SEARCH

SEARCH BY CITATION

FilenameFormatSizeDescription
cmi12145-sup-0001-si.tif2057K

Fig. S1. LPS treatment leads to podosome loss in DC after 24 h. DC were seeded on poly-l-lysine coated coverslips and then treated with LPS (100 ng ml−1) for 24 h.

A. DC were stained with Alexa Fluor 488 Phalloidin to detect F-actin. Assessment of cell morphology was performed as indicated under Experimental procedures. Arrows indicate podosome structures. Representative images are shown.

B. Bar graph shows the frequency of podosome loss related to the total population of 100 cells counted from one representative donor. Scale bar: 10 μm.

cmi12145-sup-0002-si.tif763K

Fig. S2. Internalization of heat-inactivated tachyzoites by DC. Freshly egressed tachyzoites (PRU-RFP) were heat-inactivated and then added to DC (MOI 5, 4 h). DC were stained with LAMP1 monoclonal antibody to detect acidic vacuoles as outlined in Experimental procedures. Arrows indicate RFP-expressing tachyzoite contained in a LAMP1-expressing vacuole. Scale bar: 10 μm.

cmi12145-sup-0003-si.tif1920K

Fig. S3. DC morphology following treatment with STAg. DC were seeded on poly-l-lysine coated coverslips and then treated with STAg (10 μg ml−1) for 8 h.

A. DC were stained with Alexa Fluor 488 Phalloidin to detect F-actin. Assessment of cell morphology was performed as indicated under Experimental procedures. Arrows indicate podosome structures. Representative images are shown.

B. Bar graph shows the frequency of podosome loss related to the total population of 100 cells counted from one representative donor. Scale bar: 10 μm.

cmi12145-sup-0004-si.tif3344K

Fig. S4. Morphology of DC challenged with 4-BPB-treated tachyzoites. Freshly egressed tachyzoites (PRU-RFP) were pretreated with 4-BPB (10 μM) and then added to DC (MOI 10, 5 h). DC were stained with Alexa Fluor 488 Phalloidin to detect F-actin and tachyzoites were stained with rabbit polyclonal anti-T. gondii tachyzoite antibodies to determine intracellular versus extracellular state as outlined in Experimental procedures. Arrows indicates cell-associated, extracellular tachyzoite (red + blue) in a DC exhibiting podosomes. Arrowheads indicate intracellular tachyzoites (red only) in a DC exhibiting absence of podosomes and rounded phenotype. Scale bar: 10 μm.

cmi12145-sup-0005-si.tif522K

Fig. S5. Effects of endosomal TLR activation on DC motility. DC were challenged with freshly egressed tachyzoites (PRU-RFP, MOI 1) or treated with LPS (200 ng ml−1), Poly (I:C) (1 μg ml−1), CL075 (1 μg ml−1) or complete medium (non-infected) for 6 h.

A. For each condition, plots show track analyses of 50 individual cells from one donor.

B. Velocity analysis of cells under same conditions as in (A). Data represent compiled analysis of 50 cells (± SEM) from one representative donor. Asterisks indicate significant differences (P < 0,001, Student's t-test). A small but significant reduction of velocity was observed in DC treated with Poly (I:C) and CL075.

cmi12145-sup-0006-si.tif2710K

Fig. S6. Chemokine receptor profile of Toxoplasma-infected DC. DC were challenged with freshly egressed tachyzoites (PRU-RFP, MOI 1), treated with LPS (200 ng ml−1) or maintained in complete medium (non-infected). After 6 h, the cell surface expression of CCR1, CCR2, CCR6, CCR9, CCR10 and CXCR6 was determined by flow cytometry as indicated under Experimental procedures. Histograms show the expression of chemokine receptors (coloured lines) related to the isotype control (grey lines) for the various conditions [non-infected, LPS and T. gondii (RFP+ cells)]. Data are from one representative donor. Performed on three donors with similar results.

cmi12145-sup-0007-si.avi2024K

Video S1. Podosome dissolution in DC following Toxoplasma invasion. Time-lapse analysis depicts a GFP-actin-expressing DC (green, left panel) challenged with freshly egressed RFP-expressing tachyzoites (red, left panel) as indicated under Experimental procedures. White arrow indicates parasite entering the cell. White line indicates the outline of the podosome cluster. Right panel movie shows the GFP-actin channel in greyscale highlighting actin dynamics and membrane ruffling activity at the edges of the cell. Note that, after dissolution of podosome structures following parasite invasion, podosomes do not reappear. Time stamp marker indicates time elapsed in seconds. (AVI, 5 MB).

cmi12145-sup-0008-si.avi2031K

Video S2. Podosome dynamics in DC before and after T. gondii infection. Time-lapse analysis depicts a GFP-actin-expressing DC (green, left panel) challenged with freshly egressed RFP-expressing tachyzoites (red, left panel) as indicated under Experimental procedures. White arrows indicate invading parasites. Right panel movie shows the GFP-actin channel in greyscale highlighting actin dynamics. Note abundant presence of dynamic podosome clusters previous to invasion and absence of podosome recovery after DC invasion by T. gondii. Time stamp marker indicates time elapsed in seconds. (AVI, 7 MB).

cmi12145-sup-0009-si.avi3184K

Video S3. Morphological changes in DC following invasion by T. gondii. Time-lapse analysis depicts a GFP-actin-expressing DC (green, left panel) challenged with freshly egressed RFP-expressing tachyzoites (red, left panel) as indicated under Experimental procedures. Initially, the centrally located DC exhibits podosome structures (outlined by white line, left panel). White arrow indicates invading parasite. Right panel movie shows the GFP-actin channel in greyscale highlighting actin dynamics. Note disappearance of podosomes following infection and accentuation of changes in cell morphology accompanied by migratory activation. In the background, two additional Toxoplasma-infected DC with low GFP-actin expression exhibit a migratory behaviour. Time stamp marker indicates time elapsed in seconds. (AVI, 7 MB).

cmi12145-sup-0010-si.avi2805K

Video S4. Ameboid-like migration of DC after invasion by T. gondii. Time-lapse analysis depicts a GFP-actin-expressing DC (green, left panel) challenged with freshly egressed RFP-expressing tachyzoites (red, left panel) as indicated under Experimental procedures. White arrows indicate invasion by T. gondii. Following invasion, the DC rounds up and exhibits rapid ameboid-like motility. Time stamp marker indicates time elapsed in seconds. (AVI, 5 MB).

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.