RCP-dependent α5β1 trafficking promotes formation of actin spikes at the cell front and elongated movement in 3D matrix. (A) HT1080 and A2780 cells expressing Lifeact-mEGFP were plated onto CDM for 4 h before imaging. Actin dynamics were captured as cells move in 3D using a spinning-disk confocal microscope. Arrows indicate dynamic protrusions. Zoomed images from videos are shown in the time sequence and correspond to areas indicated by dotted ROIs. Bars, 20 µm. (B) Normalized actin density at protrusions was calculated by dividing the mean integrated density at protrusions by the mean integrated density within the whole cell (n > 500/condition). (C) A2780 cells were allowed to invade through a plug of collagen and FN for 24 h before fixation. Cells were stained for actin and imaged top to bottom using a confocal microscope. Maximum projections were produced using ImageJ, and the 3D reconstructions were made using Imaris. Bars, 50 µm. (D) The 2D shape descriptors were calculated from the maximum projections images, using the particle analysis plug-in of ImageJ (n > 46/condition). (E) The 3D shape descriptors were calculated from the entire cell volume, using the 3D shape plug-in of ImageJ (n > 46). Data represent means ± SEM from at least three independent experiments. ***, P > 0.01.