Retrograde synaptopodin flow from basal junctions. (A) Frames taken from a time-lapse of synaptopodin and ZO-1. Synaptopodin retrograde flow on opposite sides of basal junctions. Arrowheads track the flow of synaptopodin puncta in two cells. Graph shows rate of retrograde flow by tracking synaptopodin puncta. Scale bar is 1 μm. (B) Structured-illumination live-microscopy of synaptopodin and vinculin showing insertion of stress fibers from two cells at basal junctions. Scale bar is 2 μm. (C) Frames taken from time-lapse of synaptopodin and vinculin. One cell is expressing synaptopodin, showing retrograde synaptopodin flow from basal junctions marked by the neighboring cell. Arrowheads track the flow of synaptopodin puncta. Scale bar is 1 μm. (D) Live imaging of synaptopodin. Left panel shows the apical plane and right panel shows the basal plane of the cell; X-Y, Y-Z, and X-Z views are shown. Synaptopodin linkers at the apical junction is highlighted in purple. The repeated and aligned synaptopodin densities are circled in blue. Yellow arrowheads mark synaptopodin at apical stress fibers in X-Z and Y-Z views. Pink arrowheads mark synaptopodin at basal stress fibers in Y-Z view. The periodic spacing of synaptopodin densities are seen in both apical and basal stress fibers. Scale bar is 2 μm.