title: Actin Purified from Maize Pollen Functions in Living Plant Cells.
abstract: A vast array of actin binding proteins (ABPs), together with intracellular signaling molecules, modulates the spatiotemporal distribution of actin filaments in eukaryotic cells. To investigate the complex regulation of actin organization in plant cells, we designed experiments to reconstitute actin-ABP interactions in vitro with purified components. Because vertebrate skeletal [alpha]-actin has distinct and unpredictable binding affinity for nonvertebrate ABPs, it is essential that these in vitro studies be performed with purified plant actin. Here, we report the development of a new method for isolating functional actin from maize pollen. The addition of large amounts of recombinant profilin to pollen extracts facilitated the depolymerization of actin filaments and the formation of a profilin-actin complex. The profilin-actin complex was then isolated by affinity chromatography on poly-L-proline-Sepharose, and actin was selectively eluted with a salt wash. Pollen actin was further purified by one cycle of polymerization and depolymerization. The recovery of functional actin by this rapid and convenient procedure was substantial; the average yield was 6 mg of actin from 10 g of pollen. We undertook an initial physicochemical characterization of this native pollen actin. Under physiological conditions, pollen actin polymerized with kinetics similar in quality to those for vertebrate [alpha]-actin and had a critical concentration for assembly of 0.6 [mu]M. Moreover, pollen actin interacted specifically and in a characteristic fashion with several ABPs. Tradescantia cells were microinjected and used as an experimental system to study the behavior of pollen actin in vivo. We demonstrated that purified pollen actin ameliorated the effects of injecting excess profilin into live stamen hair cells.
authors.affiliation: Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907.
authors.forename: H.
authors.initials: H
authors.lastname: Ren
authors.forename: B. C.
authors.initials: BC
authors.lastname: Gibbon
authors.forename: S. L.
authors.initials: SL
authors.lastname: Ashworth
authors.forename: D. M.
authors.initials: DM
authors.lastname: Sherman
authors.forename: M.
authors.initials: M
authors.lastname: Yuan
authors.forename: C. J.
authors.initials: CJ
authors.lastname: Staiger