http://hdl.handle.net/1721.1/18155 Sun, 26 May 2013 02:41:12 GMT 2013-05-26T02:41:12Z 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 Sat, 01 May 2004 00:00:00 GMT http://hdl.handle.net/1721.1/26696 2004-05-01T00:00:00Z 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 Tue, 01 Sep 1998 00:00:00 GMT http://hdl.handle.net/1721.1/26695 1998-09-01T00:00:00Z 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 Mon, 01 Jan 1996 00:00:00 GMT http://hdl.handle.net/1721.1/26694 1996-01-01T00:00:00Z 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 Sun, 01 Jan 1995 00:00:00 GMT http://hdl.handle.net/1721.1/26693 1995-01-01T00:00:00Z