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 pDisplayTM 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).