Periodic protrusions are initiated by detachment and rounding. Cells spread for 24 h and trypsinized on the microscope stage during the time-lapse recording (Video 3). (A) Composite images of DIC and Lifeact-GFP fluorescence. Media was replaced with trypsin at time 0. After ∼90 s trypsin was replaced with media to prevent complete detachment. (B) DIC and corresponding fluorescent image from region denoted by yellow rectangle (A) for two oscillation cycles that appeared immediately after cell rounding. (C) Kymographs generated at the positions and directions given by the red and purple dotted arrows shown in A. The positions of T1 and T2 show the time when trypsin was added (T1) and when trypsin was replaced by media (T2). Two red angles (I and II) show the slope of kymograph reflecting the speed of retraction during the final stage of cell rounding (I) and during the retraction of a protrusion (II). The top kymograph is from a cell transfected with Lifeact-GFP, whereas the bottom one is from a nontransfected cell. Images present one experiment out of 15 repeats. (D and E) Comparison of surface area in spread and rounded cell. Schematic (D) compares the size of a sphere required to accommodate all the cell surface from the spread state (R = 24.5 µm) to the actual size of the rounded state (r = 10.2 µm).