SUMMARY Cells in a variety of contexts migrate towards soluble chemical cues in a process known as chemotaxis. Despite nearly a century of study, the mechanistic underpinnings of chemotaxis remain incompletely understood. Spatial gradients of platelet-derived growth factor (PDGF) and other chemoattractants direct the movements of mesenchymal cells in tissues to coordinate and accelerate physiologically important processes such as wound healing, and mesenchymal chemotaxis has been implicated in pathological conditions such as cardiovascular and fibrotic diseases. Despite the central role that fibroblasts and other mesenchymal cells play in wound healing and other disease processes such as metastatic cancer and fibrosis, a rigorous understanding of mechanisms governing the directed migration of mesenchymal cells is only recently emerging. To advance further, a quantitative, integrative approach is required. Specifically, it is necessary to elucidate how the central regulatory pathways network with others and how they are coordinated with respect to subcellular location and time to affect cell behavior. In the context of directed mesenchymal cell migration, another layer of complexity is the variation of gradient conditions (midpoint concentration/surface density and steepness). Enabled by new engineering advances, we are poised to tackle these new questions related to chemotaxis and haptotaxis and to their combinatorial influence in multi-cue settings. Our Specific Aims are as follows: Aim 1: Decoding the dynamics of multiple signaling axes that shape mesenchymal chemotaxis. We will test the hypothesis that protrusion dynamics are governed by the metastable push/pull of Arp2/3 complex and NMII activities, which are insufficiently biased by a chemotactic gradient. With stable polarization of active PKC in the most-up-gradient protrusion, the inactivation of NMII there provides a ‘port in the storm’ for pro-Arp2/3 signaling to mediate more productive protrusion. Aim 2: Probing the dynamics of haptotactic sensing and signal amplification. We hypothesize that differential integrin engagement on ECM gradients drives significant cell migration bias through feedback amplification of the pro-Arp2/3 signaling axis. If so, it would imply that haptotactic gradients are able to bias pro-Arp2/3 signaling in mesenchymal cells to an extent that chemotactic gradients cannot. Aim 3: Defining gradient synergy and prioritization in multi-cue scenarios. Despite the relevance for guidance of mesenchymal cells in vivo, it is completely unknown how cells respond to co-presentation of the two gradient types in a controlled setting. Considering how chemotaxis and haptotaxis affect dynamic regulation of the actin cytoskeleton in fibroblasts, we hypothesize that the two gradients synergize when presented in a parallel orientation. By presenting the gradients in an antiparallel or orthogonal orientation, we will determine how cells prioritize the two types of cues.