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

Abstract

SummaryThe 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 a T. gondii NIMA-related kinase (Nek) ortholog that was identified in a chemical mutagenesis screen. A temperaturesensitive 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 centrosome prior to its duplication and localizes on the duplicated centrosomes facing the spindle poles in a cell-cycle-dependent manner. Mutational analysis of the activation loop suggests that localization and activity are spatio-temporally regulated by differential phosphorylation. Collectively, our results identified a novel temperature-sensitive allele for a Nek kinase and highlight its essential function in centrosome splitting in Toxoplasma. Moreover, these results conclusively show for the first time that Toxoplasma bud assembly is facilitated by the centrosome because defective centrosome splitting results in single daughter cell budding.

O -fucosylated glycoproteins form assemblies in close proximity to the nuclear pore complexes of Toxoplasma gondii

“…1B). Binding of anti-centrin antibodies to centrosomes shows that oocyst walls are permeabilized (23). Transient expression of GAP40-YFP, which highlights the inner membrane complex (24), shows that AAL binds to T. gondii nuclei throughout the tachyzoite cell cycle (Fig.…”

Apicomplexan cell cycle flexibility: centrosome controls the clutch

“…This haploid parasite replicates asexually by the simplest replication mode, termed endodyogeny, where the budding cycle (cytokinesis) is tied to only a single nuclear cycle (S/M) generating two cells per cycle. It is known that the centrosome serves as a central hub by nucleating spindle microtubule and scaffolding the components of daughter cells [3]. However, the mechanistic coordination of these two events remains largely unclear.…”

“…With a TgMAPK-L1 deficient parasite line, it was shown that TgMAPK-L1 limits centrosome core duplication to only once per cell cycle, and promotes the connection of the nuclear centrocone and the daughter basal complex [4]. Furthermore, a previously reported kinase, TgNek1-2, that plays a role in centrosome splitting, showed similar localization pattern to TgMAPK-L1 but resides towards the centrosome [3]. In the absence of TgNek1-2 activity, the centrosome duplicates but remains clustered, resulting in formation of a single daughter bud and failure of mitosis [3].…”

Targeting Toxoplasma Tubules: Tubulin, Microtubules, and Associated Proteins in a Human Pathogen

“…Toxoplasma SAS-6 is found at the centrioles during tachyzoite replication (87). Nek1 is recruited to tachyzoite centrioles prior to duplication and is essential to their subsequent separation (100). Toxoplasma expresses three centrin isoforms: all localize to centrioles, and centrin 2 and centrin 3 are found at additional subcellular sites (95,103,104).…”

“…SFA2 and SFA3 filaments connect centrioles to emergent daughters; conditional depletion of either protein blocks bud formation (88). A temperature-sensitive Nek1 mutation demonstrates that centriole separation is vital to nuclear division and counting daughters (100,124). Loss of Nek1 activity prevents completion of mitosis at the restrictive temperature.…”