The tachyzoite centrosome has a unique bipartite internal organization.

<p>The pairwise localization of several centrosome proteins was examined in tachyzoites undergoing mitosis. Detection of each protein pair was accomplished by epitope-tagging (genetic knock-in) or by using antibody raised against recombinant protein. These experiments revealed two distinct protein core complexes are contained within the tachyzoite centrosome that duplicate during the cell cycle. Two pairwise protein IFA analyses highlight these structures; the (A) upper panel is co-staining of epitope-tagged TgCEP250^myc with anti-myc (red) and anti-Centrin (green). Note that TgCEP250^myc is localized to the TgCentrin1 core structure (designated the outer core) as well as a novel second core (inner core) that was closer to the parasite nucleus in all cell cycle phases. The (B) panel is a dual epitope-tagged strain TgCEP250-L1^HA/TgCentrin1^myc co-stained with anti-HA (red) and anti-myc (green). The CEP250-related protein, TgCEP250L-1 is preferentially localized to the novel inner core that lacks TgCentrin1. (C) A recognized co-factor of TgCentrin1, TgSfi1 (red), exclusively co-localized with TgCentrin1 (green) in the outer centrosomal core (co-staining of the dual-tagged clone TgCentrin1^HA/TgSfi1^myc). Differential composition of the outer versus inner centrosome core also included outer core proteins TgSas-6 and Aurora-related kinase 1 (TgARK1) described below. (D) Duplicated centrosomal cores containing TgCEP250-L1^HA were segregated from the centrocone structure embedded in the nuclear envelope as demonstrated by co-staining with anti-TgMORN1 antibody (green). (E) The separation of the inner core from the nuclear envelope was further established by co-staining for the nuclear mesh protein, TgNMP1; analysis of dual-tagged clone TgCEP250-L1^HA/TgNMP1^myc. Note that inner core containing TgCEP250-L1^HA is localized to the cytoplasmic interface of the nucleus in the inset image. (F) <i>T</i>. <i>gondii</i> γ-Tubulin is localized to the TgCentrin1-associated outer core (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002093#pbio.1002093.g001" target="_blank">Fig. 1D</a>) that is distal from the inner core co-stained for TgCEP250-L1^HA; shown here is dual-tagged clone TgCEP250-L1^HA/γ-Tubulin^myc. (G) The spatial segregation of the bipartite core structures of the <i>Toxoplasma</i> centrosome is well resolved and aligned with the centromeres as captured by super-resolution microscopy; markers used were outer core = TgSas-6^HA, inner core = TgCEP250-L1^myc, and the anti-TgCenH3 antibody to detect the centromere/kinetochore intranuclear structure [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002093#pbio.1002093.ref025" target="_blank">25</a>]. For image panels A-D, quantitative fluorescence density plots were included. The data for each density graph was taken from a cross section of the image indicated by the white line in the enlarged merged image immediately to the left of the graph. (H) Diagram is included that summarizes the discovery of two replicating centrosome cores (one-half of the mitotic organization) with differential protein composition.