We have investigated the interactions between the actin-binding proteins gelsolin and tropomyosin, with special respect to any effects on the functional properties of gelsolin. Limited proteolysis indicated that the loop connecting the gelsolin domains G3 and G4 is involved in tropomyosin binding. Under nonpolymerizing conditions, binding of tropomyosin neither prevented the formation of a 2 : 1 actin–gelsolin complex, nor did it affect the nucleating activity of gelsolin in actin polymerization, likely as a result of competitive displacement of tropomyosin from gelsolin. To evaluate the effect of tropomyosin on the actin filament severing activity of gelsolin, we measured both filamentous actin (F-actin) viscosity and the relative number concentrations of filaments after fragmentation, either by gelsolin alone or by gelsolin–tropomyosin complexes. The interaction of gelsolin with tropomyosin caused a reduction in F-actin severing activity of up to 80% compared to gelsolin alone. Thus, being bound to gelsolin, tropomyosin prevented gelsolin from severing actin filaments. By contrast, the severing activity of gelsolin for F-actin/tropomyosin was similar to that for F-actin alone even at a tropomyosin : actin saturation ratio of 1 : 7. Thus, when bound to actin filaments, tropomyosin did not significantly inhibit the severing of filaments by gelsolin. The interaction between gelsolin and tropomyosin was largely independent of the muscle actin and tropomyosin isoforms investigated. The results obtained in the present study suggest that tropomyosin is involved in the modulation of actin dynamics not via the protection of filaments against severing, but rather by binding gelsolin in solution to prevent it from severing and to promote the formation of new actin filaments.
By forming a complex with gelsolin in solution, tropomyosin inhibits the actin filament severing activity of gelsolin. On the other hand, binding of tropomyosin to F-actin does not significantly protect filaments from being severed by gelsolin. Therefore, tropomyosin may be involved in the regulation of actin dynamics in vivo by binding gelsolin in solution and thereby preventing actin filament fragmentation.