1. Volume 20, Issue 6, Pages 731-743 (December 2016)
An Apicomplexan Actin-Binding Protein Serves as a Connector and Lipid Sensor to Coordinate Motility and Invasion 
Damien Jacot, Nicolò Tosetti, Isa Pires, Jessica Stock, Arnault Graindorge, Yu-Fu Hung, Huijong Han, Rita Tewari, Inari Kursula, Dominique Soldati-Favre 
Cell Host & Microbe 
Volume 20, Issue 6, Pages (December 2016)
DOI: /j.chom
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2. Cell Host & Microbe 2016 20, 731-743DOI: (10.1016/j.chom.2016.10.020)
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 1
GAC Is a Dynamic, Apical Protein Translocated to the Basal Pole of Motile and Invading Parasites
(A) Western blot shows TgGAC-3Ty (291 kDa) and TgGAC-mCherry-Ty (314 kDa), with TgIMC1 as loading control and ΔKu80 as parental strain.
(B) Cytosolic and apical localization at the conoid (zoom) of TgGAC-3Ty in intracellular parasites. Micronemes are stained with α-TgMIC2 antibodies.
(C) TgGAC-3Ty relocalizes from the apical (arrows) to the basal end (stars) upon A23187-induced egress. This was blocked by pretreatment with CD. Parasites were mechanically egressed.
(D) Quantification of the data presented in (C). Data are presented as mean ± SD (p value ≤ 0.001).
(E) Colocalization of TgGAC-mCherry-Ty at the CJ (arrows) in invading parasites. The CJ was visualized either first with α-TgRON4 antibodies in 0.1% saponin buffer, which permeabilized only the host cell, or using α-SAG1 antibodies in non-permeabilized conditions. Samples were subsequently permeabilized with Triton-X to gain access to TgGAC.
(F) Snapshots show TgGAC-mCherry-Ty at the CJ (arrows) in an invading parasite.
(G) Ring-like structures (arrows) of TgGAC-mCherry-Ty in motile parasites gliding in matrigel. Parasites were stimulated with A23187 prior to fixation and IFAs.
(H) In the absence of TgMyoH (48 hr ATc) or TgMyoA, the apical- (arrows) to-basal (stars) relocalization of TgGAC is abolished. Scheme shows the apicobasal flow of TgGAC (blue arrows).
(I) Live imaging shows cytosolic and apical (arrows) localization of PbGAC-GFP in the three invasive stages of P. berghei: merozoites, ookinetes, and sporozoites.
(J) 3D SIM revealed PbGAC-GFP as an apical ring-like structure (arrows) in merozoites and ookinetes. One z stack is presented. Scale bars, 2 (B, C, and E–H), 1 (I), 0.5, and 2 μm (J).
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4. Figure 2
GAC Is Vital for Gliding Motility, Invasion, and Egress from Infected Cells
(A) iMycTgGAC (287 kDa) is tightly downregulated 24 hr after ATc treatment.
(B) Cytosolic and apical localization (arrows) of iMycTgGAC in intracellular parasites. iMycTgGAC relocalizes to the basal end (stars) upon egress from the host cell.
(C) Upper panel; Airyscan confocal microscopy showed the presence of iMycTgGAC at the CJ labeled with α-TgRON4 antibodies, as presented in Figure 1E. Lower panel; isosurface 3D volume rendering of the upper panel processed with Imaris is shown.
(D) TgGAC-depleted parasites failed to form lytic plaques on a monolayer of human fibroblasts, whereas untreated TgGAC-iKD or treated ΔKu80 formed plaques of comparable sizes (7 days ± ATc).
(E–G) Parasites depleted of TgGAC (48 hr ATc) (E) were not able to glide (trails labeled with SAG1 antibodies) on gelatin-coated glass, (F) were severely impaired in all reported types of movements (p value = 0.001), and (G) were blocked in egress and invasion (p values ≤ 0.001). Data are presented as mean ± SD. Scale bars, 2 μm.
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5. Figure 3 GAC Binds to and Stabilizes F-Actin
(A) TgGAC-3Ty binds to rabbit filamentous α-actin in co-sedimentation assays. TgGRA3 and TgPRF were used as negative controls and TgMyoA was used as a positive control. P, pellet; S, supernatant.
(B) N-, but not M-TgGAC-GFPTy, binds to rabbit filamentous α-actin in co-sedimentation assays.
(C and D) Recombinant FL-TgGAC, N-TgGAC, and N-PfGAC (C) bind to filamentous PfACT1 in co-sedimentation assays and (D) increase PfACT1 polymerization (left) and decrease depolymerization (right) in a concentration-dependent manner.
(E) TgFRM1-3Ty is restricted to the apical tip of parasites and is not present at the CJ. Dashed lines represent parasite’s periphery.
(F) Conditional depletion of TgGAC (48 hr ATc) was not affecting apicoplast inheritance. Scale bars, 2 μm.
Cell Host & Microbe  , DOI: ( /j.chom )
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6. Figure 4
TgGAC Transiently Associates with the Secreted Transmembrane Adhesin TgMIC2
(A) Western blots assessed the absence of TgROM4 in TgGAC-mCherry-Ty. TgPRF was used as a loading control and a previously generated strain of TgROM4-KO was used as a control (Rugarabamu et al., 2015).
(B) TgROM4-KO resulted with the expected accumulation of TgMIC2 at the plasma membrane. Importantly, this did not affect the localization of TgGAC-mCherry-Ty at the apical end (arrows) nor induced its accumulation along the PPM.
(C) TgGAC-mCherry-Ty and TgMIC2 are detected in α-SAG1-labeled trails formed during gliding in parasites lacking TgROM4, whereas they are absent in trails of WT parasites. Importantly, TgACT is not detectable in the trails either in the presence or absence of TgROM4.
(D) Quantification of the data presented in (C). Percentages of double-stained SAG1/Ty, SAG1/MIC2, or SAG1/ACT trails are represented. Data are presented as mean ± SD (p values ≤ 0.001; ns, non-significant).
(E) In TgROM4-KO background, TgGAC-mCherry-Ty and TgMIC2 colocalized in long trails.
(F) FL-TgGAC, but not N-TgGAC, interacts with His-TgMIC2-Tail in an Ni-affinity pull-down assay.
(G) Surface plasmon resonance shows tight binding for FL-TgGAC and no binding of N-TgGAC to His-TgMIC2-Tail covalently immobilized on a CM5 sensor chip surface. The calculated Kd for FL-TgGAC is 400 nM. The binding affinity curve is shown in Figure S4D. Scale bars, 2 μm.
Cell Host & Microbe  , DOI: ( /j.chom )
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7. Figure 5 Structural Insights of GAC Reveal a Modular Protein
(A) Ab initio models of FL-TgGAC calculated by the programs DAMMIF (yellow), GASBOR (purple), and MONSA show the club shape of TgGAC. In the two-phase MONSA model, the calculated N-TgGAC contribution is represented by red and the rest of the protein by green transparent spheres. Superimposed on the MONSA model are homology models of N-TgGAC (red), the middle armadillo region (green), and the PH domain (blue). The graph represents the fits of the models to the scattering data (colors corresponding to the models). The curves have been vertically shifted to an arbitrary scale for easier visualization.
(B) GASBOR model of N-PfGAC (orange) superimposed on that of N-TgGAC (blue) and fit of the N-PfGAC model (orange line) to the SAXS data (gray dots) are shown.
(C) PIP-strip experiments revealed that TgGAC-PH and PfGAC-PH domains bind to PA. TgAPH and GST-PLC-δ1-PH were used, respectively, as PA- and PI(4,5)P2-binding controls and GST was used as a negative control.
(D) DGK1 inhibitor R59022 (30 μM) blocked the apical (arrows) to basal (stars) TgGAC relocalization upon A23187-induced egress. LPA, lysophosphatidic acid; LPC, lysophosphocholine; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PS, phosphatidylserine; S1P, Sphingosine-1-phosphate.
Cell Host & Microbe  , DOI: ( /j.chom )
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8. Figure 6 Apical Positioning of TgGAC Depends on Active TgAKMT
(A) TgGAC colocalizes with TgAKMT at the apical end.
(B) TgGAC-iKD/TgAKMT-3Ty parasites, treated 48 hr ± ATc prior to mechanical egress, were incubated 30 min in extracellular buffer. In presence of TgGAC, TgAKMT-3Ty was rapidly relocalized within the cytoplasm. Depletion of TgGAC or 1 μM CD treatment abolished this relocalization.
(C) Conditional depletion of TgAKMT resulted in the loss of apical TgGAC.
(D) No change in TgGAC-Ty expression was observed upon conditional depletion of TgAKMT-iKD (48 hr ATc). TgIMC1 was used as a loading control.
(E and F) Complementation with MycTgAKMT-WT, but not with the catalytically dead mutant MycTgAKMT-H447V, restores (E) the apical localization of TgGAC and (F) the ability to form plaques after 7 days of ATc. ΔKu80 and TgAKMT-iKO were used, respectively, as positive and negative controls.
(G) TgAKMT-KO formed smaller plaques compared to the control RH, which was partially rescued by the expression of a second copy of TgGAC.
(H and I) The apical staining (H, arrows) observed with α-H4K20Me3 and (I) multiple high molecular bands disappeared upon TgAKMT depletion (48 hr ATc).
(J) The apical ends (arrows) of P. berghei merozoites and ookinetes also were labeled with α-H4K20Me3 antibodies. Scale bars, 2 (A–C, E, and H) and 1 and 2 μm (J).
Cell Host & Microbe  , DOI: ( /j.chom )
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9. Figure 7 Model of Motility in T. gondii
Schematic representation of the switch from intracellular replication to extracellular motility. The dynamic events include (1) cytosolic relocalization of TgAKMT; (2) conoid protrusion; (3) actin nucleation and polymerization by TgFRM1, apical microneme secretion (through the conoid), and TgGAC binding to PPM via its PH domain upon PA production; (4) adhesin-receptor interaction; and (5) F-actin-adhesin connection by TgGAC and basal translocation powered by the successive actions of TgMyoH and TgMyoA. Steps (2) to (5) are expected to occur repetitively at each motile cycle where only a small fraction of TgGAC/MICs is consumed.
Cell Host & Microbe  , DOI: ( /j.chom )
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