Modulation of gelsolin function. Activation at low pH overrides Ca2+ requirement.
The activation of gelsolin by calcium has been postulated to be involved in the receptor-mediated reorganization of the actin cytoskeleton, but cytoskeletal reorganization can also occur in cells with intracellular Ca2+ clamped at nanomolar levels. Fluorescence measurements using Fura-2 show that at pH 7.4, the Ca2+ requirement for gelsolin activation in vitro is higher than previously reported, with half-maximal activation of severing and nucleation occurring at 10 microM Ca2+. The Ca2+ requirement for gelsolin activity decreases at more acid pH and is approximately 3 microM at pH 6.5. At pH below 6.0, gelsolin no longer requires Ca2+ for activity and severs actin filaments, binds two actin monomers, and nucleates filament formation in EGTA-containing solutions. The pH-activated severing activity is inhibited by mixed lipid vesicles containing phosphatidylinositol 4,5-bisphosphate. A Ca(2+)-sensitive fragment consisting of the first 135 amino acids of human cytoplasmic gelsolin also demonstrates severing activity at pH < 6.0 in the absence of Ca2+. In contrast, the gelsolin homologs severin and villin maintain Ca2+ regulation of severing activity at low pH. These differences suggest that activation of gelsolin at low pH cannot be explained merely by destabilization of F-actin. The difference in diffusion constants of gelsolin measured at pH 5.5 and 6.5, as determined by dynamic light scattering, suggests that the molecule undergoes a shape change similar to that reported upon binding Ca2+ at neutral pH. These results suggest a mechanism by which gelsolin may be activated in vivo under conditions where Ca2+ transients do not occur.
Experimental Medicine Division, Brigham and Women's Hospital, Boston, Massachusetts 02115.
J A
JA
Lamb
P G
PG
Allen
B Y
BY
Tuan
P A
PA
Janmey
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D018260:Gelsolin; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010716:Phosphatidylinositols / Q000378:metabolism; D011487:Protein Conformation; D011817:Rabbits
D000199:Actins; D002135:Calcium-Binding Proteins; D018260:Gelsolin; D008840:Microfilament Proteins; D010716:Phosphatidylinositols; D002118:Calcium