Gelsolin as a calcium-regulated actin filament-capping protein.
Various concentrations of gelsolin (25-100 nM) were added to 2 microM polymerized actin. The concentrations of free calcium were adjusted to 0.05-1.5 microM by EGTA/Ca2+ buffer. Following addition of gelsolin actin depolymerization was observed that was caused by dissociation of actin subunits from the pointed ends of treadmilling actin filaments and inhibition by gelsolin of polymerization at barbed ends. The time course of depolymerization revealed an initial lag phase that was followed by slow decrease of the concentration of polymeric actin to reach the final steady state polymer and monomer concentration. The initial lag phase was pronounced at low free calcium and low gelsolin concentrations. On the basis of quantitative analysis the kinetics of depolymerization could be interpreted as capping, i.e. binding of gelsolin to the barbed ends of actin filaments and subsequent inhibition of polymerization, rather than severing. The main argument for this conclusion was that even gelsolin concentrations (100 nM) that exceed the concentration of filament ends ( approximately 2 nM), cause the filaments to depolymerize at a rate that is similar to the rate of depolymerization of the concentration of pointed ends existing before addition of gelsolin. The rate of capping is directly proportional to the free calcium concentration. These experiments demonstrate that at micromolar and submicromolar free calcium concentrations gelsolin acts as a calcium-regulated capping protein but not as an actin filament severing protein, and that the calcium binding sites of gelsolin which regulate the various functions of gelsolin (capping, severing and monomer binding), differ in their calcium affinity.
Gremm
D
D
Institute of Physiological Chemistry, Ruhr-University Bochum, Germany.
Wegner
A
A
D051339:Actin Depolymerizing Factors; D000199:Actins / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002614:Chelating Agents / Q000494:pharmacology; D051345:Destrin; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000494:pharmacology; D018260:Gelsolin / Q000737:chemistry* / Q000502:physiology*; D007700:Kinetics; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000737:chemistry; D011485:Protein Binding; D011817:Rabbits; D013997:Time Factors
D051339:Actin Depolymerizing Factors; D000199:Actins; D002614:Chelating Agents; D051345:Destrin; D018260:Gelsolin; D008840:Microfilament Proteins; D004533:Egtazic Acid; D002118:Calcium