| Proximity of actin networks to membranes is required 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 non-adherent 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 microm, comparable to mass-weighted filament lengths.;Increasing the rigidity of actin filaments with phalloidin increases the extent of depletion, whereas shortening filaments using capping protein reduces it proportionally. Also, depletion kinetics are faster with higher actin concentrations, consistent with faster polymerization and Brownian-ratchet-driven motion. Conversely, the extent of depletion decreases with actin concentration, suggesting a thermodynamic driving force. Quantitatively, depletion kinetics and extent match existing actin kinetics, rigidity, and lengths. Surface depletion should slow membrane-associated F-actin reactions within cells another ∼10-fold beyond hydrodynamically slowed diffusion of filaments (∼10-fold).;Actin binding proteins are expected to modulate the development and extent of depletion. Preliminary experiments with alpha-actinin and Arp2/3 are presented. Requiring supported lipid bilayers to block surfaces, F-actin depletion occurs in the presence of both proteins. At early times (<5 hrs), alpha-actinin slows the development of depletion, but depletion profiles match F-actin after overnight incubation. Overnight, Arp2/3 forms large asters of F-actin that increase the extent of depletion.;To explain concentration-dependence (power-law of -1) of depletion profiles, we must modify the rigid rod model to better understand filament crowding. Computational simulation can separate probabilities of rod-rod interactions as functions of depth, rod distribution, orientation, and concentration. Using the Boltzmann distribution, calculated probabilities directly correspond to free energies of each component of crowding, allowing assessment of their relative importance. A strategy is outlined and should provide new interpretations for filament crowding.;The quality of data allows the first quantitative testing of these principles. Such steric depletion principles must 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 molecular depletion near membranes. |
