1. Structure and function of the basal complex: the unique mechanisms of Plasmodium cytokinesis
- Author
-
Morano, Alexander A
- Subjects
- Apicomplexan, Basal Complex, Cell Division, Cytokinesis, Malaria, Plasmodium, Parasitology, Molecular biology, Microbiology
- Abstract
Plasmodium falciparum is the causative agent of the most dangerous type of malaria, an ancient disease that remains a significant public health burden. Malaria pathogenesis is caused by the asexual replication of Plasmodium parasites in the red blood cells, a 48-hour cycle driving exponential increases in parasitemia, made possible by Plasmodium’s unique mode of asexual replication, schizogony, where 16-32 daughter cells are produced within a common cytoplasm. During schizogony, multiple rounds of asynchronous karyokinesis occur followed by a final, semi-synchronous round of karyokinesis paired with cytokinesis in the final stage of schizogony known as segmentation. This method of cell division requires two structures specific to the Apicomplexa: the inner membrane complex, which provides shape and stability to the daughter cells, called merozoites, and the basal complex, which is a contractile ring that functions to separate the merozoites and acts as a scaffold during construction of the IMC. In this work, we characterize the spatial and temporal complexity of the basal complex and begin to establish a mechanism of basal complex function. First, we identify Pf3D7_ 1018200, PfPPP8, as a new and essential member of the basal complex. We make use of ultrastructure expansion microscopy, long-term live-cell microscopy, and 3D-structured illumination microscopy to locate the precise basal complex defect induced by PfPPP8 depletion and thus determine that PfPPP8 is specifically required to maintain basal complex integrity and synchrony during the process of basal complex construction. In the absence of PfPPP8, basal complexes within the same parasite grow at different rates, accumulate breakage points, and collapse rather than contracting. Through bioinformatic analysis we characterize PfPPP8 as the first serine-threonine pseudophosphatase in the PPP-type phosphatase family, with similarly enzymatically inactive homologs throughout the Apicomplexa. We use PfPPP8 to identify novel basal complex proteins Pf3D7_0214700 and PfMyoJ via immunoprecipitation and develop a long-term live-cell microscopy platform to visualize the different dynamic temporal localizations of identified basal complex proteins, determining that PfMyoJ and Pf3D7_0214700 (named PfSLACR) are recruited to the basal complex at its midpoint whereas PfPPP8 is depleted during basal complex contraction. Not only does this work characterize a novel essential basal complex protein in depth, but it also demonstrates the dynamic nature of the basal complex in Plasmodium for the first time. Next, we dig deep into the structure, composition, spatial-temporal dynamics, and mechanism of the basal complex. We make extensive use of ultrastructure expansion microscopy (U-ExM) to identify PfMyoJ and PfSLACR as members of a novel basal subcompartment of the P. falciparum basal complex, with PfCINCH localized to the more apical subcompartment. Next, we optimize long-term live-cell microscopy to first identify subtle differences in the recruitment dynamics of PfSLACR and PfMyoJ, where PfSLACR arrives slightly earlier to the basal complex, and combine both live-cell microscopy and U-ExM to show that PfSLACR and PfMyoJ are removed unevenly from the basal complexes of individual merozoites within the same schizont, demonstrating a lack of radial symmetry in the process of segmentation. We demonstrate that basal complex contraction is mechanistically divergent between P. falciparum and related parasite Toxoplasma gondii, showing PfMyoJ and PfMORN1 to be both individually dispensable and dispensable in combination. Finally, we show that actin dynamics are also dispensable to the initiation, construction, and contraction of the basal complex, in the presence and absence of PfMyoJ. Thus, we establish the basal complex as a spatially complex structure, build on our earlier understanding of its temporal complexity, and make significant strides towards identifying the mechanism responsible for contraction of the basal complex. Overall, this work significantly expands our knowledge of the structure, organization, order of assembly, and mechanism of action of the basal complex, allowing for a greater understanding of Plasmodium cell division and demonstrating a surprising degree of divergence between Plasmodium and its well-studied relative Toxoplasma in this realm.
- Published
- 2024