Supporting Information - PNAS

Supporting Information Junemann et al. 10.1073/pnas.1611024113 SI Materials and Methods Plasmids. To obtain a ForG-FL (amino acids 1–1,074) GFP-fusion expression vector, cDNA from vegetative Dictyostelium discoideum AX2 laboratory WT cells was amplified by PCR and assembled in two steps into plasmid pDGFP-MCS-Neo (69) as indicated in Table S1. After sequence verification (European Nucleotide Archive accession no. LN901451), this vector served as a template for all ForG-derived expression plasmids. For expression of ForGΔDAD (amino acids 1–1,040), the 3′ half of the coding sequence was substituted with a PCR PstI/SalI fragment using the unique PstI site in the forG gene. For expression of YFP–ForG-N fusion protein in Dictyostelium, ForG-N (amino acids 1–423) was amplified by PCR as a BglII/SpeI fragment and inserted into the corresponding sites of the extrachromosomal expression plasmid pDM304-YFP. This plasmid carries an N-terminal YFP tag that was obtained by PCR from pDEXRHMCS-DYFP and carried BamHI/BglII sites (70) for insertion into the single BglII site of pDM304 (71). Similarly, the constructs YFP-ForG-NΔRBD (amino acids 119–423) and YFPForGΔRBDΔDAD (amino acids 119–1,040), both of which lack the putative N-terminal RBD, were obtained by PCR and subsequent insertion as BamHI/SpeI fragments into the BglII/SpeI sites of pDM304-YFP. For coexpression with mRFP-tagged LifeAct (30), the corresponding oligonucleotide sequences were synthesized and ligated into the shuttle vector pDM330 (71). Subsequently, the entire LifeAct-mRFP expression cassette was excised by NgoMIV and inserted into the same site of pDM304–YFP–ForG-N. For expression of RasB or RasG fusion protein, the respective coding sequences were amplified by PCR and inserted into BglII/SpeI sites of pDM304-YFP. The pan-probe for active Ras YFP-Raf1-RBD (amino acids 50–132; UniProt Q99N57) containing just the RBD of the RAF protooncogene serine/threonine protein kinase from mouse was generated by PCR amplification from cDNA and was inserted as a BglII/SpeI fragment into the respective sites of pDM304-YFP. Construction of the F-actin probe LimEΔcoil-GFP for life cell imaging has been described (72). Double-expression plasmids for in vivo interaction experiments of ForG-N with RasB or RasG by BiFC of the YFP variant Venus were adapted from Ohashi et al. (50) and constructed as follows. The coding sequence for the C-terminal beta-sheet of Venus (amino acids 211–238), followed by a 30-aa linker, a BglII site, and ForG-N sequences, was synthesized by GenArt (Life Technologies) and inserted as a BamHI/SpeI fragment into BglII/SpeI sites of expression vector pDM304 or pDM326 (71) to obtain pVC–ForG-N. This approach allowed selection of clones either with G418 (pDM304) or Blasticidin S (pDM326), respectively. The major part of Venus (amino acids 1–210) was synthesized with a 10-aa linker, followed by the RasB-WT coding sequence. This fragment was inserted into the BglII/SpeI sites of the expression cassette of the pDM344. Subsequently, the entire expression cassette was excised by NgoMIV and inserted into the same site of pVC–ForG-N to obtain pBiFC-ForG + RasB. Variants of RasB and RasG were generated by replacement of the RasB-WT coding sequence with corresponding BglII/SpeI fragments. RasB-S20N served as a negative control. For expression of ForG constructs in Escherichia coli, ForG-3P (amino acids 562–1,074) and ForG-1P (amino acids 599–1,074) were amplified by PCR and inserted into the BamHI and SalI sites of pGEX-6P-1. The ForE (Dictybase gene ID DDB_G0269626) construct ForE-4P (amino acids 1,009–1,561), previously used as a control (31), was generated in two steps due to the highly repetitive sequence elements encoding at its 5′ end. Junemann et al. www.pnas.org/cgi/content/short/1611024113

First, a fragment encoding just two proline-rich stretches (amino acids 1,042–1,561) was amplified by PCR from genomic DNA and inserted into BamHI and SalI sites of pGEX-6P-1. Then, a synthetic fragment encoding the entire FH1 domain with four proline-rich stretches was used to exchange the shorter FH1 domain by using BamHI/HindIII restriction sites. The forG KO vector was constructed by amplification of a 411base pair 5′ PstI/BamHI fragment and a 633-base pair 3′ SalI/ HindIII fragment of the forG gene from genomic DNA, and the fragments were subsequently inserted into the corresponding sites of pLPBLP (73). The rasB targeting vector was constructed by amplification of a 566-base pair 5′ PstI/BamHI fragment and a 503-base pair 3′ SalI/HindIII fragment of the rasB gene from genomic DNA, and the fragments were subsequently inserted into the corresponding sites of pLPBLP (73). Sequences of all used oligonucleotides are provided in Table S1. All original pDM vectors were obtained from the Dicty Stock Center (74). The sequences of all generated constructs were verified by sequencing. Cell Culture and Transfections. D. discoideum AX2-214 cells were cultivated at 21 °C in HL5-C medium with glucose (Formedium) and transfected by electroporation using routine protocols. Electroporation was performed with an Xcell gene pulser (Biorad) using the preset protocol 4-6 for Dictyostelium. The forG− clone 10 (strain JFL110) and rasB− (strain JFL111) cells were obtained by transfection of AX2 WT cells with the respective KO vectors linearized with BamHI and SalI. Stably transfected cells were selected with 10 μg·mL−1 Blasticidin S (InvivoGen). Gene disruption was confirmed by PCR and, in the case of forG−, also by immunoblotting. In the same way, cell lines ectopically expressing fluorescence fusion proteins were obtained by transfection with the appropriate plasmids and subsequent selection with either 10 μg·mL−1 Blasticidin S or 10 μg·mL−1 G418 (InvivoGen). ScrA− (strain JFL100) cells have been described previously (53). Protein Purification. Expression of GST-tagged proteins was induced in E. coli strain Rosetta 2 (Novagen) with 1 mM isopropyl-β-Dthiogalactoside at 21 °C for 12 h. The proteins were subsequently purified from bacterial extracts by affinity chromatography using glutathione-conjugated agarose (Macherey–Nagel), followed by size exclusion chromatography. In case of ForG-1P, ForG-3P, and ForE-4P, the GST-tag was cleaved off by PreScission protease (GE Healthcare) and removed, together with uncleaved protein, by size exclusion chromatography using an Äkta Purifier System equipped with a HiLoad 26/600 Superdex 200 column (GE Healthcare). The fractions containing the respective formin construct were pooled and dialyzed against storage buffer [150 mM KCl, 1 mM DTT, 60% glycerol, and 20 mM Hepes (pH 7.4)] and stored at −20 °C. Recombinant, tag-free Dictyostelium PFN I was purified by poly-L-proline affinity chromatography (75). Purification of recombinant heterodimeric D. discoideum Cap32/34 (referred to as CP in this work) was performed as described (76). Actin was purified from acetone powder of rabbit skeletal muscle according to standard procedures (77) and labeled on Cys374 with ATTO 488 maleimide (ATTO-TEC) or with N-(1-Pyrenyl)maleimide (Invitrogen), respectively. Pyrene-Actin Assays. Recombinant ForG-3P and ForE-4P were diluted in KMEI buffer [1× KMEI: 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 10 mM imidazole (pH 7.0)] to reach a final concentration of 1.18× KMEI in 170-μL aliquots that were placed in black, nontreated, 96-well microtiter plates (Brand GmbH). 1 of 13

Anti-foaming solution Extran AP33 (Merck) was added to a final concentration of 0.05%. Actin polymerization was initiated by injection of 30 μL of a 13.33 μM solution of 10% pyrene-labeled G-actin in G-buffer [5 mM Tris·HCl (pH 8.0), 0.2 mM ATP, 0.1 mM CaCl2, 0.5 mM DTT] into the protein mixture to reach a final concentration of 2 μM actin in 1× KMEI by the automated dispenser of a Synergy 4 fluorescence microplate reader (BioTek). After a 2-s mixing step, actin polymerization was monitored by measuring the increase of fluorescence of pyrene-actin at 364-nm excitation and 407-nm emission wave lengths for 20 min. Dilution-induced depolymerization assays were essentially performed as described before (26). Data analysis was performed using SigmaPlot 11.2 (Systat Software, Inc.) and assuming one-site competition of CP and ForG-3P for filament barbed ends to calculate the IC50 for ForG-3P. The Kd of CP for filament barbed ends was calculated assuming one-site saturation kinetics and allowed us to calculate the apparent Kd of ForG-3P by the law of mass action at half-maximal displacement of CP using the equation: 2 · ½ForG0 − 1 · KdCP KdForG = ½CP0 [ForG]0 and [CP]0 are the initial concentrations at half-maximal displacement of CP. TIRFM. TIRFM was performed on an Olympus IX-81 inverted

microscope equipped with an Apo N 60× TIRF objective and cooled Hamamatsu Orca-R2 CCD camera (Hamamatsu). The flow chambers consisted of 24 × 40-mm glass coverslips (Menzel– Gläser) that were fixed to microscope slides by double-sided adhesive tape. The flow chambers were preincubated with 10% (mol/vol) fish gelatin in 1× KMEI for 10 min and rinsed with 1× KMEI containing 10 mg/mL BSA (Sigma). Proteins were prediluted in 1× TIRF buffer [20 mM imidazole (pH 7.4), 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 50 mM DTT, 0.2 mM ATP, 15 mM glucose, 2.5 mg/mL methylcellulose, 20 μg/mL catalase, 100 μg/mL glucose-oxidase]. The assays were started by adding G-actin (1.3 μM final concentration, 23% ATTO488-labeled) to these mixtures and flushing them into the flow chambers. Images were captured every 2 s, with exposure times of 50–100 ms for at least 600 s. The pixel size corresponded to 0.21 μm due to the operation in 2 × 2 binning mode. The elongation rates of filaments were determined by manual tracking of growing barbed ends after processing the time-lapse movies by background subtraction using a 50-pixel rolling ball radius in ImageJ software (NIH). At least 20 filaments from three movies per condition were measured. The nucleation efficacies were obtained by counting and averaging the number of actin filaments in an area of 80 × 80 μm 180 s after the polymerization reaction was initiated. F-Actin Binding and Cross-Linking Assays. For high-speed sedimentation assays, G-actin in G-Buffer, ForG-3P, and CP dilutions in 1× KMEI were cleared for 60 min at 4 °C at 150,000 × g in a Beckman Optima TL-100 ultracentrifuge. The reaction mixtures were incubated for 2 h at room temperature in 1× KMEI. Subsequently, the samples were centrifuged for 60 min as mentioned above, and the pellets were brought to the original volume in 3× SDS sample buffer diluted with 1× KMEI. To quantitate the amount of ForG-3P that cosedimented with actin, the amounts of the proteins in the pellet and supernatant fractions were determined densitometrically from four independent experiments after SDS/PAGE and Coomassie Blue staining using ImageJ software. Calculation of free and bound ForG-3P was based on determining the ratio of the band intensities of the 56-kDa band from the pellet and supernatant fractions. For low-speed sedimentation, 2 μM G-actin was polymerized in presence of various ForG-3P concentrations for 2 h at room temperature in 1× Junemann et al. www.pnas.org/cgi/content/short/1611024113

KMEI, sedimented for 60 min at 15,800 × g in an Eppendorf centrifuge, and analyzed on SDS/PAGE in supernatant and pellet fractions as described above. Antibodies and Immunoblots. Polyclonal antibodies against ForG were raised by immunizing a female New Zealand White rabbit with recombinant ForG-1P (amino acids 562–1,074) following standard protocols. Immunoblotting was performed according to standard protocols using undiluted hybridoma supernatants of PsA-specific mAb MUD1 (78), contact site A-specific mAb 33-294-17 (79), cortexillin-specific mAb 241-438-1 (80), or polyclonal anti-ForG antibody serum (1:250 dilution). Primary antibodies in immunoblots were visualized with phosphatase-coupled anti-mouse (1:1,000, no. 115-055-62; Dianova) or anti-rabbit IgG (1:1,000; no. 111-055-046; Dianova). Uncropped scans of immunoblots are shown in Fig. S8. Fluorescence Microscopy and Imaging. For life-cell imaging, growthphase cells expressing fluorescent fusion proteins were seeded onto 3.5-cm-diameter glass-bottom dishes (Ibidi) and allowed to adhere on the glass surface for 20 min. The cells were then washed several times with PB buffer [17 mM Na-K-phosphate (pH 6.0]. Mutants expressing BiFC-Venus constructs were grown in LoFlo-medium (Formedium) for at least 3 h to reduce background signal from HL5-C medium, and PB was additionally supplemented with (±)-6hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox; Sigma) antioxidant to counteract bleaching. Confocal imaging was performed using either an LSM510Meta confocal microscope (Zeiss) equipped with a 63×/1.3 Plan-Neofluar objective using the 488-nm and 543-nm laser lines or a Leica TCS SP8 X microscope equipped with an HC PL AP CS2 63×/1.4 oil objective (Leica Microsystems) using the 488-nm, 496-nm, 511-nm, 565-nm, and 577-nm laser lines. Qualitative analysis of cell morphology of growth-phase cells in HL5-C medium or PB was monitored by time-lapse imaging with an inverted Olympus IX-81 microscope equipped with 100× phase-contrast optics (Olympus) and a CoolSnap EZ camera (Photometrics). Localization of YFP-tagged ForG 1–423 in agar overlay assays was performed as described (25). LY294002 (Sigma) or CK666 (Sigma) dissolved in dimethyl sulfoxide (DMSO) was added to adherent cells in PB 5–10 min before confocal imaging; the corresponding amount of DMSO was added to control cells. Data were processed using ImageJ and CorelDraw software (Corel Corporation). Analyses of F-Actin Distribution in Phagocytic Cups. Dictyostelium cells engulfing TRITC-labeled yeast particles were fixed and stained for F-actin with Alexa Fluor 488 Phalloidin (Thermo Fisher Scientific). Confocal sections of cells with partially engulfed yeast were recorded using identical settings at the LSM510Meta confocal microscope. Fluorescence intensity profiles of the F-actin signal at phagocytic cups were then measured in ImageJ with a four-pixelwide segmented line. Due to deviations of yeast size, the exact engulfment stage, and for reasons of comparability, the measured intensity profiles of the events were resampled to 100 data points, corresponding to 100% of the contour length with the resample function of MATLAB (MathWorks). Finally, the data were processed with Excel and SigmaPlot 11.2 (Systat Software, Inc.). Macropinocytosis Assays. Quantitative measurement of fluid-phase uptake was performed by standard protocol (58). In short, cells were shaken at 150 rpm for 1 h in a 30-mL HL5-C medium at a density of 6 × 106 mL−1, and TRITC-dextran (molecular mass of 65–85 kDa; Sigma) was then added to a final concentration of 2 mg/mL. Aliquots of 1 mL were taken at each time point and mixed briefly with 100 μL of Trypan Blue solution (4 mg·mL−1; Sigma) to quench fluorescence. The cells were quickly pelleted, washed twice with ice-cold PB, and lysed in 1 mL of lysis buffer [0.2% Triton X-100, 50 mM Na2HPO4 (pH 9.3)]. Fluorescence was measured directly after lysis at 544-nm excitation and 574-nm 2 of 13

emission wavelengths using a Synergy 4 microplate reader (BioTek). Obtained values were normalized to protein content, which was assessed in the microplate reader using a Pierce 660 protein assay (Thermo Fisher Scientific). For qualitative confocal microscopy sections at the Leica TCS SP8 X, the fluorescence settings were optimized with one batch of AX2 cells that were preincubated for 90 min with tracer-containing medium (1 mg·mL−1 TRITC-dextran) in glass-bottom dishes (Ibidi) at a cell density of 1.5 × 104 cm−2. After this setup step, the actual experiment with null mutants and WT control was started in parallel, and images were taken every 30 min to record tracer enrichment in the cells. Contrast of images was adjusted uniformly using ImageJ and CorelDraw software. Phagocytosis Assays. Phagocytosis of TRITC-labeled yeast particles was quantified following a standard protocol (18). In short, cells were grown overnight in a shaken suspension at 150 rpm in HL5-C medium with glucose and adjusted to a density of 2 × 106 cells per milliliter in 20 mL of medium. Subsequently, TRITC yeast was added at a sixfold excess. Aliquots of 1 mL were taken at each time point and incubated on a cooled shaker for 3 min with 100 μL of Trypan Blue solution (4 mg/mL; Sigma). Subsequently, cells were pelleted, washed once with ice-cold PB, and resuspended in 1 mL of PB for immediate measurement in a Jasco Fluo FP-6500 fluorescence spectrophotometer at 544-nm excitation and 574-nm emission wavelengths. Obtained values were normalized to respective protein content (macropinocytosis). For qualitative comparison of phagocytosis, WT and mutant cells were seeded on glass-bottom dishes (Ibidi) at a cell density of 5 × 104 cells per square centimeter and a fivefold excess of TRITC-labeled yeast particles in PB, and were imaged after 30 min of incubation with the LSM510 Meta confocal microscope. To measure phagocytosis of bacteria, a clearance assay of Witke et al. (81) was performed with modifications. Dictyostelium cells were grown overnight in shaken suspension, washed with ice-cold PB, and adjusted to a suspension 2 × 106 cells per milliliter in 10 mL of PB shaken at 150 rpm. After 30 min, 10 mL of an E. coli strain B/r suspension in PB at an optical density of around 1.4 at 600 nm (OD600) was added. At the indicated time points, 1.5-mL samples were withdrawn, supplemented with NaAzide to a final concentration of 5 mM, and vortexed for 30 s to remove surface-bound bacteria (82). Subsequently, Dictyostelium cells were pelleted for 1 min at 700 × g, and the OD600 of the supernatant containing the bacteria was measured to assess the clearance rate. Individual experiments were normalized to an initial OD600 of 0.5, and the clearance rates were additionally adjusted to protein content. Analyses of Cell Migration. Quantitative analysis of random cell motility was performed as follows: Growth-phase cells in PB

Junemann et al. www.pnas.org/cgi/content/short/1611024113

buffer were monitored every 10 s for 15 min by time-lapse imaging using an inverted Olympus IX-81 microscope equipped with 10× phase-contrast optics (Olympus) and a CoolSnap EZ camera (Photometrics). Single-cell tracks were obtained from recordings with the Track Objects plug-in of Metamorph 7 software (Molecular Devices). Total data samples of each cell line contain more than 500 tracks from three independent experiments with at least 150 individual cells each. These data were further processed to obtain the average speed of single cells using a custom-built protocol operated in Excel (Microsoft). Average speed distributions of analyzed samples are presented in box plot representations using SigmaPlot 11.2 (Systat Software, Inc.). Boxes indicate the 25th through 75th percentiles, whiskers mark the 10th and 90th percentiles, and the outliers are the fifth and 95th percentiles. Y2H Assays. For protein–protein interaction studies the MATCHMAKER GAL4 Two-Hybrid System 3 (Clontech Laboratories) was used. ForG-N (amino acids 1–423) was inserted into the pGADT7 prey vector to yield Gal4-activation domain–ForGN. Likewise, Raf1-RBD (amino acids 50–132) and Byr2-RBD (amino acids 1–236; UniProt P28829) were inserted into pGADT7 as established positive controls for active Ras (49). All GTPases were inserted into the EcoRI and BamHI sites of the pGBKT7 bait vector. The coding sequences of the constitutively active and inactive forms were synthesized by GenArt (Life Technologies) and encode for Rho- and RasGTPases corresponding to the Dictybase entries listed in Table S2. Silent mutations of internal EcoRI and BamHI sites and codon optimizations were performed when necessary. The AH109 yeast strain was cotransfected with bait and prey vector, and grown on synthetic double-dropout agar lacking leucine and tryptophan according to the manufacturer’s instructions. Several colonies were collected and grown in synthetic dropout liquid media overnight and then subjected to dropout agar plates. Protein interactions were scored by growth of cells on agar plates for 4 d at 25 °C on triple-dropout (TD) or quadruple-dropout (QD) agar plates. TD plates lacked leucine, tryptophan, and histidine, and were supplemented with 3 mM 3-amino-1,2,4-triazole to suppress the leaky HIS3-reporter gene according to the manufacturer’s instructions. QD plates lacked leucine, tryptophan, histidine, and adenine for highest stringency. Statistical Analysis. Statistical analysis was performed using SigmaPlot 11.2 software (Systat Software). Statistical significance of differences between nonnormally distributed populations was determined by the Mann–Whitney U test. When data fulfilled the criteria of normality (Shapiro–Wilk test) and equal variance (Levene’s test), statistical differences were analyzed with a two-tailed, unpaired Student’s t test. Statistical differences are reported as *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and nonsignificant.

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Fig. S1. ForG accumulates at the leading edge in 2D confinement. To visualize ForG localization in the absence of endocytic cups, the formation of these structures was physically suppressed by overlaying the cells with a thin sheet of agar. In the agar overlay, YFP-tagged ForG-N expressed in WT growth-phase cells accumulated at the leading edge. (Scale bar: 10 μm.)


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CP (nM) Fig. S2. ForG competes with capping protein, but neither bundles nor interacts with the sides of actin filaments. (A) ForG does not bundle actin filaments in low-speed sedimentation assays. After polymerizing 2 μM G-actin in presence of varying concentrations of ForG-3P for 2 h, negligible but constant amounts of the formin were found in the pellet fraction over the whole concentration range. P, pellet; S, supernatant. (B) ForG efficiently competes with CP for barbedend binding. Two micromolar actin was polymerized in the presence of the proteins indicated for 2 h before high-speed sedimentation. In the controls, most actin was found in the pellet, whereas most ForG was found in the supernatant. In the presence of CP, most of the F-actin was found in the supernatant, demonstrating efficient barbed-end capping, resulting in many short filament species that were not pelletable at 150,000 × g. Notably, in presence of CP and the formin, most actin was found in the pellet, again demonstrating very efficient displacement of CP by the formin at filament barbed ends. (C and D) ForG at filament barbed ends can be displaced only by excess CP. (C) Two micromolar actin was prepolymerized for 2 h before addition of 125 nM ForG-3P. Following 30 min of incubation, varying concentrations of CP were added to the mixture and incubated for another 30 min before high-speed sedimentation. (D) Densitometric analysis of ForG-3P bands in the pellet fraction from experiments as shown in C. Notably, complete displacement of the formin was only seen at 16-fold excess CP, suggesting high-affinity binding of ForG to filament barbed ends (n = 4). (E–G) ForG also efficiently displaces CP from filament barbed ends in dilution-induced depolymerization assays. (E) Depolymerization of 0.1 μM F-actin in the presence of a fixed concentration of CP (10 nM) and varying amounts of ForG (5–500 nM). As opposed to CP, ForG-3P alone only weakly inhibited F-actin disassembly, suggesting that it operates as a leaky capper. However, already in the presence of substoichiometric concentrations (5 nM), ForG efficiently began to replace CP from barbed ends as evidenced by the increased depolymerization rates. (F) Normalized and averaged initial slopes of depolymerization curves from experiments shown in E were plotted against the concentration of ForG-3P to calculate the IC50 value for competition with CP for filament barbed ends assuming one-site competition (n = 4, mean ± SD). (G) Assuming one-site saturation kinetics, normalized and averaged initial slopes of F-actin depolymerization curves in the presence of varying concentration of CP alone yielded a dissociation constant (Kd) of CP for barbed ends (n = 4, mean ± SD). This constant, in turn, allowed us to calculate the Kd of ForG-3P for filament barbed ends, which is about 4.4 ± 1.1 nM (Materials and Methods). AU, arbitrary units. In A–G, all concentrations for ForG-3P and CP refer to the dimers.


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DD TD +3-AT Fig. S3. ForG does not interact with Rho family GTPases. (Left) ForG-N does not interact specifically with any of the 20 Rac proteins in the Y2H assay. Yeast were transformed with the indicated constructs and selected for the presence of prey and bait plasmids by growth on double-dropout (DD) media lacking leucine and tryptophan. Interactions were assayed by growth on stringent TD media additionally lacking histidine in the presence of 3 mM 3-AT. (Right) Two putative hits obtained with RacH and RacM were found to be unspecific because these GTPases also grew on TD media in the absence of the ForG-N bait. AD, Gal4-activation domain; 3-AT, 3-amino-1,2,4-triazole; BD, Gal4-binding domain.

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Fig. S5. Genetic inactivation of the rasB gene. Inactivation of the rasB gene was confirmed by two diagnostic PCR assays to screen for disruption (KO, Left) or the presence of the WT allele (Right) using specific primer pairs.


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Time (min) Fig. S6. RasB− cells display no major defects in macropinocytosis and phagocytosis of bacteria. (A) Growth of rasB− cells in liquid medium was moderately impaired compared with WT cells. A representative growth curve of three independent experiments is shown. (B) Impaired phagocytosis of bacteria by rasB− cells as assessed by clearance of E. coli strain B/r in the supernatant. A representative curve of three independent experiments is shown. (C) Accumulation of TRITC-dextran by macropinocytosis after 90 min of incubation was visualized by confocal microscopy. (Scale bar: 10 μm.) (D) Quantification of TRITC-dextran uptake by macropinocytosis revealed no major defects in the rasB− mutant (mean ± SD, n = 2).

YFP-ForG-N (1-423)

control

+ LY294002 + 25 µM

+ 50 µM

+ 50 µM

+ 100 µM

*

*

* *

control

+ CK666

Fig. S7. Recruitment of ForG-N is PIP3- but not Arp2/3 complex-dependent. Vegetative WT cells expressing YFP–ForG-N were seeded on glass-bottom dishes in low-osmolarity phosphate buffer and imaged either in the absence or presence of the inhibitors indicated. Notably, treatment of cells with the PIP3 kinase inhibitor LY2904002 at 25 μM or 50 μM was sufficient to delocalize ForG-N completely from the membrane and render it entirely cytosolic, supporting the view that PIP3, together with Ras signaling, is required for ForG targeting (also Fig. 6E). By contrast, treatment with the Arp2/3 complex inhibitor CK666 showed no significant effect at 50 μM and 100 μM, indicating that ForG does not require the Arp2/3 complex for targeting to the cups (white stars). Confocal sections of live cells are shown. (Scale bar: 5 μm.)


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Fig. 2A 170 130

kDa 170

100

130

70 55

100 40 70 35 55 25 kDa

40

Ctx ForG kDa

kDa 170 130

170 130

100 70

100

55 70 40 55 35 25

40

15

35 PsA

csA

Fig. 2B

Fig. 2C

kDa

Fig. 5A

kDa

kDa

170

170 130

170

130

100 130

70

100

55 70 100 40 55 35

70 40 SDS-PAGE ForG

GFP Fig. S8.

Uncropped images of blots and Coomassie Blue-stained gels.


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Table S1. Oligonucleotides used in this work Primer Dictyostelium constructs GFP-ForG-FL ForG-BU ForG-2600-BglD ForG-800-NsiUp ForG-SD-FL GFP-ForGΔDAD ForG-PstI-DWN ForG-DelDAD-SD pDM304-YFP DYFP-BamHI-F1 DYFP-BglII-R1 YFP–ForG-N ForG(1–423)-BglII-F1 ForG(1–423)-SpeI-R1 YFP-ForG-NΔRBD ForG-delRBD-BU ForG(423)-SpeD YFP-ForGΔRBDΔDAD ForG-delRBD-BU ForG(1–1,040)-SpeI-R2 LifeAct-mRFP Lifeact_N-BglII-F1 Lifeact_N-SpeI-R1 ForG-KO ForG BU KO ForG PstD KO ForG HU KO ForG Sal KO Bsr-AU KO-AD4 RasB-KO RasB-5KO-BU RasB-5KO-PD RasB-3KO-H3U RasB-3KO-SD RasB-KO-AD YFP-RasB RasB-BglU RasB-SpeD YFP-RasG RasG-BglU RasG-SpeD VN210-RasB-G15V/-S20N RasB-BglU RasB-SpeD YFP-Raf1-RBD mRaf(RBD)_BglU mRaf1(RBD)_SpeD E. coli constructs ForG-3P (562–1,074) ForG BamHI 3P ForG SD ForG-1P (599–1,074) ForG BamHI 1P opt ForG SD ForE-4P (1,009–1,561) ForE-C_BU ForE CProFull_SD

Sequence

Orientation

5′-CGCGGGATCCGCATGATATTATCAATTACATTTCAATTAGATC-3′ 5′-ATCAAAGATCTCCATGGAAAAGTATCCAAAAACTATG-3′ 5′-AAGTGAATGCATTGATTTTGGTTTCCAAATC-3′ 5′-CGCGTCGACTTATTTATTTAAATTTAATTGTGATCC-3′

Forward Reverse Forward Reverse

5′-AGGACCTGCAGCAATTCAACC-3′ 5′-CGCGTCGACTTATGGATCAGCACCACCAGCAATCTTTTTA-3′

Forward Reverse

5′-ACCGGATCCAAAAATGAGTAAAGGTGAAGAACTTTTC-3′ 5′-AATAGATCTGAGTCCGGATTTGTATAGTTCATCCATG-3′

Forward Reverse

5′-ATTAGATCTATGATATTATCAATTACATTTC-3′ 5′-AATACTAGTTTAAATTTTCTTTTCAAATTCATTAA-3′

Forward Reverse

5′-ACTGGATCCTCTTCAAAATGGGTAAAAGC-3′ 5′-ACAACTAGTTTAAATTTTCTTTTCAAATTCATTAA-3′

Forward Reverse

5′-ACTGGATCCTCTTCAAAATGGGTAAAAGC-3′ 5′-CGCACTAGTTTATGGATCAGCACCACCAGC-3′

Forward Reverse

5′-GATCTAAAAATGGGTGTCGCTGACCTGATAAAGAAGTTTGAAAGCATCTCCAAGGAAGAGA-3′ 5′-CTAGTCTCTTCCTTGGAGATGCTTTCAAACTTCTTTATCAGGTCAGCGACACCCATTTTTA-3′

Forward

5′-CGCGGGATCCGCATGATATTATCAATTACATTTCAATTA-3′ 5′-CGCCTGCAGCATTATATCTTTTTCATTTG-3′ 5′-GCCAAGCTTCTTCCTCTTCTTCAAATACTTC-3′ 5′-CGCGTCGACTTGGAATAACCAAGCTACTAAACGTT-3′ 5′-CAGTTACTCGTCCTATATACG-3′ 5′-GTTCTTGAGCGACATTCATAG-3′

Forward Reverse Forward Reverse Forward Reverse

5′-CGCGGATCCCAATTCCAATAGTAAAAAGTC-3′ 5′-GCGCTGCAGGATAGTAAGTGCACTCTTACTG-3′ 5′-GCGAAGCTTGTCAAGATGATTACAGTGCTA-3′ 5′-CGCGTCGACCTTTGATCTGCCTGGCTCTTTG-3′ 5′-CTAAAGGATTAAACAATCACCACCT-3′

Forward Reverse Forward Reverse Reverse

5′-GCGAGATCTATGTCAGTTTCAAATGAATATAAAT-3′ 5′-GCGACTAGTCTAAAGGATTAAACAATCACCACC-3′

Forward Reverse

5′-GCGAGATCTATGACAGAATACAAATTAG-3′ 5′-GCGACTAGTTTATAAAAGAGTACAAGCTTTTAATGG-3′

Forward Reverse

5′-GCGAGATCTATGTCAGTTTCAAATGAATATAAA-3′ 5′-GCGACTAGTCTAAAGGATTAAACAATCACCACC-3′

Forward Reverse

5′-GTAAGATCTGATTCTTCTAAGACAAGCAATACT-3′ 5′-GATACTAGTTTAATCCAAAAAATCCACTTGCAG-3′

Forward Reverse

5′-CGCGGATCCCCAATTTCTGGTGGTGGTGCA-3′ 5′-GCGGTCGACTTATTTATTTAAATTTAATTGTGATCCTCCAGATGAATTTTCAGG-3′

Forward Reverse

5′-GCGCGGATCCGGAGCACCGCCACCGCCCCCTCCGCCACCGCCTCCGGGTGGTAAAAAAGCAGGAGCACCA-3′ 5′-GCGGTCGACTTATTTATTTAAATTTAATTGTGATCCTCCAGATGAATTTTCAGG-3′

Forward

5′-GGATCCATTTCTGGTGCTCCACCCCCACCCCCA-3′ 5′-GGCGTCGACTTAATTTTTATTTGGACTTGT-3′

Forward Reverse


Reverse

Reverse

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Table S1. Cont. Primer S. cerevisiae constructs AD–ForG-N (1–423) ForG pGADT7 EcoRI ForG pGADT7 BamHI AD-Raf1-RBD (50–132) mRaf1-GAD-RU mRaf1-GAD-BD

Sequence

Orientation

5′-GCCGAATTCATGATATTATCAATTACATTTC-3′ 5′-GGCGGATCCTTAAATTTTCTTTTCAAATTCATTAA-3′

Forward Reverse

5′-CGCGAATTCGGA GATTCTTCTAAGACAAGCAATACT-3′ 5′-CGCGGATCCTTAATCCAAAAAATCCACTTGCAG-3′

Forward Reverse

AD, Gal4-activation domain; BD, Gal4-binding domain.

Table S2. GTPase variants used for Y2H in this study GTPase RasB G15V ΔCAAX RasB S20N ΔCAAX RasC G13V ΔCAAX RasD G12V ΔCAAX RasD S17N ΔCAAX RasG G12V ΔCAAX RasG S17N ΔCAAX RasS G12V ΔCAAX RasU G25V ΔCAAX RasW G20V ΔCAAX RasX G20V ΔCAAX RasY G21V ΔCAAX RasZ G20V ΔCAAX RapA G14V ΔCAAX RapB G34V ΔCAAX RheB Q64L ΔCAAX Rac1A G12V ΔCAAX Rac1B G12V ΔCAAX Rac1C G12V ΔCAAX RacA G12V ΔC RacB G12V ΔCAAX RacC G15V ΔCAAX RacD G17V RacE G20V ΔCAAX RacF1 G12V ΔCAAX RacF2 G12V ΔCAAX RacG G12V ΔCAAX RacH M13V ΔCAAX RacI S14V ΔCAAX RacJ D18V ΔCAAX RacL G12V ΔCAAX RacM Y14V ΔCAAX RacN G62L ΔCAAX RacO G13V ΔC RacP G58V RacQ A56L ΔCAAX

Inactive

Amino acids

Dictybase gene ID

Yes

1–193

DDB_G0292998

Yes

1–185 1–183

DDB_G0281385 DDB_G0292996

Yes

1–185

DDB_G0293434

1–190 1–209 1–212 1–209 1–212 1–210 1–182 1–201 1–181 1–190 1–190 1–189 1–319 1–189 1–188 1–254 1–219 1–189 1–189 1–197 1–196 1–201 1–205 1–192 1–185 1–215 1–213 1–376 1–181

DDB_G0283537 DDB_G0270138 DDB_G0270122 DDB_G0270124 DDB_G0270126 DDB_G0270140 DDB_G0291237 DDB_G0272857 DDB_G0277041 DDB_G0277869 DDB_G0268622 DDB_G0282365 DDB_G0286555 DDB_G0279605 DDB_G0293526 DDB_G0291976 DDB_G0280975 DDB_G0269176 DDB_G0276967 DDB_G0269178 DDB_G0269240 DDB_G0277897 DDB_G0292560 DDB_G0292816 DDB_G0289103 DDB_G0278009 DDB_G0277791 DDB_G0285453 DDB_G0278011

Movie S1. YFP–ForG-N localizes to macropinosomes (refers to Fig. 1B). Time-lapse recordings of a cell coexpressing LifeAct-mRFP and YFP–ForG-N acquired by confocal microscopy. (Right) Merged channels reveal colocalization of F-actin and ForG at macropinosomes. Time is minutes and seconds. (Scale bar: 5 μm.) Movie S1


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Movie S2. YFP–ForG-N accumulates at phagocytic cups (refers to Fig. 1C). Time-lapse recordings of a YFP–ForG-N–expressing cell engulfing a TRITC-labeled yeast particle acquired by confocal microscopy. Time is minutes and seconds. (Scale bar: 5 μm.) Movie S2

Movie S3. Random migration of forG-KO cells is not impaired compared with WT cells. Time-lapse recordings of WT and forG-KO cells acquired by phasecontrast microscopy. Note the extensive ruffles and markedly exaggerated pseudopodia-like membrane extension formed by forG-KO cells, which differs considerably from WT cells mainly forming cups. Time is minutes and seconds. (Scale bar: 10 μm.) Movie S3

Movie S4. ForG promotes actin filament elongation in the presence of PFN (refers to Fig. 5C). Time-lapse recordings of single actin filaments by in vitro TIRFM. The elongation of actin filaments (1.3 μM actin, 23% Atto488-labeled) and 5 μM PFN I either in the absence or presence of 10 nM ForG-1P or 10 nM ForG-3P was recorded over a period of 10 min. Time is minutes and seconds. (Scale bar: 10 μm.) Movie S4


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Movie S5. GFP-RasG localizes to macropinosomes (refers to Fig. 6B). Time-lapse recordings of a GFP-RasG–expressing cell during uptake of TRITC-dextran containing growth medium acquired by confocal microscopy. Time is minutes and seconds. (Scale bar: 5 μm.) Movie S5

Movie S6. GFP-RasB localizes to macropinosomes (refers to Fig. 6B). Time-lapse recordings of a GFP-RasB–expressing cell during uptake of TRITC-dextran containing growth medium acquired by confocal microscopy. Time is minutes and seconds. (Scale bar: 5 μm.) Movie S6

Movie S7. Actin dynamics in phagocytosis of yeast by WT cells (refers to Fig. 7). Time-lapse recordings of two LimEΔcoil-GFP (green) expressing WT cells engulfing TRITC-labeled yeast particles (magenta) acquired by confocal microscopy. Time is minutes and seconds. (Scale bar: 5 μm.) Movie S7


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Movie S8. Actin dynamics in phagocytosis of yeast by forG− cells (refers to Fig. 7). Time-lapse recordings of LimEΔcoil-GFP–expressing forG− cells engulfing TRITC-labeled yeast particles (magenta) acquired by confocal microscopy. Note disappearance of F-actin (green) in the base of the cups at later stages of engulfment. Time is minutes and seconds. (Scale bar: 5 μm.) Movie S8

Movie S9. Actin dynamics in phagocytosis of yeast by rasB− cells (refers to Fig. 7). Time-lapse recordings of LimEΔcoil-GFP (green)–expressing rasB− cells engulfing TRITC-labeled yeast particles (magenta) acquired by confocal microscopy. This mutant resembled forG− cells. Time is minutes and seconds. (Scale bar: 5 μm.) Movie S9

Movie S10. Actin dynamics in phagocytosis of yeast by scrA− cells (refers to Fig. 7). Time-lapse recordings of LimEΔcoil-GFP (green)–expressing scrA− cells engulfing TRITC-labeled yeast particles (magenta) acquired by confocal microscopy. Note the unstable cups that failed to align efficiently along the curvature of the yeast particle. Due to impaired phagocytosis, a large excess of yeast particles had to be used to capture a sufficient number of representative events. Time is minutes and seconds. (Scale bar: 5 μm.) Movie S10


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