Supplemental Experimental Procedures Quantification of Membrane Extension

Differential membrane extensions between two successive time points (15 s apart; Figures 2 and 5) were quantified by first creating a series of segmented images that identified and tracked each migrating cell (using a threshold value set at 1.5 background in each confocal CAAX-CFP image; Orca-2 camera; Nipkow spinning disk confocal; Metamorph software, Universal Imaging, Downingtown, PA). These binary sequential images were then subtracted to identify the areas where membranes were extended. A contour analysis (Metamorph) was then used to measure the thickness of the extension as a function of the location along the cell perimeter, F(x). In order to measure the extensions at the left and right front of the cell, we tracked the center of the binarized cell as a function of time to generate a migration path. The current direction of migration was assumed to be the direction of the movement of this center (the minimal distance for direction measurements was a 3 µm displacement). The coordinates were then read into a Matlab program and the cells perimeter was segmented into a left and right front by choosing the points on the perimeter that were within −15 ° to −60 ° of this axis to the left (left front) and within 15 ° to 60 ° to the right (right front) (the angle measured from the center of the cell). The mean forward extension (in µm) were measured in the left and the right front segment. These two parameters are shown in Figure 2F. Th