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B. A. Weigele, R. C. Orchard, A. Jimenez, G. W. Cox, and N. M. Alto, 8, 532. RESOURCE AVAILABILITY Lead Contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Nat. Commun, 2017.

. Labigne-roussel, Please contact the lead author in case you require the original source data, for example time-lapse series that cannot be added to a public database. EXPERIMENTAL MODEL AND SUBJECT DETAILS HeLa and Caco-2 Cells Human epithelial HeLa cells (clone CCL-2, ATCC #11033106), HeLa cells stably expressing galectin-3-mOrange (Patricia Latour-Lambert), and intestinal epithelial Caco-2 TC7 cells (kindly provided by P. Sansonetti) were cultured in Dulbecco's modified Eagle's medium (DMEM, Thermo Fisher Scientific #10566016) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS, Sigma-Aldrich #F7524) at 37 C, 5% (HeLa) or 10% (Caco-2) CO 2 . Cell lines were checked negative for mycoplasma. Bacterial strains and culture The following streptomycin-resistant Shigella flexneri 5a M90T-Sm (GenBank #CM001474.1) derived strains harboring the pWR100 virulence plasmid, Materials Availability We are glad to share all reagents generated in this study without restriction. Please contact the lead contact (jost.enninga@pasteur.fr) without restriction. Code Availability The published article includes all datasets analyzed during this study, 1984.

. Weiner, All Shigella-containing experiments were performed with Afa-I strains, besides experiments shown in Figures 6E-6G and S7F-S7H, which were performed with poly-L-lysine-coated Shigella. For complementation, the infection cultures of Shigella icsB -(compl.) and icsB -(C306A) strains were grown in 2 mM ITPG before invasion assays. In general, bacterial dilutions were prepared for the following MOIs: live imaging 96-well format MOI 5-150 (normally MOI in range 15-40 to exclude effects of phagocytic load), live imaging 35 mm glass bottom m-Dishes (Ibidi, #81158) MOI 15 and poly-L-lysine treated bacteria MOI 50. Other bacterial pathogens, like Salmonella or E. coli InvA, were grown similarly in their corresponding medium. Salmonella infections at MOI 50 were started maximal 10 min after spinning down the bacteria in pre-heated Eppendorf tubes and washing in pre-heated EM buffer. E. coli InvA were added to imaged cells at an MOI of 50. For fixed experiments, Shigella (WT) DsRed adhered for 10 minutes at 20 C, S. flexneri strains were grown in trypticase soy broth (TCSB) with 50 mg/ml ampicillin, and cultured at 37 C on TCSB agar including 0.01% congo red to select for functional T3SS system. S. Typhimurium was grown at 37 C in lysogeny broth (LB) medium supplemented with 0.3 M NaCl and 50 mg/ml ampicillin, while E. coli InvA was cultured in LB medium with 100 mg/ml ampicillin. E. coli, 2016.

. Taylor, Cells were then transfected with the respective plasmids using X-tremeGENE 9 DNA transfection reagent (Roche, #6365779001) for 48 h (24 h for TOCA-1 and CDC42). pUC8::icsB(C306A)-ipgA construction: Mutagenesis to inactivate IcsB enzymatic activity in the pUC derivative was performed using the Q5â Site Directed mutagenesis kit (New Englands Biolabs #E0554S) as suggested by the manufacturer. Mutagenesis primers were icsB_C306_F (ATCTGAAAACgcTGCTGGTATGGCAC) and icsB_C306_R (TTACTGATTAATTTATAATTTGCC). First, plasmid was divergently amplified using mutagenesis primers and Pfu polymerase from the kit. Next, the template plasmid was digested with DpnI and the PCR product phosphorylated and ligated using the KLD enzyme mix according to the manufacturer instructions. Ligation products were transformed in chemocompentent E. coli DH5a and transformants were selected in LB agar supplemented with 100 mg/mL of ampicillin, pGFP-2XFYVE from Philippe Benarock, pmCherry-2xFYVE from Harald Stenmark, pEYFP-LAMP1 from Walther Mothes, and pEGFP-LC3 from Thomas Wollert. The plasmids pEGFP-C3-Actin, pOrange-C3-Actin, pEGFP-N1-Galectin-3, and pOrange-Galectin-3 have been described previously, vol.1, pp.3-3, 2000.

D. Cytochalasin, For each inhibitor, working concentrations were identified that did not affect cell viability in the duration of the experiment, but showed clear effects on either cell shape or actin rearrangements during infection. In general, cells were preincubated for 40 min at 37 C in EM buffer containing the corresponding inhibitor. Afterward, bacteria were added in EM buffer with the same inhibitor concentration and imaged immediately. For Cytochalasin D, cells were not pre-incubated and the inhibitor was added with the bacteria. Following siRNAs were used in siRNA transfections, Enzo Life Sciences Inc, #BML-T109-0001), 10 mM SMIFH2 (Sigma-Aldrich, #S4826), 5 mM ML141 (Tocris Bioscience, #71203-35-5; of note: we observed solubility limitations with this inhibitor, therefore the effective concentration will be lower), 25 mM ETH1864 (Tocris Bioscience, #3872, vol.10, pp.15-27632

, DNA transfection reagent. In parallel, upscaled samples for western blot quantification were prepared in 6-well plates and for live imaging at the DeltaVision wide-field microscope in 35 mm glass bottom m-Dishes (Ibidi, #81158) (Figures 4A-4C, 6C, 6D, S3A, and S6A)

. 1g, . 2b, . 3b-3d, . 4d-4f, . 5a et al., image acquisition was performed using an inverted epifluorescence Nikon Ti-E widefield microscope, Light microscopy For quantitative screening experiments (Figures 1F, vol.2

, Shigella (WT) infections were monitored as control in each experiment. To detect host protein recruitment to Shigella-induced endocytic compartments (BCV, IAMs), time-lapse movies of high resolution were recorded at 37 C on a DeltaVision Elite (GE Healthcare) widefield microscope using a 60 3 /1.42 NA oil objective and a step size of 0.25-0.35 mm (Figures 1A-1C, Working Distance (WD)) N-Plan air objective, an automatic programmable xy stage, and the Nikon perfect focus system. Cells were imaged every 1-2 min for 2-3 h inside a 37 C heating chamber, vol.2, pp.4-4

, FRAP measurement and data analysis Live-cell FRAP experiments (Figures 1D and 1E; Videos S3, S4, and S5) were performed at an inverted Perkin Elmer UltraView VOX confocal spinning disk microscope equipped with a FRAP module and Volocity software

N. A. , oil objective with a single Z plane. Cells expressing actin-GFP in 35 mm glass bottom culture dishes were imaged at 37 C in EM