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 a-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 a-actin and had a critical concentration for assembly of 0.6 pM. 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.

Soluble expression and characterization of a GFP-fused pea actin isoform (PEAc1)

“…4B). The critical concentration was a little higher than that of muscle α-actin, but consistent with the result of 0.6 µM of pollen actin [25]. The filamentous structures could be seen directly under the fluorescence microscope because of the fusion with GFP and could be specifically stained with TRITC-phalloidin ( Fig.…”

“…The low abundance of actin and the presence of proteases in most plant materials made it difficult to obtain sufficient highly purified actin for in vitro studies [22,23]. Although some methods have been developed to get relatively high yields of actin from plant pollens [24,25], it is still very difficult to purify actin (especially actin isoforms) from most of the plants [26]. Expression of proteins in E. coli has greatly facilitated our understanding of physicochemical properties of proteins including plant actin binding protein [27,28].…”

Pollen Profilin Function Depends on Interaction with Proline-Rich Motifs

“…To test the effect of actin binding proteins on F-actin and cytoplasmic organization in the complex environment of the living cell, we have developed the following model system to quantify actin-dependent nuclear positioning Ren et al, 1997). The introduction of excess profilin into T. virginiana stamen hair cells by pressure microinjection results in a rapid and dramatic reduction in the number of transvacuolar cytoplasmic strands, disrupts the F-actin cytoskeleton, and results in the displacement of the nucleus from a central position (Staiger et al, 1994).…”

“…Maize pollen actin was purified according to the method of Ren et al (1997) and stored as G-actin at 4ЊC in dialysis tubing for several days. Quartz cuvettes were loaded with 0, 0.3, or 0.45 M pollen actin in buffer G (5 mM Tris, 0.2 mM CaCl 2 , 0.01% [w/v] NaN 3 , 0.5 mM DT T, 0.4 mM ATP, pH 8.0), and profilin was added sequentially from stock solutions of 200 to 300 M in buffer G. For measurements using Mg-ATP-actin, 0.2 mM EGTA and 50 M MgCl 2 were added, and the solution was mixed for 10 min before the experiment.…”

“…At least three separate preparations of each protein were used for all data in this report. Microinjection of buffer alone or 1.4 mg/mL bovine ␥-globulin (BioRad), equivalent to the total protein concentration of 100 M profilin, had no effect on nuclear position relative to uninjected control cells Ren et al, 1997). The data include at least 30 successful injections for each treatment.…”