Intracellular pH modulation of ADF/cofilin proteins

The ADF/cofilin (AC) proteins are necessary for the high rates of actin fil ament turnover seen in vivo. Their regulation is complex enough to underlie the precision in filament dynamics needed by stimulated cells. Disassembly of actin by AC proteins is inhibited in vitro by phosphorylation of ser3 a nd pH<7.1. This study of Swiss 3T3 cells demonstrates that pH also affects AC behavior in vivo: (1) Wounded cells show pH-dependent AC translocation t o alkaline-induced ruffling membrane; (2) The Triton extractable (soluble) ADF from Swiss 3T3 cells decreases from 42+/-4% to 23+/-4% when the intrace llular pH (pH(i)) is reduced from 7.4 to 6.6; (3) Covariance and colocaliza tion analyses of immunostained endogenous proteins show that ADF partitions more with monomeric actin and less with polymeric actin when pH(i) increas es. However, the distribution of cofilin, a less pH-sensitive AC in vitro, does not change with pH; (4) Only the unphosphorylatable AC mutant (A3), wh en overexpressed as a GFP chimera, uniquely produces aberrant cellular phen otypes and only if the pH is shifted from 7.1 to 6.6 or 7.4. A mechanism is proposed that explains why AC(A3)-GFP and AC(wt)-GFP chimeras generate dif ferent phenotypes in response to pH changes. Phospho-AC levels increase wit h cell density, and in motile cells, phospho-AC increases with alkalization , suggesting a homeostatic mechanism that compensates for increased AC acti vity and filament turnover. These results show that the behavior of AC prot eins with pH-sensitivity in vitro is affected by pH in vivo. Cell Motil. Cy toskeleton 47:319-336, 2000. (C) 2006 Wiley-Liss, Inc.