Measuring actin dynamics in endothelial cells

Cytoplasmic actin distributes between monomeric and filamentous phases in c ells. As cells crawl, actin polymerizes near the plasma membrane of expandi ng peripheral cytoplasm and depolymerizes elsewhere. Thus, the finite actin filament lifetime, the diffusivity of actin monomer, and the distribution of actin between the polymer and monomer phases are key parameters in cell motility. The dynamics of cellular actin can be determined by following the evolution of fluorescence in the techniques of photoactivated fluorescence (PAF) or fluorescence recovery after photobleaching (FRAP) of microinjecte d actin derivatives. A mathematical model is discussed that measures; monom er diffusion coefficients, filament turnover rates, and the fraction of act in polymerized from measurements of the evolution of fluorescence from a ph otoactivated band [Tardy et al. (1995) Biophys. J., 69:1674-1682; McGrath e t al. (1998) Biophys. J., in press]. Applying this model to subconfluent en dothelial cells shows that similar to 40% of the actin is polymer and that these filaments turn over on average every 6 minutes. This report dicusses how PAF and FRAP can be combined with more traditional biochemistry to prob e actin cytoskeleton remodeling in endothelial cells. (C) 1998 Wiley-Liss, Inc.