Project Abstract Cell migration is a fundamental cellular process necessary for development and coopted in diseases like cancer metastasis. Our long-term goal is to elucidate the signals that control migration and cancer invasion, so that treatment strategies to reduce pathological migration and cancer metastasis can be improved. Fluctuations in cell migration forces control leading edge protrusion-retraction cycles, but we do not know what controls the force fluctuations. The overall objective here is to understand the signaling mechanisms that control and integrate the fluctuating molecular forces of cell migration. Signaling pathways can act by directing spatially-localized and coordinated fluctuations in actin, adhesion, and membrane tension paramters (instructive). Alternatively, signaling pathways may instruct some processes and act without spatiotemporal precision (permissive) in others. We will elucidate the cell migration control mechanisms by dissecting the temporal and spatial regulation of the protein kinase ERK and its signaling outputs in untransformed and cancer cells. ERK acts on multiple steps in the protrusion-retraction cycle. The disease-relevant cancer cells model a high-activity state, in which ERK activity is upregulated due to onocogenic mutations. Our central hypothesis is that ERK instructs spatially- localized synergistic fluctuations in actin assembly, adhesion lifetime, and membrane tension for edge motion and cell migration. For the first aim, we will measure the temporal fluctuations in ERK activity during edge protrusion and retraction using modified ERK biosensors. We will incorporate the experimentally-observed activity fluctuations into a computational model and experimental tests to determine which patterns dictate protrusion velocity and persistence. For the second aim, we will determine if spatiatially-organized ERK activity controls edge motion. We will test membrane and adhesion-activated ERK for the ability to induce protrusion experimentally and computationally. We will also test how the pattern of ERK retention in the membrane and adhesion domains contributes to protrusion power and width. For the third aim, we will test if ERK is controls membrane tension and adhesion lifetime for protrusion velocity. We will test signaling through Zyxin and Ezrin to actin as possible mechanisms by which ERK controls these additional for molecular forces. The proposed research is conceptually innovative because it tests the role of fluctuating ERK signals in the regulation of cell migration. It is technically innovative in the development and use of new optogenetics tools and computational models. The research is significant because it has the potential to reveal a new principle about how molecular forces are integrated to bring about motion. It will also identify scaffolds and signals that control local ERK activity fluctuations that could be adapted for new therapeutic strategies to control cell adhesion and migratio...