C. Forbes Dewey, Jr.
http://hdl.handle.net/1721.1/18154
2015-02-28T15:57:19ZA Mechanistic Model of the Actin Cycle
http://hdl.handle.net/1721.1/26696
A Mechanistic Model of the Actin Cycle
Bindschadler, M.; Osborn, E. A.; Dewey, C. F. Jr; McGrath, J. L.
We have derived a broad, deterministic model of the steady-state actin cycle that includes its major regulatory
mechanisms. Ours is the first model to solve the complete nucleotide profile within filaments, a feature that determines the
dynamics and geometry of actin networks at the leading edges of motile cells, and one that has challenged investigators
developing models to interpret steady-state experiments. We arrived at the nucleotide profile through analytic and numerical
approaches that completely agree. Our model reproduces behaviors seen in numerous experiments with purified proteins, but
allows a detailed inspection of the concentrations and fluxes that might exist in these experiments. These inspections provide
new insight into the mechanisms that determine the rate of actin filament treadmilling. Specifically, we find that mechanisms for
enhancing Pi release from the ADP-Pi intermediate on filaments, for increasing the off rate of ADP-bound subunits at pointed
ends, and the multiple, simultaneous functions of profilin, make unique and essential contributions to increased treadmilling. In
combination, these mechanisms have a theoretical capacity to increase treadmilling to levels limited only by the amount of
available actin. This limitation arises because as the cycle becomes more dynamic, it tends toward the unpolymerized state.
Biophysical Journal, 2004
2004-05-01T00:00:00ZSimultaneous Measurements of Actin Filament Turnover, Filament Fraction, and Monomer Diffusion in Endothelial Cells
http://hdl.handle.net/1721.1/26695
Simultaneous Measurements of Actin Filament Turnover, Filament Fraction, and Monomer Diffusion in Endothelial Cells
McGrath, J. L.; Tardy, Y.; Dewey, C. F. Jr; Meister, J. J.; Hartwig, J. H.
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.
Biophysical Journal, 1998
1998-09-01T00:00:00ZTheoretical Estimates of Mechanical Properties of the Endothelial Cell Cytoskeleton
http://hdl.handle.net/1721.1/26694
Theoretical Estimates of Mechanical Properties of the Endothelial Cell Cytoskeleton
Satcher, Robert L. Jr.; Dewey, C. Forbes Jr.
Current modeling of endothelial cell mechanics does not account for the network of F-actin that permeates the
cytoplasm. This network, the distributed cytoplasmic structural actin (DCSA), extends from apical to basal membranes, with
frequent attachments. Stress fibers are intercalated within the network, with similar frequent attachments. The microscopic
structure of the DCSA resembles a foam, so that the mechanical properties can be estimated with analogy to these
well-studied systems. The moduli of shear and elastic deformations are estimated to be on the order of 10^5 dynes/cm^2 . This
prediction agrees with experimental measurements of the properties of cytoplasm and endothelial cells reported elsewhere.
Stress fibers can potentially increase the modulus by a factor of 2-10, depending on whether they act in series or parallel to
the network in transmitting surface forces. The deformations produced by physiological flow fields are of insufficient
magnitude to disrupt cell-to-cell or DCSA cross-linkages. The questions raised by this paradox, and the ramifications of
implicating the previously unreported DCSA as the primary force transmission element are discussed.
Biophysical Journal, 1996
1996-01-01T00:00:00ZInterpreting Photoactive Fluorescence Microscopy Measurements of Steady-State Actin Dynamics
http://hdl.handle.net/1721.1/26693
Interpreting Photoactive Fluorescence Microscopy Measurements of Steady-State Actin Dynamics
Tardy, Y.; McGrath, J.L.; Hartwig, J.H.; Dewey, C.F.
A continuum model describing the steady-state actin dynamics of the cytoskeleton of living cells Has been
developed to aid in the interpretation of photoactivated fluorescence experiments. In a simplified cell geometry, the model
assumes uniform concentrations of cytosolic and cytoskeletal actin throughout the cell and no net growth of either pool. The
spatiotemporal evolution of the fluorescent actin population is described by a system of two coupled linear partial-differential
equations. An analytical solution is found using a Fourier-Laplace transform and important limiting cases relevant to the
design of experiments are discussed. The results demonstrate that, despite being a complex function of the parameters, the
fluorescence decay in photoactivated fluorescence experiments has a biphasic behavior featuring a short-term decay
controlled by monomer diffusion and a long-term decay governed by the monomer exchange rate between the polymerized
and unpolymerized actin pools. This biphasic behavior suggests a convenient mechanism for extracting the parameters
governing the fluorescence decay from data records. These parameters include the actin monomer diffusion coefficient,
filament turnover rate, and ratio of polymerized to unpolymerized actin.
Biophysical Journal, 1995
1995-01-01T00:00:00Z