Structure of pirl1 and related compounds and corresponding IC₅₀ in PIP₂-stimulated actin polymerization assays.

Purification and identification of SOP1 as Arp2/3 complex.
a, Purification scheme. b, Silver-stained SDS-PAGE of SOP1-containing Superdex 200 fractions and corresponding maximal actin polymerization rates from pirl1-inhibited, PIP₂-induced pyrene-actin assays supplemented with pooled pairs of adjacent column fractions (e.g. 37/38, 39/40), as in Figure 1c. c, Coomassie-stained SDS-PAGE of SOP1-containing Superdex 200 fractions (pooled fractions 38-42) and native bovine Arp2/3 complex. Lower panel shows corresponding western blot for the Arp2 subunit of Arp2/3 complex. d, Pirl1 does not directly inhibit the Arp2/3 complex. Xenopus egg extracts supplemented with pyrene-actin were treated with 5 μM pirl1 or DMSO vehicle and then stimulated with either 10 μM PIP₂ liposomes or 300 nM GST-VCA to directly activate the Arp2/3 complex.

SOP2 is the native complex of Cdc42/RhoGDI.
a, Silver-stained SDS-PAGE of SOP2-containing Superdex 200 fractions and corresponding maximal actin polymerization rates from pirl1-inhibited, PIP₂-induced pyrene-actin assays supplemented with each fraction. Asterisks indicate proteins with elution profiles correlating with SOP activity. b, Amino acid sequences of Xenopus Cdc42 and RhoGDI are shown and sequences of tryptic peptides identified by mass spectrometry in SOP2-containing fractions (51-54) are indicated in bold. c, Graph of SOP activity and densitometry of bands corresponding to Cdc42 and RhoGDI in each Superdex 200 fraction (shown in a). d, Cdc42 western blot analysis of recombinant Cdc42, two concentrations of partially-purified, native bovine Cdc42/RhoGDI complex, and SOP2-containing fraction 52. e, Recombinant Cdc42/RhoGDI complex exhibits SOP activity. Xenopus egg extracts supplemented with pyrene-actin were preincubated with 5 μM pirl1 and the indicated concentrations of recombinant Cdc42/RhoGDI complex, and PIP₂ liposomes were added to induce actin polymerization.

Pirl1 reversibly inhibits the formation of actin-rich membrane ruffles in PMA-stimulated cells.
a, BS-C-1 cells were either fixed directly (unstimulated) or simultaneously treated for 15 minutes with 250 ng/ml phorbol myristate acetate (PMA) and either DMSO vehicle (labeled “PMA”), 50 μM pirl1, compound 1, or inactive control compounds 6. Cells labeled “Pirl1 washout” were treated for 15 minutes with 50 μM pirl1 alone and then the media was removed and replaced with media lacking pirl1 for 1 hour prior to stimulation with PMA as above. All cells were fixed and stained with Alexa 488-labeled phalloidin to visualize the filamentous actin cytoskeleton. Scale bar is 50 μm. b, Quantitative analysis of the experiment shown in a. The percentage of cells exhibiting actin-rich membrane ruffles are shown for each condition. Number of cells counted for each condition are shown beneath each bar.

The biochemical suppression approach and initial characterization of suppressor activity.
a, Biochemical suppression of small molecule inhibition. A small molecule is added to cytoplasmic extract to partially inhibit the activity of interest. Separately, uninhibited extract is fractionated and individual fractions are added to the inhibited extract. Fractions that suppress compound inhibition in the activity assay are then fractionated further. The suppressor activity is purified by iterative rounds of fractionation and activity assays. b, Pirl1 inhibits PIP₂-induced actin polymerization in Xenopus egg extract. Extracts supplemented with pyrene-actin (HSS) were pre-incubated with the indicated concentrations of pirl1 or DMSO vehicle, and 10 μM PIP₂ liposomes were added (as indicated) to stimulate actin filament nucleation. Actin polymerization was detected by the fluorescence increase of pyrene-actin upon incorporation into filaments. c, Assay for suppressor activity. The indicated concentrated fractions from uninhibited extract fractionated by cation exchange chromatography were mixed with complete extract containing pirl1 (5 μM final concentration) and 10 μM PIP₂ liposomes were added to induce actin polymerization. d, The suppressor activity does not titrate pirl1 non-specifically and is PIP₂-dependent. Actin polymerization was monitored in uninhibited extracts to which fraction 9, PIP₂ liposomes, or both were added.

Pirl1 inhibits activation of Cdc42 in Xenopus egg extract and guanine nucleotide exchange on Cdc42/RhoGDI complex in purified protein assays. a, PIP₂-mediated activation of Cdc42 in Xenopus egg extract is inhibited by pirl1. Extracts were pre-treated with different concentrations of pirl1, related compounds (1-8), or DMSO vehicle as indicated. After PIP₂ liposome stimulation in the presence of GTPγS, activated Cdc42 was co-precipitated using the p21-binding domain of Pak kinase (GST-PBD) bound to glutathione-agarose beads. Cdc42 was detected by western blotting. b, Pirl1 inhibits Dbs-mediated nucleotide exchange on purified recombinant Cdc42/RhoGDI complex. 0.5 μM Cdc42/RhoGDI complex was incubated with 1 μM RhoGDI, 30 nM DH-PH domain of Dbs, [³⁵S]GTPγS, 100 μM PIP₂ liposomes and either 25 μM pirl1, compound 6, or DMSO vehicle as indicated. At each time point, protein-bound [³⁵S]GTPγS was captured by filtration and quantitated by scintillation counting. c, Pirl1 inhibits EDTA-mediated nucleotide exchange on purified recombinant Cdc42/RhoGDI. Assays were conducted as in b, except that 4 mM EDTA was used instead of Dbs. d, Dose-dependence of pirl1 and compound 6 inhibition of EDTA-mediated nucleotide exchange on purified Cdc42/RhoGDI complex. Reactions as those in c were conducted with the indicated concentrations of pirl1 or compound 6 and protein-bound GTPγS was quantified following 3 minutes of incubation. Error bars indicate standard error (n=3). e, Pirl1 does not inhibit nucleotide exchange on non-prenylated Cdc42. Assays conducted as in c, except that soluble Cdc42 was used instead of Cdc42/RhoGDI complex.