Supplemental Material

This article contains the following supporting material:

• Supplemental Video 1 - S. flexneri moves faster in the presence of WT versus RRI villin Time-lapses of GFP S. flexneri infecting MDCK cells expressing mCherry-tagged WT and RRI villin. The incorporation of villin in the actin tails that propel the bacteria allows the observation of both GFP bacteria and their associated comet tails. One frame was acquired every 5 seconds for 5 minutes. Each frame is the result of the Z projection of 4 planes separated by 1.5μm. The video is accelerated 50 times.
• Supplemental Figure 1 - Localization of the RRI mutations in the structure of the G1-G3 domains of gelsolin A, alignment of the amino-acid sequence of villin and gelsolin for the two regions mutated in RRI villin. To indicate their location in the crystal structure, the mutated amino-acids indicated in capital letters are in purple for R₁ and in pink for R2. B, structure of the G1-G3 domains of gelsolin in an inactive calcium free conformation (PDB 1DON, (Burtnick et al. , 1997)). The three domains of gelsolin depicted in different greens are annotated in the figure. C, structure of the G1-G3 domains of gelsolin interacting with actin in the presence of calcium (PDB 1RGI, (Burtnick et al. , 2004)). The same color code is used for gelsolin domains ; actin is in gray. Calcium ions bound to gelsolin and to actin are depicted in light blue and light pink spheres respectively. The calcium ion shared by gelsolin and actin is in red. The inset shows an enlarged and vertically rotated view of this shared calcium. The mutated residues in R₁ and the actin glutamic acid 167 implicated in this interaction are indicated by ball-and-stick representation.
• Supplemental Figure 2 - The severing activity of RRI villin is still sensitive to calcium. Depolymerization of F-actin in the presence of decreasing calcium concentrations (200, 100, 50 and 0μM) for WT (red to yellow) and RRI (purple to mauve) villin. Pyrene-labeled actin filaments were diluted below the critical concentration of the pointed end (100nM) in the presence of 60nM villin. No villin, control in the absence of any severing protein.
• Supplemental Figure 3 - WT and RRI villin are able to cross-link actin filaments Low speed sedimentation assay to test F-actin cross-linking ability of RRI villin. F-actin was incubated alone (lanes 1-2) or with villin WT (lanes 3-4), RRI (lanes 5-6), and Δ7 (lanes 7-8), and centrifuged at low speed (8,000 x g) for 15 minutes to pellet only cross-linked filaments. Supernatant (S) and pellet (P) fractions were visualized by Coomassie blue staining. The majority of actin is found in the pellet in the presence of WT and RRI villin whereas it remains in the supernatant in the absence of villin or with the bundling mutant, villin Δ7. Molecular weights (MW) are indicated on the last lane.