Functional dissection of NudCL1 protein in Toxoplasma gondii cell division.

Nuclear distribution protein C (NudC) is a highly conserved eukaryotic protein initially identified in Aspergillus nidulans as a dynein-mediated nuclear migration factor. However, vertebrates typically contain four paralogs sharing a common p23 domain, which engages the cochaperone HSP90 involved in protein folding and interactions. Additionally, a conserved NudC-N-terminal domain is present in two vertebrate NudC proteins. The annotated genome of Toxoplasma gondii encodes four NudC proteins based on the conservation of the p23 domain. So far, NudC proteins have not been characterized in apicomplexan parasites. We studied all four TgNudC family genes, which are all essential. Here, we focus on NudC-like 1 protein (NudC-L1), which possesses both conserved NudC and p23 domains. Surprisingly, despite its predicted fitness score of -0.47, we demonstrated that NudC-L1 is essential for the parasite lytic cycle. Depleting NudC-L1 inhibits parasite replication and leads to the development of multinucleated, unindividualized parasites, associated to elongated intercellular bridges between daughter cells. Pan Expansion Microscopy was used to assess parasite ultrastructure, revealing that NudC-L1 knockdown induces a Russian doll-like, "bud-in-bud" parasite development, with no observed defects in nuclear division. In humans, NudC protein regulates cytokinesis. In fact, Aurora B, a kinase essential for cell abscission, phosphorylates NudC at a conserved Threonine 40 residue. Remarkably, we found that TgNudC-L1 possesses this conserved Threonine residue. Moreover, previous work has demonstrated a similar Russian doll phenotype upon depletion of T. gondii Aurora kinase 1 (Ark1). This suggests that TgArk1 directly phosphorylates NudC-L1, which implies a putative role for the spindle midbody in promoting cytokinesis. This study represents the first comprehensive characterization of NudC proteins in T. gondii and has uncovered a putative connection between Ark1 and NudC-L1 in cytokinesis.