Quantitative insights into actin rearrangements and bacterial target site selection from Salmonella Typhimurium infection of micropatterned cells
Article first published online: 30 MAY 2013
© 2013 John Wiley & Sons Ltd
Volume 15, Issue 11, pages 1851–1865, November 2013
How to Cite
Vonaesch, P., Cardini, S., Sellin, M. E., Goud, B., Hardt, W.-D. and Schauer, K. (2013), Quantitative insights into actin rearrangements and bacterial target site selection from Salmonella Typhimurium infection of micropatterned cells. Cellular Microbiology, 15: 1851–1865. doi: 10.1111/cmi.12154
- Issue published online: 10 OCT 2013
- Article first published online: 30 MAY 2013
- Accepted manuscript online: 6 MAY 2013 01:56AM EST
- Manuscript Accepted: 28 APR 2013
- Manuscript Revised: 10 APR 2013
- Manuscript Received: 7 DEC 2012
- Swiss National Science Foundation. Grant Number: 310030-132997
- Swiss SystemsX.ch initiative
- a Swedish Research Council post-doctoral fellowship
- Fondation pour la Recherche Médicale en France
- Association pour la Recherche sur le Cancer
- Agence Nationale de la Recherche. Grant Number: 2010 BLAN 122902
- Centre National de la Recherche Scientifique
- Institut Curie
Fig. S1. Infection of retinal pigment epithelial cells (RPE-1) by S. Tm and of 3T3 fibroblasts by Shigella flexneri and Listeria monocytogenes. In (A), Maximum Intensity Projections of RPE-1 cells infected with a wild-type strain of S. Tm (S. Tmwt, left panel) or a Salmonella strain expressing Invasin (S. TmΔinvG, Invasin, right panel) are shown at 5 min p.i. In (B), Maximum Intensity Projections of 3T3 fibroblasts infected with S. flexneri (left panel) or L. monocytogenes (right panel) are shown at 15 min p.i. Red: actin; blue: nucleus; green: bacteria. Scale bar corresponds to 10 μm.
Fig. S2. Distribution of bacteria from infected cells analysed in Fig. 2 and schematic representation of measured cellular regions. Panel (A) is depicting Maximum Intensity Projections of bacteria from N-infected cells at different time points analysed for their F-actin distribution in Fig. 2. Panel (B) shows a schematic representation of a crossbow-shaped cell containing different actin pools: the ‘Extrados’ contains cortical F-actin, the ‘Bowstrings’ harbour contractile F-actin bundles. Newly polymerized F-actin is triggered locally by bacterial infection allowing differentiation between a non-infected side (left) and an infected side (right) of the cell. In (C), cellular regions are shown that were used to measure average intensity of F-actin to quantify F-actin ratios of Fig. 2B–D. For Fig. 2D only cells were considered that showed an infected and a non-infected side of the ‘extrados’.
Fig. S3. Analysis of the effect of Jasplakinolide on S. Tmwt-induced actin rearrangements in non-constrained cells. Representative Maximum Intensity Projections (MIP) of confocal stacks taken of unpatterned HeLa cells. The cells were treated either with 0.05% DMSO [corresponding to the amount present in 0.5 μm Jasplakinolide (Jasp)] or with increasing concentrations of Jasplakinolide for 8 min. The cells in the middle panel were infected after 3 min of pre-incubation with Jasplakinolide, for 5 min with S. Tmwt and stained with Phalloidin. The right panels show a blow ups of selected regions. The presence of actin ruffles is indicated with an arrow. Bacteria and the nucleus are shown in blue and the actin cytoskeleton in red. Scale bar: 10 μm.
Fig. S4. Analysis of the effect of Jasplakinolide on S. Tmwt-induced actin rearrangements in micropatterned cells. In (A) representative Maximum Intensity Projections (MIP) of confocal stacks taken of patterned HeLa cells, treated with 0.06 μM Jasplakinolide are shown. The cell in the left panel is not infected, while the cell in the right panel is infected with S. Tmwt for 5 min. The actin cytoskeleton is shown in red, the nucleus in blue and the bacteria in green. Scale bar: 10 μm. In (B) the Average Intensity map of the F-actin distribution for N micropatterned cells is shown. The F-actin distribution is represented with a heat map, where the regions containing most F-actin are showing the brightest colour (white). In (C), the ratio between the average F-actin intensity in the infected part of the ‘extrados’ versus the average F-actin intensity in the cytoplasm were calculated for every cell for N cells either for non-infected or for S. Tmwt-infected cells.
Fig. S5. Partitioning of actin in permeabilized S. Tmwt-infected cells. HeLa cells were mock-treated (Co), infected with S. Tm or treated with 10 μM Latrunculin B (Lat B) for 15 min, washed, and gently permeabilized in an isotonic buffer at 37°C followed by immediate separation of supernatant and cell pellet fractions (see Experimental procedures for details). Western blots in (A) show the partitioning of Beta-actin between the soluble supernatant (S) and the cell pellet (P) fractions. Quantifications in (B) show the mean ± SD from densitometric analysis of replicate samples as in (A). In (C), detection of the cytosolic protein Op18 confirms efficient plasma membrane permeabilization, while detection of the ER-integral protein Calnexin reveals that cells were not dislodged or fragmented by the present treatment.
Fig. S6. Distribution of bacteria from infected cells analysed in Fig. 3. Maximum Intensity Projections of bacteria from N-infected cells analysed for their F-actin distribution in Fig. 3 at 5 min p.i. are shown. The images are ordered corresponding to the degree of observed actin rearrangement, from top (no actin rearrangements, light green) to middle (little actin rearrangement, dark green) to bottom (extensive actin rearrangements, light blue).
Fig. S7. Relocalization of Rac1 upon infection by different strains of S. Tmwt. In (A), the average Rac1 distribution of N cells is represented with a heat map, where the regions containing most Rac1 are showing the brightest colour (yellow). HeLa cells stably expressing GFP-Rac1 were infected with either the wild-type strain (S. Tmwt) or a strain harbouring only SopE out of the four key effector proteins (S. TmSopE). In (B), the corresponding density maps are shown. Density contours are shown from 10% (red) to 90% (yellow) and represent the smallest regions that contain the percentage of analysed Rac1 structures. In (C), density maps are pairwise compared and the P-values of the comparisons are given. The P-values are calculated according to Duong et al. (2012).
Fig. S8. Schemes of anisotropic, crossbow-shaped cells. The scheme in (A) shows the anisotropic distribution of the actin cytoskeleton (cortical F-actin and contractile F-actin bundles), the microtubule organization centre (MTOC), the Golgi apparatus, secretory vesicles, lysosomes and the nucleus in the cell. In (B), the cell is schematically divided into two parts containing the ‘extrados’ (front of the cell, marked in blue) and the ‘bowstrings’ (rear of the cell, marked in red) and the structures and compartments concentrated in the respective parts are described. The model is based on data described in Thery et al. (2006) and Schauer et al. (2010).
Table S1. Bacterial strains used in this study.
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.