Simultaneous Measurements of Actin Filament Turnover, Filament Fraction, and Monomer Diffusion in Endothelial Cells

Abstract

The analogous techniques of photoactivation of fluorescence (PAF) and fluorescence recovery after photobleaching (FRAP) have been applied previously to the study of actin dynamics in living cells. Traditionally, separate experiments estimate the mobility of actin monomer or the lifetime of actin filaments. A mathematical description of the dynamics of the actin cytoskeleton, however, predicts that the evolution of fluorescence in PAF and FRAP experiments depends simultaneously on the diffusion coefficient of actin monomer, D, the fraction of actin in filaments, FF, and the lifetime of actin filaments, t (Tardy et al., 1995, Biophys. J. 69:1674–1682). Here we report the application of this mathematical model to the interpretation of PAF and FRAP experiments in subconfluent bovine aortic endothelial cells (BAECs). The following parameters apply for actin in the bulk cytoskeleton of subconfluent BAECs. PAF: D 5 3.1 6 0.4 3 1028 cm2/s, FF 5 0.36 6 0.04, t 5 7.5 6 2.0 min. FRAP: D 5 5.8 6 1.2 3 1028 cm2/s, FF 5 0.5 6 0.04, t 5 4.8 6 0.97 min. Differences in the parameters are attributed to differences in the actin derivatives employed in the two studies and not to inherent differences in the PAF and FRAP techniques. Control experiments confirm the modeling assumption that the evolution of fluorescence is dominated by the diffusion of actin monomer, and the cyclic turnover of actin filaments, but not by filament diffusion. The work establishes the dynamic state of actin in subconfluent endothelial cells and provides an improved framework for future applications of PAF and FRAP.