Summary The obligate intracellular parasite Toxoplasma gondii can cause severe opportunistic infection in humans, specially targeting the unborn child or HIV/AIDS patients. Fast rounds of host cell invasion and intracellular replication are responsible for tissue lesions observed in the parasite’s lytic cycle and caused by the tachyzoite form. Although specific drug treatments to battle Toxoplasmosis are available, severe side effects can occur and long-term application might lead to hypersensitivity. Underlying the lytic cycle is a parasite-specific replication apparatus, which forms two new daughter cells in the cytosol of the mother (endodyogeny). This process is highly regulated by parasite-specific kinases, which largely control the cell cycle. Three aurora-related kinases (Ark1-3) have previously been identified and characterized. Among these kinases, Ark3 depletion strongly affects growth of the forming daughters, but detailed actions of the kinase remain unknown. Intriguingly, the kinase has a unique subcellular localization, forming a linear structure during the division process, which extends from the duplicated centrosomes distal towards the nucleus. A similar assembly has also been reported for Plasmodium falciparum, where a similar PfArk3 dynamic is detected towards merozoite formation at the end of the cell cycle in the red blood cell. The nature of this enigmatic assembly has not further been investigated in either organism. The main goal of this proposal is to dissect the nature of the Ark3-related cluster (AC) in the parasite. Catering to this goal is our recent proximity biotinylation-based identification of two new genes whose products exhibit Ark3 dynamics during the cell division. We have named them Ark3-related component (ARC) 1 and 2. Most notably, these hypothetical proteins display severe fitness scores and support the premise that the Ark3/ARC1/2 assembly is executing an essential function at a hereto unrecognized, and completely uncharacterized division structure. We will study the AC by the following two specific aims. Under Aim1 we will revisit the Ark3 phenotype in more detail by conditional protein degradation of the kinase and compare its phenotypic effects to parasites conditionally-depleted of ARC1 and ARC2. Application of ultrastructure expansion microscopy (U-ExM) will further allow us to investigate phenotypic effects and functionally define this mysterious structure. To further map the function, under Aim2 we will reciprocally apply proximity biotinylation to ARC1 and ARC2, using the fast acting biotin ligase TurboID. Upon successful completion of this research proposal we will have gained deep understanding of Ark3 and its unconventional cluster and generate novel insides into early steps of T. gondii daughter cell formation.