed by dissolution of the nuclear envelope(Guttinger et al., 2009). In contrast, in some unicellular organisms such as the budding yeast, Saccharomyces cerevisiae, mitosis is closed: the nuclear membrane remains intact and chromosomes are separated by spindles assembled within the nucleus(Sazer et al., 2014). The mechanisms and the various regulatory molecules involved in cell division have been well studied in many eukaryotes. The cell division regulators include cyclins, cyclin-dependent kinases (CDKs), components of the anaphase promoting complex (APC), and other protein kinases and phosphatases(Chang et al., 2014; Fisher et al., 2012; Harashima et al., 2013).

An essential component of chromosome dynamics is a family of ‘Structural Maintenance of Chromosomes’ proteins, originally described in budding yeast as ‘stability of minichromosomes’ (SMC) proteins, which are implicated in chromosome segregation and condensation(Hirano, 2016; Uhlmann, 2016). Most eukaryotes have at least six genes encoding SMC proteins (each 110-170 kDa, with a central hinge region and N and C-terminal globular domains with Walker A and Walker B motifs forming the ATPase head domain). The six SMCs can be classified as subunits of condensin (SMC2 and SMC4, required for chromosomal condensation), cohesin (SMC1 and SMC3, required for chromosomal segregation) and the SMC5-SMC6 complex (involved in DNA repair and homologous recombination)(Hirano, 2016; Uhlmann, 2016).

Higher eukaryotic organisms have two different condensin complexes: condensin I and condensin II, whereas many single celled organisms such as yeast have only one condensin complex. SMC2 and SMC4 form the core structure for both condensin I and condensin II in higher eukaryotes(Hirano, 2016), and interact with three additional non-SMC components, including one kleisin(Schleiffer et al., 2003) and two heat protein subunits(Neuwald and Hirano, 2000). Kleisin Iγ (CAP-H), Heat IA (CAP-D2) and Heat IB (CAP-G) form the condensin I complex, whereas Kleisin IIβ (CAP-H2), Heat IIA (CAP-D3) and Heat IIB (CAP-G2) form the condensin II complex(Hirano, 2016; Uhlmann, 2016) (Figure 1A). Electron microscopy and protein-protein interaction studies have revealed the characteristic architecture and geometry of condensin complexes(Anderson et al., 2002; Onn et al., 2007). Condensin plays a vital role in cell division processes such as chromosomal condensation, correct folding and organization of chromosomes prior to anaphase and proper chromosome segregation and separation(Hirano, 2016; Kschonsak et al., 2017; Ono et al., 2013; Rawlings et al., 2011; Uhlmann, 2016). Indeed, both SMC and non-SMC components are necessary for its full function, for example, chromosomal condensation is not observed in the absence of kleisin, showing its critical role for complex formation and condensation(Cuylen et al., 2011; Rawlings et al., 2011).

Figure 1:
Figure 1:

Architecture of condensin (SMC2/SMC4) in Plasmodium berghei. (A) Composition of two conventional condensin complexes (condensin I and condensin II) which are comprised of heterodimeric core subunits, SMC2 and SMC4, along with non-SMC regulatory subunits, Kleisin and a pair of HEAT subunits specific for either condensin I or II (modified from Hirano T., 20168 and Uhlmann F., 201253). (B) Domain architecture of P. berghei SMC2 and SMC4. (C) Sequence coverage and amino acid identity of P. berghei SMC2 and SMC4 with H. sapiens, S. cerevisiae and A. thaliana proteins. (D) Homology-based predicted three-dimensional structures of P. berghei SMC2 and SMC4 showing coiled backbone extension without hinge domain and with ATPase head formation, required for condensin complex. See also Figure S1.

Plasmodium, the apicomplexan parasite that causes malaria, undergoes two types of atypical mitotic division during its life cycle: one in the asexual stages (schizogony in the liver and blood stages within the vertebrate host, and sporogony in the mosquito gut), and the other in male gametogenesis during the sexual stage(Arnot et al., 2011; Sinden, 1991b). Division during schizogony/sporogony resembles closed endomitosis with repeated asynchronous nuclear division without cytokinesis, resulting in a multinucleated syncytium. An intact nuclear envelope is maintained, wherein the microtubule organizing center (MTOC), known as the centriolar plaque or spindle pole body (SPB), is embedded, and rounds of mitosis and nuclear division proceed without chromosome condensation(Arnot et al., 2011; Francia and Striepen, 2014; Gerald et al., 2011; Sinden, 1991a, b; Sinden et al., 1976). In male gametogenesis, exposure of the male gametocyte to the mosquito midgut environment leads to activation, which results in endoreduplication with three rounds of rapid chromosome replication within 8-10 min and chromosomal condensation, followed by nuclear and cell division to produce eight motile male gametes (exflagellation)(Guttery et al., 2012b; Sinden, 1991b; Sinden et al., 1976; Sinden et al., 2010). During exflagellation, each condensed haploid nucleus and associated MTOC, together with a basal body, axoneme and flagellum form the microgamete that egresses from the main cellular body)(Guttery et al., 2012b; Sinden, 1991b; Sinden et al., 1976; Sinden et al., 2010).

The atypical cell division and proliferation of malaria parasites is controlled by, amongst others, unique and divergent Apicomplexa-specific CDKs, aurora-like kinases (ARKs), and mitotic protein phosphatase 1 (PP1), as well as only four APC components(Guttery et al., 2014; Roques et al., 2015; Wall et al., 2018; Ward et al., 2004; Wilkes and Doerig, 2008). However, there are no known classical group 1 cyclins, polo-like kinases (that are major regulators in mitotic entry) or classical mitotic protein phosphatases (CDC14 and CDC25) encoded in the genome(Guttery et al., 2014; Tewari et al., 2010; Ward et al., 2004; Wilkes and Doerig, 2008).

In Plasmodium, the role of condensin during cell division as well as general chromosome dynamics is unknown. Here, we investigated the location and function of the core subunit of condensin (SMC2 and SMC4) during two different mitotic division stages in the Plasmodium life cycle; during schizogony in the host blood, and during male gametogenesis in the mosquito vector. This was performed using the rodent malaria model Plasmodium berghei. For this analysis we used a combination of cell biology, proteomics, transcriptomics and reverse genetics approaches. Spatiotemporal localization using live cell imaging indicates that both SMC2 and SMC4 have a dynamic profile, with either discrete foci during schizogony or a more diffused nuclear localization during male gametogenesis. Genome-wide distribution studies using ChIP-seq experiments, suggested that both components (SMC2/SMC4) are located at or near the centromeres during schizogony, but this strong interaction is not observed during gametogenesis. Interestingly, we identified a differential composition of the condensin complex between the distinct mitotic stages, suggesting divergent mechanisms at the molecular level. Our data demonstrate that the condensin core subunits (SMC2/SMC4) have distinct functions at different stages of the parasite life cycle. Functional analyses using a conditional gene knockdown approach indicate that condensins are required for parasite proliferation and transmission.

Results

Bioinformatic analysis identifies both core subunit components (SMC2/SMC4) of condensin encoded in the Plasmodium genome

In order to identify condensin in Plasmodium we screened for condensin core subunit genes in the P. berghei genome using PlasmoDB version 42(Bahl et al., 2002). Domain analysis revealed a conserved domain architecture for both core SMC components of condensin, SMC2 and SMC4 (Figure 1A and 1B). A comparative sequence analysis revealed low sequence similarity and identity (∼29-34%, except for SMC4 with Arabidopsis thaliana (65%), Figure 1C), though there was similarity in size and overall domain structure when compared with the other studied organisms. Interestingly, we found two N-terminus ATPase domain sparsed by 44 amino acid sequences for SMC4 in P. berghei. Similar pattern has also been observed in other studied Plasmodium spp. Subsequently, we generated a 3D model of the P. berghei SMC2 and SMC4 ATPase head domain and partial coiled region using homology-based 3D structure modeling (Figure 1D). Root mean square deviation (RMSD) analysis of the 10ns molecular dynamics (MD) simulation trajectory of the proteins showed a stable conformation comparable to pre-simulation energy minimized structures. Radius of gyration analysis also confirmed stable conformation for the predicted SMC2 and SMC4 domain structures during the 10ns MD simulation (Figure S1). In this model of the SMC subunits, the N- and C-terminal ABC ATPase head and coiled-coil arms connecting the Hinge domain (Figure 1D) are present, as in other organisms. It is most likely that the heads of Plasmodium SMC2 and SMC4 undergo ATP-dependent engagement and disengagement, and may perform similar chromosomal functions as in other eukaryotes(Hirano, 2016).

Condensin core subunits are expressed at every proliferative stage of the parasite life cycle and have a centromeric location during schizogony

To locate the condensin SMC subunits during two proliferative stages (schizogony and male gametogenesis) of the Plasmodium life cycle, transgenic parasite lines were created to express GFP-tagged SMC2 and SMC4, using single crossover homologous recombination (Figure S2A). Integration PCR and western blot experiments were used to confirm the successful generation of transgenic lines (Figure S2B and S2C). We found that SMC2 and SMC4 are expressed during both schizogony and male gametogenesis. In dividing schizonts within host red blood cells, we observed discrete foci adjacent to the nuclear DNA for both SMC2 and SMC4 in the parasite cell (Figure 2A and 2B), whereas during male gametogenesis the localization was more diffuse, throughout the nucleus (Figure 2C and 2D). To further examine whether these foci are centromeric or centrosomal, we used co-localization immunofluorescence assays using anti-centrin and anti-α-tubulin together with anti-GFP antibodies. In schizonts, immunofluorescence assays with anti-centrin antibodies revealed that the SMC4 location is within close proximity to centrin suggesting non-centrosomal localization (Figure 2E). However, partial co-localization was observed with anti-α-tubulin antibodies in schizonts (Figure 2E) but not during male gametogenesis (Figure 2F). In the absence of Plasmodium centromere-specific antibodies, our preliminary results with live imaging of kinetochore/centromeric proteins NDC80GFP and SMC2mCherry expressed in the same cell, shows a centromeric localization of SMC2 protein with NDC80 (Results not shown, unpublished). In addition, we observed either a nuclear or discrete focal location adjacent to nuclear DNA for SMC4GFP throughout the parasite life cycle including female gametocytes, oocyst development and the liver stages (Figure S3), suggesting that condensin core subunits are likely involved at all proliferative stages during the parasite life cycle.

Figure 2:
Figure 2:

Temporal dynamics of condensin (SMC2 and SMC4) in two distinct Plasmodium proliferative stages (schizogony and male gametogenesis) undergoing atypical mitotic division. (A-D) Live cell imaging of SMC2GFP and SMC4GFP expressed during schizogony (100X magnification) and male gametogenesis (63X magnification). The white arrows in Panels C and D indicate discrete localization in male gametes. DIC: Differential interference contrast, Merge: Hoechst and GFP. Scale bar = 2µm. (E-F) Immunofluorescence fixed-cell imaging of SMC4GFP and colocalization with antibodies specific for centrin and α-tubulin in mitotic cells (Early schizonts 100X magnification and male gametocytes 63X magnification). The white arrow in Panel F indicates exflagellating male gamete. Scale bar = 2µm. See also Figure S2, S3

To identify the SMC2 and SMC4 DNA binding sites in a genome-wide manner, we performed ChIP-seq experiments for schizont (after 8 hours in culture) and gametocyte (6 min post activation) stages using SMC2GFP and SMC4GFP-tagged parasites. A wild type strain (WTGFP) was used as a negative control. Binding of the SMC2 and SMC4 subunits was restricted to a region close to the previous computationally annotated centromeres (centromere locations for P. berghei chromosomes were predicted based on using P. falciparum as reference and conservation of genomic sequences among Plasmodium spp.) of all 14 chromosomes at the schizont stage (Figure 3A)(Iwanaga et al., 2012). This restriction was not observed during gametocytogenesis with a random distribution of condensin core subunits, suggesting a distinct function for the core subunits in these two mitotic stages. Identical patterns were obtained between biological replicates for each condition analyzed, confirming the reproducibility of the ChIP-seq experiments.

Figure 3:
Figure 3:

ChIP-seq analysis of SMC2GFP and SMC4GFP profiles. (A) Genome-wide ChIP-seq signal tracks for SMC2GFP and SMC4GFP for all 14 chromosomes in schizont and gametocyte stages. The localization of previously annotated centromeres is indicated by blue circles. SMC2 and SMC4 proteins bind near the putative centromere in each of the 14 chromosomes (distance from centromere is shown in ±kb). (B) Zoom-in on regions associated with the ChIP-seq peak identified, low GC-content at the centers of peaks shown for chromosome 11 and chromosome 14, which suggest association of the proteins with these newly defined centromeres in the schizont stage. Signals are plotted on a normalized read per million (RPM) basis. See also Table S1.

The chromosome binding sites of SMC2 and SMC4 in the schizont were slightly away from the locations previously annotated as centromeres(Iwanaga et al., 2012). However, at the centers of the ChIP-seq peaks for all 14 chromosomes, we identified distinct regions of very low GC-content that are not present in the previously annotated centromeric regions (shown for chromosomes 11 and 14 in Figure 3B). AT-rich troughs have been associated with centromeres in yeasts(Lynch et al., 2010) The peaks are also centered within extended intergenic regions suggesting that the SMC binding sites are the first experimentally validated centromeres for all 14 P. berghei chromosomes (Table S1), and these are different to the previously computationally annotated centromeres, which are slightly different(Iwanaga et al., 2012).

The full condensin complex is present during male gametogenesis, but ancillary proteins are absent during schizogony

To examine the co-localization of SMC2 and SMC4 proteins, we generated transgenic parasite lines expressing either SMC2mCherry or SMC4GFP and crossed them genetically. The progeny, expressing both SMC2mCherry and SMC4GFP, showed co-localization of the two proteins during schizogony and gametogenesis (Figure 4A) consistent with SMC2 and SMC4 heterodimer complex formation at both stages.

Figure 4:
Figure 4:

Differential condensin complex formation during schizogony and male gametogenesis, and phylogenetic analysis of kleisin. (A) Co-localization of SMC4GFP (green) and SMC2mCherry (red). Merge: Hoechst, GFP and mCherry. Scale bar = 2µm. (B) Venn diagram displays the unique and shared proteins in the condensin complex of schizonts and gametocytes. Analysis of SMC2GFP and SMC4GFP protein complexes by tryptic digestion and liquid chromatography-mass spectrometry following GFP-specific immunoprecipitation from a lysate of schizonts maintained in culture for 8 hour and gametocytes activated for 6 min. The list of all identified proteins is provided as Table S2. (C) Different domain architecture for subunits of Condensin I and Condensin II complexes. Schematic figure displays domain composition and protein length in the respective complex subunits. (D) Maximum likelihood phylogeny based on the alignment of kleisin subunits from apicomplexan species (Plasmodium spp, Toxoplasma gondii, Cryptosporidium parvum, Babesia bovis and Eimeria tenella) and other selected organisms. Topological support from bootstrapping is shown at the nodes. The protein sequences for selected organisms have been provided in File S1. (E) Distribution of condensin components across Apicomplexa and other organisms. Presence (filled circle) or absence (empty circle) of condensin complex genes in each genome. * represent 4 Plasmodium spp., namely, P. falciparum, P. vivax, P. berghei and P. yoelii; # denotes H. sapiens, A. thaliana and D. melanogaster. See also Table S2 and File S1.

Next, we directly investigated the interaction between SMC2 and SMC4, and the presence of other interacting partner proteins, such as other condensin components (Figure 1A). We immunoprecipitated SMC2GFP and SMC4GFP from lysates of cells undergoing mitosis during either schizogony (after 8 hours of incubation in schizont culture medium in vitro, when most parasites are undergoing nuclear division) or during gametogenesis (at 6 min after activation when cells are in the last phase of cell division before cytokinesis and the chromosomes are beginning to condense). Immunoprecipitated proteins were then digested with trypsin and the resultant peptides analyzed by liquid chromatography and mass spectrometry (LC/MS). At both stages of the life cycle that were investigated, we recovered peptides from both SMC subunits confirming SMC2-SMC4 heterodimer formation during schizogony and male gametogenesis (Figure 4B). In schizonts, only SMC2 and SMC4 derived peptides were recovered, whereas from gametocyte lysates we detected kleisins and other components of canonical Condensin I and II complexes together with the SMC subunits (Figure 4B and Table S2). In some cases, condensin II HEAT subunit (CAP-G2 and CAP-D3) were observed, however since kleisin was never recovered in 5 experimental replicates of schizogony, we assume that condesin II complex formation is absent at this stage (Table S2). Analysis of the Plasmodium genome identified genes coding for both condensin I and condensin II complex subunits, in agreement with the immunoprecipitation in which we detected Kleisin Iγ (CAP-H), Heat IA (CAP-D2) and Heat IB (CAP-G) from the condensin I complex, and Kleisin IIβ (CAP-H2), Heat IIA (CAP-D3) and Heat IIB (CAP-G2) from the condensin II complex. The domain architecture of these subunits from P. berghei is shown in Figure 4C. The presence of both condensin I and II in male gametocytes is consistent with the potentially atypical chromosomal condensation has been previously observed in male gametocytes just before exflagellation(Sinden, 1991b; Sinden et al., 1976; Sinden and Hartley, 1985). In other organisms, kleisin is essential for formation of the full condensin complex, and in the absence of kleisin, chromosomal condensation is ablated(Cuylen et al., 2011; Rawlings et al., 2011). The presence of kleisins and other components of both condensin complexes in gametocytes is consistent with the potential role these complexes may play in chromosome condensation during exflagellation.

In view of the importance of kleisin to the structure and function of the condensin complexes, we examined the evolutionary relationships of kleisin among some Apicomplexa and other organisms including S. cerevisiae, A. thaliana and Homo sapiens. The phylogenetic analysis indicated that kleisin is clustered into two groups, which correspond to components of condensin I and condensin II, respectively (Figure 1A and 4D). The presence of both condensin I and II component genes only in Plasmodium and Cryptosporidium shows that requirement for both condensin complexes is not a universal feature of Apicomplexa (Figure 4E). This suggests that these two genera have unique aspects of chromosome segregation and cytokinesis.

Knockdown of condensin (SMC2 and SMC4) expression affects parasite proliferation and impairs parasite transmission

To examine further the functions of SMC2 and SMC4, we first attempted to delete the two genes. In both cases we were unable to produce gene knockout (KO) mutants (Figure S4A). Similar results have been reported previously from large scale genetic screens in P. berghei(Bushell et al., 2017; Schwach et al., 2015). Together these data indicate that the condensin subunits SMC2 and SMC4 are likely essential for asexual blood stage development (schizogony). To investigate the function of SMC2 and SMC4 during cell division in male gametogenesis, we used a promoter trap double homologous recombination (PTD) approach to down-regulate gene expression at this stage by placing each of the two genes under the control of the AMA1 promotor. AMA1 is known to be highly expressed in asexual blood stages but not during sexual differentiation. This strategy resulted in the successful generation of two transgenic parasite lines in which SMC2 and SMC4 subunit expression decreased significantly in sexual stages: Pama1smc2 (SMC2PTD) and Pama1smc4 (SMC4PTD), respectively (Figure S4B and S4C).

Since SMC2PTD and SMC4PTD had similar phenotypes, we performed a global transcriptome analysis only on SMC4PTD to identify other affected genes and regulators involved in cell division and proliferation. This analysis of SMC4PTD gametocytes 30 min after activation (when chromosome condensation and exflagellation is complete) confirmed the nearly complete ablation of smc4 gene expression (Figure 5A). For pairs of the two biological samples (wild-type [WTGFP] and SMC4PTD), the Spearman correlation coefficients of 0.97 and 0.99 respectively, demonstrated the reproducibility of this experiment. (Figure 5B). In addition to SMC4, expression of a further 104 genes was also significantly downregulated, while expression of only 5 genes was significantly upregulated (Figure 5C and Table S3). Gene Ontology (GO) enrichment analysis of the downregulated genes identified several associated with microtubule and cytoskeletal function (Figure 5D). The reduced expression levels of ten of these genes was also examined by qRT-PCR (Figure 5E). By this method, there was a statistically significant difference in the level of expression of nine of these genes when comparing samples from WTGFP and SMC4PTD (Figure 5E). Of particular interest are AP2O2 (an AP2 domain transcription factor) and HMG (putative high mobility group protein B3), which act as transcription regulators in ookinetes; a putative SET domain protein which is known to be involved in methyl group transfer from S-adenosyl-L-methionine (AdoMet) to a lysine residue in histones and most likely associated with transcriptional repression; and finally a RCC; a protein predicted to be involved in chromosome condensation and chromosomal dynamics(Bahl et al., 2002). Other genes that were significantly downregulated include FRM2, involved in cytoskeleton organization; CCRNOT2 and NOT that form the CCR4-NOT complex, a key regulator of eukaryotic gene expression; and SEC7, involved in regulation of ARF protein signal transduction (SEC7). Some of the other significantly downregulated genes, include AP2 transcription factor AP2-Sp; molecular motor kinesin-4, a putative regulator of chromosome condensation (PBANKA_0820800); and SMC1, a member of the SMC family, all known to be involved in either gene expression or chromatid segregation.

Figure 5:
Figure 5:

Global transcriptomic analysis for SMC4PTD in activated gametocytes by RNA-seq. (A) Confirmation of successful depletion of SMC4 transcript in the SMC4PTD line. (B) Log-normalized scatterplots demonstrating high correlation between replicates for genome-wide expression. Spearman correlation calculated using read counts normalized by number of mapped reads per million. (C) MA plot summarizing RNA-seq results. M: Log ratio and A: Mean average. Every gene is placed according to its log fold expression change in the SMC4PTD line as compared to the WT line (y-axis), and average expression level across replicates of both lines (x-axis). Red color indicates statistical significance of differential expression at the false positive threshold of 0.05. 105 genes are downregulated in the SMC4PTD line, and 5 genes are upregulated. (D) GO enrichment analysis of genes with log10 fold expression change of −0.5 or lower in the SMC4PTD line. (E) qRT-PCR analysis of selected genes identified as down-regulated in (C), comparing transcript levels in WT and SMC4PTD samples. Error bar = ±SEM, n= 3. Unpaired t-test was performed for statistical analysis: *p<0.05 **p<0.001, ***p<0.0001 and ****p<0.00001. See also Table S3, S5 and Figure S4.

While we were unable to detect any particular impaired phenotype in the SMC2PTD and SMC4PTD lines at the asexual blood stage (schizogony), and the parasite formed a similar number of schizonts and nuclei as compared to the WTGFP line (Figure 6A), we observed a ∼50% reduction in the number of exflagellation centers during male gametogenesis (Figure 6B). Fertilization and zygote formation leading to ookinete conversion was reduced to 10-15% (Figure 6C). The ookinete motility assay showed normal movement of SMC4PTD ookinetes as compared to WT (Video S1 and S2). In the mosquito gut on days 9, −14 and −21 post infection, we detected significantly fewer oocysts in the SMC2PTD and SMC4PTD lines (Figure 6D and 6E). Furthermore, the oocysts were considerably smaller compared to those of WTGFP (Figure 6D and Figure 6F) with unequal distribution and clusters of DNA in some of the oocysts at 14 and 21 days post infection. No sporogony or endomitosis was observed within oocysts (Figure 6G). We were also unable to detect any sporozoites in the mosquito salivary glands (Figure 6H) and hence no parasite transmission from infected mosquitoes to mice was observed for either SMC2PTD or SMC4PTD parasite lines, in bite-back experiments (Table S4) indicating that condensins are required for parasite transmission.

Figure 6:
Figure 6:

Phenotypic analysis of conditional gene expression knockdown in SMC2PTD and SMC4PTD transgenic lines at various proliferative stages during lif e cycle. (A) Average number of nuclei per schizont (mitotic division within red cell). N = 5 (minimum 100 cells). (B) Number of exflagellation centers (mitotic division during male gametogenesis) per field at 15 minutes post-activation. N = 3 independent experiments (10 fields per experiment) (C) Percentage ookinete conversion from zygotes. Minimum of 3 independent experiments (minimum 100 cells) (D) Live cell imaging of WTGFP, SMC2PTD and SMC4PTD oocysts (endomitosis in parasite within mosquito gut) at 9, 14 and 21 days post infection, using 10X and 63X magnification to illustrate differences in size and frequency. Scale bar = 5µm (63X) and 20µm (10X). (E) Number of oocysts at 9, 14 and 21 days post-infection (dpi). N = 3 independent experiments with a minimum of 5 mosquito guts. (F) Oocyst diameter at 9, 14 and 21 days post-infection (dpi). N = 3 independent experiments. (G) Number of sporozoites at 14 and 21 dpi in mosquito gut. N = 3 independent experiments with a minimum of 5 mosquito guts. (H) Number of sporozoites at 21 dpi in mosquito salivary gland. Minimum of 3 independent experiments. Error bar = ±SEM. Unpaired t-test was performed for statistical analysis: *p<0.05 **p<0.01 and ***p<0.001. See also Table S4 and Figure S4.

Discussion

Condensins are multi-subunit complexes that are involved in chromosomal condensation, organization and segregation, and have been widely studied in many eukaryotes(Hirano, 2016; Uhlmann, 2016). Their role in many unicellular protozoans such as Plasmodium is unknown. Here we describe the structure, localization and functional role of the condensin core subunits (SMC2/SMC4) in the mouse malaria-causing parasite, P. berghei using molecular dynamics, live cell imaging, protein pulldown and conditional gene knockdown approaches. Plasmodium shows atypical features of closed mitotic division during schizogony (with no observed chromosomal condensation and extensive nuclear division prior to cytokinesis) and male gametogenesis (with atypical chromosomal condensation and endoreduplication observed prior to exflagellation). Our previous studies have shown that the parasite has an unusual repertoire of proteins involved in the regulation of the cell cycle and cell proliferation: it has no identifiable centrosome, no obvious complement of cell-cycle cyclins, a small subset of APC components, a set of divergent and Plasmodium-specific CDKs, and a complete absence of polo-like kinases and CDC24 and CDC14 phosphatases, as compared to most organisms that have been studied(Arnot et al., 2011; Francia et al., 2015; Guttery et al., 2012a; Guttery et al., 2014; Roques et al., 2015; Tewari et al., 2010).

Here, using bioinformatics screening, we identified both condensin I and II complex subunit components encoded in the Plasmodium genome. The two core subunits of condensin, SMC2 and SMC4 have low sequence similarity to the proteins in model organisms but a similar protein structure was predicted by molecular modelling.

Protein localization studies in different stages of the parasite life cycle using live cell imaging of SMC2GFP and SMC4GFP show distinct patterns during the mitotic divisions of schizogony and male gametogenesis. Whereas discrete foci were detected during endomitosis in schizogony, a more diffused nuclear localization was observed during male gametogenesis. The discrete foci during schizogony were not coincident with centrin, marking the SPB but very close to it. The IFA and ChIP-seq analyses suggest that SMC2 and SMC4 form a complex that binds at or near the centromere of all 14 chromosomes, and that these protein-DNA complexes are located in the nucleus near the SPB in schizonts. Genome-wide studies of condensin distribution in mammalian or yeast cells have shown that the complex is non-randomly distributed across the chromosomes, and often found at the boundaries of topologically associating domains (TAD) within chromosome territories, which supports the proposed role in transcriptional regulation and global chromosomal organization(Kim et al., 2016; Yuen et al., 2017). The Plasmodium genome lacks classical TADs(Ay et al., 2014; Bunnik et al., 2018; Bunnik et al., 2019), and our results suggest that condensins are restricted to binding centromeric regions in the highly proliferative asexual stage, where they may have a constrained role in sister chromatid cohesion and segregation(Iwasaki and Noma, 2016).

In activated gametocytes, where the condensin complexes do not bind specifically to the centromeres of chromosomes, the proteins are distributed throughout the nucleus. This is in concordance with centromere location in many other organisms(Bachellier-Bassi et al., 2008; Fujiwara et al., 2013). The diffuse pattern observed during male gametogenesis was distinct to that in schizogony, and since both SMC2 and SMC4 are core subunits of condensin complexes, it was surprising that they had a unique distribution in the two different mitotic stages in Plasmodium. Interestingly, the protein interaction analysis showed that different complexes formed during the two mitotic divisions. We detected only the SMC2-SMC4 heterodimer in schizogony, and we observed both condensin I and condensin II complexes during male gametogenesis. These results substantiate previous electron microscopy results in which atypical and limited chromosomal condensation was observed during male gametogenesis but not in schizogony(Sinden, 1991b; Sinden et al., 1976; Sinden and Hartley, 1985). Our bioinformatics analysis shows that two parasites, Plasmodium and C. parvum, both with unusual modes of cell division(Francia and Striepen, 2014), are the only Apicomplexa where components of both condensin I and II complexes are encoded in the genomes, similar to what is observed in higher eukaryotes. In other parasites, Trypanosoma brucei encodes only condensin I complex(Hammarton, 2007) with closed mitosis and no chromosome condensation, whereas in Giardia intestinalis, both condensin complexes (condensin I and condensin II) are present and chromosome condensation occurs(Tumova et al., 2015). However, Giardia lacks one of the non-SMC HEAT subunits (HEAT IB and HEAT IIB). Another protist, Tetrahymena thermophila, exhibits noncanonical cell division between somatic and germline nucleus and has expanded paralogs of condensin I, with different kleisin components between germline (Cph1 and Cph2) and somatic cells (Cph3, Cph4 and Cph5)(Howard-Till and Loidl, 2018; Howard-Till et al., 2019).

For condensin I and II complexes, a differential localization pattern has been observed in various organisms. In the red alga Cyanidioschyzon merolae(Fujiwara et al., 2013), condensin II has a centromeric location during metaphase, whereas condensin I distributes more broadly along the chromosome arms. In higher eukaryotes including Drosophila melanogaster(Oliveira et al., 2007), Caenorhabditis elegans(Collette et al., 2011) and HeLa cells(Hirota et al., 2004; Ono et al., 2004) condensin I is present in the cytoplasm and has a nuclear localization after nuclear envelope breakdown in open mitosis, whereas the nuclear localization of condensin II is observed in interphase, it is stabilized on chromatin during prophase, and the complex remains associated with chromosomes throughout mitosis, at least in HeLa cells. Budding yeast and fission yeast which undergo closed mitosis like Plasmodium have only a single condensin complex, but there is a differential localization pattern in each species. In budding yeast, the condensin I complex is located in the nucleus throughout the cell cycle, a pattern observed for condensin II in higher eukaryotes, despite the greater protein sequence similarities of the yeast complex to higher eukaryote condensin I(Thadani et al., 2012). Also, within the nucleus, the condensin localization at the kinetochore is cell cycle dependent(Bachellier-Bassi et al., 2008). In fission yeast, the single condensin complex is predominantly cytoplasmic during interphase and nuclear during mitosis, with the location dependent on CDK phosphorylation at Thr19 of SMC4/Cut3(Sutani et al., 1999).

The present study shows that the SMC2-SMC4 complex plays an essential role during schizogony, as we and previous genome-wide functional screens were unable to disrupt the genes(Bushell et al., 2017). Our conditional knockdown approach using the promoter trap suggests that reduction in SMC2 or SMC4 affects male gametogenesis and zygote differentiation and causes total impairment of endomitotic cell division in the oocyst, thereby blocking parasite transmission.

The partial defect observed in male gamete formation (exflagellation) in the PTD parasite lines may be due to the necessity of condensin complex formation for proper chromosomal condensation during exflagellation. RNA-seq analysis of the SMC4PTD line confirmed the reduced expression of the smc4 gene and identified dysregulated transcripts that are likely either critical for gene expression, or chromosomal segregation and condensation, microtubule assembly and male gametocyte activation. This further demonstrates that SMC2 and SMC4 complexes are essential for proper chromosome condensation and separation during exflagellation. Among the significantly dysregulated genes, deletion of AP2-O2, has been shown to strongly impair ookinete and oocyst development, leading to an absence of sporozoite formation and a complete blockage of transmission(Modrzynska et al., 2017). The SET protein, which is a post translational modification protein, is essential for parasite survival(Schwach et al., 2015). RCC is predicted to be a regulator of chromosome condensation, is essential for parasite survival, and acts as anchor for both parasite kinase (CDPK7) and phosphatase (PP1)(Lenne et al., 2018). The phenotype observed in SMC4PTD parasites may therefore reflect contributions from all these differentially regulated genes.

The reduction in mature ookinete formation in the SMC4PTD line, which is the stage when meiosis takes place, suggests an important role of condensin during meiosis as well. During this stage, chromosomal condensation has been observed(Sinden and Hartley, 1985), and this may be similar to the situation in Arabidopsis, where condensin is important in chromosomal condensation during meiosis(Smith et al., 2014). A severe defect in number and size of oocyst formation in the mosquito gut was also observed, and at this stage multiple rounds of endomitotic division give rise to thousands of sporozoites. The process requires ten or more rounds of DNA replication, segregation, and mitotic division to create a syncytial cell (sporoblast) with thousands of nuclei over a period of several days(Francia and Striepen, 2014; Gerald et al., 2011). The proper organization of nuclei into individual sporoblasts is organized by putative MTOCs(Roques et al., 2019; Sinden and Strong, 1978). As condensin has been shown to play an important role in organizing MTOCs(Kim et al., 2014) it may be that in the absence of condensin the endomitotic division is impaired and no sporozoites are formed. Deletion of a number of genes that affect oocyst maturation results in a phenotype similar to that of the SMC2PTD and SMC4PTD parasite lines. For example PbMISFIT, causes reduced oocyst size(Bushell et al., 2009), and PbCYC3 has smaller oocysts but sporogony, sporozoite formation and transmission are normal(Roques et al., 2015), and disruption of the gene for a G-actin sequestering protein affects oocyst maturation and sporozoite formation(Hliscs et al., 2010). A Plasmodium mutant lacking a protein phosphatase (PbPPM5) shows a similar oocyst and sporozoite development pattern but in a global transcriptome analysis no significant differential expression of SMC4PTD was observed(Guttery et al., 2014).

In summary, the present study shows that the condensin core subunits SMC2 and SMC4 play crucial roles in the atypical mitoses of the Plasmodium life cycle. Their removal or depletion causes impaired parasite development and blocks transmission. Further analyses of non-SMC components of condensin I and II will give us better insight into the function of condensin during Plasmodium cell proliferation.

Methods

Ethics statement

All animal work done at the University of Nottingham has passed an ethical review process and has been approved by the United Kingdom Home Office. The work was carried out under UK Home Office Project Licenses (40/3344 and 30/3248).

Bioinformatics analysis

Condensin complex protein sequences were retrieved from PlasmoDB(Bahl et al., 2002), EuPathDB(Aurrecoechea et al., 2010) and from NCBI databases for model organisms (File S1). An NCBI conserved domain database (CDD) search was used to identify conserved domains. PHYRE2(Kelley et al., 2015) was used to generate 3D structure models. GROMACS 4.6.3(Van Der Spoel et al., 2005) with CHARMM27(Sapay and Tieleman, 2011) force field was used to perform molecular dynamics simulation in an aqueous environment. The energy minimization was performed using steepest descent minimization till maximum force reached below 1000KJ/mol/nm. Temperature and pressure equilibrium were done for 1ns, respectively before performing the 10 ns production simulation. ClustalW was used to generate multiple sequence alignments of the retrieved sequences(Larkin et al., 2007). ClustalW alignment parameters included gap opening penalty (GOP) of 10 and gap extension penalty (GOE) of 0.1 for pairwise sequence alignments, and GOP of 10 and GOE of 0.2 for multiple sequence alignments, gap separation distance cut-off value of 4 and the Gonnet algorithm in protein weight matrix. Other parameters like residue-specific penalty and hydrophobic penalties were “on” whereas end gap separation and use of negative matrix were set to “off”. The phylogenetic tree was inferred using the neighbor-joining method, computing the evolutionary distance using the Jones Taylor Thornton (JTT) model for amino acid substitution with the Molecular Evolutionary Genetics Analysis software (MEGA 6.0) (Tamura et al., 2013). Gaps and missing data were treated using a partial deletion method with 95% site-coverage cut-off. We performed 1000 bootstrap replicates to infer the final phylogenetic tree.

Generation of transgenic parasites

GFP-tagged vectors were designed using the p277 plasmid vector and transfected as described previously(Guttery et al., 2014). Targeted gene deletion vectors were designed using the pBS-DHFR plasmid(Tewari et al., 2010). Conditional gene knockdown constructs (SMC2PTD and SMC4PTD) were designed using Pama1 (pSS368)(Sebastian et al., 2012). P. berghei ANKA line 2.34 (for GFP-tagging) or ANKA line 507cl1 (for gene deletion and promoter swap) parasites were transfected by electroporation as described previously(Wall et al., 2018). Genotypic analysis was performed using diagnostic PCR reaction and Western blot. All of the oligonucleotides used to confirm genetically modified tag and mutant parasite lines can be found in Table S5.

ChIP-seq and Global transcriptomic analysis

For the ChIP-seq analysis, libraries were prepared from crosslinked cells (using 1% formaldehyde) and for the global transcriptome analysis, libraries were prepared from lyophilized total RNA, both using the KAPA Library Preparation Kit (KAPA Biosystems). Libraries were amplified for a total of 12 PCR cycles (15 s at 98°C, 30 s at 55°C, 30 s at 62°C) using the KAPA HiFi HotStart Ready Mix (KAPA Biosystems). Libraries were sequenced using a NextSeq500 DNA sequencer (Illumina), producing paired-end 75-bp reads. FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/), was used to analyze raw read quality. Any adapter sequences were removed using Trimmomatic (http://www.usadellab.org/cms/?page=trimmomatic). Bases with Phred quality scores below 25 were trimmed using Sickle (https://github.com/najoshi/sickle). The resulting reads were mapped against the P. berghei ANKA genome (v36) using Bowtie2 (version 2.3.4.1) or HISAT2 (version 2-2.1.0), using default parameters. Reads with a mapping quality score of 10 or higher were retained using Samtools (http://samtools.sourceforge.net/), and PCR duplicates were removed by PicardTools MarkDuplicates (Broad Institute). For the transcript analysis, raw read counts were determined for each gene in the P. berghei genome using BedTools (https://bedtools.readthedocs.io/en/latest/#) to intersect the aligned reads with the genome annotation and for the ChIP-seq to obtain the read coverage per nucleotide. For the transcriptomic analysis, read counts were normalized by dividing by the total number of mapped reads for the library. Genome browser tracks were generated and viewed using the Integrative Genomic Viewer (IGV) (Broad Institute). Proposed centromeric locations were obtained from Iwanaga and colleagues(Iwanaga et al., 2012). GC content was calculated using a sliding window of 30 bp across the peak region as described previously34. SMC2 gametocyte sample is shown at half height due to higher level of background compared to other samples. Differential expression analysis was done in two ways: (1) the use of R package DESeq2 to call up- and down-regulated genes, and (2) manual analysis, in which raw read counts were normalized by library size, and genes above a threshold level of difference in normalized read counts between conditions were called as up- or down-regulated. Gene ontology enrichment was done using PlasmoDB (http://plasmodb.org/plasmo/) with repetitive terms removed by REVIGO (http://revigo.irb.hr/).

Immunoprecipitation and Mass Spectrometry Analysis

Schizonts, following 8 hours in in vitro culture, and male gametocytes 6 min post activation were used to prepare cell lysates. Purified parasite pellets were crosslinked using formaldehyde (10 min incubation with 1% formaldehyde, followed by 5 min incubation in 0.125M glycine solution and 3 washes with phosphate buffered saline (PBS) pH7.5). Immunoprecipitation was performed using crosslinked protein and a GFP-Trap®_A Kit (Chromotek) following the manufacturer’s instructions. Proteins bound to the GFP-Trap®_A beads were digested using trypsin and the peptides were analysed by LC-MS/MS.

Phenotypic Analysis

Phenotypic analyses of the mutant parasite lines were performed at different points of parasite life cycle as described previously(Guttery et al., 2014). Briefly, infected blood was used to analyse asexual blood stages and gametocytes. Schizont culture was used to analyse different stages of asexual development. In vitro cultures were prepared to analyse activated gametocyte, exflagellation, zygote formation and ookinete development. For in vitro exflagellation studies, gametocyte-infected blood was obtained from the tails of infected mice using a heparinised pipette tip. Gametocyte activation was performed by mixing 100 µl of ookinete culture medium (RPMI 1640 containing 25 mM HEPES, 20% fetal bovine serum, 10 mM sodium bicarbonate, 50 µM xanthurenic acid at pH 7.6) to the gametocyte infected blood. Microgametogenesis was monitored at two time points to study mitotic division (6 and 15 min post activation [mpa]). The material was fixed and processed for immunofluorescence assay (IFA) using a range of different antibody markers. For mosquito transmission and bite back experiments, triplicate sets of 40-50 Anopheles stephensi mosquitoes were used. The mosquito guts were analysed on different days post infection (dpi); 9 dpi, 14 dpi and 21 dpi to check oocyst development and sporozoite formation. Parasites were visualised on a Zeiss AxioImager M2 microscope fitted with an AxioCam ICc1 digital camera (Carl Zeiss, Inc).

Quantitative RT-PCR

RNA was isolated from different parasite life stages, which include asexual stages, purified schizonts, activated and non-activated gametocytes, ookinetes and sporozoites, using an RNA purification kit (Stratagene). cDNA was prepared using an RNA-to-cDNA kit (Applied Biosystems). Primers for qRT-PCR were designed using Primer3 (Primer-blast, NCBI). Gene expression was quantified from 80 ng of total cDNA. qRT-PCR reactions used SYBR green fast master mix (Applied Biosystems) and were analysed using an Applied Biosystems 7500 fast machine. Experiments used hsp70 and arginine-tRNA synthetase as reference genes. The primers used for qRT-PCR can be found in Table S5.

Statistical analysis

Statistical analysis was performed using Graph Pad Prism 7 software. An unpaired t-test was conducted to examine significant differences between wild-type and mutant strains.

Data Availability

Sequence reads have been deposited in the NCBI Sequence Read Archive with accession number PRJNA542367.

Author contribution

RT, AAH and KGLR conceived and designed all experiments. RT, RP, SA, MB, RJW, MZ, ER, AF, DB, ED and SW performed the GFP tagging and conditional knockdown experiments; RRS performed liver stage imaging; RP, MZ, ED and RT performed protein pulldown experiments; ARB performed the mass spectroscopy; RP and DG performed the phylogenetic analysis and molecular dynamics; SA, RP XML, GB, TH, KGLR and RT performed the RNA-seq and ChIP-Seq experiments; RP, SA, AAH, KGLR and RT analyzed the data; RP, SA, AAH, KGLR and RT wrote the manuscript and all others contributed to it.

Declaration of Interests

The authors declare no competing interests.

Figure S1:
Figure S1:

Molecular dynamics simulation showing stable predicted 3D structure for SMC2 and SMC4. Related to Figure 1. Root mean square deviation (RMSD) calculation using protein backbone structure, during the 10ns production simulation for SMC2 (A) and SMC4 (B), respectively. Post 8ns and 2ns MD simulation, RMSD fluctuations becomes comparable and constant for SMC2 and SMC4 respectively. (C and D) Radius of gyration fluctuations within 2Å suggest correct and stable protein fold for SMC2 and SMC4, respectively. Additionally, during a 10ns molecular dynamics run the protein structure did not break, confirming stable predicted 3D structure.

Figure S2:
Figure S2:

Generation and genotype analysis of SMC2GFP and SMC4GFP parasite lines. Related to Figure 2. (A) Schematic representation of the endogenous smc(2/4), the GFP-tagging construct and the recombined smc(2/4) locus following single homologous recombination. Arrows 1, 2 and 3 indicate the position of PCR primers used to confirm successful integration of the construct. (B) Diagnostic PCR of SMC2GFP, SMC4GFP and WT parasites using primers IntT138 (SMC2, Arrow 1), IntT143 (SMC4, Arrow 1) and ol492 (Arrow 3). IntT138 and T1382 (SMC2, Arrow 2), IntT143 and T1432 (SMC4, Arrow 2) primers were used as control. Integration of the SMC tagging construct gives a band of 995 bp and 1006 bp for SMC2GFP and SMC4GFP parasite lines. (C) Western blot of SMC2GFP (172 kDa), SMC4GFP (198kDa) and WTGFP (29 kDa) protein to illustrate SMC2GFP and SMC4GFP in schizont stage extracts.

Figure S3:
Figure S3:

Localisation of SMC4GFP throughout the Plasmodium life cycle as detected by live cell imaging. Related to Figure 2. (A) Asexual blood stages and sexual stages at different time points. Merge: Hoechst (blue, DNA), GFP (green) and P28 (red, cell surface marker during female gamete activation, zygote and ookinete stages). (B) Sporogony in the mosquito oocyst; 9 days post infection (dpi), 14 dpi, 21 dpi and mature single sporozoite at 21 dpi. Merge: Hoechst and GFP. (C) Liver stage schizont at 60 hours post infection. Merge: DAPI (blue) and GFP (green). Scale bar = 2µM.

Figure S4.
Figure S4.

Generation of knockout and conditional knockdown of SMC2 and SMC4 genes using a dhfr drug-selectable marker or ama1 promoter trap double homologous recombination (PTD), and genotype analysis. Related to Figure 6. (A) Schematic representation of the endogenous smc(2/4) locus, the targeting gene deletion construct and the recombined smc(2/4) locus following double homologous recombination. (B) Schematic representation of the promoter trap strategy (SMC2PTD and SMC4PTD), placing smc(2/4) under the control of the blood stage ama1 promoter by double homologous recombination. Arrows 1 and 2 indicate the primers position used to confirm 5’ integration and arrows 3 and 4 indicate the primers used for 3’ integration. Primers 1 and 4 were also used for Knock-In PCR. (C) Integration PCR of the promotor trap construct into the smc(2/4) locus. Primer 1 (5’-IntPTD18 [SMC2], 5’-IntPTD008 [SMC4]) with primer 2 (5’-IntPTD) were used to determine successful 5’ integration of the selectable marker resulting in a band of 1518 and 1655 bp for SMC2PTD and SMC4PTD, respectively. Primer 3 (3’-intPTama1) and primer 4 (3’-IntPTD18 (SMC2) and 3’-IntPTD008 (SMC4)) were used to determine the successful 3’ integration of ama1 promoter resulting in a band of 1004 bp and 877 bp for SMC2PTD and SMC4PTD, respectively. Primer 1 (5’-IntPTD18 and 5’-IntPTD008) and primer 4 (3’-IntPTD18 and 3’-IntPTD008) were used to show complete knock-in of the construct with a band at 4698 bp (SMC2PTD) and 4653 bp (SMC4PTD), and the absence of a band at 1538 bp (for SMC2, endogenous) and 1493 bp (for SMC4, endogenous) resulting in complete knock-in of the construct.

Table S1: Experimentally validated coordinates of the P. berghei centromeres. Related to Figure 3.

Table S2: List of main protein hits in the SMC2GFP and SMC4GFP co-immunoprecipitation experiments. Related to Figure 4.

Table S3: List of differentially expressed genes between SMC4PTD and WT activated gametocytes. Related to Figure 5.

View this table:
Table S4:

Mosquito bite back analysis of WTGFP, SMC2PTD and SMC4PTD parasites. Related to Figure 6.

Table S5: Primers used in this study (5’ – 3’). Related to Figure 2, Figure 5 and Figure 6.

File S1:

Protein sequences used for phylogenetic analysis. Related to Figure 4.

Video S1: Ookinete motility assay for SMC4PTD parasites. Related to Figure 6.

Video S2: Ookinete motility assay for WT parasites. Related to Figure 6.

Acknowledgements

We thank Prof. Frank Uhlmann, The Francis Crick Institute, for stimulating discussions and advice on condensins and Julie Rodgers for insectary assistance. This project was funded by MRC project grants and MRC Investigators grants awarded to RT (G0900109, G0900278, MR/K011782/1) and BBSRC (BB/N017609/1). RP was supported by MRC grant MR/K011782/1. AAH was supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001097), the UK Medical Research Council (FC001097), and the Wellcome Trust (FC001097). DG was supported by the Department of Biotechnology (DBT), Government of India, grant BT/BI/25/066/2012, KGLR was supported by the National Institutes of Allergy and Infectious Diseases and the National Institutes of Health (grants R01 AI06775, R01 AI136511 and R21 AI142506-01) and the University of California, Riverside (NIFA-Hatch-225935).

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Posted June 17, 2019.
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Plasmodium condensin core subunits (SMC2/SMC4) mediate atypical mitosis and are essential for parasite proliferation and transmission
Rajan Pandey, Steven Abel, Matthew Boucher, Richard J Wall, Mohammad Zeeshan, Edward Rea, Aline Freville, Xueqing Maggie Lu, Declan Brady, Emilie Daniel, Rebecca R. Stanway, Sally Wheatley, Gayani Batugedara, Thomas Hollin, Andrew R. Bottrill, Dinesh Gupta, Anthony A. Holder, Karine G. Le Roch, Rita Tewari
bioRxiv 674028; doi: https://doi.org/10.1101/674028
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