Figure 6: Effect of CRN7-CRIB motif mutations on Cdc42 binding and rescue potential of the proteins.
From: Novel Coronin7 interactions with Cdc42 and N-WASP regulate actin organization and Golgi morphology
(a) Conserved residues in the N- and C-terminal CRIB domains boxed in yellow were mutated to alanine marked in red. The GFP tag is at the N-terminus. (b) Binding assay for GFP-CRN7 WT and CRIB mutants (Mut1 and 2, left panel; Mut3 and 4, right panel) with Cdc42 GTPase (CA and DN). Glutathione-Sepharose beads coated with GST fusions, pre-loaded with GDP (DN) or GTPγS (CA) and incubated with lysates from HEK293T cells expressing the CRN7 WT or CRIB mutants. PonceauS staining shows the GST fusion proteins. Probing was with mAb K3-184-2. (c) Bar graph showing quantification of GFP-CRN7 WT and CRIB mutants bound to Cdc42 CA and DN using ImageJ. Input set at 100% (3 independent experiments; **P < 0.01 and n.s., not significant). (d) Quantification of expression levels of endogenous CRN7 in WT fibroblasts and ectopically expressed GFP-CRN7 WT and CRIB mutants in KO fibroblasts. Cell homogenates from equal numbers of cells were analyzed by western blotting. mAb K37-142-1 detected CRN7 and mAb K3-184-2 recognized the GFP-tagged proteins. GAPDH was used for normalization. (e) Mean F-actin intensity values were derived from z-stack images of transfected cells, fixed and stained with phalloidin (n = 15 cells each, 2 independent experiments; *P < 0.05 and **P < 0.01). AU, arbitrary units. (f) Quantification of cellular spreading area at 30 and 60 min. Fixed cells stained with phalloidin (n = 25 cells each, each time point, 2 independent experiments; *P < 0.05 and **P < 0.01). (g) Percentages of cells under each condition with a fragmented or compact Golgi represented as stacked columns (n = 25 cells each, 2 independent experiments). Data shown as mean ± SD.
