PROJECT SUMMARY/ABSTRACT The objectives of our R01 award, Biophysical Control of Cell Form and Function by Single Actomyosin Stress Fibers, are to investigate contributions of cofilin-1 to front-back mechanical polarization during migration and to understand the role of stress fiber networks in confined migration. Our work takes advantage of a unique and innovative combination of single-cell biophysics, computational modeling, molecular reconstitution, and use of engineered culture platforms. These disparate methodologies all share a reliance on high-resolution optical imaging of live cells, reconstituted assemblies, or combinations thereof. Our laboratory's live-cell imaging needs are currently supported by a Nikon TE2000E2 system, which we purchased with institutional startup funds in 2006. While this system has served us very well over the past 17 years, it has been plagued of late by a series of component failures as specific pieces of hardware reach the end of their natural lifetimes. Moreover, this microscope system is now several generations out of date, complicating maintenance and making it increasingly difficult to replace components as they fail. We therefore request an Administrative Supplement for Equipment Purchases to support acquisition of a new Nikon Ti2-E inverted microscope with extended live-cell imaging capabilities and total internal reflection fluorescence (TIRF) microscopy. This purchase will critically facilitate the work in this award in three ways: (1) It will replace our existing Nikon TE2000E2 microscope system, which we purchased nearly 20 years ago, has suffered from repeated component failures, and is no longer fully serviced by the manufacturer or vendor. (2) TIRF capabilities will enable us to establish Aim 1 actin-cofilin reconstitution studies – currently restricted to collaborator Bruce Goode's laboratory at Brandeis – in the Kumar Lab at UC Berkeley. This will greatly expand our experimental bandwidth and allow cross-checking of key results across two laboratories. (3) Extended (24-48h) live-cell TIRF will enable us to image SF remodeling events during confined migration at far superior resolution than with conventional epifluorescence imaging and on a time scale longer than would be practical in a confocal or two-photon system in a core facility. We will also be able to integrate this system with an innovative microfluidic platform we are developing, which we are aiming to integrate with -omics screens to understand the molecular basis of confined migration.