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Abstract

Summary Development of polarity is well studied in cell systems such as mammalian neurons, epithelial cells and Caenorhabditis elegans zygotes. In Plasmodium the spherical and presumed apolar female gamete morphs during the 24 hours after fertilisation in the mosquito midgut into a polarised ookinete, however, little is known about how Plasmodium zygotes develop polarity. It is known that endocytosis-mediated protein transport is generally necessary for the establishment and maintenance of polarity in epithelial cells and neurons, and Rab11A GTPase is an important regulator of protein transport via recycling endosomes. Rab11A has been predicted to be involved in cytokinesis in Plasmodium falciparum and is essential in Plasmodium berghei. Here I show the expression profile of P. berghei Rab11A (PbRab11A) across the life cycle and use a promoter swap strategy to investigate the role of PbRab11A in ookinete development. By expressing PbRab11A under the clag promoter or ama-1 promoter, its expression in sexual stages is greatly reduced while its essential expression in asexual blood stages is maintained. Whilst gamete production and fertility rates remained unaffected, the ookinete conversion rates of CLAG and AMA-1 promoter swap mutants are reduced by up to 99% and 98% respectively and transmission through the mosquito is prevented. TEM analysis and immunofluorescence microscopy of developmental and structural markers show that Rab11A-CLAG promoter swap mutant zygotes lay down the Inner Membrane Complex (IMC) and are apically oriented but appear stop morphological progression and remain spherical. Western blot and transcriptome analysis of Rab11A - CLAG promoter swap mutant suggest marginal expression delay as well as deregulation of some of the transcripts of ookinete development and structural markers, and the majority of deregulated transcripts are independent of previously shown translationally stored transcripts. We predict that Rab11A is not involved in the establishment of P. berghei zygote polarity, mitosis, activation of stored mRNAs nor extensive reactivation of post-meiotic transcription but conceivably in the delivery of plasma membrane to the growing apical outgrowth which is expected to be a coordinated process of Rab11A mediated membrane trafficking and cytoskeletal dynamics. As a second line of enquiry, the establishment of polarity in P. berghei zygote with respect to the point of male and female gamete fusion is ongoing. Establishment of polarity in embryos and zygotes is dependent on the development of the axis of polarity and fertilization signals mediated by the male gamete in C. elegans and some plants. Establishment of the axis of polarity and therefore the emergence of apical complex with respect to the point of gamete fusion has not been previously studied in Plasmodium. To visualise the process of fertilization, membrane localized green fluorescence protein (GFP) expressing P. berghei male gamete producer parasites were generated. Female specific protein phosphatase with kelch-like domain (PPKL), IMC sub-compartment protein 1 (ISP1) and Glideosome-associated protein 50 (GAP50) are known to be associated with the apical complex, and are essential for P. berghei zygote to ookinete transition. Further, female specific MTOC (Microtubule Organising Center) markers - spindle pole body protein (SPBP) is also predicted to be associated with apical complex development during zygote to ookinete transition. Therefore, to investigate the emergence of the apical bud with respect to the point of gamete fusion during fertilization and through the zygote to ookinete transition, female gamete specific membrane localized mCherry expressing or PPKL/ ISP1/ GAP50/ SPBP labelled with mCherry expressing P. berghei parasite cell lines were generated in male gamete specific GFP producing P. berghei parasites. Initial fluorescent microscopy confirms the stage specific red-green fluorescence in respective transgenic P. berghei parasite lines. Further studies to confirm the hypothesis that the point of male gamete fusion cues for the point of emergence of apical bud in the zygote are ongoing.