The inset outlines a theoretical feedback loop in which protrusion and PI3K signaling reinforce each other. A pattern of light was made by focusing the light lens and blocking diffuse light in the light path. A fluorescent dextran option was used to quantify the spatial profile of excitation, and a limit was put on define the region of photoactivation. Foretinib c-Met inhibitor Image analysis All image analysis was done using MATLAB. The methods used for identification and spatiotemporal mapping of protruded/ retracted places, PI3K signaling hotspots, and extended morphological structures are described below and illustrated in Fig. S4. The protruded areas for every time period are defined as pixels related to the cell in the present image but not in the previous image and vice-versa for the retracted areas. For every protruded or retracted pixel, the angle between the cell centroid and the pixel was calculated and rounded to the nearest whole angle. Outcropping Lymph node or retraction speed was determined because the net change in number of protruded/retracted pixels over the indicated angle divided by the change over time. . This approach is simple and unambiguous in its execution, and we believe it is to be a effective way for image stacks with modest spatial and temporal resolution, as was the case here. More sophisticated protrusion mapping techniques have been described. Picture segmentation to recognize pixels related to PI3K signaling hotspots was done as previously described in detail. In short, the k means c-Met Inhibitors clustering method was applied, with k 4, and hotspots were recognized as those regions with at the very least 20 contiguous pixels within the highest strength container. . Those pixels were mapped according to their angles relative to the cell centroid, with the value given in the heat map calculated as the amount of back ground subtracted fluorescence intensities for several pixels that lie across the indicated angle. Extended morphological structures were identified as follows. Each fluorescence depth picture was thresholded, and the pixels defining the cell border were indexed according to their relative positions. The local mean distance of the cell periphery from the cell centroid was calculated for every single listed location, and pixels that were 1 um beyond the local mean were considered connected with extensive morphological structures. These structures were smoothed by a standard morphological opening procedure, and, finally, the curve of the location was enlarged by 5 pixels on each side. Pixels connected with the structures hence identified were mapped based on their angles relative to the cell centroid, with the value given in the heat map calculated as the number of pixels lying along the indicated angle. For the purposes of graphical presentation and correlation analysis, the protrusion rate, hot-spot signaling, and morphological extension measurements were smoothed using a weighted linear least squares and a primary degree polynomial design using spatial and temporal ranges of 5o and five frames, respectively.