A comparison of methods used to characterize gelation of actin in vitro

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Abstract
We have compared the meniscus depletion assay and falling ball viscometry, two means of assessing the extent of gelation in actin-based systems using mixtures of actin and the actin-binding protein filamin. We examined the effect of varying the concentrations of actin and filamin in both assays. The interaction of actin and filamin was detected only above a threshold concentration of filamin. This threshold concentration was lower for falling ball viscometry than for the meniscus depletion assay at equal actin concentrations. At constant concentrations of filamin, an increase in actin concentration caused an increase in apparent viscosity measured by the falling ball assay, but a decrease in sedimentability detected by the meniscus depletion assay. The rate of sedimentation of actin was dependent on the molar ratio of actin to filamin. At each molar ratio, the sedimentation of actin was not dependent on the specific concentrations of actin and filamin used. The apparent viscosity was dependent on both the molar ratio and the specific concentrations of actin and filamin. To relate the present results to earlier studies, we examined mixtures of actin and filamin using a macroscopic assay of gelation (tube tipping assay), and polarized light microscopy. The effect of increasing filamin concentration in the four assays was compared at three actin concentrations. Mixtures of actin and filamin whose apparent viscosities were low enough to be estimated by falling ball viscometry were optically isotropic fluids that flowed out of inverted test tubes. Mixtures of actin and filamin in the range of sensitivity of the meniscus depletion assay were either viscous fluids or gels, and were either optically isotropic or anisotropic. Thus, the four assays provide different estimates of gelation. Both the meniscus depletion assay and falling ball viscometry can be used to determine relative gelation activity, but neither can be used as a quantitative assay of gelation.