An apical membrane complex for triggering rhoptry exocytosis and invasion in Toxoplasma (2022)
Figure 5

Fig 5. TgCRMPa and TgCRMPb accumulate at the apical tip of extracellular tachyzoites, with TgCRMPa N-terminal end oriented towards the outside space.

Immunofluorescence images of extracellular tachyzoites of untagged, TgCRMPa-HA3 and TgCRMPb-HA3 parasites, incubated either with host cell monolayers for 2min, or with ionophore A23187, to induce natural or artificial conoid extrusion, respectively. Parasites were immunostained with anti-HA Abs; DNA was labeled by Hoechst. CRMPa and CRMPb consistently accumulate at the tip of extruded conoids (arrows). The apexes of A23187-treated parasites were magnified on the right, and increased in brightness and contrast to highlight the apical dots. DIC: differential interference contrast. Single focal planes are shown.

Quantification of the dot pattern upon A23187 treatment shown in (A). Values are expressed as percentage of parasites showing (dot) or lacking (no dot) the apical accumulation of TgCRMPa and TgCRMPb; n= number of parasites analyzed per line.

Immunofluorescence images of extracellular TgCRMPb-HA3 tachyzoites in presence (-ATc) or absence (+48h ATc) of TgCRMPa-FLAG3 (iKD line). Parasites were stained with anti-HA Abs to visualize CRMPb. TgCRMPb localization at the tip of the extruded conoid (arrow) disappears upon TgCRMPa depletion, but it is still detected in the cytoplasm (lower panel). DNA is labeled by Hoechst. Single focal planes are shown. DIC: differential interference contrast.

Quantification of the dot pattern shown in (C). The values are reported as in (B).

Schematic representation of the N- (top) and C-terminal (bottom) tagging of TgCRMPa with the triple HA and the miniAID. The amino acid residues where the insertion of the tag occurred are indicated with dotted lines. M: MAR domain; K: Kringle domain; EGFR: Epidermal Growth Factor Receptor. Green: predicted signal peptide; blue: transmembrane domains.

Cartoon depicting the targeting of a membrane protein to the proteasome (Pr) by the AID-degron system when the AID-fused C-terminus is exposed to the cytosol. IAA: 3-indoleacetic acid or auxin; AID: auxin-inducible degron; tv: transport vesicle.

Whole cell lysates from TIR1-expressing parental line (Ctrl), HA3-miniAID-TgCRMPa_iKD (N-term) and TgCRMPa-miniAID-HA3_iKD (C-term) lines were immunoblotted with anti-HA Abs to visualize tagged CRMPa in IAA-treated and untreated samples. CRMPa was undetectable upon 24h incubation with IAA when C-terminally, but not N-terminally, tagged with the miniAID-HA3, suggesting that the C-terminus is the one exposed towards the cytosol. TgMIC2 was used as loading control and detected with anti-MIC2 Abs. MW: molecular weight standards.

Schematic representation of the N- (top) and C-terminal (bottom) tagging of TgCRMPa with the triple HA. The amino acid residues where the insertion of the tag occurred are indicated with dotted lines. The domains are indicated as in (E).

Whole cell lysates from C-terminally and N-terminally HA3-tagged TgCRMPa lines were immunoblotted as in (G). In the "N-term" lane, in addition to the full-length and the processed form of CRMPa, a smaller band (asterisk) is also visible by anti-HA Abs staining. Untagged parasites (Ctrl) were treated in parallel. TgROP5 was used as loading control and detected with anti-ROP5 Abs. MW: molecular weight standards.

Immunofluorescence images of extracellular A23187-treated parasites expressing either N-or C-terminally HA3-tagged TgCRMPa, and immunostained as in (A). TgCRMPa accumulates at the tip of extruded conoids (arrows) in triton-permeabilized (+) or non-permeabilized (-) parasites. DIC: differential interference contrast. Single focal planes are shown.

Quantification of the dot pattern upon natural (+Human Foreskin Fibroblasts) or artificial (+A23187) conoid extrusion in parasites expressing either N- or C-terminally HA3-tagged TgCRMPa. Values are reported as in (B).