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Gelsolin displaces phalloidin from actin filaments. A new fluorescence method shows that both Ca2+ and Mg2+ affect the rate at which gelsolin severs F-actin

1994; Elsevier BV; Volume: 269; Issue: 52 Linguagem: Inglês

10.1016/s0021-9258(20)30078-8

1083-351X

Philip G. Allen, Paul A. Janmey,

Silymarin and Mushroom Poisoning

W e describe an assay for measuring both the extent and kinetics of the severing of F-actin, based on the enhanced fluorescence emission of tetramethylrhodaminephalloidin bound to F-actin.The enhanced fluorescence is lost after exposure to active gelsolin by displacement of the phalloidin from actin during severing.This assay requires small amounts of actin and gelsolin, can be used to measure reaction times ranging from 1 to los s, and does not require covalent modification of either protein.The rate of fluorescence loss is linearly related to the concentrations of both actin and gelsolin.However, the apparent rate constant of the reaction is highly dependent on the divalent cation concentration, varying between lo4 and 1 0 ' M-' s-' when the [Ca2+] varies between 20 and 200 p ~. Addition of Mg2' increases the apparent rate constant at equivalent Ca2+ concentration.These results suggest that in vitro the rate-limiting step in the severing process is the activation of gelsolin by the binding of Ca2+ and Mg2' to several low affinity (Kd = 100 p) sites on gelsolin.While activation of gelsolin by Ca2+ is a slow process, the binding and severing of actin occurs at a rate approaching the diffusion limit.Gelsolin is a calcium-activated, polyphosphoinositide-inhibited actin-binding protein which can sever actin filaments in vitro (1-3).Gelsolin has been implicated as a controlling element in cell movement in vivo, as cells which express increased levels of gelsolin move at faster rates than control cells (4).The cortex of cells is rich in actin filaments, which can have the properties of either gels or liquids depending on filament length and degree of cross-linking.A regulatable actin severing protein such as gelsolin which shortens filaments and introduces breaks into cross-linked structures may be important in regulating changes in the cytoplasmic architecture that accompany cell crawling, cytokinesis, shape changes, and exocytosis (5).Gelsolin can bind actin monomers and sever and cap the high affinity end of actin filaments.Gelsolin requires elevated Ca2+(1) or H' (6) to sever F-actin, and the initial characterization indicated two Ca2+ binding sites with an affinity of 1 PM (7, 8).Recent studies, however, report two classes of Ca2+ binding sites with affinities of 10-20 PM (9,lO) and 100-1000 1" (9,111.Consistent with these reports, the half-maximal extent of gelsolin severing and nucleation in vitro occurs a t 10-20 VM Ca2+Grants AR 38910 (to P. A. J.) and AL 07680 (to P. G.