The dynamics of protrusion and retraction establish changes in cell shape and directionality. By applying the spatiotemporal dynamics of cell protrusion/retraction and Lu AA21004 PI3K signaling monitored by total internal reflection fluorescence microscopy, we show that randomly migrating fibroblasts reorient polarity through PI3K dependent pivoting and branching of protrusions. PI3K inhibition didn’t affect the initiation of newly branched lumps, nor did it stop protrusion induced by photoactivation of Rac. Somewhat, PI3K signaling increased after, maybe not before, the onset of local protrusion and was required for the lateral spreading and stabilization of nascent branches. Throughout chemotaxis, the division experiencing the larger chemoattractant focus was favored, and, therefore, the cell reoriented to be able to align with the external gradient. increases after, not before, the initiation of protrusion induced spontaneously or by liberation of photoactivatable Rac. Finally, it is found pro-protein that biasing the part and pivot re-orientation mechanism allows chemotactic fibroblasts to align migration directionality with the external gradient. migration directionality to be aligned by chemotactic fibroblasts. lamellipodial branching in fibroblasts is not a normal mechanism of motility but alternatively a stochastic process that resets migration polarity. The essential function of PI3K signaling in this process is not in the creation of new protrusions but rather to promote lateral spreading and distribution of the state. Reorientation of cell migration by control of motility dynamics across disparate time scales We previously showed that PI3K signaling, monitored by total internal reflection fluorescence microscopy in migrating fibroblasts expressing the GFP AktPH biosensor, is localized in protrusive structures during both random migration and chemotaxis, and, thus, the pattern of PI3K signaling correlates with general direction of cell migration. Furthermore, PI3K signaling is transient, with local regions growing and dying out, with a characteristic time scale of 15 min in randomly migrating cells, the dynamics are globally coupled, in the perception that the emergence of a hotspot buy Linifanib tends to be briefly followed or preceded by the death of another. Here, for exactly the same cohort of randomly migrating cells, we mapped the radial protrusion/retraction velocity alongside the places of PI3K signaling hotspots and regions of fingerlike morphological extension as a function of time and angular position. These spatiotemporal routes show unique character on long and short time scales. Although specific protrusion and signaling events tend to be relatively short lived, consistent with the previous analysis, they are almost exclusively limited to long lived morphological extensions of the cell. Thus, protrusion and retraction occur along well-defined tracks inside the spatiotemporal road. Accordingly, over the cell population, PI3K signaling and protrusion are absolutely related, but the correlation of morphological extension with either protrusion or signaling is even greater.