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© The Rockefeller University Press, 0021-9525/2001//1035 $5.00
The Journal of Cell Biology, Volume 153, Number 5, , 2001 1035-1048

Local Photorelease of Caged Thymosin β4 in Locomoting Keratocytes Causes Cell Turning

^a Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
^b Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
^c Department of Physiology, University of Wisconsin at Madison, Madison, Wisconsin 53706
^d Center for Biomedical Imaging Technology, University of Connecticut Health Center, Farmington, Connecticut 06030
Department of Cell and Developmental Biology, University of North Carolina, 108 Taylor Hall, CB 7090, Chapel Hill, NC 27599-7090.(919) 966-1856(919) 966-5703

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 β4 (Tβ4) was photoactivated in a defined local region of locomoting fish scale keratocytes and the resulting perturbation of locomotion was studied. Purified Tβ4 was produced in an inactive form by "caging" with ([n-nitroveratryl]oxy)chlorocarbamate. In vitro spectrophotofluorometric assays indicated that caged Tβ4 did not change the normal actin polymerization kinetics, whereas photoactivated Tβ4 significantly inhibited actin polymerization. With an a priori knowledge of the cytoplasmic diffusion coefficient of Tβ4 as measured by fluorescence recovery after photobleaching experiments, the rapid sequestration of actin monomers by uncaged Tβ4 and the consequent reduction in the diffusional spread of the Tβ4–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 Tβ4 in keratocytes could potentially elicit a regional locomotory response. Indeed, when caged Tβ4 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 Tβ4 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 Tβ4.

Key Words: thymosin β4 • caged compounds • cell locomotion • FRAP • keratocyte

© 2001 The Rockefeller University Press

Abbreviations used in this paper: A, unsequestered G-actin; GST, glutathione S-transferase; NVOC, ([n-nitroveratryl]oxy)chlorocarbamate; T, free Tβ4; TA, actin–β4 complex; Tβ4, thymosin β4.

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