TRACER DIFFUSION THROUGH F-ACTIN - EFFECT OF FILAMENT LENGTH AND CROSS-LINKING

We have determined diffusion coefficients for small (50- to 70-nm diam eter) fluorescein-thiocarbamoyl-labeled Ficoll tracers through F-actin as a function of filament length and cross-linking. fx45 was used to regulate filament length and avidin/biotinylated actin or ABP-280 was used to prepare cross-linked actin gels. We found that tracer diffusio n was generally independent of filament length in agreement with theor etical predictions for diffusion through solutions of rods. However, i n some experiments diffusion was slower through short (less than or eq ual to 1.0 mu m) filaments, although this result was not consistently reproducible. Measured diffusion coefficients through unregulated F-ac tin and filaments of lengths > 1.0 mu m were more rapid than predicted by theory for tracer diffusion through rigid, random networks, which was consistent with some degree of actin bundling. Avidin-induced cros s-linking of biotinylated F-actin did not affect diffusion through unr egulated F-actin, but in cases where diffusion was slower through shor t filaments this cross-linking method resulted in enhanced tracer diff usion rates indistinguishable from unregulated F-actin. This finding, in conjunction with increased turbidity of 1.0-mu m filaments upon avi din cross-linking, indicated that this cross-linking method induces F- actin bundling. By contrast, ABP-280 cross-linking retarded diffusion through unregulated F-actin and decreased turbidity. Tracer diffusion under these conditions was well approximated by the diffusion theory. Both cross-linking procedures resulted in gel formation as determined by falling ball viscometry. These results demonstrate that network mic roscopic geometry is dependent on the cross-linking method, although b oth methods markedly increase F-actin macroscopic viscosity.