Protein Phosphatase 1 regulates atypical chromosome segregation and cell polarity during mitotic and meiotic division in Plasmodium sexual stages

PP1 is a conserved serine/threonine phosphatase that regulates many aspects of mitosis and meiosis, often working in concert with other phosphatases, such as CDC14 and CDC25, in model eukaryotes. However, the malaria parasite, Plasmodium spp. lacks the CDC14 and CDC25 genes. The proliferative stages of the parasite life cycle include sexual development within the mosquito vector, with male gamete formation characterized by an atypical rapid mitosis, with three rounds of DNA synthesis, successive spindle formation with clustered kinetochore, and a meiotic stage during zygote to ookinete development following fertilization. It is unclear how PP1 is involved these unusual processes. Using real-time live-cell imaging, conditional gene knockdown, RNAseq and proteomic approaches, we show that Plasmodium PP1 is involved in both chromosome segregation during mitotic exit, and establishment of cell polarity during these sexual stages, suggesting that PP1 inhibitors may block parasite transmission. Gametocytes (non-activated 0 min and activated 30 min-) were collected from from PP1PTD and WT lines. For RNA extraction, parasite samples were passed through a plasmodipur column to remove host DNA contamination prior to RNA isolation. Total RNA was extracted from activated gametocytes and schizonts of WT-GFP and PP1PTD parasites (two biological replicates each) using an RNeasy purification kit (Qiagen). RNA was vacuum concentrated (SpeedVac) and transported using RNA stable tubes (Biomatrica). Strand-specific mRNA sequencing was performed of total RNA and using TruSeq stranded mRNA sample prep kit LT (Illumina), as previously described55. Libraries were sequenced using an Illumina Hiseq 4000 sequencing platform with paired-end 150 bp read chemistry. The quality of the raw reads was assessed using FATSQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc). Low-quality reads and Illumina adaptor sequences from the read ends were removed using Trimmomatic R56. Processed reads were mapped to the P. beghei ANKA reference genome (release 40 in PlasmoDB- http://www.plasmoddb.org) using Hisat257 (V 2.1.0) with parameter “—rna-strandness FR”. Counts per feature were estimated using FeatureCounts. Raw read counts data were converted to counts per million (cpm) and genes were excluded if they failed to achieve a cpm value of 1 in at least one of the three replicates performed. Library sizes were scale-normalized by the TMM method using EdgeR software59 and further subjected to linear model analysis using the voom function in the limma package. Differential expression analysis was performed using DeSeq. Genes with a fold-change greater than two and a false discovery rate corrected p-value (Benjamini-Hochberg procedure) < 0.05 were considered to be differentially expressed. Genes with fold change greater than 2 and p-value less than 0.05 were considered as significantly differentially expressed.