JCS018432 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Recruitment of GFP-SNX1 is restored using SL1344-ΔsigD bacteria complemented with SigD on a plasmid. Cells expressing GFP-SNX1 were infected with a SL1344-ΔsigD mutant that additionally carried a plasmid encoding SigD and its chaperone SigE (SL1344-ΔsigD-pSigDE), and imaged live. Selected frames and a region of interest (ROI) of a representative movie are shown; time is in seconds. Scale bar: 10 µm.

• Movie 1 -

  Movie 1. 3D-view of endogenous SNX1 at a plasma membrane ruffle. MDCK cells were infected with SL1344 for 15 minutes, then fixed, immunolabeled with anti-SNX1 (green), and treated with TRITC-phalloidin (red) and DAPI (blue). Optical z-slices were acquired (z=5.37 µm, 12 sections) and data sets were deconvolved (98% confidence), 3D-rendered and animated using Volocity software. Resulting movies were exported as.avi file before conversion to.mov file.

• Movie 2a
• Movie 2b
• Movie 2c
• Movie 2d -

  Movies 2a-d. Recruitment of SNX1 to sites of Salmonella invasion induces formation of highly dynamic long-range tubules. MDCK cells expressing GFP-SNX1 fusion protein were infected with the S. Typhimurium strain SL1344 at the time point that is readily identifiable by the increase in lucency in the fluorescent channel. Images were captured every 20 seconds for ∼25 minutes. Movies were compiled using Volocity software and exported at ten frames per second. (a) fluorescent channel and (b) phase contrast, (c) magnification of a region of interest (ROI) of movie in (a), (d) phase contract of same ROI.

• Movie 3 -

  Movie 3. 3D-view of GFP-SNX1-meshowrks at sites of infection. HeLa cells expressing GFP-SNX1 were infected with SL1344 for 15 minutes, then fixed and treated with TRITC-phalloidin (red) and DAPI (blue). Optical z-slices were acquired (z=4.76 µm, 29 sections) and data sets were deconvolved (98% confidence), 3D-rendered, animated and exported using Volocity software.

• Movie 4a
• Movie 4b -

  Movie 4. No pronounced recruitment of GFP-SNX1 is observed upon infection with the SL1344-ΔsigD mutant. Cells transfected with GFP-SNX1 were infected with a SL1344 mutant lacking the inositol phosphatase SigD (ΔsigD). Images were captured every 20 seconds. Movies were compiled using Volocity software and exported at ten frames per second. (a) fluorescent channel and (b) phase contrast.

• Movie 5a
• Movie 5b -

  Movie 5. Recruitment GFP-SNX1 is restored using SL1344-ΔsigD bacteria complemented with SigD on a plasmid. Cells transfected with GFP-SNX1 were infected with a SL1344-ΔsigD mutant complemented with a plasmid encoding for SigD and its chaperone SigE (SL1344-ΔsigD-pSigDE), and imaged live. (a) fluorescent channel and (b) phase contrast.