Figure 2:

Metavinculin tail domain wild-type (WT) and cardiomyopathy (CM) mutants exhibit similar F-actin binding but differ in the ability to form higher order actin assemblies. (A) Representative SDS-PAGE results obtained from high speed F-actin co-sedimentation (100K g, 30 min) assays in the presence of Vt, MVt or MVtp WT and CM proteins (S - supernatant, P - pellet). (B) Quantification of protein fractions present in pellets representing individual sub-populations of Vt, MVt or MVtp constructs bound to F-actin. Error bars represent standard deviation (SD) (n=2, 2 replicates for each n). (C) Representative SDS-PAGE results obtained from low speed F-actin co-sedimentation (12K g, 15 min) assays in the presence of Vt, MVt or MVtp WT and CM proteins. (D) Quantification of actin fractions in pellets representing sub-populations of F-actin present in bundles or in higher-order assemblies induced by Vt, MVt or MVtp constructs. Error bars represent SD (n=2, 5 replicates for each n). Vt and MVt or MVtp WT (labeled as MVt, MVtp), as well as all MVtp CM mutants (labeled as A934V, ΔL954 and R975W), bind actin filaments similarly. However, while Vt drives almost the entire population of actin filaments into bundles or higher-order assemblies (~95%), MVt or MVtp WT display little assembly formation (~15–20%). In contrast, increased F-actin assembly formation is observed for MVtp CM mutants (~30–40%) compared to MVtp WT. Statistical significances in panels B (with respect to Vt) and D (with respect to MVtp) are indicated by; non-significant (n.s.), p < 0.05 (*), p < 0.01 (**).