SIMULTANEOUS MEASUREMENTS OF ACTIN FILAMENT TURNOVER, FILAMENT FRACTION, AND MONOMER DIFFUSION IN ENDOTHELIAL-CELLS

The analogous techniques of photoactivation of fluorescence (PAF) and fluorescence recovery after photobleaching (FRAP) have been applied pr eviously to the study of actin dynamics in living cells. Traditionally , separate experiments estimate the mobility of actin monomer or the l ifetime of actin filaments. A mathematical description of the dynamics of the actin cytoskeleton, however, predicts that the evolution of fl uorescence in PAF and FRAP experiments depends simultaneously on the d iffusion coefficient of actin monomer, D-1 the fraction of actin in fi laments, FF, and the lifetime of actin filaments, tau (Tardy et at., 1 995, 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 B AECs. PAF: D = 3.1 +/- 0.4 x 10(-8) cm(2)/s, FF = 0.36 +/- 0.04, tau = 7.5 +/- 2.0 min. FRAP: D = 5.8 +/- 1.2 x 10(-8) cm(2)/s, FF = 0.5 +/- 0.04, tau = 4.8. +/- 0.97 min. Differences in the parameters are attr ibuted to differences in the actin derivatives employed in the two stu dies and not to inherent differences in the PAF and FRAP techniques. C ontrol experiments confirm the modeling assumption that the evolution of fluorescence is dominated by the diffusion of actin monomer, and th e cyclic turnover of actin filaments, but not by filament diffusion. T he work establishes the dynamic state of actin in subconfluent endothe lial cells and provides an improved framework for future applications of PAF and FRAP.