Genetic impairment of parasite myosin motors uncovers the contribution of host cell membrane dynamics to Toxoplasma invasion forces
© Bichet et al. 2016
Received: 2 August 2016
Accepted: 8 October 2016
Published: 9 November 2016
The several-micrometer-sized Toxoplasma gondii protozoan parasite invades virtually any type of nucleated cell from a warm-blooded animal within seconds. Toxoplasma initiates the formation of a tight ring-like junction bridging its apical pole with the host cell membrane. The parasite then actively moves through the junction into a host cell plasma membrane invagination that delineates a nascent vacuole. Recent high resolution imaging and kinematics analysis showed that the host cell cortical actin dynamics occurs at the site of entry while gene silencing approaches allowed motor-deficient parasites to be generated, and suggested that the host cell could contribute energetically to invasion. In this study we further investigate this possibility by analyzing the behavior of parasites genetically impaired in different motor components, and discuss how the uncovered mechanisms illuminate our current understanding of the invasion process by motor-competent parasites.
By simultaneously tracking host cell membrane and cortex dynamics at the site of interaction with myosin A-deficient Toxoplasma, the junction assembly step could be decoupled from the engagement of the Toxoplasma invasive force. Kinematics combined with functional analysis revealed that myosin A-deficient Toxoplasma had a distinct host cell-dependent mode of entry when compared to wild-type or myosin B/C-deficient Toxoplasma. Following the junction assembly step, the host cell formed actin-driven membrane protrusions that surrounded the myosin A-deficient mutant and drove it through the junction into a typical vacuole. However, this parasite-entry mode appeared suboptimal, with about 40 % abortive events for which the host cell membrane expansions failed to cover the parasite body and instead could apply deleterious compressive forces on the apical pole of the zoite.
This study not only clarifies the key contribution of T. gondii tachyzoite myosin A to the invasive force, but it also highlights a new mode of entry for intracellular microbes that shares early features of macropinocytosis. Given the harmful potential of the host cell compressive forces, we propose to consider host cell invasion by zoites as a balanced combination between host cell membrane dynamics and the Toxoplasma motor function. In this light, evolutionary shaping of myosin A with fast motor activity could have contributed to optimize the invasive potential of Toxoplasma tachyzoites and thereby their fitness.
KeywordsMembrane dynamics Cortical actin dynamics Myosins Forces Cell invasion Macropinocytosis Protozoan parasite Toxoplasma
Non-professional phagocytes such as epithelial, endothelial, and fibroblast cells provide intracellular niches to a plethora of single-celled invasive microbes. The proliferation of these intracellular microbes including non-parasitic and parasitic prokaryotes or eukaryotes can cause diseases with devastating sociological or economic impact on livestock and humans. A common mechanism among the sequences that set intracellular niches for microbes is the remodeling of both the host cell lipid bilayer and the cortical cytoskeleton. This rearrangement provides the membrane and drives the force required to engulf the microbe within subcellular compartments. Most bacteria enter non-phagocytic cells within a few minutes by either “discreet zippering” phagocytosis or “conspicuous triggering” macropinocytosis, although this oversimplified categorization has been questioned . The general concept distinguishes (1) the restrained host cell membrane protrusions firmly adhering to and extending like a zipper around the bacteria surface to form a tight-fitting phagosome from (2) the diffuse host cell membrane ruffles emerging at the vicinity of the bacteria once triggered by the first wave of bacterial effectors, and further shaping spacious macropinosomes/phagosomes [2, 3]. Once sealed, these subcellular compartments acquire and lose endocytic markers, thereby creating a microenvironment that bacteria have to control or escape . In contrast, a group of flagellated protozoan parasites clustered in the Apicomplexa phylum has evolved a distinct strategy to colonize their host cells that relies upon both their own driving force and their ability to avoid the host cell endocytic pathway once internalized within vacuoles. Early studies on major members of this phylum, specifically Toxoplasma gondii and Plasmodium spp., have highlighted the lack of host cell contribution when the parasite invasive stages, also called zoites, actively invade their respective host cells in a process completed within a few seconds [5–8]. Invasion starts with the insertion in the host cell plasma membrane (PM), by the zoite, of a multi-subunit complex (identified as the apical major antigen 1 (AMA1)-rhoptry neck (RON) complex and possibly enlarged with the recently discovered claudin-like apicomplexa microneme protein (CLAMP) . This macromolecular complex connects the two cells by forming a circular tight junction (TJ) [10–13] that will act as a door of entry. The zoite then tracts itself into a PM invagination that arises below the TJ  and then evolves as a non-fusogenic parasitophorous vacuole (PV) that will support zoite growth and multiplication . Our recent kinematic analysis has allowed tracking of the RON complex during its secretion and assembly into the PM and its establishment of a traction bridge with the host PM and its associated cortical actin lattice [16, 17]. In this scheme, the invasive force is thought to be provided by the single-headed unconventional myosin A (MyoA) of the apicomplexan-specific myosin class XIV [8, 18, 19]. Accordingly, the general expectation was that MyoA-deficient T. gondii parasites would lose their ability to enter the host cells and would not be viable. Yet, using a conditional recombination system, it was possible to maintain MyoA-lacking (ΔMyoA) parasites in vitro [13, 20]. It was then proposed that another class XIV myosin, namely myosin C (MyoC), a spliced variant of the single gene encoding MyoB and MyoC isoforms, would compensate for MyoA loss of function . On the other hand, mutants for additional components of the motor complex including actin —which cannot be compensated by paralogs being encoded by a single copy gene — were also engineered and have led to the proposal of alternative mechanisms for zoite motility and cell invasion based on zoite hydrodynamic forces  but which need to be validated. However, it has not been investigated so far whether a contribution from the host cell could potentially account for the residual invasiveness of MyoA-deficient Toxoplasma. Yet, phagocytosis-mediated uptake of live zoites into phagocytes followed within a few minutes by egress from the early phagosome into a second vacuole, namely the PV, has already been reported for motor-competent virulent  and avirulent  tachyzoites. We therefore decided to re-evaluate the role of T. gondii zoite motors during invasion by applying high resolution live and fixed imaging in conjunction with functional assays to compare how motor-competent and MyoA- or MyoB/C-deficient tachyzoites access a growth-compatible PV in non-phagocytic cells. Together, our data undoubtedly position the MyoA motor at the center of the Toxoplasma tachyzoite invasive force. Further, this study reveals that MyoA-deficient zoites enter host cells by a yet undescribed process that shares only initial features with macropinocytosis. This in-depth analysis of how motor-impaired Toxoplasma interact with mammalian cells to either succeed or fail at entering them calls for a new Toxoplasma-host cell invasion paradigm based on a balanced contribution between zoite motor engagement and host cell membrane/cortex dynamics.
MyoA-deficient tachyzoites enter their non-phagocytic host cells with a significantly slower kinetics than MyoA+ tachyzoites
Real-time tracking of the host cell PM and cortical actin demonstrates that MyoA- but not MyoB/C-deficient tachyzoites are pushed into host cells through actin-powered PM protrusions that encircle the zoite body
Movie 1 ΔMyoA tachyzoite entering HeLa cell expressing the CAAX-mC PM reporter by progressive wrapping through host cell PM ruffles. Scale bar: 5 μm.
Movie 2 ΔMyoA tachyzoite entering U2OS cell expressing the PDFGTM-GFP PM reporter by progressive wrapping through PM ruffles. Note the retraction of the ruffles immediately behind the internalized tachyzoite. Scale bar: 5 μm.
Movie 3 ΔMyoA tachyzoite entering HeLa cell expressing the CAAX-mC PM reporter by progressive wrapping through PM ruffles. Scale bar: 5 μm.
Movie 4 Two attached ΔMyoA tachyzoite sisters engulfed in the same event by U2OS cell expressing the PDFGTM-GFP PM reporter through sustained progression of PM ruffles. Scale bar: 5 μm.
Movie 5 ΔMyoB/C tachyzoites invading HeLa cell expressing the CAAX-mC PM reporter through a typical static or capped TJ. Scale bar: 5 μm.
Movie 6 ΔMyoA tachyzoite entering U2OS cell expressing the LifeAct-GFP F-actin reporter. Note that F-actin elongates around the parasite and disassembles once internalization is complete. Scale bar: 5 μm.
Movie 7 ΔMyoA tachyzoite entering HeLa cell expressing the LifeAct-GFP F-actin reporter. Note that F-actin accumulates at the TJ and elongates around the parasite while disassembling once internalization is complete. Scale bar: 5 μm.
Movie 8 ΔMyoA tachyzoite entering HeLa cell co-expressing the CAAX-mC and LifeAct-GFP constructs. Note that PM and F-actin dynamics are tightly coupled during zoite engulfment. Scale bar: 5 μm.
Movie 9 ΔMyoA tachyzoite entering U2OS cell co-expressing the PDGFRTM-mC and LifeAct-GFP constructs. Note that PM and F-actin dynamics are tightly coupled in the ruffles that allow the zoite to be pushed into a PM-derived PV which is not associated with F-actin. The tachyzoite resides and further develops in this PV. Scale bar: 5 μm.
Host cell PM ruffles force ΔMyoA tachyzoites to pass through the TJ into a primary PV which traffics in the cytoplasm and allows subsequent Toxoplasma development
Movie 10 ΔMyoA tachyzoite entering U2OS cell expressing the PDGFRTM-GFP. Note the typical PM wrapping and the fate of the primary vacuole. The parasite remains and develops within the primary PV. Scale bar: 5 μm.
Movie 11 3D reconstruction from z stacks (2 m) capturing ΔMyoA tachyzoite entering an HFF cell in presence of 10 kDa fluorescent dextran (Dx, red). Dx serves as endosome and macropinosome markers while the ROP2 staining (green) indicates that the parasite has secreted the rhopty protein to enter a typical vacuole.
The unique mode of ΔMyoA zoite entry relies on host cell actin polymerization but not on late effectors of macropinocytosis, consistent with the absence of macropinosome formation
Since host cell F-actin was dynamically visualized in the expanding PM protrusions enwrapping ΔMyoA zoites, we next interrogated how interfering with host cell actin dynamics affected the rate of ΔMyoA zoite entry. To this end, we exposed U2OS and spontaneously arising retinal pigment epithelial (ARPE-19) cells to a cocktail of cell-permeant actin poisons that quickly arrests actin dynamics and myosin II-based reorganization but preserves the existing steady-state actin organization, in contrast to most actin inhibitors described so far. The cocktail acts through the concerted activities of jasplakinolide, latrunculin B, and Y27632 (JLY): it is effective within seconds for periods longer than 10–15 min after drug removal . These unique features gave us the opportunity to selectively block host cell actin turnover in fetal calf serum (FCS)-starved U2OS and ARPE-19 cells independently of Toxoplasma.
Movie 12 ΔMyoA tachyzoite entering PtK1 cell. Note the extreme constriction at the TJ site. Scale bar: 5 μm.
Long-lasting compressive forces applied by the host cell ruffles on the zoite apex induce critical hydrostatic pressure leading to Toxoplasma irreversible membrane damage
Movie 13 ΔMyoA tachyzoite failing to enter HBMEC cell. Note that the zoite is almost split in two parts at the constriction site and is eventually released with marked structural alterations. Scale bar: 5 μm.
Movie 14 ΔMyoA tachyzoite failing to enter U2OS cell co-expressing the PDGFRTM-GFP and mC-actin constructs. Note the narrowness of the PM fold surrounding the zoite apex and supported by F-actin. As a result compressive forces are applied on the zoite and induce large membrane bleb before zoite lysis. Scale bar: 5 μm.
Movie 15 ΔMyoA tachyzoite failing to enter HeLa cell expressing the CAAX-mC PM reporter. Note the rearward thrust of the tachyzoite when apically engaged in a PM tunnel and the blebbing of the zoite membrane when trapped in the PM ruffles. Scale bar: 5 μm.
Movie 16 ΔMyoA tachyzoite failing to enter HeLa cells co-expressing the CAAX-mC and LifeAct-GFP constructs. Note that the PM tube extending beneath the zoite is enriched in F-actin. Scale bar: 5 μm.
Movie 17 ΔMyoA tachyzoite failing to enter HeLa cell expressing the CAAX-mC PM reporter. Note the impressive length and dynamics of the membrane tunnel that forms underneath the apex of the parasite trapped in the PM and whose membrane blebs during PM tunnel retraction. Scale bar: 5 μm.
Movie 18 ΔMyoA tachyzoite failing to enter HeLa cell expressing the MyrPalm-GFP PM reporter. Note the impressive length and dynamics of the membrane tunnel that forms underneath the apex of the parasite, which is trapped in the PM inner invagination and is released after tunnel retraction. Scale bar: 5 μm.
Movie 19 ΔMyoA tachyzoite failing to enter U2OS cell expressing the PDGFRTM-GFP PM reporter. Note the impressive length and dynamics of the PM tunnel that forms underneath the apex of the parasite, which is eventually released after tunnel retraction and is no longer attached to the cell. Scale bar: 5 μm.
Movie 20 ΔMyoA tachyzoite failing to enter U2OS cell expressing the LifeAct-GFP F-actin reporter. Note that the PM tunnel that folds underneath the parasite is enriched in F-actin. Scale bar: 5 μm.
Until recently, the force driving the entry of Toxoplasma tachyzoites into non-professional phagocytes was thought to exclusively rely on the functional interplay between parasite F-actin and myosin motors while the host cell remained essentially passive [5, 6]. This study alters this view by reporting that tachyzoites devoid of MyoA motor can still access a typical PV following their polarized contact with the target cell due to an active contribution of the latter. This work also highlights the need to assess in depth the contribution of host cell membrane dynamics to the high-speed process of wild-type Toxoplasma entry into cells.
Host cell actin-based forces promote the formation of PM protrusions that drive a fraction of the ΔMyoA tachyzoite population into a typical PV through a process with unique features
In the absence of the MyoA motor, we found that tachyzoites can rely on local host cell PM protrusions that fold over the distal part of the zoite body and apply forces eventually moving the tachyzoite through a TJ into the nascent PV. Such observations explain the in vitro viability of the ΔMyoA mutant. Because of the drastic reduction in the rate of internalized ΔMyoA parasites when host cells are chemically impaired in actin polymerizing/depolymerizing machineries, we conclude that host cell actin turnover is necessary to internalize these mutants. In line with this requirement, dynamic tracking of the PM/PV and of actin cytoskeleton remodeling revealed a selective accumulation of F-actin underneath the TJ and within the PM projections wrapping around the zoite. While Toxoplasma survival is expected to be prevented if the PM projections sealed into a macropinosome-like compartment that would rapidly integrate the endocytic pathway , ΔMyoA parasites were indeed not found within macropinosomes. Furthermore, the lack of effect of inhibitors directed against the PI3K or against myosin II and ROCK, all known to target late effectors required for macropinosome or phagosome closure [32, 33, 38–41], allowed us to propose a novel strategy used by Toxoplasma tachyzoites to enter cells that shares initial but not late features with macropinocytosis.
TJ assembly in the host cell PM/cortex associates with PM/cortex dynamic remodeling
The MyoA-deficient Toxoplasma is the first mutant providing the unique opportunity to uncouple TJ insertion into the host cell PM from zoite motor activity. In the absence of MyoA, we found that the early steps of invasion, before mobilization of motor activity, i.e., rhoptry secretion and/or TJ folding, were associated with confined host cell PM ruffle-like protrusions. The flattening out of PM reservoirs is now universally recognized as an actin assembly-driven mechanism regulated by membrane tension to adjust cell shape and properties [42, 43]. Insertion of hydrophobic proteins in the PM can act as wedges known to create local changes in curvature in tight interplay with actin rearrangements . Interestingly, further characterization of several host actin regulatory factors that have been identified as required during cell invasion using high throughput RNA interference screening should enable one to shed light on the host cell cortex remodeling during Toxoplasma entry . Therefore, we propose that the PM protrusions emerging around ΔMyoA mutants result from the local change of PM tension occurring upon insertion of the RON complex. In addition the local dismantling of the host cell cortical actin meshwork following secretion of the actin-severing rhoptry protein toxofilin at the onset of entry  is likely to contribute to a shift in membrane tension. Future engineering of a double MyoA-toxofilin knock out Toxoplasma would help to precisely decipher whether toxofilin activity can not only (1) fuel the TJ anchoring response as we proposed in, but also (2) be involved in more complex changes in PM dynamics including those promoting ruffle-like protrusions. If we hypothesize that TJ induces PM remodeling, we could expect the PM responses to be limited when the TJ remains transient as occurs with motor-functional tachyzoites, whereas these changes should be exacerbated in time and nature when tachyzoites have no MyoA motor to engage generation of traction forces at the TJ. Note that several SEM micrographs have documented protrusions around the apex of motor-competent zoites in endothelial and leukocyte cells [47, 48], neutrophils included  during what was thought to be active invasion. Importantly, while neutrophils are prototypic cells for PM unwrinkling and tube-like protrusions [50, 51], our SEM analysis revealed that fibroblasts also formed small PM ruffles around the apex of motor-competent parasites, thus reinforcing the concept of a general early and transient local response from the host cell PM and cortex when the TJ builds up regardless of subsequent motor engagement. SEM also strongly supported a key contribution of PM remodeling during internalization of MyoA-deficient zoites. It is worth mentioning the recent biophysical model of plasmodial merozoite invasion of the erythrocyte that assumes a modification of PM curvature triggered by the TJ insertion in the red blood cell and proposes this shift to account for the early enwrapping of the zoite . The triggers underlying changes in PM might be even more diverse, as a pioneer study using optical tweezers showed that randomly attached merozoites, i.e., those that have not released RONs, were already able to induce red blood cell deformation . In this framework, analyzing if and how MyoA-deficient Plasmodium merozoites triggered sufficient changes in the red blood cell PM and underlying cytoskeletal cortex to be successfully enwrapped will certainly be informative.
Multiple and intertwined PM responses following TJ assembly can lead to either successful or failed internalization events
The membrane dynamics that operates at the TJ site includes not only the PM outer protrusions but also a tight-fitting inner PM invagination that surrounded the apex of the mutant zoite ahead from the TJ. The dynamics of the two processes worked in tandem as they concomitantly formed, retracted, and withdrew. Quantitative analysis of the video sequences revealed that when PM ruffles could not stretch enough over the zoite body, they instead trapped the zoite into the PM infolding to eventually cause a huge membrane bleb in the zoite. These observations suggest that PM outer protrusions apply compressive forces on the apex of the zoite that can harm the zoite. In support of this view is the increased rate of internalization (i.e., decrease in the rate of failed events) when host cell contractile activities are inhibited. In addition, in some abortive events, a tube of PM several micrometers long that spun and dragged the parasite formed underneath the TJ being enriched in F-actin and associated actin proteins, in particular myosin II. In that setting the zoite stayed engaged in the PM inner tight invagination and showed a RON2-positive ring that suggested a TJ had formed. Collectively these data point to a complex interplay between effector and regulatory factors of the host cell actomyosin-driven contractile activities to promote folding of the PM tube.
This study positions T. gondii tachyzoite MyoA as a central provider of parasite invasive force and highlights a previously unseen mode of entry for intracellular microbes, relying on host cell membrane dynamics following zoite tight polarized contact. It provides a precise explanation for the marked reduction in invasiveness for ΔMyoA tachyzoites. Future structural and functional studies are necessary to understand how the RON complex insertion dictates the fast remodeling of the PM and how its tension/stretching properties are changed and appropriate curvatures promoted to allow (1) PV folding and (2) ruffles/tube assembly and dynamics. An interesting candidate is the CLAMP, appropriately localized to the cell zoite TJ throughout invasion, as published in the course of the reviewing process by the team of Lourido . Finally, given the harmfulness of the host cell PM remodeling on the MyoA-deficient zoite, we propose to consider host cell invasion as the competitive contribution of both a Toxoplasma motor function and a complex host cell membrane dynamics. The shaping of MyoA during evolution, with a low affinity for ADP  that correlates with fast motor activity, may have contributed to optimize the invasive potential of Toxoplasma tachyzoites and thereby their fitness.
Cells, parasites, and reagents
All media and products used for cell culture were from Gibco-Life Technologies (St Aubin, France) unless specified. Cells in culture were strictly screened for mycoplasma contamination monthly with the MycoAlert Kit (Lonza Rockland, Rockland, ME, USA) and cured with Plasmocin™ (InvivoGen, San Diego, CA, USA) if necessary. Human foreskin fibroblasts (HFFs), human epithelial cervical cancer cells (HeLa), human ARPE-19 retinal epithelial cells, and human U2OS osteosarcoma epithelial cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with GlutaMAX, 10 % heat-inactivated FCS, penicillin (100 U/mL), streptomycin (100 mg/mL), and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). When specific fluorescent HeLa cell lines were used, they were grown in the presence of the appropriate antibiotics (puromycin or G418) used for selection: these lines stably express either the lipid- (non raft) CAAX or the lipid- (raft) MyrPalm PM targeting domain in fusion with mCherry and GFP, respectively . Rat kangaroo kidney epithelial cells (PtK1) were cultured in Ham’s F12 medium (Sigma-Aldrich, Lyon, France) containing 25 mM HEPES, 10 % fetal bovine serum (FBS), and antibiotics. Human brain endothelial cells (HCMEC/D3) were grown in Endothelial Basal Medium-2 (Lonza Walkersville, Walkersville, MD, USA) supplemented with 5 % FBS, 10 mM HEPES, antibiotics, 1 % chemically defined lipid concentrate (Invitrogen Ltd., Paisley, UK), 1.4 μM hydrocortisone, 5 μg/mL ascorbic acid, and 1 ng/mL basic fibroblast growth factor (Sigma-Aldrich, St Louis, MO, USA). T. gondii strains (lox-MyoA, ΔMyoA, ΔMyoB/C) were propagated on HFF cells. All cultures were maintained at 37 °C and 5 % CO2 atmosphere. Antibodies used in this study included the homemade affinity purified rabbit anti-T. gondii toxofilin , affinity purified mouse anti-T. gondii RON4 antibodies , mouse monoclonal anti-T. gondii P30 antibodies (Novocastra, Leica Biosystem, Nanterre, France), polyclonal rabbit anti-p34-Arc/ARPC2 (ref 07-227 batch 32474, Upstate, Millipore, Molsheim, France), mouse monoclonal anti-cortactin p80/p85 (clone 4 F11, ref 05-180, batch 28747, Upstate, Millipore, Molsheim, France), anti-non-muscle myosin II (ref M8064, Sigma-Aldrich, Lyon, France), mouse anti-Ty antibodies (ref 200-301-W45, Rockland Immunochemicals Inc., Limerick, PA, USA), anti-recombinant ROP2 serum (gift of J.F. Dubremetz). Secondary antibodies used were highly cross-adsorbed goat anti-mouse, goat anti-rabbit, or goat anti-rat antibodies conjugated with Alexa Fluor® 488, Alexa Fluor® 568, Alexa Fluor® 633, or Alexa Fluor® 660 (Life Technologies, Thermo Fisher, Waltham, MA, USA). The micropinocytosis marker Alexa Fluor® 594 dextran (10 kDa, ref D22913) was obtained from Life Technologies, Thermo Fisher, Waltham, MA, USA. Inhibitors used in this study included the actin drugs jasplakinolide (ref J4580) and latrunculin B (ref L5288), the ROCK inhibitor Y27632 (ref Y0503), the myosin II ATPase inhibitor blebbistatin (ref B0560), the PI3K inhibitor wortmannin (ref W1628), and the DNA stain Hoechst 33258. All were purchased from Sigma-Aldrich (Lyon, France).
Transient expression of PM and actin fluorescent reporters
In addition to the PM reporters that were stably expressed in HeLa cells (see above), we used transient expression of additional host cell PM and actin markers. U2OS and HeLa cells were routinely transfected separately or in pair combination with various constructs. The list of constructs included pDisplay TM plasmid encoding the PDGFR trans-membrane domain (Life Technologies, Thermo Fisher, Waltham, MA, USA) in fusion with GFP (gift from V. Heussler, Institut Cell Biology, Bern (CH), Bern, Switzerland) or in fusion with mCherry (mC) (homemade), pCMVmCherry-actin (gift of V. Delorme-Walker, Scripps, La Jolla, CA, USA), and pCMVLifeAct-TagGFP2 (Ibidi, Biovalley, Nanterre, France).
Videomicroscopy, confocal microscopy, and image acquisition
Parasites were collected within a few hours following spontaneous egress from the HFF monolayers and washed in Hanks’ Balanced Salt Solution (HBSS) supplemented with 1 % FCS (HBSS-FCS). Time-lapse video microscopy was conducted in Chamlide chambers (LCI Corp., Seoul, Korea) installed on an Eclipse Ti inverted confocal microscope (Nikon France Instruments, Champigny sur Marne, France) with a temperature- and CO2-controlled stage and chamber (LCI Corp., Seoul, Korea), equipped with a CoolSNAP HQ2 camera (Photometrics, Roper Scientific, Lisses, France) and a CSU X1 Yokogawa spinning disk (Roper Scientific, Lisses, France), three lasers (with excitation wavelength λ 491, 561, and 642 nm), and three dichroic mirrors. The microscope was piloted using Metamorph software (Universal Imaging Corporation, Roper Scientific, Lisses, France), and images of parasite-cell interaction were acquired with settings including 1 or 2 frames/s for up to 40 min, and 1 frame/30 s for up to 6 h when assessing the viability of internalized parasites. Depending on the experiment, one to two laser wavelengths were used sequentially for each time point to monitor separately or simultaneously the dynamics of PM and actin fluorescent reporters. When needed, the chamber was perfused with syringe-pumped HBSS-FCS medium for 1 min at a medium flow rate of about 20–30 μL/min. Confocal imaging was performed on the same device using the three lasers with sections ranging from 0.250–0.3 μm.
Image stacks for every event of interest were prepared and annotated with time, bar scale, and arrows with Metamorph software from the raw image data file. Next ImageJ software (Rasband, W.S., ImageJ, US National Institutes of Health, Bethesda, MD, USA, http://imagej.nih.gov/ij/, 1997–2014) was used to add some additional labels on the time lapses, movies, and maximal projections from confocal z stacks. In some video sequences, the “Manual tracking” plug-in was used to track in time the spatial xy positions of the parasite’s constriction site .
Immunofluorescence labeling of ΔMyoA tachyzoites interacting with host cells
We analyzed the modalities of interaction between ΔMyoA parasites and a variety of epithelial, fibroblastic, endothelial, and osteosarcoma cells that were grown in complete medium at 70–90 % confluency on poly-L-lysine-coated glass coverslips. Cell monolayers were washed with HBSS-FCS, and newly released ΔMyoA parasites were rapidly centrifuged (1.5 min, 250 g) on top of the monolayers to synchronize the contact between the two partner cells. Samples were next incubated for 10–20 min under culture conditions before paraformaldehyde (PFA) fixation (2 % in phosphate-buffered saline (PBS), 30 min, room temperature (RT)). Free aldehydes were quenched in NH4Cl (50 mM, 10 min), and cells were incubated in blocking buffer (2 % bovine serum albumin in PBS, 30 min, RT), then with anti-P30 antibodies (20 min, RT) (Novocastra, Nanterre, France) followed by Alexa Fluor® conjugated anti-mouse antibodies (30 min, RT) (Molecular Probes, Life Technologies, St Aubin, France). Samples were next permeabilized with 0.5 % Triton X-100 (5 min, RT), incubated again with the blocking buffer and then with Alexa Fluor® conjugated phalloidin to stain F-actin (2 μM, 45 min, RT) or sequentially with antibodies against proteins of interest (see list above) followed by relevant Alexa Fluor® conjugated secondary antibodies (1 h, RT). In some assays, cell permeabilization was performed straight after fixation prior to immunostaining. In the assays performed with both ΔMyoA tachyzoites and the fluorescent Alexa594 dextran (500 μg/mL, 10 kDa), ARPE-19 and HFF cells were fixed after 10 or 20 min of interaction and permeabilized with Triton X-100 (0.01 % in PBS, 5 min, RT). Mouse serum against ROP2 and anti-mouse Alexa Fluor® conjugated secondary antibodies were used to stain the PV membrane . Cells were mounted in Mowiol® 4-88 (Sigma-Aldrich, St Louis, MO, USA) and analyzed within 24 h by confocal microscopy using the Eclipse Ti inverted microscope.
Scanning electron microscopy
ARPE-19 cells were grown at 80 % confluency for 24 h on poly-l-lysine-coated glass coverslips and incubated with either RH-MyoA, LoxMyoA, or ΔMyoA tachyzoites for 4–5 min (MyoA+) and 15–20 min (ΔMyoA). After cell fixation in 2.5 % glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) (1 h, 4 °C), samples were washed in 0.1 M cacodylate buffer (pH 7.2) (12 h, 4 °C), ethanol dehydrated, and critical point dried in CO2 atmosphere with an Emitech K850 apparatus (Quorum Technologies, Laughton, UK). Coverslips containing the infected monolayers were attached to SEM aluminum holders and were gold coated using a JEOL SEM instrument, JPC-1200 (JEOL, Freising, Germany) and analyzed with the Scanning Electron Microscope SU3500 (Hitachi, Tokyo, Japan). Digital images were recorded, and photocompositions were realized with ImageJ and Photoshop software.
About 2 × 104 U2OS and ARPE-19 cells were plated in a 96-well plate to obtain 80 % confluency 24 h later. Cells were starved in 0.01 % FCS for 12–16 h prior to the assay and were pre-treated as follows: (1) with JLY as described in  with 20 μM Y27632 for 10 min prior to addition of 8 μM jasplakinolide and 5 μM latrunculin B for an additional 10 min before extensive washing in medium and immediate contact with ΔMyoA tachyzoites, (2) with jasplakinolide alone (1 μM) for 15 min and treated as indicated for JLY inhibitors, (3) with blebbistatin (25 μM), Y27632 (20 μM) separately or in combination for 15 min with the drugs kept during the invasion assays due to the high reversibility of both compounds, and (4) with wortmannin (10 μM) for 30 min that was washed out prior to the invasion assay. ΔMyoA tachyzoites were settled on top of the cells by gentle centrifugation (2 min, 250 g) and incubated at 37 °C and 5 % CO2 for 15 min before PFA fixation. Following sequential labeling of extra- and intracellular tachyzoites with anti-TgP30 and anti-TgGRA1 antibodies and differential fluorophore-coupled secondary antibodies, respectively, prior and post TX-100 permeabilization, Hoechst 33242 was added to label all nuclei (mammalian cells and parasites), and the samples were automatically scanned at a magnification of × 20 under an Olympus Scan^R automated inverted microscope (3 wells per condition, 16 fields of acquisition per well). Image processing with the Cell^R software successively included signal-to-noise ratio optimization to allow cell nuclei segmentation, channel-associated image detection, and image subtraction (extracellular zoites subtracted from extra- plus intracellular tachyzoites), and intracellular tachyzoite segmentation using an edge detection algorithm. The whole assay including imaging procedure was applied to samples of untreated pre-starved ARPE-19 and HFF cells that were incubated in complete medium supplemented with 20 % FCS but no phenol red with ΔMyoA tachyzoites to which Alexa 594 dextran (500 ug/mL) was added for 10 min after the parasite centrifugation step. Samples were fixed in PFA, permeabilized, and stained with anti-T. gondii ROP2 antibodies to observe macropinosomes and nascent PVs. Control of the efficiency of macropinocytosis inhibitors was performed under the conditions used for invasion assays and assessed by quantifying the number of macropinosomes positive for fluorescent Alexa594 dextran (500 μg/mL, 10 kDa) as described . Statistics were performed with GraphPad Prism software. To check whether JLY treatment was cytotoxic for the U2OS cells, we assessed viability by pre-loading them with cell-permeant calcein-AM (0.5 μM final dilution) and Hoechst 33258 (1.5 μM final dilution) reagents (15 min, RT) before JLY exposure. Drugs were removed and the amount of green (i.e., live) and blue (i.e., total) fluorescent cells was measured over a 2-h period using semi-automatic imaging (×20) with an Olympus Scan^R automated inverted microscope under conditions similar to those used for the invasion assays in P96-well plates (N = 1 experiment). Reversibility of the JLY effect on cell invasion was measured under the same conditions (N = 3 experiments).
Apical major antigen 1
Claudin-like apicomplexa microneme protein
Dulbecco’s modified Eagle’s medium
Epidermal growth factor
Fetal calf serum
Human brain endothelial cells
Human foreskin fibroblast
- JLY cocktail:
Platelet-derived growth factor receptor trans-membrane domain-green fluorescent protein
- ROCK inhibitor:
Rho-associated protein kinase inhibitor
- RON protein:
Rhoptry neck protein
- ROP protein:
Scanning electron microscopy
We are grateful to Pr Pascale Gueirard (Faculté Medecine et Pharmacie, Clermont-Ferrand, France) and Geraldine Toutirais (PTME, Museum National d’Histoire Naturelle, Paris) from the electron microscopy and microanalysis facilities for their assistance with SEM. We thank G. Milon (Institut Pasteur, Paris), V. Delorme-Walker (Scripps Institute, La Jolla, CA), D. Ferguson (University of Oxford, Oxford, UK), G. Charras (UCL, London), and A. Chitnis for careful reading and editing of the manuscript.
IT’s laboratory has been funded by the Fondation pour la Recherche Médicale (FRM-DEQ20100318279).
Availability of data and material
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
IT designed and analyzed the experiments; IT and MB performed the videomicroscopy assays and processed the video sequences. BT performed the quantitative invasion and pharmacological assays. VG assisted in parasite and cell culture and transfection. IF and IT performed the SEM experiments. MM provided the Toxoplasma mutant and critical analysis throughout the study. IT wrote the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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