An apical membrane complex for triggering rhoptry exocytosis and invasion in Toxoplasma (2022)
Figure 6

Fig 6. CRMP and Nd complexes differentially regulate rhoptry secretion at the exocytic site in Toxoplasma gondii.

Immunofluorescence images of intracellular (upper panel) and extracellular (lower panel) tachyzoites co-expressing TgCRMPa-HA3 and TgNd6-TY2. Extracellular parasites were incubated with host cell monolayers for 2min prior to fixation. Parasites were stained with anti-HA and anti-TY Abs to label CRMPa and Nd6, respectively. Nd6, but not CRMPa, accumulates at the tachyzoite apex in intracellular parasites (arrowheads), while both proteins localize at the tip of the extruded conoid in extracellular parasites (arrows). DNA is labeled by Hoechst. Single focal planes are shown. DIC: differential interference contrast.

Ultrastructure Expansion Microscopy of extracellular tachyzoites, either untagged or co-expressing TgCRMPa-HA3/TgCRMPa-TY2 and TgCRMPb-HA3 together or in pairwise combination with TgNd6-TY2. Parasites were treated with A23187 prior to fixation and preparation for U-ExM, and stained with anti-HA, anti-TY and anti-α/β tubulin Abs to label CRMPs, Nd6/CRMPa and microtubules, respectively. Shown are maximum intensity projections of z-stack confocal images. CRMPs overlap with Nd6 at the tip of the extruded conoid (yellow selection). A magnified image of the apical tip of each parasite is shown on the right.

Extent of colocalization between TgCRMPa-TY2 and TgCRMPb-HA3, and TgCRMPs-HA3 with TgNd6-TY2 in the apical dot shown in B. Pearson's correlation coefficient was measured using the Fiji-JACoP plugin. Values are expressed as mean ± SD. Three-five parasites per line were analyzed.

Quantification of the dot pattern for TgCRMPa-HA3 and TgCRMPb-HA3 in TgNd9_iKD lines. CRMPs accumulation at the apical tip of extracellular parasites was measured as in Fig EV4A. TgCRMPs were still found in the apical dot in absence of TgNd9 (+ATc). Numbers are expressed as percentage of parasites showing (dot) or lacking (no dot) the tip accumulation of TgCRMPa and TgCRMPb. The number of parasites (n) analyzed for each line is reported at the top of the column.

Immunofluorescence and DIC images of an extracellular parasite and of parasites in early, middle and late stages of host cell invasion. Parasites co-expressing TgCRMPa-HA3 and TgNd6-TY2 were incubated with host cell monolayers and fixed after 2, 3, and 5min. Parasites were immunostained with anti-HA, anti-TY and anti-RON5 Abs to label CRMPs, Nd6 and the moving junction, respectively. DNA is labeled by Hoechst. In contrast with TgNd6 (arrow), the apical accumulation of TgCRMPa observed in extracellular parasites (arrow), disappears upon entering the host, a step marked by the formation of the moving junction (arrowhead), and for the entire process. Non-specific anti-TY labeling of mitochondria was detected for both untagged and tagged lines (Fig EV6). Single focal planes are shown.

Model depicting the proposed role for the TgCRMP complex in rhoptry exocytosis. Upon contacting the host cell via a host ligand, CRMPs participate in the activation of the signaling pathway targeting the AV-RSA system, and leading to the fusion events required for the timely discharge of rhoptry contents. Rh: rhoptry; AV: apical vesicle; RSA: rhoptry secretory apparatus.

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