New α-actinin-4-sstFRET522 force sensor targets to contractomeres and supports actin assembly. (A) Reconstitution actin assembly assay using stripped membranes and recombinant α-actinin-4-sstFRET522 (see Methods). Upper right image shows targeting of α-actinin-4-sstFRET522 to contractomeres on stripped membranes. Upper middle image shows actin assembly on contractomeres. Right images show membrane fragments under phase contrast microscopy. Lower middle image shows overlapping α-actinin-4-sstFRET522 and actin (circled). Scale bar is 2 μm. (B) Expression of α-actinin-4-CFP and α-actinin-4-venus, with CFP or venus inserted at the same internal site as α-actinin-4-sstFRET522 (see Method) in MDCK cells. Scale bar is 5 μm. (C) FRET between α-actinin-4-CFP and α-actinin-4-venus did not show difference between cytoplasm, junction, and contractomere. Wilcoxon-Mann-Whitney test P > 0.1 between cytoplasm (n = 16), junction (n = 8), and contractomere (n = 8). Scale bar is 5 μm.