Apicomplexan F‐actin is required for efficient nuclear entry during host cell invasion
2019; Springer Nature; Volume: 20; Issue: 12 Linguagem: Inglês
10.15252/embr.201948896
ISSN1469-3178
AutoresMario Del Rosario, Javier Periz, Georgios Pavlou, Oliver Lyth, María Fernanda Latorre Barragán, Sujaan Das, Gurman S. Pall, Johannes Felix Stortz, Leandro Lemgruber, Jamie Whitelaw, Jake Baum, Isabelle Tardieux, Markus Meissner,
Tópico(s)Bartonella species infections research
ResumoArticle4 October 2019Open Access Source DataTransparent process Apicomplexan F-actin is required for efficient nuclear entry during host cell invasion Mario Del Rosario Mario Del Rosario orcid.org/0000-0002-0430-1463 Wellcome Centre For Integrative Parasitology, Institute of Infection, Immunity & Inflammation, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow, UK Search for more papers by this author Javier Periz Javier Periz Wellcome Centre For Integrative Parasitology, Institute of Infection, Immunity & Inflammation, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow, UK Search for more papers by this author Georgios Pavlou Georgios Pavlou Institute for Advanced Biosciences, CNRS, UMR5309, INSERM U1209, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Oliver Lyth Oliver Lyth Department of Life Sciences, Imperial College London, London, UK Search for more papers by this author Fernanda Latorre-Barragan Fernanda Latorre-Barragan orcid.org/0000-0002-9280-705X Wellcome Centre For Integrative Parasitology, Institute of Infection, Immunity & Inflammation, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow, UK Faculty of Science, Food Engineering and Biotechnology, Technical University of Ambato, Ambato, Ecuador Search for more papers by this author Sujaan Das Sujaan Das Wellcome Centre For Integrative Parasitology, Institute of Infection, Immunity & Inflammation, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow, UK Search for more papers by this author Gurman S Pall Gurman S Pall Wellcome Centre For Integrative Parasitology, Institute of Infection, Immunity & Inflammation, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow, UK Search for more papers by this author Johannes Felix Stortz Johannes Felix Stortz orcid.org/0000-0002-5928-1850 Wellcome Centre For Integrative Parasitology, Institute of Infection, Immunity & Inflammation, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow, UK Search for more papers by this author Leandro Lemgruber Leandro Lemgruber Wellcome Centre For Integrative Parasitology, Institute of Infection, Immunity & Inflammation, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow, UK Search for more papers by this author Jamie A Whitelaw Jamie A Whitelaw CRUK Beatson Institute, Glasgow, UK Search for more papers by this author Jake Baum Jake Baum orcid.org/0000-0002-0275-352X Department of Life Sciences, Imperial College London, London, UK Search for more papers by this author Isabelle Tardieux Isabelle Tardieux orcid.org/0000-0002-5677-7463 Institute for Advanced Biosciences, CNRS, UMR5309, INSERM U1209, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Markus Meissner Corresponding Author Markus Meissner [email protected] orcid.org/0000-0002-4816-5221 Wellcome Centre For Integrative Parasitology, Institute of Infection, Immunity & Inflammation, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow, UK Experimental Parasitology, Department for Veterinary Sciences, Ludwig-Maximilians-University Munich, Munich, Germany Search for more papers by this author Mario Del Rosario Mario Del Rosario orcid.org/0000-0002-0430-1463 Wellcome Centre For Integrative Parasitology, Institute of Infection, Immunity & Inflammation, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow, UK Search for more papers by this author Javier Periz Javier Periz Wellcome Centre For Integrative Parasitology, Institute of Infection, Immunity & Inflammation, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow, UK Search for more papers by this author Georgios Pavlou Georgios Pavlou Institute for Advanced Biosciences, CNRS, UMR5309, INSERM U1209, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Oliver Lyth Oliver Lyth Department of Life Sciences, Imperial College London, London, UK Search for more papers by this author Fernanda Latorre-Barragan Fernanda Latorre-Barragan orcid.org/0000-0002-9280-705X Wellcome Centre For Integrative Parasitology, Institute of Infection, Immunity & Inflammation, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow, UK Faculty of Science, Food Engineering and Biotechnology, Technical University of Ambato, Ambato, Ecuador Search for more papers by this author Sujaan Das Sujaan Das Wellcome Centre For Integrative Parasitology, Institute of Infection, Immunity & Inflammation, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow, UK Search for more papers by this author Gurman S Pall Gurman S Pall Wellcome Centre For Integrative Parasitology, Institute of Infection, Immunity & Inflammation, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow, UK Search for more papers by this author Johannes Felix Stortz Johannes Felix Stortz orcid.org/0000-0002-5928-1850 Wellcome Centre For Integrative Parasitology, Institute of Infection, Immunity & Inflammation, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow, UK Search for more papers by this author Leandro Lemgruber Leandro Lemgruber Wellcome Centre For Integrative Parasitology, Institute of Infection, Immunity & Inflammation, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow, UK Search for more papers by this author Jamie A Whitelaw Jamie A Whitelaw CRUK Beatson Institute, Glasgow, UK Search for more papers by this author Jake Baum Jake Baum orcid.org/0000-0002-0275-352X Department of Life Sciences, Imperial College London, London, UK Search for more papers by this author Isabelle Tardieux Isabelle Tardieux orcid.org/0000-0002-5677-7463 Institute for Advanced Biosciences, CNRS, UMR5309, INSERM U1209, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Markus Meissner Corresponding Author Markus Meissner [email protected] orcid.org/0000-0002-4816-5221 Wellcome Centre For Integrative Parasitology, Institute of Infection, Immunity & Inflammation, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow, UK Experimental Parasitology, Department for Veterinary Sciences, Ludwig-Maximilians-University Munich, Munich, Germany Search for more papers by this author Author Information Mario Del Rosario1, Javier Periz1, Georgios Pavlou2, Oliver Lyth3, Fernanda Latorre-Barragan1,4, Sujaan Das1, Gurman S Pall1, Johannes Felix Stortz1, Leandro Lemgruber1, Jamie A Whitelaw5, Jake Baum3, Isabelle Tardieux2 and Markus Meissner *,1,6 1Wellcome Centre For Integrative Parasitology, Institute of Infection, Immunity & Inflammation, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow, UK 2Institute for Advanced Biosciences, CNRS, UMR5309, INSERM U1209, Université Grenoble Alpes, Grenoble, France 3Department of Life Sciences, Imperial College London, London, UK 4Faculty of Science, Food Engineering and Biotechnology, Technical University of Ambato, Ambato, Ecuador 5CRUK Beatson Institute, Glasgow, UK 6Experimental Parasitology, Department for Veterinary Sciences, Ludwig-Maximilians-University Munich, Munich, Germany *Corresponding author. Tel: +49 89 21803622; E-mail: [email protected] EMBO Reports (2019)20:e48896https://doi.org/10.15252/embr.201948896 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The obligate intracellular parasites Toxoplasma gondii and Plasmodium spp. invade host cells by injecting a protein complex into the membrane of the targeted cell that bridges the two cells through the assembly of a ring-like junction. This circular junction stretches while the parasites apply a traction force to pass through, a step that typically concurs with transient constriction of the parasite body. Here we analyse F-actin dynamics during host cell invasion. Super-resolution microscopy and real-time imaging highlighted an F-actin pool at the apex of pre-invading parasite, an F-actin ring at the junction area during invasion but also networks of perinuclear and posteriorly localised F-actin. Mutant parasites with dysfunctional acto-myosin showed significant decrease of junctional and perinuclear F-actin and are coincidently affected in nuclear passage through the junction. We propose that the F-actin machinery eases nuclear passage by stabilising the junction and pushing the nucleus through the constriction. Our analysis suggests that the junction opposes resistance to the passage of the parasite's nucleus and provides the first evidence for a dual contribution of actin-forces during host cell invasion by apicomplexan parasites. Synopsis Nuclei of apicomplexan parasites represent a major obstacle that needs to be squeezed through the junctional ring bridging the host cell and the parasite during invasion. This study proposes a push-and-pull mechanism whereby F-actin machinery eases nuclear passage. Super-resolution microscopy imaging reveals that F-actin forms a highly dynamic, cytosolic network in extracellular parasites. F-actin independently accumulates at the posterior pole and the junctional ring during invasion. F-actin forms a continuous network between the junction and the posterior ring that surrounds the nucleus. Mutant parasites with dysfunctional acto-myosin exhibits decreased junctional and perinuclear F-actin and reduced nuclear passage through the junction. Introduction The phylum Apicomplexa consists of more than 5,000 species, most of them obligate intracellular parasites, including important human and veterinary pathogens, such as Plasmodium (malaria) or Toxoplasma (toxoplasmosis). During their complex life cycles, apicomplexan parasites move through different environments to disseminate within and between hosts and to invade their host cell 1. Therefore, the invasive stages, called zoites, evolved a unique invasion device, consisting of unique secretory organelles and the parasites’ acto-myosin system, the Glideosome, localised in the narrow space (~30 nm) between the plasma membrane and the inner membrane complex (IMC) 2. Zoites actively enter the host cell by establishing a tight junctional ring (TJ) at the point of contact between the two cells. The TJ is assembled by the sequential secretion of unique secretory organelles (micronemes and rhoptries), leading to the insertion of rhoptry neck proteins (RONs) into the host cell plasma membrane (PM) and underneath 3. On the extracellular side, the exposed domain of the RON2 member binds the micronemal transmembrane protein AMA1 exposed on the parasite surface, resulting in the formation of a stable, junctional complex 3. The TJ is further anchored to the host cell cortex by de novo formation of F-actin through the recruitment of actin-nucleating proteins 45. During host cell invasion, the parasites use their acto-myosin motor to pass through the TJ. However, the exact role and orientation of the parasite's acto-myosin system is still under debate 6 and intriguingly, mutants for key component of this system show residual motile and invasive capacities 789, the latter reflecting in large part an alternative and host cell actin-dependant mode of entry 10. According to the Glideosome model, the force generated for motility and invasion relies exclusively on F-actin polymerised at the apical tip of the parasite by the action of Formin-1 and translocated within the narrow space (~30 nm) between the IMC and PM of the parasite 11. However, recent studies suggest that the parasite can also use other motility systems, such as a secretory-endocytic cycle that produces retrograde membrane flow 12, similar to the fountain flow model suggested for other eukaryotes 1314. In support of the linear motor model, was the detection of parasite F-actin underneath the junction formed by invading parasites when using an antibody preferentially recognising apicomplexan F-actin. Furthermore, the detection of cytosolic locations, predominantly around the nucleus 15, suggests additional roles of this cytoskeletal protein during invasion. While it was assumed a major role of F-actin in driving Apicomplexa zoite gliding motility and cell invasion, recent studies demonstrated the pivotal role of F-actin in multiple other processes, such as apicoplast inheritance 16, dense granule motility 17 and likely nuclear functions through the control of expression of virulence genes in malaria parasites 18. However, building a comprehensive model for F-actin dynamics, localisation and function in apicomplexan parasites has been hampered for decades by the lack of tools enabling reliable F-actin detection. Interestingly, several studies suggested that F-actin is interacting with subpellicular microtubules 19 and/or the subpellicular matrix of the IMC 2021. Furthermore, components of the Glideosome, such as GAPM proteins 22 or the invasion-critical myosin, MyoH were demonstrated to interact with microtubules 23, suggesting a coordinated action of the actin and microtubule cytoskeleton during host cell invasion. With the adaptation of nanobodies specifically recognising F-actin, it is now possible to analyse F-actin dynamics in apicomplexan parasites 2425 leading to the identification of distinct cytosolic networks of dynamic actin in both T. gondii and P. falciparum 112425. We originally identified two F-actin polymerisation centres from where most of the F-actin flow occurs in intracellular parasites 24, later expanded to three based on further studies 1125. One polymerisation centre can be found at the apical tip, corresponding well to the described location of Formin-1, and one close to the parasite's Golgi complex, corresponding to the location of Formin-2 1125. In T. gondii, these three polymerisation centres of F-actin correlate well with the location of Formins-1, 2 and 3 in T. gondii 11. Formins-2 and 3 appear to have overlapping functions during the intracellular development of the parasite for material exchange and formation of an intravacuolar network in intracellular parasites, while Formin-1 (FH-1) has been explicitly implicated in the apical polymerisation of F-actin required for gliding motility and cell invasion 11. In this study, although not directly shown, the authors suggested that FH-1 polymerises F-actin at the apical tip of the parasite that is exclusively transported by the action of the Glideosome within the subalveolar space to the posterior pole. Interestingly, at least in intracellular parasites, the majority of F-actin dynamics appears to occur within the cytosol of the parasite close to the parasite's Golgi, forming a continuous dynamic flow from centre to the periphery of the parasite and from the apical to the basal pole 2425. In addition, measurement of peripheral F-actin flow demonstrated that it can occur bidirectional from the apical to the posterior pole and vice versa 25. In good agreement with a cytosolic oriented F-actin system, previous reports demonstrated parasite F-actin to be associated with the subpellicular microtubules that are connected to and stabilise the IMC 19202126. Furthermore, gliding-associated proteins identified via co-immunoprecipitation with the Glideosome were recently found to play important roles in stabilising the IMC and directly connecting it to the subpellicular microtubules 2227, suggesting that a close connection exists between parasite F-actin, the Glideosome and the subpellicular network. Here we compared F-actin dynamics in WT and mutant parasites for the acto-myosin system and correlated its dynamic location with the phenotypic consequences during host cell invasion. Interestingly, in unstimulated parasites, F-actin flow in extracellular parasites appears to be bidirectional, with the majority of events occurring in a retrograde direction, which is similar to measurements performed on intracellular parasites 25. The activation of calcium signalling results in a shift towards retrograde F-actin flow, resulting in F-actin accumulation at the posterior pole. Comparison of F-actin dynamics in parasite mutants for the acto-myosin system helped to identify the nucleus as a major obstacle for efficient host cell invasion that needs to be squeezed through the junction using F-actin dynamics to translocate and potentially deform the nucleus through this process. Our analysis strongly suggests a push-and-pull mechanism for nuclear entry during host cell invasion, in analogy to the dynamics observed during migration of other eukaryotes through a constricted environment 28. Results Rational for selecting parasite mutants in order to analyse F-actin dynamics during host cell invasion Over recent years, conditional mutagenesis systems have been employed to generate and characterise a whole assortment of parasite mutants affected in host cell invasion. These mutants can be affected in different, unrelated pathways, such as microneme secretion, formation of the tight junction or host cell entry. Since we were interested in analysing F-actin dynamics during the invasion process, we chose parasite mutants that were previously characterised to be significantly affected, but not blocked in host cell entry and linked to the parasites acto-myosin system. We excluded all mutants that have been described to be completely blocked in the initiation of invasion, such as MyoH 23 or the micronemal protein MIC8 29, since no invasion event can be expected that can be analysed. Similarly, we also excluded mutants involved in actin dynamics that have not been implicated in host cell invasion, such as FH-2,3 11 or myosins not involved in host cell invasion, such as MyoI 30. Therefore, we chose parasite actin 79, the actin depolymerisation factor ADF 31 and myosin A (MyoA), which has been used previously to successfully identify the contribution of the host cell for parasite invasion 10. F-actin flow is mainly organised within the cytosol of extracellular parasites and is modulated by Calcium and cGMP signalling We previously imaged F-actin flow in intracellular parasites and were able to discriminate two major polymerisation centres, one at the apical tip and one close to the apicoplast, where F-actin is formed and subsequently transported to the posterior end of the parasite 24. Indeed, recent studies on Toxoplasma and Plasmodium demonstrated that Formin-2 is localised to the apicoplast of the parasite and required for most of the intracellular F-actin dynamic 1125, while Formin-1 is localised at the apical tip, where it is thought to be exclusively required for parasite motility and invasion. In good agreement, in intracellular parasites, F-actin appears to be formed at two polymerisation centres, localised at the apical tip and close to the Golgi region of the parasite, indicating that FH-1 and FH-2 are acting as nucleators during intracellular parasite development. Intriguingly, the majority of F-actin dynamics in intracellular parasites occurs within the cytosol of the parasite with F-actin flow occurring in a bidirectional manner (retrograde and anterograde) 2425. We were interested to determine if during the transient extracellular life and at the time of cell invasion, the patterns of actin dynamics differ from the intracellular ones and how they were impacted upon disruption of the Glideosome or factors critically involved in F-actin regulation. Since transient transfections are typically associated with overexpression of the target protein hence with significant uncontrolled impact on a dynamic equilibrium 24, we generated T. gondii lines stably expressing Cb-EmeraldFP. We ensured that the expression of the chromobody was comparable between the different parasite lines and ensured that its expression does not lead to phenotypic alterations and misleading analysis. We established transgenic parasites expressing Cb-EmeraldFP in WT parasites, a null mutant for myosin A (MyoA), the core motor of the Glideosome 16 and a conditional mutant for the critical actin regulator, actin depolymerisation factor ADF, adfcKD 31. Using live 3D-structured illumination microscopy (3D-SIM), we compared F-actin dynamics and found two discernible F-actin polymerisation centres (Fig 1A) as previously seen in intracellular parasites 24. Time-lapse analysis demonstrated that the majority of F-actin dynamics occurs in the cytosol of the parasite with some F-actin flow detectable at the periphery occurring in retrograde and anterograde orientation, similar to intracellular parasites (see also 25). In good agreement with the localisation of FH-1 and FH-2, two polymerisation centres can be detected. F-actin flow starts from the apical tip (1st polymerisation centre, yellow arrow in Fig 1A) to the second polymerisation centre close to the Golgi (red arrow, Fig 1A) to the posterior pole of the parasite (Fig 1A, Movie EV1). While disruption of myoA did not result in significant changes of F-actin localisation or dynamics in resting parasites (Fig 1A, Movie EV1), depletion of ADF completely abrogated actin dynamics, F-actin accumulation being observed at both apical and posterior parasite poles (Fig 1A, Movie EV1). Figure 1. F-actin, formed at two major nucleation centres, forms a dynamic, continuous network within the cytosol of the parasite Stills depicting actin flow (see Movie EV1) in extracellular RH, myoA KO and adf KD parasites expressing Cb-EmeraldFP. Continuous F-actin flow can be seen from the apical tip, to the Golgi region towards the posterior pole of the parasite. No difference can be observed between RH and myoA KO. For adf KD, the flow is completely abrogated. Red arrowhead marks the Golgi region, while the yellow arrowhead marks the apical nucleation centre. The time lapse is presented in Movie EV1. Skeletonisation processing of (A) depicting areas where F-actin dynamics/flow is prevalent. Individual signals for F-actin form a continuous, dynamic network that connects apical and posterior pole of the parasite. Red arrowhead marks the Golgi region nucleation centre, while the yellow arrowhead marks the apical nucleation centre. The images represent a projection of frames for each timepoint to provide a better overview of the dynamics of F-actin. The time lapse is presented in Movie EV1. Kymograph analysis of (A) and (B). The kymograph was generated by tracing a line in the middle of the parasite's body (red line). The colour-coded kymograph represents forward movement (red), backward movement (green) and static F-actin (blue). The results demonstrate continuous exchange of F-actin from the apical to the posterior pole of the parasite within the cytosol of the parasite. This cytosolic exchange is similar in RH and myoA KO parasites, while completely abolished in the case of adf KD. Top three rows: Parasites expressing Cb-EmeraldFP along with myoA-SNAP before and after Ca2+ ionophore (A23187) or BIPPO treatment. The parasites were also treated with 2 μM of cytochalasin D or DMSO (as control) for 30 min. After the addition of A23187, preferential relocalisation of actin can be observed at the apical tip, while the addition of BIPPO caused F-actin accumulation at the basal end. 4th row: myoAKO parasites expressing Cb-EmeraldFP before and after treatment with A23187 and BIPPO. While no apical or basal accumulation of F-actin is observed, some peripheral location of F-actin occurs in the presence of A23187 or BIPPO, preferentially in the apical half of the parasite. Bottom row: adf cKD parasites expressing Cb-EmeraldFP before and after treatment with Ca2+ ionophore or BIPPO. No apparent change in F-actin localisation can be seen upon treatment. The time lapse is presented in Movie EV2. Skeletonisation of movies shown in (D). Before the addition of A23187 or BIPPO, RH and myoA KO parasites behave similar with no accumulation of F-actin at the apical tip. After treatment with calcium ionophore A23187, preferential accumulation of F-actin at the apical tip can be observed for RH (red arrowhead), while BIPPO presented preferential accumulation in the basal end (red arrowhead). In myoA KO, some relocalisation of actin is observed at the periphery of the parasite (yellow arrowhead). In the case of adf cKD, no relocalisation occurs. The images represent a projection of frames for each timepoint to provide a better overview of the dynamics of F-actin. Quantification of actin accumulation before and after treatment with Ca2+ ionophore or BIPPO. The parasites were counted for F-actin accumulation after adding Ca2+ ionophore or BIPPO. Numbers were generated by counting total number of parasites and then number of parasites with actin accumulation on the apical or basal tip. A minimum of 200 parasites were counted upon 3 biological replicates. Two-way ANOVA was used for statistical analysis and Tukey's multiple comparison test. ****P < 0.0001. Error bars represent standard deviation. Data information: Numbers indicate minutes:seconds, and scale bars represent 5 μm. Source data are available online for this figure. Source Data for Figure 1 [embr201948896-sup-0011-SDataFig1.zip] Download figure Download PowerPoint To map the actin structures visualised with the chromobody, we employed skeletonisation processing 32, which converts F-actin signals into individual pixels, that can be traced to obtain information about the localisation and dynamics of F-actin. The resulting skeletonised movie can be projected to show where the majority of (detectable) F-actin dynamics occur during the timeline of the movie (Fig 1B, Movie EV1). This analysis confirmed that parasite F-actin formed at the apical tip (yellow arrowhead) and Golgi region (red arrowhead), converge within the cytosol of the parasite and reach the basal pole of the parasite. Interestingly, both peripherical and cytoplasmic flow appear to be connected and do not occur independently from each other (Fig 1B). Note that both WT and myoAKO parasites displayed similar signals, whereas the ADF—conditional mutant did not. Next, kymograph analysis was performed to analyse individual F-actin flow events using KymographClear and KymographDirect 33 confirming the presence of a continuous dynamic F-actin network in cytosolic location that connects the apical pole, Golgi area and basal pole of the parasite, in good agreement with the location of the 3 Formins previously described in T. gondii 11. KymographClear is a FIJI plugin that generates a 3 colour-coded kymograph, where each colour labels either forward movement (red), backward movement (green) or static (blue) as shown (Fig 1C and Appendix Fig S1). This kymograph can then be read into the KymographDirect software, which uses automatic detection to trace trajectories of moving particles in the image. Values are then assigned to these trajectories based on both intensity and speed 33. The intensity can be assessed in specified tracks over time as the average intensity over time shown in Fig 1C. Both the RH and myoA KO presented dynamic signals connecting the apical pole (close to the point 0), Golgi region (between 2 and 3 μm) and basal pole. In the case of the adf KD, only static signals could be detected at the apical and basal pole of the parasite, confirming the lack of F-actin dynamics in these mutants. Next, we used this analysis to investigate intensity profiles of actin signal travelling in the periphery of the parasite (Appendix Fig S1B–D). Interestingly, actin dynamics along the periphery is similar between WT and myoA KO with bidirectional actin flow events as revealed by the kymographs, while confirming previous results with the adf KD mutants, which shows a lack of F-actin dynamics across the entire parasites body (first half (−), Appendix Fig S1A–C). Together, the data presented in this work point that similarly to intracellular parasites, the F-actin is highly dynamic in extracellular parasites displaying two major polymerisation centres, one at the apical tip and another at the Golgi region of the parasite. Of note, this analysis underlines that there is actin flow coming from the cytoplasm towards the periphery and shaping a highly dynamic network that connects the apical and basal pole of the parasite, therefore independently of any F-actin network positioned between the zoite's PM and IMC. Tosetti et al 11 suggested that the activation of a Ca2+-signalling cascade would lead to increased retrograde transport of F-actin, eventually accumulating posteriorly. However, we found that the treatment of parasites with the calcium ionophore A23187 induced anterior accumulation of F-actin in wild-type parasites in the majority of cases (Fig 1D–F; Appendix Fig S1A; Movie EV2), while BIPPO triggered a predominantly basal F-actin accumulation (Fig 1D–F; Appendix Fig S1A; Movie EV2). BIPPO is a drug capable of inhibiting 3′,5′-cyclic nucleotide phosphodiesterases (PDEs) responsible in blocking the breakdown of cyclic nucleotides such as cAMP and cGMP. This mechanism is believed to be responsible in the activation of microneme secretion and egress by modulating the signal pathway of PKG-dependent processes 34. Once MyoA null mutants were submitted to treatments with A23187 or BIPPO, the F-actin remained rather evenly distributed within the cytoplasm albeit with some peripheral enrichment in the first half of the parasite closer to the apical end (Fig 1D–F; Movie EV2). In contrast, depletion of ADF led to the expected phenotype with F-actin accumulating at the apical and basal pole of the parasite, independently of A23187 or BIPPO addition (Fig 1D–F; Movie EV2). Together, these data suggest that calci
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