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. 2020 May 29;9(6):1355. doi: 10.3390/cells9061355

Figure 1.

Figure 1

Intellectual disability-causing mutations in CYFIP frequently cause constitutive activation of WAVE regulatory complex (WRC). (A) Quantification of lamellipodia formation. Indicated, EGFP-tagged CYFIP1 constructs (or EGFP as control) were transfected into B16-F1 CYFIP1/2 KO cells (clone #3), and lamellipodia formation was assessed after staining of the actin cytoskeleton with ATTO 594-labelled phalloidin. Lamellipodial actin networks that were generally small, narrow, irregular, or displayed multiple ruffles were defined as ‘‘immature lamellipodia’’, as opposed to regular, fully developed lamellipodia [7]. n gives number of cells analyzed, and data correspond to arithmetic means ± SEM from at least three independent experiments. Statistical significance was assessed for differences between the percentages of cells with “no lamellipodia” phenotype. n.s.: not statistically significant; ** p < 0.01; *** p < 0.001 (two-sample, two-sided t-test). In all cases, statistical analysis was done for the A site mutant alone compared to each individual mutation introduced into the A site-mutated background. (B) Cell morphologies of CYFIP1/2 KO cells (clone #3) expressing respective constructs, as indicated. Panels in top row show stainings of the actin cytoskeleton with phalloidin, and bottom row images show fluorescence of the same cells derived from EGFP fluorescence. Scale bar, 10 µm. (C) Close up view of the interface between CYFIP and the C-region of WAVE, required for transinhibition of WAVE’s WCA domain. Mutations in CYFIP presumably causing constitutive activation of WRC through abrogation of the transinhibitory WCA-domain interaction are shown. (D) Surface conservation of CYFIP. Note that high sequence conservation is found in a larger area of CYFIP contacting the C-region of WAVE, and in which many activating mutations are located.