Loss of a conserved MAPK causes catastrophic failure in assembly of a specialized cilium-like structure in Toxoplasma gondii

Abstract

Primary cilia are important organizing centers that control diverse cellular processes. Apicomplexan parasites like Toxoplasma gondii have a specialized cilium-like structure called the conoid that organizes the secretory and invasion machinery critical for the parasites' lifestyle. The proteins that initiate the biogenesis of this structure are largely unknown. We identified the Toxoplasma orthologue of the conserved kinase ERK7 as essential to conoid assembly. Parasites in which ERK7 has been depleted lose their conoids late during maturation and are immotile and thus unable to invade new host cells. This is the most severe phenotype to conoid biogenesis yet reported, and is made more striking by the fact that ERK7 is not a conoid protein, as it localizes just basal to the structure. ERK7 has been recently implicated in ciliogenesis in metazoan cells, and our data suggest that this kinase has an ancient and central role in regulating ciliogenesis throughout Eukaryota.

FIGURE 1:

A cartoon comparing the (A) metazoa cilium and (B) Toxoplasma apical complex.

FIGURE 2:

ERK7 is apically localized. (A) A 0.5-μm confocal slice of immunofluorescence using antibodies for 3xHA-tagged ERK7 (green), β-tubulin (blue), and the apical cap marker ISP1 (red). Note that anti-tubulin does not stain the conoid, likely due to antigen accessibility. Arrows indicate daughter buds. Scale bar: 3 μm. Maximum intensity projection of SIM stacks of (B) ERK7-3xHA (green) and GFP-α-tubulin (magenta), and (C) ERK7-3xHA (green) and anti-ISP1 (magenta). Scale bars: 5 μm.

FIGURE 3:

Loss of ERK7 blocks parasite invasion and egress. (A) ERK7^AID parasites were incubated as indicated with IAA and lysates were probed with anti-FLAG and anti–β-tubulin as a loading control. (B) Quantification of three replicate plaque assays with parental, ERK7^AID, and ERK7^AID(wt-comp) strains +/− IAA. (C) Phase image of ERK7^AID parasites grown 24 h +/− IAA. (D) Parasites as in C were grown 18 h more until vacuoles began to rupture. (E) Quantification of egress from n = 3 replicates grown as indicated in IAA. (F) Quantification of attached and invaded parasites from ERK7^AID and ERK7^AID(wt-comp) strains grown +/− IAA. (G) Quantification of Gaussia luciferase activity secreted by the indicated strains grown +/− IAA. (H) ERK7^AID and ERK7^AID/IAA expressing an actin chromobody (magenta) and secreting Venus into the PV (green) were imaged at the indicated times after ionophore treatment. Scale bars: 10 μm. (I) Quantification of PV Venus signal of parasites as in H relative to the first frame (n = 93 −IAA; 81 +IAA). Error bars are SD.

FIGURE 4:

The apical complex is disrupted in parasites lacking ERK7. TEM of (A) ERK7^AID parasites develop normal conoids [C] and well organized secretory organelles (micronemes [M] and rhoptries [R]), while those grown (B) for 18 h in IAA have no identifiable conoid and disorganized secretory organelles. TEM of negatively stained detergent-extracted parasites reveal normal microtubule structures in ERK7^AID parasites (n = 20) (C) including the distinctive rings and conoid spiral of the apical complex, which are lost when parasites are grown for 18 h in IAA (n = 60; D). Complementation of ERK7^AID with wild-type ERK7 rescues all phenotypes (n = 20; E, F).

FIGURE 5:

Loss of ERK7 results in disorganized apical structures. (A–E) ERK7^AID parasites were grown in +/− IAA and imaged using the indicated markers. All images are maximum intensity projections of confocal stacks. Scale bars: 3 μm. White arrows indicate the apical complex GFP-tubulin foci in mature parasites, which is missing in +IAA parasites (gray arrows). (F, G) ERK7^AID parasites were grown for 6 h in +/− IAA and imaged (GFP-tubulin, green; SAS6L, magenta). Scale bars: 4 μm.