BETA-THYMOSINS ARE NOT SIMPLE ACTIN MONOMER BUFFERING PROTEINS - INSIGHTS FROM OVEREXPRESSION STUDIES

beta-Thymosins are the currently favored candidates for maintaining th e large actin monomer (G-actin) pool in living cells, To determine if beta-thymosin behaves like a simple G-actin buffering agent in the com plex environment of a cell, we overexpressed thymosin beta 10 (T beta 10) in NIH3T3 cells and determined the effect on the monomer/polymer e quilibrium, T beta 10 is the predominant beta-thymosin isoform in the NIH3T3 cell line, and it is present in approximately equal molar ratio to profilin and cofilin/actin depolymerizing factor, two other well c haracterized actin monomer binding proteins, Clonal cell. lines that o verexpressed three times more T beta 10 had 23-33% more polymerized ac tin than control cells, and the filaments appeared thicker after stain ing with fluorescent phalloidin. There was no change in total actin, p rofilin, and cofilin/actin depolymerizing factor content, The overexpr essing cells were more motile; they spread faster and had higher chemo tactic and wound healing activity, Assuming that there is no compensat ory inactivation pf the. other classes of monomer binding proteins, ou r paradoxical observation can be accounted for quantitatively by a par allel in vitro study (Carlier, M.-F., Didry, D., Erk, I., Lepault, J., Van Troys, L., Vanderkekove, J., Perelroizen, I., Yin, H. L., Doi, Y. , and Pantaloni, D., (1996) J. Biol. Chem. 271, 9231-9239), beta-Thymo sin at levels comparable with that found in the overexpressing cells b inds actin filaments and decreases the critical concentration (C-c) fo r actin polymerization, This reduces the monomer buffering ability of beta-thymosin, so that above a certain threshold an incremental increa se in thymosin does not lead to a corresponding increase in G-actin. F urthermore, the decrease in C-c reduces the buffering capacity of the other actin monomer binding proteins, As a consequence, an increase in beta-thymosin does not necessarily result in a proportionate increase in actin monomer content in a complex environment containing other ac tin monomer binding proteins, The outcome depends on the level of beta -thymosin expression relative to the composition of the other actin mo nomer binding protien, Our results suggest that beta-thymosins are not simple actin buffering proteins and that their biphasic action may ha ve physiological significance.