Contractomeric α-actinin-4 experiences actomyosin force. (A) A working hypothesis showing contractomeric α-actinin might experience actomyosin-dependent force. (B) Design and expression of an α-actinin-4 FRET force sensor (α-actinin-4-sstFRET522). Recombinant α-actinin-4-sstFRET522 is able to bundle actin filaments (lower panels, see Methods). (C) Expression of α-actinin-4-sstFRET522 in MDCK cells. Left panel is Western blot for α-actinin-4 using an antibody recognizing both the endogenous α-actinin-4 (orange asterisk) and α-actinin-4-sstFRET522 (blue asterisk). Right panel is Western blot for venus recognizing only α-actinin-4-sstFRET522. Markers are 135 kD in pink and 100 kD in purple. (D) Homodimer of α-actinin-4-sstFRET522 is the predominant species containing the force sensor. Left panel shows a Western blot of an anti-venus immunoprecipitation (IP) experiment using an antibody recognizing both the endogenous α-actinin-4 (orange arrow) and α-actinin-4-sstFRET522 (blue arrow). Samples were loaded in duplicates. Markers are 135 kD in pink and 100 kD in purple. Quantitation of Western blot band intensities is used to calculate the predicted and actual fractions of α-actinin-4-sstFRET522 homodimers and hetero-dimers (see Materials and methods). Heterodimers are mostly unstable, containing <1% of total α-actinin-4-sstFRET522 expressed in cells. (E) FRET readings as proxies for tensile and compressive forces. FRET measurements (see Materials and methods) showing low FRET at protrusions (n = 11) and high FRET, corresponding to compressive force, at the junctions (n = 13) using α-actinin-4-sstFRET522 as the tension sensor. Control sensor with the tension-sensing module inserted at the C-terminal (α-actinin-1-sstFRET-C) is non-responsive to location changes. Control α-actinin-1-sstFRET-C at protrusions (n = 10) and at junction (n = 20) show the same background FRET measurement as α-actinin-4-sstFRET522 (n = 11) at protrusions. Another control sensor with the tension-sensing module within a rigid spectrin α-helix repeat (α-actinin-1-M-sstFRET) is also non-responsive to location changes, with FRET measurement at protrusions (n = 9) the same as at junctions (n = 24). Wilcoxon-Mann-Whitney test P < 0.0001 between ruffle and junction measurements. Scale bars are 5 μm. (F) FRET measurements (see Methods) showing α-actinin-4-sstFRET522 background FRET (same α-actinin-1-sstFRET-C FRET) at protrusions and higher FRET at the junctions and contractomeres (middle graph). Left panels show the areas used for contractomere and junction measurements are circled in yellow. N (young junction) = 13, n (ruffle) = 18, n (mature junction) = 20, n (contractomere) = 16. Scale bar is 10 μm. Right graph shows that inhibition of actomyosin dynamics reduces α-actinin-4-sstFRET522 FRET at the contractomere to background FRET level (same α-actinin-1-sstFRET-C FRET). N (untreated) = 22, n (ML-7) = 26, n (LatB) = 19, n (CytoD) = 30. Wilcoxon-Mann-Whitney test P < 0.0001 between untreated and drug treated groups.