Figure 5: The CRIB motifs in mammalian CRN7 and their impact on the rescue activity of the protein.
From: Novel Coronin7 interactions with Cdc42 and N-WASP regulate actin organization and Golgi morphology
(a) Sequence alignment of human CRN7-CRIB domains (NT and CT) with CRIB domains from human Coronin 1C and D. discoideum CRN7. The CRIB consensus shown on top. Similar amino acids boxed in yellow. Highly conserved amino acids marked in red. (b) In vitro binding assay for full length GFP-CRN7WT with Rac1 and Cdc42 GTPases in their CA/Q61L and DN/T17N forms. Glutathione-Sepharose beads coated with GST alone and GST fusions pre-loaded with GDP (DN) or GTPγS (CA) and incubated with lysates from HEK293T cells expressing GFP-CRN7. PonceauS staining shows the GST fusion proteins. GFP-CRN7 detected with GFP-specific mAb K3-184-2. (c) Quantification of GFP-CRN7 bound to Rac and Cdc42 proteins (CA and DN) using ImageJ. Input set at 100% (3 independent experiments; **P < 0.01). (d) Lysates from HEK293T cells transiently co-expressing GFP-CRN7 and Myc-tagged Cdc42 GTPase CA and DN forms used for co-IP. GFP-CRN7 pulled down using GFP microbeads. Precipitated Cdc42 detected with anti-Myc mAb 9E10. Immunoprecipitated GFP-CRN7 detected with mAb K3-184-2. GFP used for control. *indicates shorter exposure for the input, arrows point to Myc-Cdc42. (e) Co-localisation analysis of CRN7 and Cdc42 mutant proteins. GFP-CRN7 in green; blue, nuclei stained with DAPI, Myc-tagged GTPases, red. Yellow, regions of co-localisation. Scale bar = 10 μm. (f) The graph shows a higher CRN7 and Cdc42 DN colocalization coefficient (R) as calculated by Pearson’s correlation coefficient using the ImageJ plugin ‘Colocalization Finder’ (n = 25 cells, unpaired two-tailed t-test, **P < 0.01). (g) Mean F-actin intensity values from z-stack images of transfected cells, fixed and stained with phalloidin (n = 10 cells each, 2 independent experiments; *P < 0.05). AU, arbitrary units. (h) Quantification of cellular spreading area for transfected cells. Fixed cells stained with phalloidin (n = 12 cells each, each time point, 2 independent experiments; *P < 0.05). (i) Percentages of various transfected cells with a fragmented or compact Golgi (n = 32 cells each, 2 independent experiments). Data shown as mean ± SD.
