Local photorelease of caged thymosin β4 in locomoting keratocytes causes cell turning

The broad aim of this work was to explore the feasibility of using light-directed perturbation techniques to study cell locomotion. Specifically, a caged form of thymosin beta4 (T beta4) was photoactivated in a defined local region of locomoting fish scale keratocytes and the resulting perturbation of locomotion was studied. Purified T beta4 was produced in an inactive form by caging with ([n-nitroveratryl]oxy)chlorocarbamate. In vitro spectrophotofluorometric assays indicated that caged T beta4 did not change the normal actin polymerization kinetics, whereas photoactivated T beta4 significantly inhibited actin polymerization. With an a priori knowledge of the cytoplasmic diffusion coefficient of T beta4 as measured by fluorescence recovery after photobleaching experiments, the rapid sequestration of actin monomers by uncaged T beta4 and the consequent reduction in the diffusional spread of the T4-actin complex were pre-dieted using Virtual Cell software (developed at the Center for Biomedical Imaging Technology, University of Connecticut Health Center). These simulations demonstrated that locally photoactivating T beta4 in keratocytes could potentially elicit a regional locomotory response. Indeed, when caged T beta4 was locally photoactivated at the wings of locomoting keratocytes, specific turning about the irradiated region was observed, whereas various controls were negative. Additionally, loading of exogenous T beta4 into both keratocytes and fibroblasts caused very rapid disassembly of actin filaments and reduction of cellular contractility. Based on these results, a mechanical model is proposed for the turning behavior of keratocytes in response to photoreleased T beta4.