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 subpopulations of both cultured fibroblasts and keratinocytes. To investigate the physical interaction between annexin II and actin at the cell periphery, in vitro reconstitution experiments were carried out with keratins used as a control. Annexin II, isolated by immunoaffinity column chromatography, was found to exist as globular structures measuring 10 to 25 nm in diameter by rotary shadowing, similar to a previous report. We believe that these structures represent its polymeric forms. By negative staining, monomeric annexin II was detectable as tapered rods, measuring 6 nm in length and 1 to 2 nm in diameter. When annexin II was mixed with actin in 3 mM piperazine-N, N-bis-2-ethanesulfonic acid (PIPES) buffer with 10 mM NaCl2, 2 mM MgCl2 and 0.1 mM CaCl2, thick twisting actin bundles formed, confirming previous reports. This bundling was much reduced when calcium was removed. In the presence of 5 mM ethylenediamine 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 annexin II was present. These results suggest that annexin II, in conjunction with Ca2+, may be involved in a flexible system accommodating changes in the membrane cytoskeletal framework at the cell periphery in keratinocytes.
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Department of Medicine, University of Chicago, Illinois 60637.
Bystol
M E
ME
Tranvan
A
A
D016475:3T3 Cells; D000199:Actins / Q000378:metabolism*; D000818:Animals; D017306:Annexin A2 / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D015603:Keratinocytes / Q000378:metabolism; D007633:Keratins / Q000378:metabolism*; D051379:Mice
D000199:Actins; D017306:Annexin A2; D007633:Keratins; D002118:Calcium