Characterization of a MOB1 Homolog in the Apicomplexan Parasite Toxoplasma gondii

SIMPLE SUMMARY: Monopolar spindle One Binder1 (MOB1) proteins regulate key cellular functions, namely cell multiplication and cell division. The unicellular parasite Toxoplasma gondii transitions between several morphological stages, with the need to control the number of parasites in its cellular environment. We hypothesized that MOB1 proteins could participate in the regulation of the T. gondii life cycle, having identified one MOB1 protein (TgMOB1) coded in its genome. However, this study shows that TgMOB1 presents divergent features. While in organisms studied to date the lack of MOB1 has led to cell division defects, this did not occur in T. gondii in vitro cultures where mob1 was not an essential gene. Additionally, the identification of TgMOB1 proximity interacting partners detected novel MOB1 interactors. Still, TgMOB1 localizes to the region between the new-forming nuclei during cell division, and T. gondii parasites multiply slower with TgMOB1 overexpression and faster when there is a lack of TgMOB1, indicating an intricate role for TgMOB1 in T. gondii. This study uncovers new features of the T. gondii biology, a zoonotic parasite and model organism for the phylum Apicomplexa, and highlights the complex roles MOB1 proteins may assume, with possible implications for disease processes. ABSTRACT: Monopolar spindle One Binder1 (MOB1) proteins are conserved components of the tumor-suppressing Hippo pathway, regulating cellular processes such as cytokinesis. Apicomplexan parasites present a life cycle that relies on the parasites’ ability to differentiate between stages and regulate their proliferation; thus, Hippo signaling pathways could play an important role in the regulation of the apicomplexan life cycle. Here, we report the identification of one MOB1 protein in the apicomplexan Toxoplasma gondii. To characterize the function of MOB1, we generated gain-of-function transgenic lines with a ligand-controlled destabilization domain, and loss-of-function clonal lines obtained through CRISPR/Cas9 technology. Contrary to what has been characterized in other eukaryotes, MOB1 is not essential for cytokinesis in T. gondii. However, this picture is complex since we found MOB1 localized between the newly individualized daughter nuclei at the end of mitosis. Moreover, we detected a significant delay in the replication of overexpressing tachyzoites, contrasting with increased replication rates in knockout tachyzoites. Finally, using the proximity-biotinylation method, BioID, we identified novel members of the MOB1 interactome, a probable consequence of the observed lack of conservation of some key amino acid residues. Altogether, the results point to a complex evolutionary history of MOB1 roles in apicomplexans, sharing properties with other eukaryotes but also with divergent features, possibly associated with their complex life cycle.