Simultaneous Measurements of Actin Filament Turnover, Filament Fraction, and Monomer Diffusion in Endothelial Cells
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Author(s)
McGrath, J. L.
Tardy, Y.
Dewey, C. F. Jr
Meister, J. J.
Hartwig, J. H.
Date Issued
September 1998
Publisher
Biophysical Society
Citation
Biophysical Journal , 75, p. 2070-2078 (1998)
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.
Description
Biophysical Journal, 1998
Subjects
Endothelial Cells
Actin Filament Turnover
Monomer Diffusion
Filament Fraction
MIT Department
Massachusetts Institute of Technology. Department of Mechanical Engineering
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