Supplementary Figure 2: CYRI-B interacts directly with active Rac1.
From: Fam49/CYRI interacts with Rac1 and locally suppresses protrusions
a – Summary of yeast 2-hybrid derived interacting regions between CYRI-B and constitutively active Rac1G12V. Confidence score A = best to D = low (see Methods). b – Schematic of clones from yeast two-hybrid screen (a). Overlaps define core Rac1-binding domain (RBD) of CYRI-B aa30-236. c - Schematic of GFP and GST constructs. eCFP-CRIB-PBD represents the CRIB region of PAK1, used as a positive control. d-f – MBP trap beads were loaded with recombinant WT or mutant maltose binding protein MBP-Rac1 protein and incubated with recombinant GST-CRIB-PBD, GST-CYRI-B RBDWT and GST-CYRI-B RBDR161D. Ponceau staining shows MBP-Rac1 (d). Binding (arrowhead) represents ratio relative to Rac1WT (e-f). (n = 3 independent experiments). See Supplementary Fig. 7. g-h - GST-pulldowns showing specific binding of CYRI-B RBD to Rac1. Lysate from CHL-1 cells expressing GFP (negative control), eCFP-CRIB-PBD, GFP-Rhotekin (reporter for Rac1 or RhoA activity respectively) or GFP-RBDWT mixed with immbolized GST, GST-Rac1WT, GST-Rac1Q61L, GST-RhoAQ63L, GST-Cdc42Q61L. Probed for GFP and GST (g). Binding (arrowhead) was assessed relative to GST (h). (n = 3 independent pull-down). See Supplementary Fig. 7. i - Control PLA of COS-7 cells co-expressing CYRI-BWT -HA and MYC-Rac1Q61L using anti-HA or anti- MYC antibodies. PLA signal (yellow), F-actin (magenta) and nuclei (blue). Scale bar = 50 μm. j - Cell area for each field of view (fov); average cell area was similar between conditions. (Myc-WT/WT-HA n = 34 fov; Myc-WT/R160-161D-HA n = 35 fov; Myc-T17N/WT-HA n = 34 fov; Myc-T17N/ R160-161D-HA n = 55 fov; Myc-Q61L/WT-HA n = 39 fov; Myc-Q61L/ R160-161D-HA n = 36 fov). k-l - Proximity ligation assay COS-7 cells on laminin and co-expressing pLIX-mVenus-CYRI-B-HA (WT or mutant R160/161D) and MYC-tagged Rac1 as indicated. (k). Randomized fields of view across 2 independent experiments. PLA signal was thresholded using Fiji and compared with cell area (see Methods) (l). One-way ANOVA with Dunn’s post-test between CYRI-BWT and MYC-Rac1 as indicated. Two-tailed Mann-Whitney test between CYRI-BWT and CYRI-BR160/161D for each MYC-Rac1 construct. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. (anti-HA n = 34 ; anti-Myc n = 33 ; Myc-WT/WT-HA n = 40 ; Myc-WT/R160/161D-HA n = 30 ; Myc-T17N/WT-HA n = 63 ; Myc-T17N/ R160/161D-HA n = 34 ; Myc-Q61L/WT-HA n = 22 ; Myc-Q61L/R160/161D-HA n = 40). Scale bar = 50 μm. Bar and scatter plots show data points with mean and s.e.m.
