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Original Article |
Correspondence to: Ken Jacobson, Department of Cell and Developmental Biology, University of North Carolina, 108 Taylor Hall, CB 7090, Chapel Hill, NC 27599-7090. Tel:(919) 966-5703 Fax:(919) 966-1856 E-mail:frap{at}med.unc.edu.
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 ß4 (Tß4) was photoactivated in a defined local region of locomoting fish scale keratocytes and the resulting perturbation of locomotion was studied. Purified Tß4 was produced in an inactive form by "caging" with ([n-nitroveratryl]oxy)chlorocarbamate. In vitro spectrophotofluorometric assays indicated that caged Tß4 did not change the normal actin polymerization kinetics, whereas photoactivated Tß4 significantly inhibited actin polymerization. With an a priori knowledge of the cytoplasmic diffusion coefficient of Tß4 as measured by fluorescence recovery after photobleaching experiments, the rapid sequestration of actin monomers by uncaged Tß4 and the consequent reduction in the diffusional spread of the Tß4–actin complex were predicted using Virtual Cell software (developed at the Center for Biomedical Imaging Technology, University of Connecticut Health Center). These simulations demonstrated that locally photoactivating Tß4 in keratocytes could potentially elicit a regional locomotory response. Indeed, when caged Tß4 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 Tß4 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 Tß4.
Key Words: thymosin ß4, caged compounds, cell locomotion, FRAP, keratocyte
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