DESCRIPTION: The mechanism by which a cell interacts with its physical environment underlies fundamental cell functions of cell adhesion and migration. Understanding these relationships has broad ranging implications from informing the design of next generation engineered tissues to aiding in the understanding of tumor metastases. Cell migration has long been studied in the context of a petri dish; however, recent work has shown that cells migrate in vivo through a unique mechanism, lobopodial migration, not observed in standard in vitro cell culture. This application seeks to develop a model of in vivo migration, and examine two fundamental unanswered questions related to migratory morphologies. This grant seeks to merge the disciplines of regenerative medicine, materials science and cell biology to determine a mechanism by which MSCs sense and respond to the curvature of nanofibers present in the extracellular matrix and leading to altered migratory morphology. Specifically, this application seeks to identify if MSCs are capable of actively wrapping around a nanofiber and if they can then sense the diameter of the nanofiber through focal adhesion or vesicle-stabilizing mechanisms leading to altered migratory morphology. Preliminary evidence in support of this grant has indicated that there is a correlation between fiber diameter and focal adhesion size/maturity. The fiber diameters corresponding to the largest adhesions also demonstrated increased RhoA activity and cytoskeletal stiffness. Additionally, nanofiber diameter demonstrated a correlation with the vesicle-stabilizing protein Arfaptin 2; when fiber diameter approached the typical size of an Arfaptin 2 decorated vesicle the level of Rac-1 activation increased in an Arfaptin 2 specific manner (Arfaptin 2 is known to associate with active Rac-1). Specific Aim 1 will produce nanofiber substrates that demonstrate a range of diameters from 1.0?m - 100nm; while maintaining consistency with all other geometric parameters of a nanofiber substrate and will examine the activation and localization of the GTPases cdc42, Rac-1 and RhoA to determine the migratory morphology on each fiber diameter. Additionally, Specific Aim 1 will use time-correlated single photon counting to determine whether the membrane near the nanofiber is under tension or compression. Specific Aim 2 examines the role of the focal adhesion proteins FAK and Src on sensing the diameter of nanofibers leading to altered migratory morphology. Specific Aim 3 examines the role of the vesicle-stabilizing proteins, Arfaptin 2 and Caveolin 1&2, on sensing the diameter of nanofibers leading to altered migratory morphology. Successful completion of this application will provide design guidelines for future biomaterial architectures for applications from wound healing to tissue regeneration and advance biology through identification of an intracellular sensing mechanism of the extracellular matrix and expanded current knowledge of why unique migratory morphologies are observed in vivo.