Experimental Model and Subject Details

Unless otherwise stated, axenic Dictyostelium strains were derived from the MRC-Ax2 axenic strain (DBS0236849) provided by the Kay laboratory and were routinely cultured in filter sterilized HL-5 medium (Formedium) at 22°C. RacG mutants and corresponding parental strain (from the Devreotes group, Johns Hopkins, Ax2D) were kind gifts from Francisco Rivero (University of Hull) [42]. Growth rates were measured by seeding cells at 0.5 × 10⁵/mL in HL-5 and counting cell number twice daily for three days. Growth rate was then calculated by fitting an exponential growth curve using Graphpad Prism software. Cells were transformed by electroporation: 6 × 10⁶ cells were resuspended in 0.4 mL of ice-cold E-buffer (10 mM KH₂PO₄ pH 6.1, 50 mM sucrose) and transferred to 2 mm electroporation cuvette containing DNA (0.5 μg for extrachromosomal plasmids, 15 μg for knockout vectors). Cells were then electroporated at 1.2 kV and 3 μF capacitance with a 5 Ω resistor in series using a Bio-Rad Gene Pulser II. After 24 h transformants were selected in either 20 μg/mL hygromycin (Invitrogen), 10 μg/mL G418 (Sigma-Aldrich) or 10 μg/mL blasticidin (Melford).

Nonaxenic mutants were generated from the DdB (Wel) subclone of NC-4 shown to be the lab isolate with fewest duplications and parent strain of Ax2 [61]. The published DdB strain and corresponding NF1 mutants were gifts from Rob Kay (MRC-LMB, Cambridge) [37]. All DdB-derived strains were routinely maintained on lawns of Klebsiella aerogenes bacteria in SM agar plates (Formedium) at 22°C. 24 h prior to all experiments, cells were washed free of bacteria and transferred to HL5 medium supplemented with 20% fetal calf serum to axenically adapt [38]. DdB and its derivatives were transformed as described in [59]: 2 × 10⁶ cells were resuspended in 100 μl ice-cold H40 buffer (40mM HEPES, 1mM MgCl₂ pH 7.0) and 10μg DNA in 2mm cuvettes and exposed to 2 pulses of 400V 5 s apart and 3 μF capacitance in a square-wave electroporator (BTX EMC399, Harvard Apparatus). Cells were then grown in Petri dishes in a suspension of K. aerogenes in SorMC buffer (15mM KH₂PO₄, 2mM Na₂HPO₄, 50μM MgCl₂, 50 μM CaCl₂, pH 6.0), and transformants selected with G418 (Sigma-Aldrich) using either 5 μg/mL for knockouts or 10 μg/mL for extrachromosomal vectors.

BAR domain containing proteins were identified by multiple BLAST searches using Dictybase (www.dictybase.org) [62]. Coding sequences were then amplified by PCR from vegetative Ax2 cDNA adding compatible restriction sites for subcloning into the BglII/SpeI sites of the N- and C-terminal GFP-fusion Dictyostelium extrachromosomal expression vectors pDM1043 and pDM1045 (non-axenically selectable versions of the pDM modular expression system (Veltman et al., 2009)). Truncation and point mutants of RGBARG were also generated by PCR and expressed using pDM450 [63]. The rbgA (DDB_G0269934) knockout construct was generated by PCR fusion of ∼1Kb 5′ and 3′ recombination arms with the floxed blasticidin selection cassette from pDM1079, as described in detail in (Paschke et al., 2018). To select bacterially-grown cells, an identical construct was made using the G418 selection cassette from pDM1082. After transformation, independent clones were obtained by dilute plating in 96 well plates. Disruption of the RGBARG locus was screened by PCR from genomic DNA isolated from 1 × 10⁶ cells lysed in 100 μl 10mM Tris- HCl pH8.0, 50 mM KCl, 2.5mM MgCl₂, 0.45% NP40, 0.45% Tween 20 and 0.4 mg/mL Proteinase K (NEB). After 5 min incubation at room temperature, the proteinase K was denatured at 95°C for 10 min prior to PCR analysis. The Ras binding domain (RBD) of RAF1-GFP construct used as an active Ras reporter was a gift from Gareth Bloomfield [37]. The PTEN-mCherry/PH_PkgE-GFP, PH_PkgE-RFP/GFP-MyoIB and RBD-GFP/PAK1_CRIB-GFP co-expression constructs were all gifts from Peggy Paschke (Beatson Institute, Glasgow).