- Research article
- Open Access
The toxoplasma-host cell junction is anchored to the cell cortex to sustain parasite invasive force
© Bichet et al.; licensee BioMed Central. 2014
- Received: 27 September 2014
- Accepted: 10 December 2014
- Published: 31 December 2014
The public health threats imposed by toxoplasmosis worldwide and by malaria in sub-Saharan countries are directly associated with the capacity of their related causative agents Toxoplasma and Plasmodium, respectively, to colonize and expand inside host cells. Therefore, deciphering how these two Apicomplexan protozoan parasites access their host cells has been highlighted as a priority research with the perspective of designing anti-invasive molecules to prevent diseases. Central to the mechanism of invasion for both genera is mechanical force, which is thought to be applied by the parasite at the interface between the two cells following assembly of a unique cell-cell junction but this model lacks direct evidence and has been challenged by recent genetic studies. In this work, using parasites expressing the fluorescent core component of this junction, we analyze characteristic features of the kinematics of penetration of more than 1,000 invasion events.
The majority of invasion events occur with a typical forward rotational progression of the parasite through a static junction into an invaginating host cell plasma membrane. However, if parasites encounter resistance and if the junction is not strongly anchored to the host cell cortex, as when parasites do not secrete the toxofilin protein and, therefore, are unable to locally remodel the cortical actin cytoskeleton, the junction travels retrogradely with the host cell membrane along the parasite surface allowing the formation of a functional vacuole. Kinetic measurements of the invasive trajectories strongly support a similar parasite driven force in both static and capped junctions, both of which lead to successful invasion. However, about 20% of toxofilin mutants fail to enter and eventually disengage from the host cell membrane while the secreted RhOptry Neck (RON2) molecules are posteriorally capped before being cleaved and released in the medium. By contrast in cells characterized by low cortex tension and high cortical actin dynamics junction capping and entry failure are drastically reduced.
This kinematic analysis newly highlights that to invade cells parasites need to engage their motor with the junction molecular complex where force is efficiently applied only upon proper anchorage to the host cell membrane and cortex.
- Cortical actin
- Host cell Invasion
Toxoplasmosis is a worldwide spread zoonosis caused by the protozoan Apicomplexa parasite Toxoplasma gondii that imposes serious economic loss in livestock. It is also a concern in human health since about a third of the population is thought to silently carry parasites, which under immunosuppressive conditions, revert to replicative parasites called tachyzoites. Subsequent uncontrolled expansion of the tachyzoite population is commonly responsible for cerebral, cardiac and pulmonary life-threatening diseases. Because tachyzoites only multiply in a parasitophorous vacuole (PV) that derives from the host cell plasma membrane (PM) invagination at the time of entry , tachyzoite invasiveness is thus a primary determinant of Toxoplasma infection outcome. Such strict dependence on host cells has impelled decades of research to decipher the molecular mechanisms of the invasion event and eventually to design anti-invasion strategies as pharmacological or immunological approaches to control infection and to prevent diseases . Other Apicomplexa zoites, in particular the etiological agents of malaria, that is, Plasmodium spp parasites, invade host cells and use a similar strategy to this end; therefore, the long-lasting interest in host cell invasion and the pressing need to progress in this research go much beyond Toxoplasma.
While the active participation of a membrane-associated contractile system of Apicomplexa zoites during host cell entry was emphasized in the 1980s – and later assigned to a conserved actin-myosin (MyoA)-based force –, a contribution of the host cell through cortical actin dynamics has been more recently unmasked ,. To establish an intimate contact with a permissive host cell, zoites secrete at their apical pole a protein complex from vesicles called the rhoptries (RhOptry Neck (RON) complex), that assembles as a ring into the host cell PM and beneath – and that connects with de novo-nucleated host cell actin filaments . Therefore, the current model specifies that zoites trigger the transient buildup of a unique tight interface, called a junction, between the two cells that serves as a door of entry and that seems optimally anchored to the host cell cortical actin cytoskeleton to act as a traction site for the parasite motor-based force production ,. The T. gondii rhoptry protein toxofilin that loosens the host cell cortical actin meshwork at the onset of invasion has been proposed to promote local availability of actin monomers for actin assembly at the junction .
Although the recent localization of actin juxtaposed to the RON-positive ring in Plasmodium merozoite invading an erythrocyte – is in line with the zoite motor force scheme, such observation has not been confirmed for T. gondii tachyzoites. In addition, the ‘force transmitting’ function of two molecules that back up the model by acting as physical bridges between the RON ring and the parasite motor, namely AMA1 and the glycolytic enzyme aldolase ,, has been recently questioned – while no other potential linkers to fulfill the role have been identified. Moreover, an actin/myoA-independent mode of entry has been evoked as an alternative strategy since T. gondii tachyzoites devoid of actin or myoA showed unexpected residual gliding and invasive capabilities ,. Finally, recent theoretical modeling of erythrocyte invasion by Plasmodium merozoite highlighted the possibility that host cell membrane projections induced by the parasite could promote its firm positioning on the red blood cell surface as well as its subsequent internalization, thereby shifting the model towards collaboration for force production between the two partners . While traction forces between Plasmodium sporozoites and substrates have been measured using reflection interference contrast and traction force microscopy , no direct evidence for a traction force exerted by the zoite at the junction has ever been demonstrated for Plasmodium or Toxoplasma. These major flaws of the model are in large part due to the difficulty of tracing actin/myoA dynamics or junction components in live cells during the high-speed entry process, which lasts tens of seconds and involves tiny amounts of molecules.
In this context, we decided to inspect in detail the force origin and features powering parasite entry into the host cell by simultaneously tracking the tachyzoite apex, the tachyzoite-cell junction and the host PM during the penetration event. To this end, we used tachyzoites expressing a fluorescent functional RON2 (RHΔKu80:Ron2mCherry)  that marks the junction site being the RON complex core component, which spans the host cell plasma bilayer . Kinematic analysis of parasite pre-invasive and invasive behaviors revealed a major scenario that includes (1) a minimal impulse of a few microns per second speed followed by (2) a brief decrease in motion coinciding with RONs release and insertion into the host cell PM, and then (3) a rotational progression at a few tens of microns per second into the nascent PV while the junction remains quasi stationary. However, when parasites encounter some higher resistance that impedes progression or when the junction is not properly anchored, the latter flows retrogradely along the parasite surface and the host PM eventually encloses the zoite in a growth permissive PV. Kinematic measurements during host cell entry strongly argue for a similar parasite driven force in static and capped junctions. Importantly, when the junction is not well anchored to the host actin cortex the secreted RON2 molecules are displaced without the PM to the posterior pole and are then shed from the zoite, which as a consequence disengages from the host cell PM. Accordingly, constitutively blebbing cells that display low cortex tension and high cortical actin dynamics provide the optimal conditions for stable junction and successful invasion. Collectively, these data demonstrate that the zoite applies a motor force onto the RON2-containing junction that leads to invasion when the latter is properly anchored to the host cell cortex.
A minimum impulse of the tachyzoite is typically associated with penetration into host cells
A major burst of RON2 molecules traffics to the conoid tip and inserts into the host cell PM in a few seconds preceding penetration
The RON2-positive junction is capped with the host PM to the parasite rear pole when forward motion into the cell is interrupted
Kinematic modeling during cell penetration reveals typical changes in body curvature that are linked with two parasite rotations independently of junction scenario
Deficient anchorage of the junction favors junction-PM capping but also leads to invasion failure, a situation exacerbated with parasites lacking the host cortical actin depolymerizer, toxofilin
Low cortex tension and high actin dynamics promote stable anchoring of the junction during entry by both toxofilin+and toxofilin tachyzoites
While host-cell invasion is a vital process for most Apicomplexa parasites that has been explored for decades, the mechanistic model that prevailed all these years has recently shown its weaknesses and limitations. A primary issue concerns the relevance of the tight junction formed between the parasite and a permissive cell as a traction point for force transmission during cell invasion. We chose to re-examine this question using time-lapse imaging, tracking of the parasite apex and careful analysis of the kinematics of entry to quantitatively analyze invasion sequences under different parasite and host cell settings. First, we used Toxoplasma expressing a fluorescent version of RON2, the junction core component  reported as crucial for invasion ,. RON2 inserts into the host cell PM and bridges the two cells during invasion: while it firmly binds to the zoite surface-exposed AMA1 protein, the role of this partnership has been controversial ,, and will only be resolved with a full understanding of the junction function(s). The parasites encoding RON2mCherry in replacement of RON2 express a functional RON2  and behave similarly as the parental strain, thereby enabling us to document dynamically that a burst of RON2 molecules traffics from the rhoptry to the tip of the extruded conoid, and subsequently inserts into the host cell plasma bilayer to initiate PV folding. Although RON2 trafficking was associated with parasite motion of a few μm/second, the RON2 insertion step into the host cell PM occurred when the parasite had almost stopped. Next, simultaneous tracking of the zoite apex and the RON2 secreted subset informed on the fate of the junction during the penetration phase. This kinematic analysis revealed that the vast majority of zoites typically progressed forward into the nascent PV by passing the whole body through the junction at an average speed of about 0.24 μm/second: in that situation, the junction’s spatial coordinates remained almost stationary, a feature consistent with a junction firmly anchored to the host cell-cortex and capable of sustaining a parasite motor propelling force. Moreover, analysis of the parasite trajectories using Maltlab software identified two changes in parasite body curvature in all penetration events, the second one ending the entry process. While the latter likely contributes to the fission process during PV separation, the first change in curvature corresponds to the screw-like behavior reported long ago , probably directed by the peculiar microtubule network organization of the zoite. These features again support an entry process driven by the parasite force(s).
An additional line of evidence for a propulsive force came from the visualization of junctions being capped towards the rear end of the tachyzoite in about 8% to 20% events. When we simultaneously used live-fluorescent labeling of the parasite and the host cell, we observed that the secreted RON2 molecular subset and the host cell PM rearward capping movement were coupled to eventually enclose the parasite in a growth permissive PV. In addition, kinematic analysis of the parasite motion showed that the changes in RC were also conserved in these events. These findings indicate that while the RON2 was properly organized within the lipid bilayer, the anchorage of the junction was weaker than for the static one and made the whole junction and cell PM more amenable to displacement while the zoite continued to pull. Indeed, the capping response precisely coincided with the arrest of the parasite on its way in, as exemplified by the tachyzoite hitting a PV-containing zoite. Finally, based on the duration of the penetration phase, and the fact that the sum of the speed of the parasite and of the capped junction was comparable during both scenarios, we propose that the zoite tracts onto the junction similarly in all events while premature immobilization allowed the visualization of the traction activity. Although it seems unlikely, the possibility theoretically remains that the capping of RON2 coupled to the cell PM would result from a still unknown host cell local response to the parasite-induced tension that would drag the junction and PM along the body to enclose the parasite without parasite motor requirement. However, this is even less likely if we consider the events where parasites progressed into the PM that did not invaginate but instead were pushed outwards (that is, by the parasite-exerting force), and thereby was not subjected to host cell viscosity/elasticity constraints. Crucially, such membrane evagination could be associated with junction and membrane capping, a situation only explained by the parasite pulling onto the membrane and a weaker anchorage of the junction (see Figure 6E and Additional file 15: Movie 14).
Local higher resistance beneath the PM commonly results from denser/thicker cytoskeleton lattices and primarily from the contractile cortical actin-myosin meshwork. Optimized Atomic Force Microscopy (AFM) has indeed revealed the spatial inhomogeneity of viscoelasticity over a cell . These relate to regions of distinct membrane and cortical tensions providing specific nanomechanical properties, and defined by cortex thickness, actin dynamic, actin crosslinking and contractility . Therefore, it is expected that, in the same cell, tachyzoites can penetrate by the classical forward progression and static junction or instead can trigger junction retrograde capping when they encounter a stiffer cortex area. In addition, we reported here that junction capping was more frequently observed in fibroblasts which display strong ventral and dorsal actin stress fibers that result in high membrane tension . Of note, the highest rate of capped junction was repeatedly observed with the HFF cell line, which is the only growth limited cell line used throughout the study and, therefore, theoretically subject to aging. Aging is typically associated with an increase in cortex stiffening known to arise from an altered polymerization of the cortical actin cytoskeleton . Conversely, in M2 cells that display a cortex of lower resistance since they lack the cortical actin cross-linker filamin, the entry with static junction was the rule including for toxofilin-deficient parasites (88.6%) that lack the ability to loosen a thick actin lattice . Future studies on junction function will certainly gain from the increasing knowledge on membrane microdomains and nanoelastic properties at a single cell level. Of note, Coppens and Joiner elegantly proposed in the early 2000s that cholesterol in the host cell PM is necessary to trigger secretion of bulb rhoptry products that associate with cell entry . The RONs discovery and their localization at the junction site , with the availability of the RON2mCherry strain now allow assessing in real time whether the host cell PM cholesterol is required for proper (1) secretion of the RON complex, (2) insertion of this complex into the host cell PM or (3) anchoring function of the junction.
Finally, key insights came from the observation of invasion failure, which while exceptional with wild type tachyzoites (less than 2%) is markedly increased in Δ toxofilin tachyzoites (up to 18%). While parasites were readily engaged in penetration, as judged by RON2 and PM folding, they failed to pull back the junction with the host cell PM after being stopped. Instead, they slowly disengaged from the cell and could twirl or pause again as free parasites. These new observations first contradict the current statement that RON2 assembly and the so-called junction structuring in the host cell PM commit parasite to invasion ,. Secondly they clearly point to a role of toxofilin in providing the junction with the mechanical properties required for an efficient traction force. Indeed, the significant decrease in the amount of Δ toxofilin mutants that failed to invade M2 cells agrees well with the necessity of both cortical disassembly and free actin availability, provided through the toxofilin activity to complete invasion. Finally, when invasion aborted, a wave of RON2 molecules was capped towards the posterior end of the zoite where the material accumulated before being released in the medium. This is strongly suggestive of a motor engagement to the RON2-labeled junction on the parasite side. Whether a default in actin-mediated anchorage of the junction affects the stability of the RON2 ring in the cell bilayer inducing RON2 dislodgment upon force application awaits confirmation.
This work provides a series of compelling evidence for the traction point property of the junction when the zoite applies a force, and is reminiscent of the focal adhesions acting as transmission sites for actomyosin-generated forces during cell motility onto a substrate. However, we are still missing the molecular identity of the functional junction to elucidate how force is exerted at this site. In this context, it will also be of future interest to dissect how the motor incompetent parasites can get access to the intracellular milieu including under constraints driven by three-dimensional tissue-like microenvironments, which strongly impact cell membrane and cortex mechanical properties. In addition, this work brings new tools to further investigate what is (are) the trigger(s) and the mechanisms responsible for RON2/RONs trafficking within the rhoptry neck and for their subsequent organization into the host cell lipid bilayer. The role of the conoid, a missing appendage in non-coccidian including Plasmodium, in the RONs organization during release and assembly should be examined in concert with the expected mechanical perturbations in the bilayer these should cause. Such changes in membrane and cortex tension might drive various local membrane responses at the micro-domain scale, some being, then, side effects of the entry process per se. Indeed, thin filopodia-like projections extending at the junction site along the tachyzoite were occasionally observed (see Additional file 18: Movie 17 and Figure 9A) although they differed from the membrane folds enwrapping the merozoite to provide force during red blood cell penetration . It is noteworthy that Plasmodium zoites do not express toxofilin homologues but since they also face the cortical lattice of the host cell during penetration, they might use alternative strategies to overcome this constraint. Because the architecture of the red blood cell skeleton is quite different from those of nucleated cells, local targeting of a protein such as spectrin by the merozoite blood stage might be sufficient to trigger a cascade of events that leads to profound membrane and cortex rearrangements coincident with entry and underlying the characteristic echinocytosis process. Moreover, the slender hepatic stage of Plasmodium (that is, the sporozoite) might not need to loosen the cell cortex to the extent that the Toxoplasma bulky tachyzoite does, or/and might use different pathways to this end, such as proteolysis of key actin-membrane adaptors. Therefore, future comparative studies on the mechanisms by which Plasmodium sp. and Toxoplasma zoites infect host cells will undoubtedly highlight similarities and singularities in the fine tuning of the invasion strategies selected by the two parasites.
Cells and parasites
All media and products used for cell culture were from Gibco-Life technologies (St Aubin, France). HFF, NRK and HeLa were grown in (Dulbecco’s) modified Eagle’s medium ((D)MEM) supplemented with glutamax (Gibco), heat-inactivated FCS (10%), penicillin (100 U/ml), streptomycin (100 mg/ml) and 10 mM HEPES. PtK1 were cultured in Ham’s F12-medium (Sigma-Aldrich, L'isle d'Abeau Chesnes, St Quentin Fallavier, France) containing 25 mM HEPES, 10% FBS and antibiotics. All cultures were maintained at 37°C and 5% CO2 atmosphere. T. gondii strains (RH-GFP, RH-ΔKu80-RON2mCherry, RH-ΔKu80-Δtoxofilin-RON2mCherry) were propagated on HFF cells as described . To characterize the RH-ΔKu80-RON2mCherry strain, we compared (1) the pre-invasive trajectories and (2) the duration of the invasion event of RON2-tachyzoites and RON2mCherry using the techniques described in the section below referring to image analysis. In addition, using laser scanning confocal microscopy, we quantified the progeny per vacuole (n = 500 vacuoles in triplicates per assay, two independent growth assays) of the two strains developing on a HFF monolayer after 30 minutes of contact followed by extensive washing of the culture to remove extracellular parasites and by 28 hours of culturing.
Molecular cloning, transfection and cell line selection
The RHΔKu80:Δtoxofilin:Ron2mCherry strain was generated by first replacing the endogenous toxofilin locus with the hxgprt gene by homologous recombination. The Multisite Gateway Pro 3-fragment Recombination system was used to clone the HXGPRT cassette (amplified with primers HX-B4r:
AACCTTGCATTCAAACCCG and HX-B3r: GGGGACAACTTTATTATACAAAGTTGT
GATCCCCCTCCACCGCGGTGTCACTG) flanked by the 1 kb 5′(attB1toxo: GGGGACAA
GGGG AC AAC TTTGTATAGAAAAGTTGGGTGGTTCGACGCGTCGACGCCT) and the 1 kb 3′ (attB3toxo: GGGGACAACTTTGTATAATAAAGTTGCGAATCTGTTTGGGAT
GGCTTTGAC/ attB2toxo: GGGGACCACTTTGTACAAGAAAGCTGGGTATGTAGGGT
TCCACTGTCCTGCGG) up and downstream the toxofilin coding sequence. The target sequence was amplified (Phusion High Fidelity DNA polymerase, NEB, Ipswich, MA, USA) and 107 parasites were electroporated with 25 μg of the PCR product. Recombinant parasites were selected with 25 μg/ml mycophenolic acid and 50 μg/ml xanthine and cloned by limiting dilution. Then, the ron2 gene was fused to the mcherry coding sequence as described in  except that the 1.1 kb fragment corresponding to the 3’end of the RON2 gene was cloned into the mcherry-LIC-DHFTRS vector. Clones were selected with 500 ng/ml pyrimethamine and cloned by limiting dilution.
Generation of CAAX-mCherry retroviruses is described in . Generation of PHPLCΔ-GFP retroviruses is described in . To generate cells stably expressing a GFP tagged membrane marker, we excised MyrPalm from a plasmid acquired from MyrPalm-CFP (plasmid 14867, ) and inserted it into pRetroQAcGFPC1. Retroviruses were then generated by transfecting the plasmids into 293-GPG cells for packaging (a kind gift from Prof. D. Ory, Washington University) . For generation of stable cell lines, retroviral supernatants were used to infect wild type HeLa cells or M2 cells. The cells were selected in the presence of 500 ng/ml puromycin or 1 μg/ml G418 for two weeks.
Videomicroscopy and image acquisition
Parasites were collected within a few hours following spontaneous egress from the HFF monolayers and washed in HBSS supplemented with 1% 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 spinning disk (Yokogawa, Roper Scientific, Lisses, France). The microscope was piloted using Metamorph software (Universal Imaging Corporation, Roper Scientific, Lisses, France) and images were acquired with settings including 1 frame/second for 20 minutes, with one to two laser wavelengths depending on the experiment.
Images stacks for every event of interest (pre-invasive motion, cell penetration, entry failure, and so on) were prepared and annotated with time, 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 , 1997–2014). and the ‘manual tracking’ plugin were used to simultaneously track the spatial positions of the parasite’s apex, the RON2 fluorescently labeled junction and the fluorescently labeled host cell PM at the junction over time. The xy Cartesian coordinates allowed reconstituting trajectories of interest. Saving these data as Excel files and integrating the time interval between consecutive frames provided both the instantaneous and mean speeds of the parasite of interest. Retrieved data of the instantaneous velocities and the frames they referred to were next imported to KaleidaGraph Software (Synergy Software, Reading, PA, USA) to plot the parasite’s speed as a function of time.
Kinematic analysis of the parasite trajectory during cell penetration
Among the 150 cases showing a static junction, 24 and 27 were recorded on HFF and NRK fibroblasts, respectively, and 11, 36 and 52 on HeLa, M2 and Ptk-1 epithelial cells, respectively. With regard to the capped junction events, three and four were obtained on NRK and HFF fibroblasts, respectively, while two, four and eleven were observed on M2, HeLa and Ptk-1 epithelial cells, respectively.
Immunofluorescence labeling of invading tachyzoites
To catch parasites in the process of penetrating into HeLa cells, we performed invasion assays as previously described . After paraformaldehyde fixation (2% in PBS, 30 minutes), free aldehydes were quenched in NH4Cl (50 mM, 10 minutes), and cells were incubated in blocking buffer (2% BSA in PBS, 30 minutes), then with anti-P30 antibodies (20 minutes, 23°C) (Novocastra, Nanterre, France) followed by Alexa Fluor® 660 conjugated secondary antibodies (30 minutes, 23°C) (Molecular Probes, Life Technologies, St Aubin, France). Samples were next permeabilized with 0.5% Triton-X 100 (five minutes, 23°C), exposed again to the blocking buffer and incubated with Alexa Fluor® 488 conjugated phalloidin to stain F-actin (45 minutes, 23°C). In other assays, cell permeabilization was performed after fixation and affinity-purified anti-RON4 antibodies were used before Alexa Fluor® 488 conjugated secondary antibodies. Cells were mounted in Mowiol® 4–88 (Sigma Aldrich, St Louis, MO, USA) and analyzed by confocal microscopy using the Eclipe Ti inverted microscope.
We thank Gary Ward (University of Vermont, Burlington, VT, USA) for providing us with the compound 130038 enhancing tachyzoite motility, and Alexandre Bougdour (University of Joseph Fourier, Grenoble, France) for the gift of mcherry-LIC-DHFTRS vector. I.T. laboratory has been funded by the “Fondation pour la Recherche Médicale” agency (FRM-DEQ20100318279) and the Domaines d’Intérêts Majeurs” DIM MALINF for a grant for equipment contributing to the Imaging facility used in this work.
- Suss-Toby E, Zimmerberg J, Ward GE: Toxoplasma invasion: the parasitophorous vacuole is formed from host cell plasma membrane and pinches off via a fission pore. Proc Natl Acad Sci U S A. 1996, 93: 8413-8418. 10.1073/pnas.93.16.8413.PubMed CentralPubMedView ArticleGoogle Scholar
- Bargieri D, Lagal V, Tardieux I, Ménard R: Host cell invasion by apicomplexans: what do we know?. Trends Parasitol. 2012, 28: 131-135. 10.1016/j.pt.2012.01.005.PubMedView ArticleGoogle Scholar
- Russell DG, Sinden RE: The role of the cytoskeleton in the motility of coccidian sporozoites. J Cell Sci. 1981, 50: 345-359.PubMedGoogle Scholar
- Russell DG: Host cell invasion by Apicomplexa: an expression of the parasite’s contractile system?. Parasitology. 1983, 87: 199-209. 10.1017/S0031182000052562.PubMedView ArticleGoogle Scholar
- King CA: Cell motility of sporozoan protozoa. Parasitol Today. 1988, 4: 315-319. 10.1016/0169-4758(88)90113-5.PubMedView ArticleGoogle Scholar
- Dobrowolski JM, Sibley LD: Toxoplasma invasion of mammalian cells is powered by the actin cytoskeleton of the parasite. Cell. 1996, 84: 933-939. 10.1016/S0092-8674(00)81071-5.PubMedView ArticleGoogle Scholar
- Meissner M, Schlüter D, Soldati D: Role of Toxoplasma gondii myosin A in powering parasite gliding and host cell invasion. Science. 2002, 298: 837-840. 10.1126/science.1074553.PubMedView ArticleGoogle Scholar
- Foth BJ, Goedecke MC, Soldati D: New insights into myosin evolution and classification. Proc Natl Acad Sci U S A. 2006, 103: 3681-3686. 10.1073/pnas.0506307103.PubMed CentralPubMedView ArticleGoogle Scholar
- Gonzalez V, Combe A, David V, Malmquist NA, Delorme V, Leroy C, Blazquez S, Ménard R, Tardieux I: Host cell entry by apicomplexa parasites requires actin polymerization in the host cell. Cell Host Microbe. 2009, 5: 259-272. 10.1016/j.chom.2009.01.011.PubMedView ArticleGoogle Scholar
- Gaji RY, Huynh MH, Carruthers VB: A novel high throughput invasion screen identifies host actin regulators required for efficient cell entry by Toxoplasma gondii. PLoS One. 2013, 8: e64693-10.1371/journal.pone.0064693.PubMed CentralPubMedView ArticleGoogle Scholar
- Lebrun M, Michelin A, El Hajj H, Poncet J, Bradley PJ, Vial H, Dubremetz JF: The rhoptry neck protein RON4 re-localizes at the moving junction during Toxoplasma gondii invasion. Cell Microbiol. 2005, 7: 1823-1833. 10.1111/j.1462-5822.2005.00646.x.PubMedView ArticleGoogle Scholar
- Besteiro S, Michelin A, Poncet J, Dubremetz JF, Lebrun M: Export of a Toxoplasma gondii rhoptry neck protein complex at the host cell membrane to form the moving junction during invasion. PLoS Pathog. 2009, 5: e1000309-10.1371/journal.ppat.1000309.PubMed CentralPubMedView ArticleGoogle Scholar
- Straub KW, Cheng SJ, Sohn CS, Bradley PJ: Novel components of the Apicomplexan moving junction reveal conserved and coccidia-restricted elements. Cell Microbiol. 2009, 11: 590-603. 10.1111/j.1462-5822.2008.01276.x.PubMed CentralPubMedView ArticleGoogle Scholar
- Straub KW, Peng ED, Hajagos BE, Tyler JS, Bradley PJ: The moving junction protein RON8 facilitates firm attachment and host cell invasion in Toxoplasma gondii. PLoS Pathog. 2011, 7: e1002007-10.1371/journal.ppat.1002007.PubMed CentralPubMedView ArticleGoogle Scholar
- Bargieri D, Lagal V, Andenmatten N, Tardieux I, Meissner M, Ménard R: Host cell invasion by apicomplexan parasites: the junction conundrum. PLoS Pathog. 2014, 10: e1004273-10.1371/journal.ppat.1004273.PubMed CentralPubMedView ArticleGoogle Scholar
- Delorme-Walker V, Abrivard M, Lagal V, Anderson K, Perazzi A, Gonzalez V, Page C, Chauvet J, Ochoa W, Volkmann N, Hanein D, Tardieux I: Toxofilin upregulates the host cortical actin cytoskeleton dynamics, facilitating Toxoplasma invasion. J Cell Sci. 2012, 125: 4333-4342. 10.1242/jcs.103648.PubMed CentralPubMedView ArticleGoogle Scholar
- Angrisano F, Riglar DT, Sturm A, Volz JC, Delves MJ, Zuccala ES, Turnbull L, Dekiwadia C, Olshina MA, Marapana DS, Wong W, Mollard V, Bradin CH, Tonkin CJ, Gunning PW, Ralph SA, Whitchurch CB, Sinden RE, Cowman AF, McFadden GI, Baum J: Spatial localisation of actin filaments across developmental stages of the malaria parasite. PLoS One. 2012, 7: e32188-10.1371/journal.pone.0032188.PubMed CentralPubMedView ArticleGoogle Scholar
- Zuccala ES, Gout AM, Dekiwadia C, Marapana DS, Angrisano F, Turnbull L, Riglar DT, Rogers KL, Whitchurch CB, Ralph SA, Speed TP, Baum J: Subcompartmentalisation of proteins in the rhoptries correlates with ordered events of erythrocyte invasion by the blood stage malaria parasite. PLoS One. 2012, 7: e46160-10.1371/journal.pone.0046160.PubMed CentralPubMedView ArticleGoogle Scholar
- Riglar DT, Richard D, Wilson DW, Boyle MJ, Dekiwadia C, Turnbull L, Angrisano F, Marapana DS, Rogers KL, Whitchurch CB, Beeson JG, Cowman AF, Ralph SA, Baum J: Super-resolution dissection of coordinated events during malaria parasite invasion of the human erythrocyte. Cell Host Microbe. 2011, 9: 9-20. 10.1016/j.chom.2010.12.003.PubMedView ArticleGoogle Scholar
- Starnes GL, Coincon M, Sygusch J, Sibley LD: Aldolase is essential for energy production and bridging adhesin-actin cytoskeletal interactions during parasite invasion of host cells. Cell Host Microbe. 2009, 5: 353-364. 10.1016/j.chom.2009.03.005.PubMed CentralPubMedView ArticleGoogle Scholar
- Tyler JS, Treeck M, Boothroyd JC: Focus on the ringleader: the role of AMA1 in apicomplexan invasion and replication. Trends Parasitol. 2011, 27: 410-420. 10.1016/j.pt.2011.04.002.PubMed CentralPubMedView ArticleGoogle Scholar
- Giovannini D, Späth S, Lacroix C, Perazzi A, Bargieri D, Lagal V, Lebugle C, Combe A, Thiberge S, Baldacci P, Tardieux I, Ménard R: Independent roles of apical membrane antigen 1 and rhoptry neck proteins during host cell invasion by apicomplexa. Cell Host Microbe. 2011, 10: 591-602. 10.1016/j.chom.2011.10.012.PubMedView ArticleGoogle Scholar
- Bargieri DY, Andenmatten N, Lagal V, Thiberge S, Whitelaw JA, Tardieux I, Meissner M, Ménard R: Apical membrane antigen 1 mediates apicomplexan parasite attachment but is dispensable for host cell invasion. Nat Commun. 2013, 4: 2552-10.1038/ncomms3552.PubMed CentralPubMedView ArticleGoogle Scholar
- Shen B, Sibley LD: Toxoplasma aldolase is required for metabolism but dispensable for host-cell invasion. Proc Natl Acad Sci U S A. 2014, 111: 3567-3572. 10.1073/pnas.1315156111.PubMed CentralPubMedView ArticleGoogle Scholar
- Andenmatten N, Egarter S, Jackson AJ, Jullien N, Herman JP, Meissner M: Conditional genome engineering in Toxoplasma gondii uncovers alternative invasion mechanisms. Nat Methods. 2013, 10: 125-127. 10.1038/nmeth.2301.PubMed CentralPubMedView ArticleGoogle Scholar
- Egarter S, Andenmatten N, Jackson AJ, Whitelaw JA, Pall G, Black JA, Ferguson DJ, Tardieux I, Mogilner A, Meissner M: The toxoplasma Acto-MyoA motor complex is important but not essential for gliding motility and host cell invasion. PLoS One. 2014, 9: e91819-10.1371/journal.pone.0091819.PubMed CentralPubMedView ArticleGoogle Scholar
- Dasgupta S, Auth T, Gov NS, Satchwell TJ, Hanssen E, Zuccala ES, Riglar DT, Toye AM, Betz T, Baum J, Gompper G: Membrane-wrapping contributions to malaria parasite invasion of the human erythrocyte. Biophys J. 2014, 107: 43-54. 10.1016/j.bpj.2014.05.024.PubMed CentralPubMedView ArticleGoogle Scholar
- Münter S, Sabass B, Selhuber-Unkel C, Kudryashev M, Hegge S, Engel U, Spatz JP, Matuschewski K, Schwarz US, Frischknecht F: Plasmodium sporozoite motility is modulated by the turnover of discrete adhesion sites. Cell Host Microbe. 2009, 6: 551-562. 10.1016/j.chom.2009.11.007.PubMedView ArticleGoogle Scholar
- Frixione E, Mondragón R, Meza I: Kinematic analysis of Toxoplasma gondii motility. Cell Motil Cytoskeleton. 1996, 34: 152-163. 10.1002/(SICI)1097-0169(1996)34:2<152::AID-CM6>3.0.CO;2-D.PubMedView ArticleGoogle Scholar
- Håkansson S, Morisaki H, Heuser J, Sibley LD: Time-lapse video microscopy of gliding motility in Toxoplasma gondii reveals a novel, biphasic mechanism of cell locomotion. Mol Biol Cell. 1999, 10: 3539-3547. 10.1091/mbc.10.11.3539.PubMed CentralPubMedView ArticleGoogle Scholar
- Leung JM, Rould MA, Konradt C, Hunter CA, Ward GE: Disruption of TgPHIL1 alters specific parameters of Toxoplasma gondii motility measured in a quantitative, three-dimensional live motility assay. PLoS One. 2014, 9: e85763-10.1371/journal.pone.0085763.PubMed CentralPubMedView ArticleGoogle Scholar
- Carey KL, Westwood NJ, Mitchison TJ, Ward GE: A small-molecule approach to studying invasive mechanisms of Toxoplasma gondii. Proc Natl Acad Sci U S A. 2004, 101: 7433-7438. 10.1073/pnas.0307769101.PubMed CentralPubMedView ArticleGoogle Scholar
- Hanssen E, Dekiwadia C, Riglar DT, Rug M, Lemgruber L, Cowman AF, Cyrklaff M, Kudryashev M, Frischknecht F, Baum J, Ralph SA: Electron tomography of Plasmodium falciparum merozoites reveals core cellular events that underpin erythrocyte invasion. Cell Microbiol. 2013, 15: 1457-1472. 10.1111/cmi.12132.PubMedView ArticleGoogle Scholar
- Dvorak JA, Miller LH, Whitehouse WC, Shiroishi T: Invasion of erythrocytes by malaria merozoites. Science. 1975, 187: 748-750. 10.1126/science.803712.PubMedView ArticleGoogle Scholar
- Mondragon R, Frixione E: Ca(2+)-dependence of conoid extrusion in Toxoplasma gondii tachyzoites. J Eukaryot Microbiol. 1996, 43: 120-127. 10.1111/j.1550-7408.1996.tb04491.x.PubMedView ArticleGoogle Scholar
- Del Carmen MG, Mondragón M, González S, Mondragón R: Induction and regulation of conoid extrusion in Toxoplasma gondii. Cell Microbiol. 2009, 11: 967-982. 10.1111/j.1462-5822.2009.01304.x.PubMedView ArticleGoogle Scholar
- Lee SH, Hayes DB, Rebowski G, Tardieux I, Dominguez R: Toxofilin from Toxoplasma gondii forms a ternary complex with an antiparallel actin dimer. Proc Natl Acad Sci U S A. 2007, 104: 16122-16127. 10.1073/pnas.0705794104.PubMed CentralPubMedView ArticleGoogle Scholar
- Muñiz-Hernández S, Carmen MG, Mondragón M, Mercier C, Cesbron MF, Mondragón-González SL, González S, Mondragón R: Contribution of the residual body in the spatial organization of Toxoplasma gondii tachyzoites within the parasitophorous vacuole. J Biomed Biotechnol. 2011, 2011: 473983-10.1155/2011/473983.PubMed CentralPubMedView ArticleGoogle Scholar
- Cunningham CC, Gorlin JB, Kwiatkowski DJ, Hartwig JH, Janmey PA, Byers HR, Stossel TP: Actin-binding protein requirement for cortical stability and efficient locomotion. Science. 1992, 255: 325-327. 10.1126/science.1549777.PubMedView ArticleGoogle Scholar
- Stossel TP, Condeelis J, Cooley L, Hartwig JH, Noegel A, Schleicher M, Shapiro SS: Filamins as integrators of cell mechanics and signalling. Nat Rev Mol Cell Biol. 2001, 2: 138-145. 10.1038/35052082.PubMedView ArticleGoogle Scholar
- Beck JR, Chen AL, Kim EW, Bradley PJ: RON5 is critical for organization and function of the Toxoplasma moving junction complex. PLoS Pathog. 2014, 10: e1004025-10.1371/journal.ppat.1004025.PubMed CentralPubMedView ArticleGoogle Scholar
- Lamarque MH, Roques M, Kong-Hap M, Tonkin ML, Rugarabamu G, Marq JB, Penarete-Vargas DM, Boulanger MJ, Soldati-Favre D, Lebrun M: Plasticity and redundancy among AMA-RON pairs ensure host cell entry of Toxoplasma parasites. Nat Commun. 2014, 5: 4098-10.1038/ncomms5098.PubMedView ArticleGoogle Scholar
- Harvey KL, Yap A, Gilson PR, Cowman AF, Crabb BS: Insights and controversies into the role of the key apicomplexan invasion ligand, Apical Membrane Antigen 1. Int J Parasitol. 2014, 44: 853-857. 10.1016/j.ijpara.2014.08.001.PubMedView ArticleGoogle Scholar
- Mahaffy RE, Park S, Gerde E, Käs J, Shih CK: Quantitative analysis of the viscoelastic properties of thin regions of fibroblasts using atomic force microscopy. Biophys J. 2004, 86: 1777-1793. 10.1016/S0006-3495(04)74245-9.PubMed CentralPubMedView ArticleGoogle Scholar
- Salbreux GCG, Paluch E: Actin cortex mechanics and cellular morphogenesis. Trends Cell Biol. 2012, 22: 536-545. 10.1016/j.tcb.2012.07.001.PubMedView ArticleGoogle Scholar
- Burridge K, Wittchen ES: The tension mounts: stress fibers as force-generating mechanotransducers. J Cell Biol. 2013, 200: 9-19. 10.1083/jcb.201210090.PubMed CentralPubMedView ArticleGoogle Scholar
- Dulińska-Molak I, Pasikowska M, Pogoda K, Lewandowska M, Eris I, Lekka M: Age-related changes in the mechanical properties of human fibroblasts and its prospective reversal after anti-wrinkle tripeptide treatment. Int J Pept Res Ther. 2014, 20: 77-85. 10.1007/s10989-013-9370-z.PubMed CentralPubMedView ArticleGoogle Scholar
- Coppens I, Joiner K: Host but not parasite cholesterol controls Toxoplasma cell entry by modulating organelle discharge. Mol Biol Cell. 2003, 14: 3804-3820. 10.1091/mbc.E02-12-0830.PubMed CentralPubMedView ArticleGoogle Scholar
- Srinivasan P, Beatty WL, Diouf A, Herrera R, Ambroggio X, Moch JK, Tyler JS, Narum DL, Pierce SK, Boothroyd JC, Haynes JD, Miller LH: Binding of Plasmodium merozoite proteins RON2 and AMA1 triggers commitment to invasion. Proc Natl Acad Sci U S A. 2011, 108: 13275-13280. 10.1073/pnas.1110303108.PubMed CentralPubMedView ArticleGoogle Scholar
- Roos DS, Donald RG, Morrissette NS, Moulton AL: Molecular tools for genetic dissection of the protozoan parasite Toxoplasma gondii. Methods Cell Biol. 1994, 45: 27-63. 10.1016/S0091-679X(08)61845-2.PubMedView ArticleGoogle Scholar
- Harris AR, Peter L, Bellis J, Baum B, Kabla AJ, Charras GT: Characterizing the mechanics of cultured cell monolayers. Proc Natl Acad Sci U S A. 2012, 109: 16449-16454. 10.1073/pnas.1213301109.PubMed CentralPubMedView ArticleGoogle Scholar
- Harris AR, Charras GT: Experimental validation of atomic force microscopy-based cell elasticity measurements. Nanotechnology. 2011, 22: 345102-10.1088/0957-4484/22/34/345102.PubMedView ArticleGoogle Scholar
- Violin JD, Zhang J, Tsien RY, Newton AC: A genetically encoded fluorescent reporter reveals oscillatory phosphorylation by protein kinase C. J Cell Biol. 2003, 161: 899-909. 10.1083/jcb.200302125.PubMed CentralPubMedView ArticleGoogle Scholar
- Ory DS, Neugeboren BA, Mulligan RC: A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes. Proc Natl Acad Sci U S A. 1996, 93: 11400-11406. 10.1073/pnas.93.21.11400.PubMed CentralPubMedView ArticleGoogle Scholar
- Trajectory analysis using the ImageJ software and the ‘ManualTracking’ plugin [http://imagej.nih.gov/ij/]
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