Supplementary Figure 1 Screen for polypeptides that affect cellular actin filaments. (a) Table summarizing results from all polypeptides tested. Source shows organism, gene, and amino acid numbers used. Effect on F- actin is qualitative analysis of gross changes in cellular phalloidin intensity and distribution. In addition to human Gelsolin-GS1 and Salmonella SpvB described in this paper candidate proteins included: GFP-P2A (self-cleavable peptide) tagged constitutively active human Cofilin(S3A) (ref. 26), Vibrio cholerae MARTX VC actin crosslinking domain (refs. 20,21), Salmonella SipA actin crosslinking minimal domain (ref. 22), Photorhabdus luminescens TccC3 actin stabilizing ADP ribosylation domain (ref. 23), and Clostridium botulinum C2I ADP ribosylation domain (refs. 24,25). (b) Representative micrographs from the screen. HeLa cells were transiently transfected with GFP-tagged constructs or latrunculin A then fixed and stained for actin filaments (594-phalloidin, red) and nuclei (DAPI, blue). Due to these being transient transfections, expression levels varied from cell to cell. N = 1 (cofilin, MARTX VC), 2 (SipA), or 4 (TccC3, C2I) independent experiments. Scale, 50 m.
Supplementary Figure 2 SpvB targets a conserved residue on actin. SpvB ADP-ribosylates a conserved arginine on actin monomers (R177 in human beta actin)(ref. 8). Lineup shows cytoplasmic and muscle actins from diverse eukaryotes, with this residue (highlighted in yellow) conserved in all actin homologs. Accordingly, DeAct- SpvB functions in all eukaryotes we have tested thus far. NCBI Reference sequence numbers are shown in parentheses.
Supplementary Figure 3 Expression of DeActs in cultured cells. (a) DeActs decrease average cell surface area in HeLa cells. Graphs shows mean ± SEM. Statistical significance: one-way ANOVA and Dunn s multiple comparison post hoc test, **p<0.01; N = 3 independent experiments. (b) Live cell imaging to count HeLa cells expressing GFP, DeAct-GS1 or DeAct-SpvB at 24hr and 48hr post-transfection. Fold increase in GFP+ cells reflects the combination of proliferation and new expression minus cell death. *p<0.05, N = 4 independent experiments (150-200 GFP+ cells per experiment). (c) Wound healing of rat embryonic fibroblasts transiently transfected with DeActs or GFP control and scratch-wounded at time zero. Note that no defect in would healing is observed upon DeAct-GS1 transfection, most probably due to the fraction of untransfected and low DeAct-GS1 expressing cells. N = 2 independent experiments. (d) HeLa cells co-expressing the focal adhesion marker tagrfp-paxillin (red; ref. 28) and GFP, DeAct-GS1 or DeAct-SpvB (green). GFP expressing cells were treated with 10 µm latrunculin B for 30 minutes. Scale, 20 µm. (e) DeActs decrease the number of focal adhesions per cell, visualized by paxillin overexpression. Treatment with latrunculin B has the same effect. Graphs show mean ± SEM. Statistical significance: one-way ANOVA and Dunn s multiple comparison post hoc test, ***p<0.001, **p<0.01 (N = 2 independent experiments for all conditions, n = 17, 20, 27, 28 cells, left to right). (f-g) Live-imaging of HeLa cells co-expressing GFP, DeAct-GS1 or DeAct-SpvB and MARCKS-tagRFP-T shows that DeActs decrease the number of dynamic filopodia (e) and the total number of filopodia per cell (f). Graphs show mean ± SEM. Statistical significance: one-way ANOVA and Dunn s multiple comparison post hoc test, ***p<0.001, **p<0.01, (N = 3, 2, 2, 2 independent experiments, n = 16, 10, 12, 7 cells, left to right). (see also Supplementary Video 1). (h-i) DeAct-GS1 induced loss of actin filaments in mature OLs does not affect cell area. Trend lines in i shows nonlinear (exponential) fit; each data point is one cell. Data are from a single experiment representative of N = 3 independent experiments.
Supplementary Figure 4 Rapid induction of DeActs with third-generation TetON promoter. (a) Mechanism of action and construct design for doxycycline/tet-inducible DeActs using Clontech Tet-On 3G inducible expression vector. In presence of the tetracycline-regulated transactivator (Tet-On 3G, blue), doxycycline (Dox) induces dose-dependent expression from the TetON 3G promoter. Tet-On 3G HeLa cells (stably expressing Tet-On 3G transactivator; Clontech) were transfected with Tet-ON_GFP, TetON_DeAct-GS1, or TetON_DeAct-SpvB in (b-h). (b-c) Rapid induction of DeAct-Gsn expression. 24 hr post transfection, expression of TetON_DeAct-GS1 was induced by addition of 100 ng/ml Dox, and cells were imaged every 20 minutes. (b) shows frames from live cell imaging. (c) shows normalized GFP fluorescence following addition of Dox at time 0; average of 10 cells from one representative experiment of N = 3 independent experiments. (d) Live cell imaging of HeLa cells expressing TetON DeActs. Rate of single cell motility was measured after detection of GFP expression, imaging every 20 min for at least 3 hr. Statistical significance: one-way ANOVA and Dunnett s multiple comparison post hoc test, ***p<0.001, *p<0.05, n = 9, 23, 10 GFP+ cells, left to right. (e) Representative micrographs of cells expressing TetON DeActs. 24 hr post transfection, cells were treated with or without Dox as noted for 6 hr, then fixed and stained with Alexa 594-phalloidin and CellMask Blue to visualize cell morphology. (f-h) Cells were transfected with TetON_GFP (f), TetON_DeAct-Gsn (g) or TetON_DeAct-SpvB (h) as above and treated with or without Dox as noted overnight, then fixed and stained with Alexa 594-phalloidin. Scatter plots show actin filament level (phalloidin) as a function of DeAct expression; each data point is a single cell from one representative experiment of N = 2 independent experiments. Note that DeAct-SpvB expression intensities (h) are not comparable to GFP and DeAct-GS1 (f,g) due to low expression induced by 5 ng/ml Dox. Graphs in c,d show mean ± SEM. All scale bars, 50 m.
Supplementary Figure 5 Identification of transfected cells with dual-promoter, inducible DeActs. (a) Construct design for dual-promoter constructs with constitutive mcherry expression (CMV promoter) and inducible DeAct expression on the opposite strand. Left, dual-promoter TetON_DeAct-GS1; right, TetON_SpvB tagged with Escherichia coli dihydrofolate reductase destabilization domain (DHFRdd) with R12Y, G67S, Y100I mutations (ref. 11). (b-c) HeLa TetON 3G cells (stably expressing Tet transactivator protein) were transfected with dual-promoter CMV_mCherry/TetON_DeAct-GS1 (b) or CMV_mCherry/TetON_DHFRdd-SpvB constructs (c). After 24 hr DeAct expression was induced with 100 ng/ml Dox, then cells were fixed after 18 hr and stained for actin filaments with Alexa 647-phalloidin. (d-e) HeLa Tet-On 3G transfected as above were imaged every hour after addition of Dox. Note rapid induction of DeAct-GS1 visualized by GFP expression (d), and induction of attenuated (DHFRdd-tagged) SpvB demonstrated by rapid cell rounding (e). All scale bars, 50 m. b-e are representative micrographs from N = 3 independent experiments.
Supplementary Figure 6 Expression of DeActs in neurons (a) A representative example of dynamic axonal branches from a 5 minute time lapse acquisition. Closed arrow heads indicate growth and open arrow heads represent no growth or retraction (see also Supplementary Video 3). Scale, 10 µm. (b-c) Cortical neuronal migration after in utero electroporation with GFP, DeAct-GS1, or DeAct-SpvB. Scale, 50 µm. (c) Quantification of neuronal morphologies of GFP-positive neurons which migrated past the IZ. 1, leading and trailing process; 2, leading process only; 3, no processes; 4, trailing process only (N = 3 embryos from 3 different litters, n = 104, 135, 38 cells GFP, DeAct-GS1, or DeAct- SpvB). Graphs show mean ± SEM. Statistical significance: one-way ANOVA and Dunnett s multiple comparison post hoc test, *p<0.05.