Project Summary Lifelong lens transparency and flexible shape, required for focusing light onto the retina, relies upon epithelial and fiber cells whose shapes and organizations depend on filamentous (F-) actin networks. Epithelial cells contain three distinct F-actin networks: lateral cell junctions, basal stress fibers, and unique apical polygonal arrays. These networks consist of tropomyosin (Tpm) isoforms that stabilize F-actin, as well as non-muscle myosin IIA (NMIIA), and are thought to generate contractile or tensile forces to stabilize epithelial deformation and integrity during whole lens shape changes, but this has not been tested. Epithelial cells differentiate into long, thin fiber cells that form complex membrane interlocking protrusions and paddle-like domains that change with maturation and depth. Fiber cell membrane protrusions are supported by F-actin networks stabilized by fiber cell Tpm3.5, which regulates F-actin cross-linkers. In Tpm3.5-depleted lenses, the flexible crosslinker, a- actinin1, is increased on membranes, whereas the stiff crosslinker fimbrin (plastin) is decreased. Tpm3.5- depleted lenses have decreased whole lens stiffness and resiliency suggesting that more flexible F-actin networks allow greater fiber cell membrane deformation to result in softer lenses. However, the mechanistic links between F-actin networks, membrane deformation, cellular architecture, and whole lens shape change are unclear. The objective of this proposal is to determine how the F-actin networks in epithelial and fiber cells control membrane deformations and cellular shapes to confer whole lens transparency and flexibility. To address this, we will use mouse lenses to test gene function and primate lenses as a model for human lens shape change. Aim 1 will test the hypothesis that distinct F-actin and NMIIA networks control epithelial deformation and stability during whole lens shape changes. Tpm isoforms associated with epithelial F-actin networks will be determined, and fluorescent-tagged Tpms, F-actin, NMIIA and cell membranes visualized by live cell confocal microscopy to investigate network dynamics and cell deformation during whole lens shape changes. F-actin network functions will be targeted by pharmacological (mouse and primate) or genetic (mouse) approaches. Aim 2 will test the hypothesis that Tpm3.5-regulated F-actin networks in fiber cells confer membrane deformation and lens flexibility in a depth-dependent fashion during whole lens shape change. Fiber cell shape deformations under mechanical strain will be visualized by multiphoton imaging of fluorescent- labeled membranes in live lenses (mouse), membrane structures examined by scanning electron microscopy of lenses fixed under deformation (mouse and primate), and whole lens stiffness measured as a function of lens age. This work will elucidate the fundamental basis by which F-actin networks establish lens epithelial stability and fiber cell deformability to sustain lifelong l...