Synaptopodin is required for mechanotransduction at the cell junction. (A) Deconvolved optical section at the apical junction showing tension-induced α-actinin-4 recruitment (yellow arrowheads) is reduced in synaptopodin knockdown monolayers (white arrowheads). (B) Quantitation of junctional synaptopodin and α-actinin-4 in A before (U) and after (P) cyclic pressure. Means are represented by horizontal lines. (C) Plotting junctional levels of synaptopodin and α-actinin-4 before (no P) and after (Low P) cyclic pressure shows similar slope in tension-induced response in parental and synaptopodin knockdown cells. (A–C) The images and quantitation shown are from a single representative experiment out of three experiments. (D) Wide-field immunofluorescence images showing cytoplasmic localization of vinculin in maturing synaptopodin knockdown cells before and after cyclic pressure. Junctional localization of vinculin in mature synaptopodin knockdown monolayers is also compromised. (E) Western blots showing that redistribution of vinculin and α-actinin-4 to the TX-100–insoluble fraction induced by intercellular tension is compromised in maturing synaptopodin knockdown cells. (F) Deconvolved z-composites showing spreading of synaptopodin knockdown cells on collagen-coated polyacrylamide gel. (G) Measurements of wound diameters showing wound-induced contractility is absent in synaptopodin and α-actinin-4 monolayers. Error bars are standard errors. n = 3. (H) Phase-contrast image of wound edge of MDCK cells after 30 min of wound expansion. (I) Dark-field images of wound showing expanded wound in WT but not in α-actinin-4 knockdown cells. Bars: (A, D, and F) 10 µm; (H) 50 µm; (I) 500 µm.