THE BUNDLING OF ACTIN WITH POLYETHYLENE-GLYCOL 8000 IN THE PRESENCE AND ABSENCE OF GELSOLIN

Actin filament and bundle formation occur in the cytosol under conditi ons of very high total macromolecular concentration. In this study we have utilized the inert molecule polyethylene glycol 8000 (PEG) as a m eans of simulating crowded conditions in vitro. Column-purified Ca-act in was polymerized in the absence and presence of gelsolin (to regulat e mean filament lengths between 50 and 5000 mers) and PEG (2-8%) using various concentrations of KCI and/or 2 mM divalent cations. Bundling was characterized by the scattered light intensity and mean diffusion coefficients obtained from dynamic light scattering, as well as by flu orescence and phase-contrast microscopy. The minimum concentration of KCI required for bundling decreases both with increasing concentration of PEG at a fixed mean filament length, and with decreasing filament length at a fixed concentration of PEG. In the absence of divalent cat ion, bundling is reversible on dilution, as determined by intensity le vels, diffusion coefficients, and microscopy. However, with either 2 m M Mg2+ or Ca2+ added, bundling is irreversible under conditions of hig her PEG concentrations or longer filaments, indicating that osmotic pr essure effects cannot fully explain actin bundling with PEG. Weaker di valent cation-binding sites on actin as well as disulfide bonds appear to be involved in the irreversible bundling.