Identification of CD2AP and EVL as the actin-regulating activities at the adherens junctional complex. (a) Biochemical reconstitution of actin assembly on high salt/CHAPS-treated membranes with α-actinin-4 and EVL. (b) Quantitation of actin puncta showing recovery of actin assembly on high salt/CHAPS-treated membranes (LPHSCh) supplemented with α-actinin-4 and EVL (P < 0.0001) but not with α-actinin-4 or EVL alone. (c) Biochemical reconstitution of actin assembly on high salt–treated membranes with α-actinin-4 alone or together with CD2AP. (d) Quantitation of actin puncta showing inhibition of actin assembly by CD2AP (P < 0.0001). (e) F-actin spin-down assay showing cosedimentation of α-actinin-4 and CD2AP with F-actin. (f) CD2AP did not interfere with α-actinin-4 recruitment to the membrane junctional complex. High salt–stripped membranes were incubated with rhodamine-labeled α-actinin-4 in the absence or presence of 1 µM CD2AP. (g) Actin depolymerization assay by dilution of membrane junctional actin puncta into buffer in the presence or absence of the severing protein cofilin.