How differential distributions of actin-bindingproteins are formed in living cells.

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We examined the distribution and dynamics of actin-binding proteins in Ng108-15 cells. EGFP/fascin localized in filopodia. Pseudo-unphosphorylated mutant, in which 39-serine of fascin was replaced to asparatic acid, localized in filopodia while pseudo-phosphorylated mutant, in which 39-serine was replaced to alanine, showed dispersed distribution, suggesting that localization of fascin is regulated by phosphorylation. EGFP/smooth muscle type tropomyosin localized only in stress fibers, while EGFP/nonmusle type tropomyosin localized in stress fibers, filopodia, and lamellipodia. EGFP/caldesmon localized in all the actin structures.Reconstituted characterization of purified proteins revealed that fascin formed tight F-actin bundles with polarity. Drebrin bound to F-actin with a stoichiometry of 5 actin versus 1 drebrin molecule. Drebrin inhibited the actin-binding of caldesmon, actin-binding of tropomyosin, and actin-binding of fascin.These results suggest that competitive binding between drebrin and tropomyosin/caldesmon, between drebrin and fascin, and between fascin and tropomyosin/caldesmon may cause the differential localization fascin, drebrin, and tropomyosin/caldesmon, resulting in different organization of actin structure.