The Toxoplasma gondii centrosome is the platform for internal daughter budding as revealed by a Nek1 kinase mutant

Abstract

The pathology and severity of toxoplasmosis results from the rapid replication cycle of the apicomplexan parasite Toxoplasma gondii. The tachyzoites divide asexually through endodyogeny, wherein two daughter cells bud inside the mother cell. Before mitosis is completed, the daughter buds form around the duplicated centrosomes, and subsequently elongate to serve as the scaffold for organellogenesis and organelle partitioning. The molecular control mechanism of this process is poorly understood. Here, we characterized an ortholog of NIMA-related kinase (Nek) in T. gondii identified in a chemical mutagenesis screen. A temperature sensitive mutant, V-A15, possesses a Cys316Arg mutation in TgNek1 (a novel mutant allele in Neks), which is responsible for growth defects at the restrictive temperature. Phenotypic analysis of V-A15 indicated that TgNek1 is essential for centrosome splitting, proper formation of daughter cells, and faithful segregation of genetic material. In vitro kinase assays showed that the mutation abolishes the kinase activity of TgNek1. TgNek1 is recruited to the spindle pole prior to mitosis and localizes to the duplicated centrosomes on the face of the spindle poles in a cell-cycle-dependent manner. Mutational analysis of the activation loop suggests localization and activity are spatio-temporally regulated by differential phosphorylation. Collectively, our results identified a novel temperature sensitive allele for a Nek kinase, which in Toxoplasma highlights its essential function in centrosome splitting. Moreover, these results conclusively show for the first time that Toxoplasma bud assembly is facilitated by the centrosome since defective centrosome splitting results in single daughter cell budding.