Failure to heal wounds and scarring (the opposite) constitute major medical challenges, afflicting African Americans to the greatest extent. These pathologies often arise from dysregulation of cell repopulation of the (missing) tissue. During tissue (re)generation and engineered repair stromal fibroblasts, endothelial cells, and stem cells migrate into the provisional matrix to form both supporting matrix and vasculature. The initial cel immigration is directional, driven by signals, `cues', that arise from within the wound bed. However, once within the tissue, the cells must distribute without evident stimuli gradients. Cells can locomote productively even in isotropic environments, suggesting that a major component is cell-intrinsic. Our long-term goal is to determine how cells establish and then maintain progressive motility to repopulate tissues in response to external stimuli. Adherent cells undertake two forms of motility based on both adhesive properties and tightness of weave (or pore size) of the matrix. The key controls that convert actin cytoskeletal effects to locomotion and provide directionality have been defined by others and us during current and prior grant periods. Less frequently, as these cells transiently shift to fit through small pores and when encountering low-adhesive matrices (such as the initial fibrin-rich clots), the cells compact and move forward by following blebs, in amoeboid motility. We propose that these two modes of motility are actually inter-related using a key, still to-be-defined regulator of cytoskeletal-membrane interactions. The findings during the current grant period found that alpha-actinin-4 (ACTN4) can bridge the actin cytoskeleton to the membrane and that its interaction with actin filament is regulated by both growth factor-triggered tyrosyl-phosphorylations and calpain-mediated cleavages, both of which modulate actin-binding capabilities. Further, ACTN4 can either bind actin filaments or phospho-inositides, suggesting a mechanism coupling the anti-parallel dimer of ACTN4 to bridge the cytoskeleton to the membrane. Thus, we hypothesize that the ACTN4 is a master regulator of cytoskeleton-membrane connectivity, and that it is asymmetrically regulated by calpain cleavages during both mesenchymal and amoeboidal motility. We will determine whether: I. ACTN4 is `locked' into an actin-bound conformation by calpain-2 to bring the rear of the cell forward. II. ACTN4 bridging of the membrane is loosened by a unique tandem but dependent phosphorylation motif. III. Amoeboid motility involves asymmetric signaling responses integrating PIPs and ACTN4 enabling blebbing. Accomplishment of these investigations will define molecular bases of the spatial restriction of receptor signaling pathways and resultant biophysical responses critical to migration into cell repopulation, providing missing information vital for understanding tissue regeneration. This knowledge will enable the design on a subcellular scale of `smart' scaffolds and materials for cell and tissue engineering of intrinsic and applied cells directing the synthesis of both the matrx and the vascular bed that is required to support tissue function.