Deciphering the spatiotemporal organization and regulation of Plasmodium falciparum schizogony

Despite decades-long’ efforts to combat malaria, it is still responsible for the death of over half a million people each year. All clinical symptoms of malaria are caused by the rapid asexual proliferation of parasites of the genus Plasmodium in the blood of patients. Here, Plasmodium replicates via schizogony: an atypical form of replication, where nuclei multiply asynchronously. Only once approximately twenty nuclei are formed, daughter cells assemble. However, the coordination of DNA replication and nuclear division as well as the molecular determinants of asynchronous nuclear multiplication are unknown. In my thesis, I investigated the organization and regulation of asynchronous nuclear multiplication during schizogony in Plasmodium falciparum (P. falciparum), which causes the most severe form of human malaria. I first determined that P. falciparum proliferates via alternating rounds of DNA replication and nuclear division, with nuclei seemingly acting as ‘cells-within-cells’. By showing that the episomally expressed replication fork protein PCNA1::GFP transiently accumulates only in those nuclei that undergo DNA replication, I established a marker for DNA replication compatible with life-cell imaging. In combination with the marker 3xNLS::mCherry for the nuclei, imaging of PCNA1::GFP allowed me to track DNA replication and nuclear division in nuclei of single parasites as they underwent schizogony. I then quantified the overall dynamics of nuclear multiplication as well as the dynamics and organization of the individual DNA replication and nuclear division events. To establish a mathematical model of nuclear multiplication, I collaborated with P. Binder, showing that DNA replication is influenced by a limiting factor and that asynchronous nuclear divisions enable rapid parasite proliferation. I also investigated the molecular basis of the transient accumulation of PCNA1::GFP in nuclei during S-phase and found that this is most likely caused by association of PCNA1 with the DNA during replication. As PCNA1 shuttles between the cytoplasm and the nucleus, I also analyzed sequence motifs potentially important for the nucleo-cytoplasmic transport of PCNA1 and found that PCNA1 may contain a classical nuclear export signal (NES), which should facilitate nuclear export via the export receptor exportin-1. Yet, this NES may not be functional, as chemical inhibition of exportin-1 in an engineered cell line which is sensitive to the exportin-1 inhibitor leptomycin B (LMB), did not affect nuclear accumulation or export of PCNA1::GFP. Together, I characterized the spatiotemporal organization of nuclear multiplication, defining the basic organization and regulation of the cell cycle in P. falciparum in the blood stage of infection. This may not only help to uncover novel targets for malaria intervention but also to expand our understanding of the unusual cell cycle biology of an early-branching eukaryote