Live-cell fluorescence imaging of microgametogenesis in the human malaria parasite Plasmodium falciparum

Formation of gametes in the malaria parasite occurs in the midgut of the mosquito and is critical to onward parasite transmission. Transformation of the male gametocyte into microgametes, called microgametogenesis, is an explosive cellular event and one of the fastest eukaryotic DNA replication events known. The transformation of one microgametocyte into eight flagellated microgametes requires reorganisation of the parasite cytoskeleton, replication of the 22.9 Mb genome, axoneme formation and host erythrocyte egress, all of which occur simultaneously in <20 minutes. Whilst high-resolution imaging has been a powerful tool for defining stages of microgametogenesis, it has largely been limited to fixed parasite samples, given the speed of the process and parasite photosensitivity. Here, we have developed a live-cell fluorescence imaging workflow that captures the entirety of microgametogenesis. Using the most virulent human malaria parasite, Plasmodium falciparum, our live-cell approach captured early microgametogenesis with three-dimensional imaging through time (4D imaging) and microgamete release with two-dimensional (2D) fluorescence microscopy. To minimise the phototoxic impact to parasites, acquisition was alternated between 4D fluorescence, brightfield and 2D fluorescence microscopy. Combining live-cell dyes specific for DNA, tubulin and the host erythrocyte membrane, 4D and 2D imaging together enables definition of the positioning of newly replicated and segregated DNA. This combined approach also shows the microtubular cytoskeleton, location of newly formed basal bodies, elongation of axonemes and morphological changes to the erythrocyte membrane, the latter including potential echinocytosis of the erythrocyte membrane prior to microgamete egress. Extending the utility of this approach, the phenotypic effects of known transmission-blocking inhibitors on microgametogenesis were confirmed. Additionally, the effects of bortezomib, an untested proteasomal inhibitor, revealed a clear block of DNA replication, full axoneme nucleation and elongation. Thus, as well as defining a framework for broadly investigating microgametogenesis, these data demonstrate the utility of using live imaging to validate potential targets for transmission-blocking antimalarial drug development. Author summary Upon transmission of Plasmodium, the causative agents of malaria, male and female gametes form in the mosquito midgut in a remarkably rapid and complex cellular transformation. Microgametogenesis, or male gamete formation, describes the formation of eight haploid microgametes from a single gametocyte in only 15 minutes. Simultaneous egress from the host erythrocyte, substantial cytoskeletal rearrangement and DNA replication occurs during this characteristically explosive transformation. Whilst microgametogenesis has been extensively studied using various microscopy techniques, efforts to date have been limited by the need for complex transgenic parasite generation and fixed-parasite protocols. Here, we have developed a live-cell fluorescence imaging approach to study microgametogenesis, overcoming both the laborious staining steps and spatiotemporal limitations of existing approaches. Crucially, our newly devised workflow utilises widefield fluorescence microscopy, commercially available stains and an open-access analysis software and is hence accessible to the wider malaria research community. We demonstrate the ability to retrieve and quantify volumetric data from our live-cell approach and highlight the applicability of our live-cell approach to transmission-blocking drug discovery. Using our workflow, we have elucidated the cellular phenotypes of both existing and unknown inhibitors of microgametogenesis. Using this approach, we find the proteasome to be a potential microgametogenesis-blocking cellular target.