Apicomplexan parasites are responsible for severe human diseases, including Plasmodium spp. causing malaria, Toxoplasma gondii causing encephalitis and birth defects, and Cryptosporidium parvum causing diarrhea. Drug resistance and/or poor specificity are constantly undermining therapeutic regimens to treat these diseases. In order to identify new drug targets, the P.I.'s lab focuses on enhancing the understanding of cell biological processes wherein the parasite differs from its host. One such process is cell division since it is morphologically distinct from mammalian cell division and lies at the core of the pathology associated with apicomplexan diseases. Deciphering parasite cell division is also of interest in understanding the evolution of cell division in different eukaryotic lineages. Specifically, under this proposal daughter budding will be studied using Toxoplasma tachyzoite cell division as a simple and accessible model. Tachyzoites divide by internal budding, wherein two daughter cells are assembled inside the mother cell. The daughter cytoskeletons form around the duplicated centrosomes, and subsequently elongate to serve as scaffold for organelle genesis and partitioning. How the cytoskeleton building blocks assemble on the centrosome is not well understood. However, it is now established that many components are unique to the parasite and are not shared with the mammalian host. Furthermore, halfway through assembly of this cytoskeleton scaffold a contractile force starts to taper the daughters toward the basal end. The basal complex responsible for this contraction is the functional ortholog of the mammalian contractile ring, but interestingly, its constriction is independent of actin polymerization. In fact, the motor that powers the basal complex is still unknown. In sum, despite our basic knowledge of the structural components driving cytokinesis, we still lack detailed information on how it is powered and how the various steps are controlled and coordinated. Under this proposal the researchers will dissect putative phosphorylation controls of cytokinesis. Through several recent studies of cytokinesis several kinases and phosphatases with apparent critical roles in different cell division steps have already been identified. The functions of these enzymes will be dissected by knock-out studies as well as kinase substrate identification studies. Independent of this first goal, the enigmatic mechanism underlying basal complex constriction will be unraveled. Candidate motor proteins will be experimentally validated next to the pursuit of a candidate independent approach. The latter entails the chemical genetic Bio-ID approach and, in conjunction with super-resolution microscopy, will lead to the molecular definition of the basal complex architecture. Upon completion of this proposal the researchers expect to have characterized critical phosphorylation controls at the different stages in the cell division process, and to have identified the mechanism driving basal complex constriction. Both these milestones will provide specific drug targets serving as jump off points for future work.
Fast lytic rounds of cell division underlie the pathogenesis of diseases caused by apicomplexan parasites, including the malaria parasite, whereas in this application the model apicomplexan Toxoplasma gondii will be used to dissect the control and mechanism of cell division. Toxoplasma cell division is driven by assembly of the cortical cytoskeleton nucleating on the centrosome, a process not targeted by any drug currently in clinical use. To identify promising new drug targets and understand the unique biology, the work will focus on dissecting the control and progression of assembly of the unique cytoskeleton by kinases and phosphatases as well as aim to identify the mechanism of constriction of the basal complex, the functional ortholog of the contractile ring, which proceeds through an unconventional and as yet unknown mechanism.
