PIP3 induces negative curvature at the leading edge. AX2 cells were transfected with a vector that expresses the F-actin marker Lifeact-mRFP and the PIP3 marker PH-CRAC-GFP. Cells were grown axenically in HL5 medium and incubated overnight in LoFlo medium to reduce autofluorescence. Cells were transferred to an acid-washed glass-bottom dish (MatTek Corporation) and allowed to migrate randomly without stimulation. Images were acquired sequentially on a spinning disk microscope in the following order: mRFP, GFP, trans. Exposure time per channel was 90 ms, and one set of images was collected every 2 s.
