ACTIN PURIFIED FROM MAIZE POLLEN FUNCTIONS IN LIVING PLANT-CELLS

A vast array of actin binding proteins (ABPs), together with intracell ular signaling molecules, modulates the spatiotemporal distribution of actin filaments in eukaryotic cells. To investigate the complex regul ation 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 unpredictab le binding affinity for nonvertebrate ABPs, it is essential that these in vitro studies be performed with purified plant actin. Here, we rep ort the development of a new method for isolating functional actin fro m 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 com plex was then isolated by affinity chromatography on poly-L-proline-Se pharose, and actin was selectively eluted with a salt wash. Pollen act in was further purified by one cycle of polymerization and depolymeriz ation. The recovery of functional actin by this rapid and convenient p rocedure 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 ac tin polymerized with kinetics similar in quality to those for vertebra te alpha-actin and had a critical concentration for assembly of 0.6 mu M. Moreover, pollen actin interacted specifically and in a characteri stic fashion with several ABPs. Tradescantia cells were microinjected and used as an experimental system to study the behavior of pollen act in in vivo. We demonstrated that purified pollen actin ameliorated the effects of injecting excess profilin into live stamen hair cells.