JCS040345 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. RhoG and Elmo2NT are recruited to SGEF-induced dorsal ruffles. HUVEC were transfected with myc-SGEF and either GFP-RhoG or GFP-Elmo2NT, and immunostained using anti-myc antibody. RhoG and Elmo2NT colocalise with SGEF in dorsal ruffles (best seen in merged images). Bar, 10 μm.

• Supplemental Figure S2 -

  Fig. S2. Endogenous RhoG is recruited to Yersinia cell contact site. (A) HUVEC were infected with Yersinia strain WA-C for 30 minutes and endogenous RhoG was detected with immunofluorescence using anti-RhoG antibody (green fluorescence). Boxes indicate areas that were enlarged about threefold. Bar, 6 μm (upper panel) or 2 μm (lower panel). (B) HUVEC were transfected with RhoG siRNA and immunostained using anti-RhoG antibody. No specific RhoG signal could be detected in the RhoG knock down HUVEC. Bar, 6 μm.

• Supplemental Figure S3 -

  Fig. S3. Intracellular localisation of YopE and YopEΔMLD. (A) For disruption of the Golgi apparatus, Brefeldin A (Sigma-Aldrich, Munich, Germany) was added to GFP-YopER144A transfected HUVEC for 60 minutes at a concentration of 5 μg/ml and Golgi was immunostained with anti-GS27 antibody. GS27 staining was dispersed and the Golgi-specific localisation of YopE was abolished (compare with Fig. 7). Merged image is the overlay of green and red channel. Bar, 10 μm. (B) Bacterially translocated and transfected YopER144A localise to the same compartments in HUVEC. Cells were either infected with Yersinia strain WA-C(pTTSS+pYopER144A) for 60 minutes and then immunostained for YopE or transfected with GFP-YopER144A. Bar, 10 μm. (C) Cytoplasmic localisation of YopEΔMLD. HUVEC were transfected with GFP-YopER144AΔMLD and stained for F-Actin with Alexa-568-labelled phalloidin to visualize the cell border. Merged image is the overlay of green and red channel. Bar, 10 μm.

• Supplemental Figure S4 -

  Fig. S4. YopEΔMLD inactivates Cdc42 and TC10. (A) Cos-7 cells were infected with indicated Yersinia strains for 2 hours and subjected to GST-PAK-CRIB pull-down assay. Endogenous Cdc42 was detected with an anti-Cdc42 antibody. (B) Cos-7 cells were transfected with GFP-TC10 and then infected with indicated Yersinia strains for 2 hours. Active GFP-TC10 was pulled down with GST-PAK-CRIB and detected by western blotting using anti-GFP antibody.

• Supplemental Figure S5 -

  Fig. S5. YopE reduces Elmo2NT recruitment to Yersinia. HUVEC were transfected with GFP-Elmo2NT and then infected with WA-C(pTTSS+pYopER144A) or WA-C(pTTSS+pYopE). GFP-Elmo2NT recruitment was quantified after 15 and 45 minutes of infection. Each bar represents mean±s.d. (error bars) of three different experiments with at least 60 cells analysed per experiment.

• Movie 1 -

  Movie 1. HUVEC were transfected with GFP-RhoG and infected with Yersinia strain WA-314. Frames were acquired every 5 seconds in the green channel and recorded for 23 minutes. Video shows protracted and alternating GFP-RhoG recruitment to the bacteria cell contact site. Fig. 3 shows single frames of the video.