CHARACTERIZATION OF GELSOLIN TRUNCATES THAT INHIBIT ACTIN DEPOLYMERIZATION BY SEVERING ACTIVITY OF GELSOLIN AND COFILIN

Gelsolin is a calcium-activated actin-binding protein with six subdoma ins. The N-terminal (G1) domain is essential for actin-filament-severi ng activity while other domains within G2-3 position the protein on th e filament side allowing G1 to sever. In order to generate reagents ca pable of competitively inhibiting endogenous gelsolin and, potentially , other actin filament regulatory protein, we expressed several trunca tes of gelsolin in Escherichia coli, and analyzed how they affected th e in vitro activity of two different actin-binding proteins, gelsolin and cofilin. A Ca2+-sensitive truncate containing G2-6 inhibited the F -actin-depolymerizing activities of both gelsolin and cofilin. while a G2-3 truncate was less effective. Using two independent assays, our r esults support the idea that gelsolin truncates inhibit actin filament severing and do not markedly affect actin subunit dissociation kineti cs, Cosedimentation assays in the presence of calcium demonstrate that the G2-6 truncate binds to F-actin more strongly than the G2-3 trunca te consistent with a protection mechanism by conformational change of F-actin and/or competitive binding to actin filaments which depends up on the presence of actin filament binding domains.