THE PLANT CELL, Vol 9, Issue 8 1445-1457, Copyright © 1997 by American Society of Plant Biologists

RESEARCH ARTICLE

Actin Purified from Maize Pollen Functions in Living Plant Cells

H. Ren, B. C. Gibbon, S. L. Ashworth, D. M. Sherman, M. Yuan and C. J. Staiger
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907

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.

This article has been cited by other articles:

				F. Chaudhry, C. Guerin, M. von Witsch, L. Blanchoin, and C. J. Staiger Identification of Arabidopsis Cyclase-associated Protein 1 as the First Nucleotide Exchange Factor for Plant Actin Mol. Biol. Cell, August 1, 2007; 18(8): 3002 - 3014. [Abstract] [Full Text] [PDF]

				M. Takaine and I. Mabuchi Properties of Actin from the Fission Yeast Schizosaccharomyces pombe and Interaction with Fission Yeast Profilin J. Biol. Chem., July 27, 2007; 282(30): 21683 - 21694. [Abstract] [Full Text] [PDF]

				X. HE, Y.-M. LIU, W. WANG, and Y. LI Distribution of G-actin is Related to Root Hair Growth of Wheat Ann. Bot., July 1, 2006; 98(1): 49 - 55. [Abstract] [Full Text] [PDF]

				E. Yokota, M. Tominaga, I. Mabuchi, Y. Tsuji, C. J. Staiger, K. Oiwa, and T. Shimmen Plant Villin, Lily P-135-ABP, Possesses G-Actin Binding Activity and Accelerates the Polymerization and Depolymerization of Actin in a Ca2+-Sensitive Manner Plant Cell Physiol., October 1, 2005; 46(10): 1690 - 1703. [Abstract] [Full Text] [PDF]

				A. Michelot, C. Guerin, S. Huang, M. Ingouff, S. Richard, N. Rodiuc, C. J. Staiger, and L. Blanchoin The Formin Homology 1 Domain Modulates the Actin Nucleation and Bundling Activity of Arabidopsis FORMIN1 PLANT CELL, August 1, 2005; 17(8): 2296 - 2313. [Abstract] [Full Text] [PDF]

				K. Yi, C. Guo, D. Chen, B. Zhao, B. Yang, and H. Ren Cloning and Functional Characterization of a Formin-Like Protein (AtFH8) from Arabidopsis Plant Physiology, June 1, 2005; 138(2): 1071 - 1082. [Abstract] [Full Text] [PDF]

				S. Huang, R. C. Robinson, L. Y. Gao, T. Matsumoto, A. Brunet, L. Blanchoin, and C. J. Staiger Arabidopsis VILLIN1 Generates Actin Filament Cables That Are Resistant to Depolymerization PLANT CELL, February 1, 2005; 17(2): 486 - 501. [Abstract] [Full Text] [PDF]

				M. L. Preuss, D. R. Kovar, Y.-R. J. Lee, C. J. Staiger, D. P. Delmer, and B. Liu A Plant-Specific Kinesin Binds to Actin Microfilaments and Interacts with Cortical Microtubules in Cotton Fibers Plant Physiology, December 1, 2004; 136(4): 3945 - 3955. [Abstract] [Full Text] [PDF]

				X. Fan, J. Hou, X. Chen, F. Chaudhry, C. J. Staiger, and H. Ren Identification and Characterization of a Ca2+-Dependent Actin Filament-Severing Protein from Lily Pollen Plant Physiology, December 1, 2004; 136(4): 3979 - 3989. [Abstract] [Full Text] [PDF]

				S. Huang, L. Blanchoin, F. Chaudhry, V. E. Franklin-Tong, and C. J. Staiger A Gelsolin-like Protein from Papaver rhoeas Pollen (PrABP80) Stimulates Calcium-regulated Severing and Depolymerization of Actin Filaments J. Biol. Chem., May 28, 2004; 279(22): 23364 - 23375. [Abstract] [Full Text] [PDF]

				S. Huang, L. Blanchoin, D. R. Kovar, and C. J. Staiger Arabidopsis Capping Protein (AtCP) Is a Heterodimer That Regulates Assembly at the Barbed Ends of Actin Filaments J. Biol. Chem., November 7, 2003; 278(45): 44832 - 44842. [Abstract] [Full Text] [PDF]

				Y. Fu, G. Wu, and Z. Yang Rop GTPase-dependent Dynamics of Tip-localized F-Actin Controls Tip Growth in Pollen Tubes J. Cell Biol., March 5, 2001; 152(5): 1019 - 1032. [Abstract] [Full Text] [PDF]

				D. R. Kovar, P. Yang, W. S. Sale, B. K. Drobak, and C. J. Staiger Chlamydomonas reinhardtii produces a profilin with unusual biochemical properties J. Cell Sci., January 12, 2001; 114(23): 4293 - 4305. [Abstract] [Full Text] [PDF]

				E. B. Blancaflor and S. Gilroy Plant cell biology in the new millennium: new tools and new insights Am. J. Botany, November 1, 2000; 87(11): 1547 - 1560. [Abstract] [Full Text]

				D. R. Kovar, B. K. Drøbak, and C. J. Staiger Maize Profilin Isoforms Are Functionally Distinct PLANT CELL, April 1, 2000; 12(4): 583 - 598. [Abstract] [Full Text]

				B. C. Gibbon, D. R. Kovar, and C. J. Staiger Latrunculin B Has Different Effects on Pollen Germination and Tube Growth PLANT CELL, December 1, 1999; 11(12): 2349 - 2364. [Abstract] [Full Text]

				V. E. Franklin-Tong Signaling and the Modulation of Pollen Tube Growth PLANT CELL, April 1, 1999; 11(4): 727 - 738. [Full Text]

				S. R. Clarke, C. J. Staiger, B. C. Gibbon, and V. E. Franklin-Tong A Potential Signaling Role for Profilin in Pollen of Papaver rhoeas PLANT CELL, June 1, 1998; 10(6): 967 - 980. [Abstract] [Full Text]

				B. C. Gibbon, L. E. Zonia, D. R. Kovar, P. J. Hussey, and C. J. Staiger Pollen Profilin Function Depends on Interaction with Proline-Rich Motifs PLANT CELL, June 1, 1998; 10(6): 981 - 994. [Abstract] [Full Text]

				E. Yokota and K.-i. T. a. T. Shimmen Actin-Bundling Protein Isolated from Pollen Tubes of Lily . Biochemical and Immunocytochemical Characterization Plant Physiology, April 1, 1998; 116(4): 1421 - 1429. [Abstract] [Full Text]