Synaptopodin consists of three functional domains. (A) Western blot showing coimmunoprecipitation of synaptopodin internal aa 245–600 with endogenous synaptopodin, β-catenin, and α-actinin-4, whereas synaptopodin C-terminal aa 600–929 disrupted interactions of endogenous synaptopodin, β-catenin, and α-actinin-4. (B) Wide-field immunofluorescence images showing junctional localization of synaptopodin N-terminal aa 1–600 and C-terminal aa 600–929 in mature monolayers. However, only aa 1–600 redistributed from cell junction to cytoplasm when cells were grown on soft silicone substrates (white asterisks). (C) Wide-field immunofluorescence images showing synaptopodin FL aa 1–929 (blue arrowheads) and N-terminal aa 1–600 (white arrowheads) targeting to the cell junction in response to cyclic pressure. Expression of synaptopodin C-terminal aa 600–929, which colocalizes with α-actinin-4 (pink arrowheads), caused cells to round up when mechanical force was applied to the junction (yellow arrowheads). (D) Coomassie blue staining showing a major band copurifies with the synaptopodin C-terminal aa 600–929 fragment from MDCK cells. Mass spectroscopy identifies the band as myosin II. (E) The three domains of synaptopodin. Bars, 10 µm.