Plasmodium falciparum CRK4 links early mitotic events to the onset of S-phase during schizogony.

Plasmodium falciparum proliferates through schizogony in the clinically relevant blood stage of infection. During schizogony, consecutive rounds of DNA replication and nuclear division give rise to multinucleated stages before cellularization occurs. Although these nuclei reside in a shared cytoplasm, DNA replication and nuclear division occur asynchronously. Here, by mapping the proteomic context of the S-phase-promoting kinase PfCRK4, we show that it has a dual role for nuclear-cycle progression: PfCRK4 orchestrates not only DNA replication, but in parallel also the rearrangement of intranuclear microtubules from hemispindles into early mitotic spindles. Live-cell imaging of a reporter parasite showed that these microtubule rearrangements coincide with the onset of DNA replication. Together, our data render PfCRK4 a key factor for nuclear-cycle progression, linking entry into S-phase with the initiation of mitotic events. In part, such links may compensate for the absence of canonical cell cycle checkpoints in P. falciparum. IMPORTANCE The human malaria parasite Plasmodium falciparum proliferates in erythrocytes through schizogony, forming multinucleated stages before cellularization occurs. In marked contrast to the pattern of proliferation seen in most model organisms, P. falciparum nuclei multiply asynchronously despite residing in a shared cytoplasm. This divergent mode of replication is, thus, a good target for therapeutic interventions. To exploit this potential, we investigated a key regulator of the parasite's unusual cell cycle, the kinase PfCRK4 and found that this kinase regulated not only DNA replication but also in parallel the rearrangement of nuclear microtubules into early mitotic spindles. Since canonical cell cycle checkpoints have not been described in P. falciparum parasites, linking entry into S-phase and the initiation of mitotic events via a kinase, may be an alternative means to exert control, which is typically achieved by checkpoints.

Asynchronous nuclear cycles in multinucleated Plasmodium falciparum facilitate rapid proliferation.

Malaria-causing parasites proliferate within erythrocytes through schizogony, forming multinucleated stages before cellularization. Nuclear multiplication does not follow a strict geometric 2n progression, and each proliferative cycle produces a variable number of progeny. Here, by tracking nuclei and DNA replication, we show that individual nuclei replicate their DNA at different times, despite residing in a shared cytoplasm. Extrapolating from experimental data using mathematical modeling, we provide strong indication that a limiting factor exists, which slows down the nuclear multiplication rate. Consistent with this prediction, our data show that temporally overlapping DNA replication events were significantly slower than partially overlapping or nonoverlapping events. Our findings suggest the existence of evolutionary pressure that selects for asynchronous DNA replication, balancing available resources with rapid pathogen proliferation.

An extended DNA-free intranuclear compartment organizes centrosome microtubules in malaria parasites.

Proliferation of Plasmodium falciparum in red blood cells is the cause of malaria and is underpinned by an unconventional cell division mode, called schizogony. Contrary to model organisms, P. falciparum replicates by multiple rounds of nuclear divisions that are not interrupted by cytokinesis. Organization and dynamics of critical nuclear division factors remain poorly understood. Centriolar plaques, the centrosomes of P. falciparum, serve as microtubule organizing centers and have an acentriolar, amorphous structure. The small size of parasite nuclei has precluded detailed analysis of intranuclear microtubule organization by classical fluorescence microscopy. We apply recently developed super-resolution and time-lapse imaging protocols to describe microtubule reconfiguration during schizogony. Analysis of centrin, nuclear pore, and microtubule positioning reveals two distinct compartments of the centriolar plaque. Whereas centrin is extranuclear, we confirm by correlative light and electron tomography that microtubules are nucleated in a previously unknown and extended intranuclear compartment, which is devoid of chromatin but protein-dense. This study generates a working model for an unconventional centrosome and enables a better understanding about the diversity of eukaryotic cell division.