Identification of synaptopodin as a mechanosensitive junctional protein. (A) Peptides (green font) from α-actinin-4 cross-linking experiment matching synaptopodin sequence. Basic residues lysine (K) and arginine (R) are underlined. (B) Deconvolved optical section at the apical junction showing colocalization of synaptopodin, α-actinin-4, E-cadherin, and β-catenin in mature monolayer. Yellow arrowheads show colocalization of synaptopodin, α-actinin-4, and E-cadherin. (C) Deconvolved optical section at the apical junction showing colocalization of synaptopodin, α-actinin-4, E-cadherin, and actin (phalloidin) at latrunculin-resistant junctional puncta (orange arrowheads). (D) Deconvolved optical section at the apical junction showing tension-induced junctional accumulation of synaptopodin and α-actinin-4 (orange arrowheads). (E) Quantitation of junctional synaptopodin, α-actinin-4, and actin before (U) and after (P) cyclic basal 2 mmHg pressure in young monolayers. Means are represented by horizontal lines. (F) Correlation of junctional α-actinin-4 and E-cadherin before (No P) and after (Low P) cyclic basal 2 mmHg pressure in young monolayers. (G) Correlation of synaptopodin and α-actinin-4 junctional levels before (No Pressure) and after (Low Pressure) cyclic basal 2 mmHg pressure in young monolayers. (H) Correlation of synaptopodin and actin junctional levels before (No Pressure) and after (Low Pressure) cyclic basal 2 mmHg pressure in young monolayers. (I) Correlation of α-actinin-4 and actin junctional levels before (No Pressure) and after (Low Pressure) cyclic basal 2 mmHg pressure in young monolayers. (D–I) The images and quantitation shown are from a single representative experiment out of six experiments. Bars, 2 µm.