PROJECT SUMMARY/ABSTRACT Actomyosin stress fibers (SFs) enable cells to tense the extracellular matrix (ECM), a process key to cell shape determination, motility, and morphogenesis. Over the past 15+ years, including the past period of R01 support, we have made significant contributions to the field’s understanding of SF mechanics and contributions to cell structure. Our work is particularly notable for the use of femtosecond laser nanosurgery (FLN), which has enabled us to show that the three canonical SF subtypes – dorsal fibers, transverse arcs, and ventral fibers – collectively enforce a front-back tension gradient that underlies two-dimensional (2D) mesenchymal migration. We also showed that the SF network architecture can mechanically reinforce individual SFs, which has significant implications for symmetry breakage during directed migration and force propagation through cell monolayers. With this intellectual foundation in place, our renewal application turns to two important questions: How is polarization of tension in the SF network encoded by molecular signals classically understood to establish front-back polarity? And how does our knowledge of 2D SF networks translate to confined migration geometries like those found in tissue? We will address these questions through two specific aims, both of which build upon publications from this award. In Specific Aim 1, we will investigate mechanistic contributions of cofilin-1 to establishment and maintenance of SF front-back tension polarization during migration. We hypothesize that cofilin-1 establishes front-back polarization of SF tension by promoting the assembly and contractile maturation of transverse arcs. By combining biophysical, engineering, and cell biological tools, we will identify key molecular and force-based signals that modulate recruitment of cofilin-1 to developing transverse arcs. In an innovative new collaboration with Dr. Bruce Goode (Brandeis) we will reconstitute actin bundles in microfluidic devices and quantify the relationship between tensile force and cofilin- 1 engagement. In Specific Aim 2, we will dissect contributions of SF networks to migration in confined geometries where the ECM imposes axial cues and sterically precludes elaboration of 2D SF networks. We hypothesize that increasing confinement redirects SF assembly from the 2D dorsal fiber-transverse arc-ventral fiber assembly pathway towards de novo parallelized SF assembly. We will combine microengineered culture platforms, single-cell mechanical tools, and superresolution imaging to probe confinement-induced changes in SF assembly, architecture, and mechanics. Aim 2 will leverage two established, productive collaborations: With Dr. Ulrich Schwarz (U. Heidelberg), we will develop multiscale computational models that relate SF network architecture and mechanics to cell migration in confined spaces. With neurosurgeon Dr. Manish Aghi (UCSF), we will test the clinical value of our observations by asking if conf...