IN-VITRO MODULATION OF FILAMENT BUNDLING IN F-ACTIN AND KERATINS BY ANNEXIN-II AND CALCIUM

In our preliminary subcellular localization experiment we demonstrated that annexin II co-localized with submembranous actin in subpopulatio ns of both cultured fibroblasts and keratinocytes. To investigate the physical interaction between annexin II and actin at the cell peripher y, in vitro reconstitution experiments were carried out with keratins used as a control. Annexin II, isolated by immunoaffinity column chrom atography, was found to exist as globular structures measuring 10 to 2 5 nm in diameter by rotary shadowing, similar to a previous report. We believe that these structures represent its polymeric forms. By negat ive staining, monomeric annexin II was detectable as tapered rods, mea suring 6 nm in length and 1 to 2 nm in diameter. When annexin II was m ixed with actin in 3 mM piperazine-N,N-bis-2-ethanesulfonic acid (PIPE S) buffer with 10 mM NaCl2, 2 mM MgCl2 and 0.1 mM CaCl2, thick twistin g actin bundles formed, confirming previous reports. This bundling was much reduced when calcium was removed. In the presence of 5 mM ethyle nediamine tetra-acetic acid (EDTA) in 5 mM tris, pH 7.2, keratins were found to form a network of filaments, which began to disassemble when the chelator was removed and became fragmented when 0.1 mM CaCl2 was added. Keratins under the same conditions did not fragment when annexi n II was present. These results suggest that annexin II, in conjunctio n with Ca2+, may be involved in a flexible system accommodating change s in the membrane cytoskeletal framework at the cell periphery in kera tinocytes.