An Apicomplexan Actin-Binding Protein Serves as a Connector and Lipid Sensor to Coordinate Motility and Invasion

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1 Article An Apicomplexan Actin-Binding Protein Serves as a Connector and Lipid Sensor to Coordinate Motility and Invasion Graphical Abstract Authors Damien Jacot, Nicolò Tosetti, Isa Pires,..., Rita Tewari, Inari Kursula, Dominique Soldati-Favre Correspondence inari.kursula@uib.no (I.K.), dominique.soldati-favre@unige.ch (D.S.-F.) In Brief Host cell invasion by apicomplexan parasites involves gliding motility that is powered by rearward translocation of secreted transmembrane adhesins by the parasite actomyosin system. Jacot et al. identify a connector that bridges this actomyosin system to parasite adhesinhost receptor complexes, ensuring the propelling of these intracellular parasites into host cells. Highlights d The Apicomplexa require a glideosome-associated connector (GAC) for motility and invasion d GAC is an actin-binding protein that stabilizes parasite F- actin d d GAC binds to the adhesin MIC2 and phosphatidic acid to coordinate motility An apical lysine methyltransferase is crucial for the apical positioning of GAC Jacot et al., 2016, Cell Host & Microbe 20, December 14, 2016 ª 2016 Elsevier Inc.

2 Cell Host & Microbe Article An Apicomplexan Actin-Binding Protein Serves as a Connector and Lipid Sensor to Coordinate Motility and Invasion Damien Jacot, 1 Nicolò Tosetti, 1 Isa Pires, 2 Jessica Stock, 3 Arnault Graindorge, 1 Yu-Fu Hung, 2 Huijong Han, 2 Rita Tewari, 3 Inari Kursula, 2,4, and Dominique Soldati-Favre 1,5, 1 Department of Microbiology & Molecular Medicine, University of Geneva, 1 Rue Michel-Servet, 1211 Geneva, Switzerland 2 Biocenter Oulu and Faculty of Biochemistry and Molecular Medicine, University of Oulu, Aapistie 7, Oulu, Finland 3 School of Life Sciences, Queens Medical Centre, University of Nottingham, Nottingham NG2 7UH, UK 4 Department of Biomedicine, University of Bergen, Jonas Lies vei 91, 5009 Bergen, Norway 5 Lead Contact Correspondence: inari.kursula@uib.no (I.K.), dominique.soldati-favre@unige.ch (D.S.-F.) SUMMARY Apicomplexa exhibit a unique form of substratedependent gliding motility central for host cell invasion and parasite dissemination. Gliding is powered by rearward translocation of apically secreted transmembrane adhesins via their interaction with the parasite actomyosin system. We report a conserved armadillo and pleckstrin homology (PH) domaincontaining protein, termed glideosome-associated connector (GAC), that mediates apicomplexan gliding motility, invasion, and egress by connecting the micronemal adhesins with the actomyosin system. TgGAC binds to and stabilizes filamentous actin and specifically associates with the transmembrane adhesin TgMIC2. GAC localizes to the apical pole in invasive stages of Toxoplasma gondii and Plasmodium berghei, and apical positioning of TgGAC depends on an apical lysine methyltransferase, TgAKMT. GAC PH domain also binds to phosphatidic acid, a lipid mediator associated with microneme exocytosis. Collectively, these findings indicate a central role for GAC in spatially and temporally coordinating gliding motility and invasion. INTRODUCTION Active host cell entry, egress, and migration across non-permissive biological barriers are crucial steps in the lytic cycle of Apicomplexa and are powered by gliding motility. A phylum-specific actomyosin system, called the glideosome, sustains the forward movement of the parasites by the concomitant rearward translocation of transmembrane adhesins attached to the extracellular matrix (reviewed in Heintzelman, 2015). These adhesins (such as AMA1 4 and MICs in T. gondii and AMA1, TRAP, MTRAP, and CTRP in Plasmodium) are apically discharged at the parasite plasma membrane (PPM) by regulated, secretory organelles called micronemes. Host cell invasion involves an additional complex of rhoptry neck proteins (RONs) apically released onto the host plasma membrane, which serves as a receptor for the microneme protein AMA1 and its homologs to form the circular/moving junction (CJ) (Besteiro et al., 2011; Lamarque et al., 2014; Riglar et al., 2011). The translocation of the CJ by the glideosome propels the parasite into the host cell (Figure S1A). In T. gondii, myosin H (TgMyoH), anchored to an apical microtubule complex composing the conoid, serves as the first translocator of the CJ from the tip of the parasite to the apical end of the inner membrane complex (IMC). There, TgMyoA, confined to the limited space between the PM and the IMC, takes the relay down to the basal end of the parasite (Frénal et al., 2010, 2014; Graindorge et al., 2016; Meissner et al., 2002). TgMyoA is dispensable, but its deletion severely impacts parasite motility and leads to the compensatory relocalization of TgMyoC along the IMC from its restricted position at the basal polar ring. This rescues enough motility to sustain parasite survival (Andenmatten et al., 2013; Frénal et al., 2014). In contrast to TgMyoA, TgMyoH is indispensable (Graindorge et al., 2016). In Plasmodium, MyoA is the only motor reported to act at the CJ (Baum et al., 2006b). According to the current model, actin and actin-binding proteins critically assist parasite motility. A unique feature of apicomplexan actin is that it is largely maintained in a heterogeneous mixture of sequestered actin monomers and short filaments of variable length (Dobrowolski et al., 1997; Schmitz et al., 2005; Skillman et al., 2011, 2013). T. gondii actin polymerizes in an isodesmic manner, resulting in very short and heterologous filaments (Skillman et al., 2013). Importantly, temporal regulation of actin polymerization during gliding must be tight, and only a limited set of key players in actin dynamics, including profilin (PRF), actin depolymerization factor (ADF), and formins (FRMs), have been identified and characterized in Apicomplexa. Both PRF and ADF (ADF1 in Plasmodium species [spp.]) act primarily to sequester globular actin (G-actin) (Mehta and Sibley, 2010; Sch uler et al., 2005; Singh et al., 2011; Skillman et al., 2012). Conditional depletion of both proteins in T. gondii affects gliding motility, invasion, and egress, presumably by impacting actin turnover (Mehta and Sibley, 2011; Plattner et al., 2008). Investigation of PRF in the intraerythrocytic stage of P. berghei Cell Host & Microbe 20, , December 14, 2016 ª 2016 Elsevier Inc. 731

3 A TgGAC mcherry-ty ΔKu80 TgGAC-3Ty B TgGAC-3Ty D % TgGAC 260 α-mic2 Merge Zoom Zoom TgGAC relocalization no TgGAC relocalization 0 + CD - CD 50 E TgIMC1 72 C / α-imc1 TgGAC-3Ty - CD - CD + CD 1 μm α-ron4 0.1% Saponin 0.1% Triton-X Merge - A A23187 non-permeabilized 0.1% Triton-X Merge F G + A s mcherry 11s 13s H TgMyoH-iKD/ TgGAC-3Ty - ATc + A23187 TgMyoA-KO/ TgGAC-3Ty I Schizont PbGAC-GFP DIC GFP Hoechst/GFP + ATc + A23187 Conoid IMC TgMyoH TgMyoA Merozoite DIC GFP Hoechst/GFP J subpellicular microtubules PbGAC-GFP plasma membrane Ookinete Merozoite Ookinete DIC GFP α-p28/hoechst/gfp α-gfp/hoechst α-gfp α-p28/hoechst Zoom Sporozoite DIC GFP Hoechst/GFP 732 Cell Host & Microbe 20, , December 14, 2016 (legend on next page)

4 also revealed a consistent and critical role in invasion and egress (Pino et al., 2012). Apicomplexa lack the ARP2/3 complex, one of the major actinnucleating machineries in other organisms (Baum et al., 2006a), and, instead, nucleation of actin filaments is mediated by two formins in Plasmodium spp. and three in T. gondii. TgFRM1 and TgFRM2 act in concert at the parasite pellicle to transiently generate short filaments necessary for motility (Daher et al., 2010). TgFRM3 is restricted to coccidians and its deletion does not impact the parasite lytic cycle (Daher et al., 2012). In P. falciparum, PfFRM1 is at the apical end of merozoites and follows the CJ during host cell invasion, whereas PfFRM2 localizes diffusely throughout the parasite cytoplasm (Baum et al., 2008). In addition to the essential role of actin in motility (Drewry and Sibley, 2015), actin dynamics, together with myosin F, also are linked to the inheritance of a vestigial plastid called the apicoplast and the positioning of other organelles (Andenmatten et al., 2013; Heaslip et al., 2016; Jacot et al., 2013; Mueller et al., 2013). In Apicomplexa, motility is tightly controlled in space and time, as parasites become immediately motile upon egress and stop moving equally fast following internalization. Phosphorylation has been demonstrated to play a prominent role in the apical release of micronemes, a prerequisite to initiate motility and to control components of the glideosome, such as TgMyoA (Gaji et al., 2015; Sharma and Chitnis, 2013; Tang et al., 2014). Recently, phosphoinositide metabolism and specifically phosphatidic acid (PA) have been identified as key lipid mediators of the signaling leading to microneme secretion (Brochet et al., 2014; Bullen et al., 2016). Extracellular stimuli triggering microneme exocytosis culminate with the activation of phosphoinositide-phospholipase C and the generation of the second messengers diacylglycerol (DAG) and IP 3. DAG is then converted to PA by the diacylglycerol kinase-1 (DGK1) and controls microneme release (Bullen et al., 2016). An apical lysine methyltransferase (TgAKMT), localized to the parasite apical end, is also a key factor controlling motility (Heaslip et al., 2011). Deletion of TgAKMT did not affect microneme secretion and, hence, its contribution to motility remained mysterious. Intriguingly, in the presence of signals stimulating egress, TgAKMT redistributes from the apical pole to the cytosol, suggesting that a temporal control of its localization is relevant. The connection between the adhesin-receptor complexes and the actomyosin system was attributed to aldolase (ALD) due to its ability to bind both F-actin and the cytosolic tails of several adhesins, including TgMIC2, its Plasmodium ortholog TRAP, and AMA1 (Buscaglia et al., 2003; Jewett and Sibley, 2003; Sheiner et al., 2010; Shen and Sibley, 2014; Starnes et al., 2006, 2009). A recent study demonstrated that depletion of TgALD causes toxic accumulation of fructose-1,6-bisphosphate, but, in the absence of extracellular glucose, no impairments in motility and invasion were observed (Shen and Sibley, 2014). In consequence, the connection between micronemal adhesins and the glideosome remains an open question that challenges the current capping model for apicomplexan motility and invasion. Here we identified an essential factor for gliding, invasion, and egress named glideosome-associated connector (GAC). GAC dynamically relocalizes from the apical to the basal end of gliding parasites and localizes at the CJ of invading T. gondii parasites. During motility, TgGAC transiently associates with the transmembrane adhesin TgMIC2 and binds to the tail of MIC2 in vitro. GAC possesses a pleckstrin homology (PH) domain that binds to PA. Importantly, TgGAC binds to and stabilizes actin filaments and its recruitment at the conoid depends on TgAKMT activity. Overall, these findings offer a coherent and comprehensive view of the essential molecular machine that orchestrates gliding motility in Apicomplexa. RESULTS GAC Is a Dynamic Apical Protein Translocated to the Basal End of Motile and Invading Parasites by Myosins GAC encodes a large armadillo-repeat-containing protein highly conserved among members of the Apicomplexa phylum but absent in other alveolates (Tewari et al., 2010) (Figure S1B; Table S1). The TgGAC (EuPathDB: TGME49_312630) locus was modified by single homologous recombination to insert either an epitope tag or a red fluorescent protein at the TgGAC C terminus. In intracellular TgGAC-3Ty and parasites, TgGAC localized to the cytosol as well as to the conoid (Figures 1A, 1B, and S1C). TgGAC-3Ty was fully cytosolic, based on fractionation and digitonin proteinase K protection assays (Figures S1D and S1E). Upon Ca 2+ ionophore- (A23187) induced microneme secretion and parasite egress, TgGAC accumulated at the basal end. TgGAC relocalization was abolished by Cytochalasin D (CD), an inhibitor of actin polymerization known to block parasite motility (Dobrowolski and Sibley, 1996)(Figures 1C, 1D, 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 (314 kda), with TgIMC1 as loading control and DKu80 as parental strain. (B) Cytosolic and apical localization at the conoid (zoom) of TgGAC-3Ty in intracellular parasites. Micronemes are stained with a-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 at the CJ (arrows) in invading parasites. The CJ was visualized either first with a-tgron4 antibodies in 0.1% saponin buffer, which permeabilized only the host cell, or using a-sag1 antibodies in non-permeabilized conditions. Samples were subsequently permeabilized with Triton-X to gain access to TgGAC. (F) Snapshots show at the CJ (arrows) in an invading parasite. (G) Ring-like structures (arrows) of 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 mm (J). Cell Host & Microbe 20, , December 14,

5 and S1C). Basal end accumulation of TgGAC was transient and depended on parasite motility. During host cell invasion, TgGAC was present at the CJ, as demonstrated under differential permeabilization conditions, by colocalization with TgRON4 and by labeling the parasite-host interface with antibodies recognizing surface antigen 1 (SAG1) (Figure 1E). This was confirmed in invading parasites by timelapse video microscopy using (Figure 1F; Movie S1). In extracellular parasites gliding in a 3D matrigel, formed ring-like structures along the parasite length (Figure 1G). This suggests a common mechanism for migration across biological barriers and host cell invasion. The sensitivity of TgGAC apicobasal translocation to CD implies the involvement of F-actin and, indirectly, the participation of a myosin motor. Together, TgMyoH and TgMyoA span the entire length of the parasite and logically could power this transport. Epitope tagging of endogenous TgGAC in the TgMyoH-iKD (Graindorge et al., 2016) and disruption of TgMyoA in TgGAC- 3Ty parasites established that the absence of these motors does not alter the apical localization of TgGAC (Figures S1F S1H) but abolishes its translocation to the basal end upon Ca 2+ stimulation (Figure 1H). In conclusion, TgGAC is a soluble, dynamic component of the CJ that translocates as a ring-like structure from the apical end to the basal end in motile and invasive parasites upon the successive action of the TgMyoH and TgMyoA glideosomes. In malaria parasites, GAC exhibits >% sequence identity with TgGAC, suggestive of a similar function (Table S1). In P. berghei, endogenous PbGAC (EuPathDB: PBANKA_ ) fused to GFP was soluble and expressed at all life cycle stages examined (Figures S1I S1L). PbGAC-GFP locates to the cytosol at all stages and shows a distinct accumulation at the extreme apical end of the invasive blood stage merozoites, motile ookinetes, and sporozoites (Figure 1I). Refined investigation by 3Dstructured illumination microscopy (SIM) revealed that PbGAC- GFP forms a ring-like structure at the apical end of both merozoite and ookinete (Figure 1J; Movie S2). TgGAC Is Essential for Gliding Motility, Invasion, and Egress without Impacting Microneme Secretion A conditional knockdown (TgGAC-iKD) was generated in T. gondii by promoter replacement (Figures S2A and S2B). Depletion of inducible TgGAC (imyctggac) was achieved in 24 hr upon anhydrotetracycline (ATc) treatment (Figure 2A). Importantly, imyctggac relocalized from the apical end to the basal end upon parasite egress (Figure 2B). High-resolution Airyscan confocal microscopy revealed the presence of imyctggac at the CJ labeled with TgRON4 (Figure 2C). TgGAC-iKD parasites failed to form lytic plaques after 7 days of ATc treatment, indicating a critical impairment in one or more steps of the lytic cycle (Figure 2D). Deeper investigation of TgGAC depletion revealed a complete block in gliding motility with an impact on the three forms of 2D movement (Håkansson et al., 1999)(Figures 2E and 2F; Movie S3). This block led to only 10% and 2% residual egress and invasion, respectively (Figure 2G; Movie S4; Table S2). In contrast, intracellular parasite replication was unaffected (Figure S2C). The phenotype of TgGAC-iKD was functionally fully rescued by a second cdna copy of TgGAC (Figures S2D S2F). Conoid protrusion, host cell attachment, and microneme secretion (assessed by the release and post-exocytosis processing of TgAMA1, TgMIC2, and TgMIC6) were unaffected (Movie S3, right; Figures S2G and S2H). In summary, TgGAC-depleted parasites are unable to egress and glide and are blocked at the attachment step during invasion. These phenotypes are identical to those observed upon depletion of TgMyoH (Graindorge et al., 2016). GAC Binds to and Stabilizes Actin Filaments The dynamic nature of GAC and its implication in glideosome function are compatible with a role as a connector bridging the adhesin tails to the actomyosin system. The first indication that TgGAC binds to F-actin was obtained from extracellular TgGAC-3Ty parasites treated with 1 mm jasplakinolide (stabilizer of F-actin), which resulted in acrosome-like TgGAC-positive actin projections through the conoid (Shaw and Tilney, 1999) (Figure S3A). Direct evidence was obtained from co-sedimentation of TgGAC-3Ty from parasite lysates with purified rabbit F-actin (Figure 3A). Three partly overlapping regions of TgGAC were expressed as second copies in T. gondii tachyzoites (Figure S3B). The middle region (M-TgGAC-GFPTy) localized apically, N-TgGAC-GFPTy was cytosolic, while C-TgGAC- GFPTy was poorly expressed (Figures S3C and S3D). N-TgGAC-GFPTy, but not M-TgGAC-GFPTy, co-sedimented with rabbit F-actin (Figure 3B). C-TgGAC-GFPTy was not soluble and could not be assessed. In parallel, GST-N-TgGAC, GST-M- TgGAC, and full-length His-FL-TgGAC were recombinantly produced in Escherichia coli and shown to recapitulate the same F-actin-binding properties (Figures S3E and S3F). To assess the effect of GAC on the divergent parasite actin, recombinant PfACT1 was used for co-sedimentation and actin polymerization assays. Of relevance, actin is encoded by a single gene in Apicomplexa, with the exception of Plasmodium spp. that encode two actin isoforms, ACT1 and ACT2, of which the latter is only expressed at the gametocyte stage (Dobrowolski et al., 1997; Wesseling et al., 1989). FL-TgGAC, N-TgGAC, and P. falciparum N-PfGAC bound filamentous PfACT1 (Figures 3C and S3G). Unexpectedly, FL-TgGAC and N-PfGAC also enhanced polymerization of PfACT1 and decreased the rate of depolymerization (Figure 3D). However, the effect of GAC on the initial rate of polymerization was very small (Figures 3D and S3H S3K), suggesting a function in stabilization via binding to pre-existing short filaments rather than de novo nucleation. Relevant here, TgFRM1 and TgFRM2 were reported to participate in nucleation and elongation of actin filaments and proposed to act along the parasite pellicle (Daher et al., 2010). This redundancy and the contrasting localizations of the two formins described in P. falciparum (Baum et al., 2008) prompted us to revisit the localization of these two actin nucleators in T. gondii. 3Ty epitope tags were added to the C termini of TgFRM1 and TgFRM2 by recombination at the respective loci. Although TgFRM1 was previously localized along the pellicle when controlled by an inducible promoter, the endogenous tagged protein was restricted to the apical tip of the parasite. Importantly, in invading parasites TgFRM1 did not appear to follow the CJ (Figure 3E), in contrast to what was reported in Plasmodium merozoites (Baum et al., 2008). Also contrasting with the previous report based on antibodies raised against TgFRM2, endogenously tagged TgFRM2 localized in the vicinity 734 Cell Host & Microbe 20, , December 14, 2016

6 A ΔKu80 TgGAC-iKD ATc +48h - +24h +48h B TgGAC-iKD C TgGAC-iKD imyctggac α-myc α-myc α-ron4 Merge D TgCAT α-myc / α-cat TgGAC-iKD - ATc TgGAC-iKD + ATc 72 ΔKu80 + ATc F G Egress Invasion E TgGAC-iKD - ATc + ATc % Immobile Twirling 20 Helical 0 Circular ΔKu80 TgGAC-iKD -ATc TgGAC-iKD +ATc % of egressed parasites A23186 DMSO % of invaded parasites TgGAC-iKD - ATc TgGAC-iKD + ATc ΔKu80 + ATc 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 a-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 DKu80 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 mm. of the apicoplast and was not implicated in motility and invasion (N.T., unpublished data). In this context, conditional depletion of TgGAC did not affect apicoplast inheritance, indicative of its dedicated role in motility (Figure 3F). In conclusion, TgFRM1 likely initiates actin polymerization at the tip of the parasite, while GAC acts along the pellicle, stabilizing the actin filaments carried by the myosins to generate motion. TgGAC Transiently Associates In Vivo with the Transmembrane Adhesin TgMIC2 and Binds to TgMIC2 Tail In Vitro To determine whether GAG bridges the glideosome to the extracellular matrix and host cell surface, we investigated the potential interaction between TgGAC and TgMIC2. In vivo, the binding of GAC to the tails is expected to be transient, as GAC does not appear to decorate the micronemes. To be productive, the interaction should ideally take place at the PPM and only in the context of the adhesins associated to their respective receptors. Concordantly, the tails of secreted adhesins engaged to their receptors are likely to undergo modifications, such as phosphorylation or oligomerization to provide an additional level of regulation. Once secreted, the adhesins are rapidly cleaved within their transmembrane domain by the rhomboid protease TgROM4, allowing the parasite to disengage from host receptors (Figure S1A). Accordingly, knockout (KO) of TgROM4 leads to adhesin accumulation at the PPM (Rugarabamu et al., 2015; Shen et al., 2014). To capture this expected short-lived interaction, TgROM4 was knocked out using CRISPR/Cas9 in both and TgGAC-iKD (Figures 4A and S4A). In the absence of TgROM4, TgMIC2 accumulated at the PPM of extracellular parasites but did not alter TgGAC localization (Figure 4B). Incubating parasites with heparin, which is a receptor for TgMIC2 (Carruthers et al., 2000), or with TgRON2 peptides recognized by TgAMA1 (Tonkin et al., 2011), had no impact on TgGAC localization. In parasites lacking TgROM4 and gliding on gelatincoated slides, uncleaved TgMIC2 was translocated to the basal end and left behind in trails positive for SAG1. Compellingly, in this mutant, TgGAC, but not actin, was massively detected along with TgMIC2 in the trails (Figures 4C and 4D), where TgMIC2 colocalized with TgGAC (Figure 4E). In wild-type (WT) parasites, neither TgMIC2 nor TgGAC was readily detectable in Cell Host & Microbe 20, , December 14,

7 A B F-actin TgGAC- 3Ty TgGAC-3Ty P S P S TgGRA3 TgPRF TgMyoA Rabbit α-actin / α-gra3 / α-prf/ α-myoa / Coomassie blue F-actin N-TgGAC GFPTy F-actin M-TgGAC GFPTy D fluorescence (AU) fluorescence (AU) TgPRF N-TgGAC-GFPTy P - S - P + S + Rabbit α-actin / α-prf/ Coomassie blue M-TgGAC-GFPTy P - S - P + S + TgPRF Rabbit α-actin / α-prf/ Coomassie blue nm FL-TgGAC 50 nm FL-TgGAC 250 nm FL-TgGAC 500 nm FL-TgGAC 800 nm FL-TgGAC t (s) 0 nm N-PfGAC 50 nm N-PfGAC 250 nm N-PfGAC 500 nm N-PfGAC 800 nm N-PfGAC t (s) fluorescence (AU) fluorescence (AU) C FL-TgGAC SAG1-positive trails. The same observations were reproduced in TgGAC-iKD and TgGAC-iKD/ROM4-KO (Figures S4B and S4C). Taken together, this indicates that, in the absence of transmembrane adhesin cleavage by TgROM4, the interaction between TgGAC and TgMIC2 in gliding parasites is strong enough to tear TgGAC from the parasite. Next, recombinant His-FL-TgGAC was shown to bind directly to the cytosolic tail of TgMIC2 in pull-down assays using immobilized GST or GST-TgMIC2-Tail (Figure S4D). To avoid interfer- F-buffer G-buffer M S P S P S P S P S P S P S P FL- TgGAC Coomassie blue M Coomassie blue N-TgGAC PfACT1 F-buffer G-buffer S P S S P P S P S P S P S P N- TgGAC 55 PfACT1 Coomassie blue N-PfGAC F-buffer G-buffer S P S P S P S P M S P S P t (s) t (s) 736 Cell Host & Microbe 20, , December 14, 2016 E F α-gap45 Merge α-cpn60 TgFRM1-3Ty α-ron4 Merge TgGAC-iKD +ATc 48h α-act α-cpn60 N- PfGAC PfACT1 Figure 3. GAC Binds to and Stabilizes F-Actin (A) TgGAC-3Ty binds to rabbit filamentous a-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 a-actin in co-sedimentation assays. (C and D) Recombinant FL-TgGAC, N-TgGAC, and N-PfGAC (C) bind to filamentous PfACT1 in cosedimentation 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 mm. ence by the large GST tag, the assays were repeated using immobilized His- TgMIC2-Tail and purified FL-TgGAC and N-TgGAC. Importantly, FL-TgGAC, but not N-TgGAC, interacted with His- TgMIC2-Tail (Figures 4F and 4SE). Furthermore, surface plasmon resonance using FL-TgGAC and immobilized His- TgMIC2-Tail confirmed tight binding with ak d in the sub-micromolar range (Figures 4G and S4F). These results establish the dual role of GAC in binding and stabilizing F-actin along the pellicle and in connecting myosin motors to the tails of TgMIC2. Dissection of GAC Structure and Functional Domains To obtain preliminary structural insight into its function, we used a combination of small-angle X-ray scattering (SAXS) and homology modeling to characterize the 3D structure of GAC. Ab initio model building using three different software resulted in virtually identical models (Figure 5A), with a maximum dimension of about 27 nm for the full-length protein and 16 nm for both N-GACs (Figure S5A). FL-TgGAC is a club-shaped molecule with a giant armadillo-repeat region forming the wider end and a PH domain sitting at the narrow top, far from the actin-binding N-GAC (Figure 5A). The shapes of N-TgGAC and N-PfGAC (Figures 5B and S5B) suggest it could wind around an actin filament, interacting with at least two to three protomers, which is in line with a filament-stabilizing function. In addition, the PH domain of GAC suggests it may interact with membrane phosphatidylinositol lipids. Importantly, microneme exocytosis recently was linked to PA signaling at the PPM in connection with an acylated PH

8 A C TgROM4-wt TgROM4-KO TgROM4-WT TgROM4-KO TgROM4-KO TgROM4 72 α-mic2 α-mic2 B 25 TgPRF α-rom4 / α-prf + A A23187 TgROM4-WT α-mic2 Non-permeabilized 0.1% Triton-X D TgROM4-KO α-mic2 Non-permeabilized % Triton-X F α-act Flow through Wash 1 Wash 2 Wash 3 Elution 1 Elution 2 FL TgGAC α-act Flow through Wash 1 Wash 2 Wash 3 Elution 1 Elution 2 N TgGAC % Ty ns MIC2 Actin Coomassie gel G His-TgMIC2 Tail Coomassie gel His-TgMIC2 Tail TgROM4-wt TgROM4-KO E /TgROM4-KO α-mic2 Merge Figure 4. TgGAC Transiently Associates with the Secreted Transmembrane Adhesin TgMIC2 (A) Western blots assessed the absence of TgROM4 in. 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 TgGACmCherry-Ty at the apical end (arrows) nor induced its accumulation along the PPM. (C) and TgMIC2 are detected in a-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, 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 K d for FL-TgGAC is 0 nm. The binding affinity curve is shown in Figure S4D. Scale bars, 2 mm. domain-containing protein (APH), conserved across the Apicomplexa phylum. APH is present at the outer micronemal surface, acts as a PA sensor, and participates in microneme exocytosis (Bullen et al., 2016). The PH domains of TgGAC and PfGAC fused to GST (GST-TgGAC-PH and GST-PfGAC-PH) were produced recombinantly, purified, and probed on phosphoinositide Cell Host & Microbe 20, , December 14,

9 A B C GST TgAPH GST TgGAC-PH GST PfGAC-PH GST GST PLC-δ1-PH D LPA LPC PI PI(3)P PI(4)P PI(5)P PE PC S1P PI(3,4)P 2 PI(3,5)P 2 PI(4,5)P 2 PI(3,4,5)P 3 PA PS Blank - R R59022 α-gap45 Merge α-gap45 Merge 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-d1-PH were used, respectively, as PA- and PI(4,5) P 2 -binding controls and GST was used as a negative control. (D) DGK1 inhibitor R59022 (30 mm) 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. strips (PIP-strip) using GST-TgAPH (Bullen et al., 2016) and GST as positive and negative controls, respectively (Figure S5C). TgGAC-PH and PfGAC-PH selectively bound to PA and not substantially to any other PIP. PIP-strips probed with GST showed no binding, while the human GST-tagged phospholipase C-d1 PH domain (GST-PLC-d1-PH, provided by the manufacturer as a control) selectively bound PI(4,5)P2 (Figure 5C). Pharmacological inhibition of the PA-producing enzyme DGK1 by compound R59022 blocks microneme secretion and parasite motility (Bullen et al., 2016). Accordingly, under egress conditions, R59022 abolished the relocalization of TgGAC (Figure 5D). In conclusion, GAC is a modular protein with its N-terminal domain binding to F-actin, the middle part containing the determinant for targeting to the conoid, and the C-terminal PH domain binding to PA, presumably produced at the PPM at the time of egress and invasion and orientating GAC toward the membrane. Apical Positioning of TgGAC Depends on an Active Lysine Methyltransferase In T. gondii, TgAKMT previously was reported to assist parasite motility, invasion, and egress without affecting exocytosis (Heaslip et al., 2011; Sivagurunathan et al., 2013). TgAKMT is present at the apical tip of the parasite, and endogenously tagged TgAKMT-3Ty in TgGAC-iKD colocalized with TgGAC at the conoid (Figures 6A and S6A). Upon depletion of TgGAC or CD treatment, the cytosolic relocalization of TgAKMT in motile parasites was blocked (Figure 6B) (Heaslip et al., 2011). However, we ruled out an interaction between TgAKMT and TgGAC based on coimmunoprecipitation (coip) using both intracellular and extracellular parasites. Reciprocally, we assessed the impact of TgAKMT on TgGAC function by generating a conditional knockdown of TgAKMT (Figures S6B S6D). Depletion of TgAKMT recapitulated the severe phenotypes previously reported with conventional TgAKMT-KO (Heaslip et al., 2011)(Figure S6E). Strikingly, in the absence of TgAKMT, apical accumulation of TgGAC was lost (Figure 6C), while its overall level of expression remained unchanged (Figure 6D). Complementation of TgAKMT-iKD with either wild-type MycTgAKMT-WT or the catalytically dead enzyme Myc-TgAKMT-H447V (Heaslip et al., 2011) revealed that the lysine methyltransferase activity of TgAKMT is a prerequisite to restore apical localization of TgGAC (Figure 6E). Concordantly, only complementation with Myc- TgAKMT-WT resulted in the recovery of lytic plaques (Figures 6F and S6F). Consequently, the role of TgAKMT in glideosome function is intimately linked to TgGAC position and possibly to GAC function. 738 Cell Host & Microbe 20, , December 14, 2016

10 A C TgGAC-iKD/TgAKMT-3Ty D TgAKMT-iKD/TgGAC-Ty - ATc + ATc ΔKu80 ATc + TgAKMT-iKD TgGAC-Ty + - TgGAC-Ty TgIMC1 α-myc Zoom/Merge B E TgGAC-iKD/TgAKMT-3Ty in EC buffer - ATc + ATc - ATc / + CD 72 / α-imc1 TgAKMT-iKD +ATc F MycTgAKMT H447V / α-gap45 / α-gap45 / α-gap45 G α-myc α-myc TgAKMT-iKD +ATc ΔKu80 +ATc H TgAKMT-KO/ TgGAC-GFPTy TgAKMT-KO Myc TgAKMT-H447V Myc TgAKMT-wt TgAKMT-iKD/TgGAC-Ty +ATc MycTgAKMT wt TgAKMT-iKD - ATc RH α-h4k20me3 I ΔKu80 TgAKMT-iKD ATc + - J +ATc α-act/merge α-h4k20me3 α-act/merge PbGAC-GFP DIC Hoechst α-gfp α-h4k20me3 Merge DIC Hoechst α-gfp α-h4k20me3 Merge α-h4k20me3 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 mm 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. DKu80 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 a-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 a-h4k20me3 antibodies. Scale bars, 2 (A C, E, and H) and 1 and 2 mm (J). Cell Host & Microbe 20, , December 14,

11 GAC MyoA Extracellular matrix Host receptors AKMT Formin 1 MICs/AMA1 MyoH Actin Micronemes AKMT GAC Apical polar rings Preconoidal ring GAC ring GAC ring in movement Subpellicular microtubules Parasite plasma membrane Intracellular replicating T. gondii PPM PPM extracellular matrix/host IMC PM GAC (cytosolic) IMC Extracellular moving T. gondii 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. In support of this view, we generated a CRISPR/Cas9-mediated TgAKMT-KO (Figure S6G), and we demonstrated that overexpression of TgGAC in this mutant partially restored the phenotype resulting from TgAKMT deletion (Figure 6G). To investigate the range of potential substrates of TgAKMT, we used a-h4k20me3 antibodies reported to stain the nucleus and the apical tip of T. gondii (Xiao et al., 2010). Pertinently, the apical staining and several large proteins completely disappeared upon conditional depletion of TgAKMT (Figures 6H and 6I). However, immunoprecipitated was not detectable with a-h4k20me3 antibodies. The same antibodies also labeled the apical end of both P. berghei merozoites and ookinetes, suggestive of a potentially conserved recruitment of GAC to its site of action by a yet unknown mechanism but dependent on protein methylation (Figure 6J). DISCUSSION Gliding motility is a unique and vital attribute of the obligate intracellular Apicomplexa lifestyle. The function of GAC validates the capping model of motility and invasion but also reveals unexpected findings. Although armadillo-repeat proteins are common in cytoskeletal networks, GAC is, to the best of our knowledge, a unique example of an armadillo domain directly binding to and stabilizing F-actin. The super-helical structure of the F-actin-binding N-terminal armadillo domain of GAC suggests that the mechanism of filament stabilization might involve binding to two to three actin protomers in a very short filament. In this context, we revisited the localization of TgFRM1, previously associated with gliding motility and invasion (Daher et al., 2010). Endogenously epitope-tagged TgFRM1 is confined to the conoid, a localization consistent with that of PfFRM1 (Baum et al., 2008). However, in contrast to PfFRM1, which was tentatively localized to the CJ of invading merozoites, TgFRM1 does 7 Cell Host & Microbe 20, , December 14, 2016 not translocate with this moving structure and remains attached to the apical pole. A separate investigation excluded the participation of TgFRM2 in motility (N.T., unpublished data). In light of these results, FRM1 is optimally positioned to play a central role in nucleating short actin filaments necessary to engage GAC into the glideosome and transport the released pulses of microneme adhesins toward the posterior pole. GAC, in turn, appears to play a crucial role along the pellicle in stabilizing short filaments nucleated by FRM1. Translocation of TgGAC along the parasite length is ensured by the concerted action of TgMyoH and TgMyoA (Figure 7). Interestingly, in extracellular parasites gliding in a 3D environment, a ring-like structure reminiscent of the CJ was observed. This suggests that gliding and invading parasites actually rely on the same fundamental mechanism. TgGAC associates with TgMIC2 in vivo as detected in the trails when TgROM4 cleavage was prevented. This interaction was confirmed in vitro to be tight, with a sub-micromolar Kd. Curiously, this direct binding was not confirmed by coip using parasite lysates, indicating additional levels of regulation, possibly involving transient changes in the properties of the tails that regulate a timely accessibility to TgGAC in vivo. In the in vivo context, the association of adhesins with TgGAC is likely transient, and binding of adhesins to their receptors might intuitively be a prerequisite, which would elegantly prevent an unproductive recruitment of TgGAC. In addition, phosphorylation of some adhesin tails already has been reported to affect parasite invasion, suggesting one way to control TgGAC specificity of interaction (Leykauf et al., 2010; Tham et al., 2015; Treeck et al., 2009). Oligomerization of the adhesins as reported for TgMIC2, triggered by their interactions with their receptor, might be an alternative scenario (Ce re de et al., 2002; Harper et al., 2004). Importantly, PA produced in the inner PPM leaflet acts as lipid mediator of motility and invasion by controlling microneme secretion (Bullen et al., 2016). The affinity of the GAC PH

12 domain to PA offers an ideal mechanism to coordinate in space and time the recruitment of GAC at the PPM and to ensure a selective and tight binding to the adhesin tails. The functional relationship between the lysine methyltransferase activity of TgAKMT and the apical positioning of TgGAC sheds light on the enigmatic role of this enzyme in motility and invasion. In the presence of egress-stimulating signals, TgAKMT redistributes from the apical complex to the cytosol and, following reinvasion, is recruited back to the conoid (Heaslip et al., 2011). This suggests that TgAKMT cycles between the cytosol (motile) and the conoid (immotile) to either free or replenish the pool of TgGAC that is progressively consumed during motility (Figure 7). However, the significance of the relocalization of TgAKMT in motile parasites is not understood (Heaslip et al., 2011). Importantly, the lack of TgAKMT relocalization in the absence of TgGAC likely reflects a TgGACdependent perturbation of actin dynamics. Relocalization of TgAKMT might reflect a switch of post-translational modification (PTM), from methylation to possibly phosphorylation, that is reminiscent of the described methyl-phospho switch. These PTMs can be mutually exclusive, resulting in an interconversion between inactive and active states (Biggar and Li, 2015). The range of TgAKMT substrates appears to include numerous proteins present at the apical pole. The highly conserved nature of GAC in Apicomplexa evokes a common function. This is supported by the fact that Plasmodium spp. possess a conoid-like structure (Wall et al., 2016) and that PbGAC is found in all motile stages of P. berghei. In conclusion, GAC qualifies as the connector in place of aldolase, previously thought to play a central role in motility and invasion. GAC is a dynamic protein that translocates from the apical to the posterior pole in motile parasites, binds F-actin, and bridges the actomyosin system to transmembrane adhesins. Concordantly, GAC is crucial for gliding motility, a central process for host cell invasion and parasite dissemination. The GAC PH domain binds to PA, a lipid mediator that might plausibly coordinate adhesin release with activation of the actomyosin system. The dual binding of GAC to PA and MIC tails might enhance the specificity and affinity of the interaction. The far less well-understood regulation implicating lysine methylation plays an instrumental role in potentiating TgGAC function. Collectively, these findings consolidate the capping model and shed light on the regulation of gliding motility in Apicomplexa. EXPERIMENTAL PROCEDURES Animals All animal work at Nottingham has passed an ethical review process and was approved by the United Kingdom Home Office. Work was carried out in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986 and in compliance with European Directive 86/609/EEC for the protection of animals used for experimental purposes under UK Home Office Project Licenses /3344 and 30/3248. Gliding Assay in Matrigel Freshly egressed parasites ( ml) were mixed with the same volume of Matrigel (Geltrex A ), pipetted on coverslips placed in 24-well plates, and incubated for 30 min at 37 C and 5% CO 2 prior to stimulation with ml DMEM containing 6 mm calcium ionophore (A23187). After 5 min, medium was removed and samples were fixed for 10 min with paraformaldehyde (PFA)/glutaraldehyde (GA). Samples were processed as described for immunofluorescence assays (IFAs). Trail-Gliding Assay Freshly egressed /ROM4-WT and / ROM4-KO parasites were washed twice with DMEM, resuspended in DMEM with 3 mm calcium ionophore (A23187), and placed on coverslips in 24-well plates. The plate was centrifuged for 1 min at 1,200 rpm and incubated 15 min at 37 C and 5% CO 2. Samples were fixed for 10 min with PFA/GA. Fixed cells were blocked 30 min with 2% BSA/PBS, incubated with a-sag1 antibodies diluted in 2% BSA/PBS for 20 min, and washed three times with PBS. Cells were fixed with 1% formaldehyde/pbs for 3 min and washed once with PBS. Permeabilization using 0.2% Triton X-/PBS was performed for 20 min. IFAs were performed as described for IFAs. For the trails co-stained for TgGAC and TgMIC2, IFAs were performed following standard protocol. Rabbit Muscle Actin Co-sedimentation Assay Rabbit muscle actin co-sedimentation assays were performed using the Actin Binding Protein Spin-Down Assay Biochem Kit: rabbit skeletal muscle actin from Cytoskeleton, according to the manufacturer s protocol. All centrifugation steps were performed at 130,000 3 g. T. gondii total extracts were lysed in the actin buffer provided in the kit with three additional freeze and thaw cycles followed by two sonication cycles. Biochemical Assays To assess the binding of GAC to PfACT1, a co-sedimentation assay was performed. PfACT1 in G buffer (10 mm HEPES [ph 7.5], 0.2 mm CaCl 2, 0.5 mm ATP, and 0.5 mm TCEP) in the absence or presence of 50, 250, 500, and 800 nm FL- or N-TgGAC or N-PfGAC was polymerized overnight at room temperature (RT) by adding F buffer to final concentrations of 50 mm KCl, 4 mm MgCl 2, and 1 mm EGTA, in addition to the G buffer components, in a volume of ml. Filaments were sedimented at 435,000 3 g for 1 hr at RT. Pellets and supernatants were separated and the pellets resuspended in G buffer. Both pellet and supernatant fractions were analyzed by SDS-PAGE and Coomassie brilliant blue staining. Fluorescence spectroscopy was used to assess the effects of FL-TgGAC and N-PfGAC on parasite actin polymerization and depolymerization kinetics. Polymerization of 4 mm PfACT1, of which 5% was labeled with pyrene, was initiated by adding F buffer as above to a total reaction volume of 150 ml. Reactions were set up in triplicate in the absence or presence of 50, 250, 500, and 800 nm FL-TgGAC or N-PfGAC. The increase in pyrene fluorescence upon polymerization was measured for 2 hr at 25 C using a Tecan M0 Pro fluorescence plate reader with excitation and emission wavelengths of 365 and 7 nm, respectively. Depolymerization of 10 mm overnight-polymerized pyrene-labeled PfACT1 was initiated by diluting with F buffer to a final concentration of 0.1 mm in the absence or presence of 50, 250, 500, and 800 nm FL-TgGAC or N-PfGAC. The change in pyrene fluorescence upon depolymerization was measured at 25 C, as above. For both polymerization and depolymerization assays, several independent experiments with triplicate samples were performed using different batches of actin and GACs. Protein-Lipid Overlay Assay PIP-strip (Echelon Biosciences) assays were carried out according to the manufacturer s instructions and as previously described (Bullen et al., 2016). Three experiments were performed from three different batches of recombinant proteins. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, six figures, three tables, and four movies and can be found with this article online at AUTHOR CONTRIBUTIONS D.J., D.S.-F., N.T., I.P., I.K., and R.T. designed the experiments. J.S. performed the experiments with P. berghei, I.P. performed the biochemical assays involving PfACT1, Y.-F.H. and H.H. prepared samples for the biochemical and structural analyses and performed the experiments on recombinant MIC2, and Y.-F.H. and I.K. analyzed the SAXS data. D.J., N.T., and A.G. performed all the other experiments. D.J., D.S.-F., and I.K. wrote the manuscript. Cell Host & Microbe 20, , December 14,