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

Abstract

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 live-cell 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.

Fig. 1. Location of PP1 during asexual blood stage schizogony and its association with kinetochore (NDC80) and glideosome (MyosinA).

a Live-cell imaging of PP1-GFP (Green) showing its location during different stages of intraerythrocytic development and in free merozoites. DIC differential interference contrast, Hoechst stained DNA (blue), Merge green and blue images merged. A schematic guide showing the locations of PP1GFP foci during segmentation of merozoites is depicted in right-hand panel. b Live-cell imaging showing location of PP1–GFP (green) in relation to the kinetochore marker NDC80-mCherry (red) and DNA (Hoechst, blue). Merge: green, red and blue images merged. A schematic guide showing the locations of PP1GFP foci with NDC80-mCherry during segmentation of merozoites is depicted in right-hand panel. c Live imaging showing the location of PP1–GFP (green) in relation to inner membrane complex marker MyoA-mCherry (red) and DNA (Hoechst, blue) during different stages of intraerythrocytic development and in extracellular merozoites. A schematic guide showing the locations of PP1GFP foci with MyoA-mCherry during segmentation of merozoites is depicted in the three right-hand panels. Merge: green, red and blue images merged. Sch-E (Early schizont), Sch-M (Middle schizont), Sch-L (Late schizont) and Sch-S (Segmented schizont). In all panels, scale bar = 5 µm.

Fig. 2. The location of PP1 and its association with the kinetochore during chromosome segregation in male gametogony.

a Live-cell imaging of PP1-GFP during male gametogony showing an initial diffused localisation before activation and focal points after activation in the later stages (shown as minutes post activation). Panels are DIC, Hoechst (blue, DNA), PP1-GFP (green) and Merge (green and blue channels). b Live-cell imaging of parasite line expressing both PP1-GFP and NDC80-mCherry showing location of PP1 (green) and NDC80 (red) in male gametocytes at different time points after activation. A schematic guide showing the locations of PP1GFP foci with DNA and NDC80-mCherry during male gametogony is depicted in the right panel. Merge/DNA is green, red and blue (Hoechst, DNA) channels. In both panels, scale bar = 5 µm.

Fig. 3. PP1-GFP localisation during zygote formation, ookinete development and sporogony inside the mosquito gut.

a Live-cell imaging showing PP1-GFP location in male and female gametes, zygote and during ookinete development (stages I–V and mature ookinete). A cy3-conjugated antibody, 13.1, which recognises the P28 protein on the surface of zygotes and ookinetes, was used to mark these stages. Panels: DIC (differential interference contrast), PP1-GFP (green, GFP), 13.1 (red), Merged: Hoechst (blue, DNA), PP1-GFP (green, GFP) and P28 (red). Scale bar = 5 μm. Insets show, at higher magnification, the PP1-GFP signal on the zygote and developing apical end of early stage ‘retorts’, and in the nucleus of late retorts and ookinete stage. b Live-cell imaging of PP1-GFP (green) in relation to NDC80-mCherry (red) and Hoechst staining (blue, DNA) in zygote and ookinete stages. c Live-cell imaging of PP1-GFP in developing oocysts in mosquito guts at 7-, 14- and 21-days post-infection and in a sporozoite. Panels: DIC, Hoechst (blue, DNA), PP1-GFP (green), Merged (blue and green channels). d Live-cell imaging of PP1-GFP in relation to NDC80 in developing oocysts and in a sporozoite. Panels: DIC (differential interference contrast), PP1-GFP (green), NDC80-mCherry (red), Merge (Hoechst, blue, DNA; PP1-GFP, green; NDC80-mCherry, red). In all panels, scale bar = 5 µm.

Fig. 4. PP1 has an important role during male gamete formation and zygote-ookinete development.

a qRT-PCR analysis of pp1 transcription in PP1PTD and WT-GFP parasites, showing the downregulation of pp1. Each bar is the mean of three biological replicates ±SD. b Male gametogony (exflagellation) of PP1PTD line (black bar) and WT-GFP line (white bar) measured as the number of exflagellation centres per field. Mean ± SD; n = 3 independent experiments. c Ookinete conversion as a percentage for PP1PTD and WT-GFP parasites. Ookinetes were identified using P28 antibody as a surface marker and defined as those cells that differentiated successfully into elongated ‘banana shaped’ ookinetes. Round cells show zygotes that did not start to transform and ‘retorts’ could not differentiate successfully in ookinetes. Mean ± SD; n = 3 independent experiments. d Representative images of round cells, retorts and fully differentiated ookinetes. Scale bar = 5 μm. e Total number of GFP-positive oocysts per infected mosquito in PP1PTD and WT-GFP parasites at 7-, 14- and 21-days post infection (dpi). Mean ± SD; n = 3 independent experiments. f Representative images of mosquito midguts on day 14 showing them full of oocysts in WTGFP and no oocyst in PP1PTD. Scale bar = 200 μm for ×10 magnification and 50 μm for ×63 magnification. g Total number of sporozoites in oocysts of PP1PTD and WT-GFP parasites at 14 and 21 dpi. Mean ± SD; n = 3 independent experiments. h Total number of sporozoites in salivary glands of PP1PTD and WT-GFP parasites. Bar diagram shows mean ± SD; n = 3 independent experiments. Unpaired t-test was performed for statistical analysis. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 5. Ultrastructure analysis of PP1PTD gametocytes shows defects in nuclear pole and axoneme assembly during male gametogony.

Electron micrographs of WTGFP (a–c, g–i) and PP1PTD (d–f, j–l) male gametocytes at 6 min (a–f) and 30 min (g–i) post activation (pa). Scale bars represent 1 µm (a, d, g, j) and 100 nm in all other micrographs. a Low power micrograph of a WTGFP male gametocyte with two nuclear poles (NP) associated with the nucleus (N). b Enlargement of the enclosed area showing the nuclear pole (NP) with adjacent basal body (B) and associated axoneme (A). c Cross section of an axoneme showing the 9 + 2 microtubule arrangement. d Low power micrograph of PP1PTD male gametocyte showing the central nucleus (N) with a nuclear pole and associated basal body (enclosed area). e Enlargement of the enclosed area showing the nuclear pole (NP) with adjacent basal body (B) and associated axoneme (A). f Cross section through an axoneme showing the 9 + 2 arrangement of microtubules. g Low power micrograph of a 30-min pa WTGFP male gametocyte showing the nucleus with areas of condensed chromatin. Note the cross sectioned free male gametes (Mg). A – axoneme. h Periphery of a male gametocyte undergoing exflagellation with the nucleus (N) associated with flagellum (F) protruding from the surface. i Cross section of a free male gamete showing the 9 + 2 microtubules of the flagellum (F) and electron dense nucleus (N). j Low power micrograph of a PP1PTD male gametocyte at 30 min pa showing the central nucleus (N) with a nuclear pole and an increased number of axoneme profiles (A) within the cytoplasm. k Detail of the periphery of a nucleus showing the nuclear pole (NP), basal body (B) and associated axoneme (A). l Cross section of an axoneme showing the 9 + 2 microtubule arrangement.

Fig. 6. Transcriptome of PP1PTD mutant reveals important roles of PP1 in parasite cell cycle, motor protein function and cell polarity during gametocyte biology.

Volcano plots showing significantly down-regulated (blue Log₂ fold ≤ −1, q value < 0.05) and up-regulated genes (brown Log₂ fold ≥1, q value < 0.05) in PP1PTD compared to wild-type lines in non (0 min)-activated gametocytes (a) and 30 min activated gametocytes (b). Non-differentially regulated genes are represented as black dots. c Expression heat maps showing affected genes from specific functional classes of proteins such as kinases, phosphatases, motor proteins and proteins associated with parasite motility, entry into the host cell, ookinete surface and cell cycle. Genes are ordered based on their differential expression pattern in non-activated gametocytes. d Validation by qRT-PCR of a few genes randomly selected based on the RNA-seq data. All the experiments were performed three times in duplicate with two biological replicates. *p ≤ 05.

Fig. 7. Interacting partners of PP1 during asexual schizont and sexual gametocyte stages.

a List of proteins interacting with PP1 during schizont and gametocyte stages. b Venn diagram showing common interacting partners in schizonts and gametocytes with some additional proteins specific to gametocytes.