A conserved ankyrin repeat-containing protein regulates conoid stability, motility and cell invasion in Toxoplasma gondii

Abstract

Apicomplexan parasites are typified by an apical complex that contains a unique microtubule-organizing center (MTOC) that organizes the cytoskeleton. In apicomplexan parasites such as Toxoplasma gondii, the apical complex includes a spiral cap of tubulin-rich fibers called the conoid. Although described ultrastructurally, the composition and functions of the conoid are largely unknown. Here, we localize 11 previously undescribed apical proteins in T. gondii and identify an essential component named conoid protein hub 1 (CPH1), which is conserved in apicomplexan parasites. CPH1 contains ankyrin repeats that are required for structural integrity of the conoid, parasite motility, and host cell invasion. Proximity labeling and protein interaction network analysis reveal that CPH1 functions as a hub linking key motor and structural proteins that contain intrinsically disordered regions and coiled coil domains. Our findings highlight the importance of essential protein hubs in controlling biological networks of MTOCs in early-branching protozoan parasites.

Conditional Knockdown of Proteins Using Auxin-inducible Degron (AID) Fusions in Toxoplasma gondii

“…Set up the Q5 PCR reaction using pYFP-mAID-3HA, Floxed HXGPRT (Addgene #87259) (Brown et al ., 2017) or pYFP-AID-3HA, Floxed HXGPRT (Addgene #87260) (Long et al ., 2017b) plasmids as the template (Figure 2F). A standard 500 μl reaction consists of:…”

“…Incubation times needed to achieve complete protein knockdown will vary based on protein localization, stability, and abundance. As little as 15 min has been used to achieve complete knockdown of soluble cytosolic (m)AID-fusions such as YFP (Brown et al, 2017; Long et al, 2017b). Following knockdown, parasites can be examined for phenotypes directly, harvested for follow-up assays, or lysed for analysis of (m)AID-tagged protein levels.…”

“…The T. gondii line RH TIR1-3FLAG (genotype: RHΔ hxgprt Δ ku80; TUB1:TIR1-3FLAG, SAG1:CAT ) (Brown et al ., 2017; Long et al ., 2017b) has been submitted to the ATCC for distribution. To apply the auxin degron system to other T. gondii strains, the plasmid pTUB1:OsTIR1-3FLAG, SAG1:CAT (Addgene #87258) is available for transfection.…”

“…We recently engineered an RHΔ hxgprt Δ ku80 line of T. gondii to stably express TIR1 from Oryza sativa (RH TIR1-3FLAG) (Brown et al ., 2017; Long et al ., 2017a). In this background, we were able to use CRISPR/Cas9 genome editing (Shen et al ., 2014; Sidik et al ., 2014; Shen et al ., 2017) to tag essential T. gondii genes of interest with AID-3HA or mini-AID(mAID)-3HA, regulate their expression with auxin, and identify phenotypes associated with their loss (Brown et al ., 2017; Long et al ., 2017a and 2017b). …”

CRISPR-mediated Tagging with BirA Allows Proximity Labeling in Toxoplasma gondii

“…(2015) adapted BioID for use in T. gondii , identifying several novel protein components of the inner membrane complex (IMC). BioID has since been employed in T. gondii research to identify interactors of kinases (Gaji et al ., 2015), calmodulins (Long et al ., 2017a), and to define the protein repertoire of other cellular compartments including the parasitophorous vacuole (Nadipuram et al ., 2016), sutures of the IMC (Chen et al ., 2017), and the apical complex (Long et al ., 2017b). Here we will describe the protocol for generating a BirA gene fusions using CRISPR/Cas9 tagging (Shen et al ., 2014; Shen et al ., 2017), in vivo BirA biotin labeling and purification of biotinylated proteins from parasites, and identification of captured biotinylated proteins by mass-spectrometry.…”