Filament rigidity causes F-actin depletion from nonbinding surfaces

Abstract

Proximity to membranes is required of actin networks for many key cell functions, including mechanics and motility. However, F-actin rigidity should hinder a filament's approach to surfaces. Using confocal microscopy, we monitor the distribution of fluorescent actin near nonadherent glass surfaces. Initially uniform, monomers polymerize to create a depletion zone where F-actin is absent at the surface but increases monotonically with distance from the surface. At its largest, depletion effects can extend >35 m, comparable with the average, mass-weighted filament length. Increasing the rigidity of actin filaments with phalloidin increases the extent of depletion, whereas shortening filaments by using capping protein reduces it proportionally. In addition, depletion kinetics are faster with higher actin concentrations, consistent with faster polymerization and faster Brownian-ratchet-driven motion. Conversely, the extent of depletion decreases with actin concentration, suggesting that entropy is the thermodynamic driving force. Quantitatively, depletion kinetics and extent match existing actin kinetics, rigidity, and lengths. However, explaining depletion profiles and concentration dependence (power-law of ؊1) requires modifying the rigid rod model. Within cells, surface depletion should slow membrane-associated F-actin reactions another Ϸ10-fold beyond hydrodynamically slowed diffusion of filaments (Ϸ10-fold). In addition, surface depletion should cause membranes to bend spontaneously toward filaments. Such depletion principles underlie the thermodynamics of all surface-associated reactions with mechanical structures, ranging from DNA to filaments to networks. For various functions, cells must actively resist the thermodynamics of depletion.confocal microscopy ͉ cytoskeleton ͉ fluorescence ͉ polymer physics ͉ polymer depletion

Encapsulation of Active Cytoskeletal Protein Networks in Cell-Sized Liposomes

“…To prevent nonspecific membrane adhesion of actin, we included 5 mol % PEGylated lipids in the lipid mixture, which act as a steric brush. The membranes were indeed passivated successfully, as shown by confocal imaging (Figure A): while the actin network (green) is homogeneous throughout the liposome, near the membrane (red) an empty depletion zone devoid of actin is observed, as expected for rigid actin filaments near nonbinding surfaces . The depletion of actin close to the membrane is clearly visible in a fluorescence intensity cross-section of the liposome, as shown in Figure B.…”

“…The membranes were indeed passivated successfully, as shown by confocal imaging (Figure 5A): while the actin network (green) is homogeneous throughout the liposome, near the membrane (red) an empty depletion zone devoid of actin is observed, as expected for rigid actin filaments near nonbinding surfaces. 69 The depletion of actin close to the membrane is clearly visible in a fluorescence intensity cross-section of the liposome, as shown in Figure 5B. To create well-defined actinÀmembrane anchoring, we used biotinÀstreptavidin bonds, which are nearly permanent.…”

Observation and Kinematic Description of Long Actin Tracks Induced by Spherical Beads

“…The purified actin monomers were equilibrated with G-buffer (2 mM Tris-HCl, pH 8.0, 0.2 mM ATP, 0.5 mM DTT, and 0.2 mM CaCl 2 ). The protein was rapidly frozen in liquid nitrogen and stored at À80 C. Alexa 488-labeled G-actin was prepared according to the procedure in Fisher and Kuo (33), using Alexa 488-succinimidyl ester (Invitrogen, Carlsbad, CA). ADF/cofilin and the verprolin central acidic domain (VCA) of N-WASP were purchased from Cytoskeleton (Denver, CO).…”

How cofilin severs an actin filament

“…Naturally, the corresponding Euler buckling load of cofilin-decorated filaments is also reduced fivefold with respect to native filaments, which may have consequences for force generation, motility and filament turnover under load. The depletion of filaments from non-binding surfaces (Fisher and Kuo 2009) will also be affected.…”