Small Espin: A Third Actin-bundling Protein and Potential Forked Protein Ortholog in Brush Border Microvilli

An ∼30-kD isoform of the actin-binding/ bundling protein espin has been discovered in the brush borders of absorptive epithelial cells in rat intestine and kidney. Small espin is identical in sequence to the COOH terminus of the larger (∼110-kD) espin isoform identified in the actin bundles of Sertoli cell–spermatid junctional plaques (Bartles, J.R., A. Wierda, and L. Zheng. 1996. J. Cell Sci. 109:1229–1239), but it contains two unique peptides at its NH₂ terminus. Small espin was localized to the parallel actin bundles of brush border microvilli, resisted extraction with Triton X-100, and accumulated in the brush border during enterocyte differentiation/migration along the crypt–villus axis in adults. In transfected BHK fibroblasts, green fluorescent protein–small espin decorated F-actin–containing fibers and appeared to elicit their accumulation and/or bundling. Recombinant small espin bound to skeletal muscle and nonmuscle F-actin with high affinity (K_d = 150 and 50 nM) and cross-linked the filaments into bundles. Sedimentation, gel filtration, and circular dichroism analyses suggested that recombinant small espin was a monomer with an asymmetrical shape and a high percentage of α-helix. Deletion mutagenesis suggested that small espin contained two actin-binding sites in its COOH-terminal 116–amino acid peptide and that the NH₂-terminal half of its forked homology peptide was necessary for bundling activity.