Actin Polymerization Induced by GTPS in Permeabilized Neutrophils Is Induced and Maintained by Free Barbed Ends

doi:10.1074/jbc.270.47.28075

Actin Polymerization Induced by GTPS in Permeabilized Neutrophils Is Induced and Maintained by Free Barbed Ends (*)

From the (1)Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6018 Department of Cell and Developmental Biology and
(2) Department of Physiology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6018

^§ To whom correspondence should be addressed. Tel.: 215-898-4559; Fax: 215-898-8780.

Abstract

To address the mechanisms through which agonists stimulate actin polymerization, we examined the roles of monomer sequestering proteins and free barbed ends on actin polymerization induced by guanosine 5′-3-O-(thio)triphosphate (GTPS) in neutrophils permeabilized with streptolysin O. Addition of profilin (without GTPS) caused a net decrease in F-actin. Thus, merely making profilin available in the cell was not sufficient to induce actin polymerization. On the other hand, addition of profilin hardly affected the polymerization induced by GTPS, while thymosin β or DNase I decreased this polymerization. These data suggested that GTPS induced polymerization by increasing the availability of barbed ends. In the presence of cytochalasin B, profilin did inhibit polymerization induced by GTPS, demonstrating that GTPS did not inhibit profilin's monomer sequestering ability.

The F-actin induced by GTPS was not limited by a time-dependent loss of G-actin or G-proteins from permeabilized cells since, following stimulation with suboptimal concentrations of GTPS, addition of more GTPS induced further polymerization. Barbed ends remained free after F-actin reached plateau since (a) cytochalasin B caused depolymerization of induced F-actin and (b) profilin did not depolymerize induced F-actin unless the cells were first treated with cytochalasin to cap barbed ends. The data indicate that GTPS maintains an increased level of F-actin by keeping at least a few barbed ends available for polymerization.

Footnotes

↵* This work was supported by National Institutes of Health Grants AI 19883 (to S. H. Z.) and AR40840 (to V. N., and D. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵⁽⁾ The abbreviations used are:

Tβ4

thymosin β

SLO

streptolysin O

TRITC

tetramethylrhodamine isothiocyanate

GTPS

guanosine 5′-3-O-(thio)triphosphate
↵⁽⁾S. H. Zigmond, unpublished results.
↵⁽⁾M. Tardif, S. Huang, T. Redmond, D. Safer, M. Pring, and S. H. Zigmond, unpublished observation.
- Received June 21, 1995.