Skip to main content
The Plant Cell logoLink to The Plant Cell
. 1997 Aug;9(8):1445–1457. doi: 10.1105/tpc.9.8.1445

Actin Purified from Maize Pollen Functions in Living Plant Cells.

H Ren 1, B C Gibbon 1, S L Ashworth 1, D M Sherman 1, M Yuan 1, C J Staiger 1
PMCID: PMC157010  PMID: 12237391

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.

Full Text

The Full Text of this article is available as a PDF (2.4 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Andersland J. M., Jagendorf A. T., Parthasarathy M. V. The Isolation of Actin from Pea Roots by DNase I Affinity Chromatography. Plant Physiol. 1992 Dec;100(4):1716–1723. doi: 10.1104/pp.100.4.1716. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Carlier M. F., Laurent V., Santolini J., Melki R., Didry D., Xia G. X., Hong Y., Chua N. H., Pantaloni D. Actin depolymerizing factor (ADF/cofilin) enhances the rate of filament turnover: implication in actin-based motility. J Cell Biol. 1997 Mar 24;136(6):1307–1322. doi: 10.1083/jcb.136.6.1307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chen X., Sullivan D. S., Huffaker T. C. Two yeast genes with similarity to TCP-1 are required for microtubule and actin function in vivo. Proc Natl Acad Sci U S A. 1994 Sep 13;91(19):9111–9115. doi: 10.1073/pnas.91.19.9111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cooper J. A., Pollard T. D. Methods to measure actin polymerization. Methods Enzymol. 1982;85(Pt B):182–210. doi: 10.1016/0076-6879(82)85021-0. [DOI] [PubMed] [Google Scholar]
  5. Fedorov A. A., Pollard T. D., Almo S. C. Purification, characterization and crystallization of human platelet profilin expressed in Escherichia coli. J Mol Biol. 1994 Aug 19;241(3):480–482. doi: 10.1006/jmbi.1994.1522. [DOI] [PubMed] [Google Scholar]
  6. Ghosh G., Mukherjee J., Biswas S., Pal A. Actin-like protein from Mimosa pudica L. Indian J Biochem Biophys. 1987 Dec;24(6):336–339. [PubMed] [Google Scholar]
  7. Giehl K., Valenta R., Rothkegel M., Ronsiek M., Mannherz H. G., Jockusch B. M. Interaction of plant profilin with mammalian actin. Eur J Biochem. 1994 Dec 1;226(2):681–689. doi: 10.1111/j.1432-1033.1994.tb20096.x. [DOI] [PubMed] [Google Scholar]
  8. Hennessey E. S., Drummond D. R., Sparrow J. C. Molecular genetics of actin function. Biochem J. 1993 May 1;291(Pt 3):657–671. doi: 10.1042/bj2910657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Katakami Y., Katakami N., Janmey P. A., Hartwig J. H., Stossel T. P. Isolation of the phosphatidylinositol 4-monophosphate dissociable high-affinity profilin-actin complex. Biochim Biophys Acta. 1992 Jul 31;1122(2):123–135. doi: 10.1016/0167-4838(92)90314-4. [DOI] [PubMed] [Google Scholar]
  10. Kim E., Miller C. J., Reisler E. Polymerization and in vitro motility properties of yeast actin: a comparison with rabbit skeletal alpha-actin. Biochemistry. 1996 Dec 24;35(51):16566–16572. doi: 10.1021/bi9623892. [DOI] [PubMed] [Google Scholar]
  11. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  12. Liu X., Yen L. F. Purification and characterization of actin from maize pollen. Plant Physiol. 1992 Jul;99(3):1151–1155. doi: 10.1104/pp.99.3.1151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lopez I., Anthony R. G., Maciver S. K., Jiang C. J., Khan S., Weeds A. G., Hussey P. J. Pollen specific expression of maize genes encoding actin depolymerizing factor-like proteins. Proc Natl Acad Sci U S A. 1996 Jul 9;93(14):7415–7420. doi: 10.1073/pnas.93.14.7415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ma Y. Z., Yen L. F. Actin and Myosin in pea tendrils. Plant Physiol. 1989 Feb;89(2):586–589. doi: 10.1104/pp.89.2.586. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. McCurdy D. W., Williamson R. E. An actin-related protein inside pea chloroplasts. J Cell Sci. 1987 Apr;87(Pt 3):449–456. doi: 10.1242/jcs.87.3.449. [DOI] [PubMed] [Google Scholar]
  16. Meagher R. B. Divergence and differential expression of actin gene families in higher plants. Int Rev Cytol. 1991;125:139–163. doi: 10.1016/s0074-7696(08)61218-8. [DOI] [PubMed] [Google Scholar]
  17. Nefsky B., Bretscher A. Yeast actin is relatively well behaved. Eur J Biochem. 1992 Jun 15;206(3):949–955. doi: 10.1111/j.1432-1033.1992.tb17005.x. [DOI] [PubMed] [Google Scholar]
  18. Pardee J. D., Spudich J. A. Purification of muscle actin. Methods Cell Biol. 1982;24:271–289. doi: 10.1016/s0091-679x(08)60661-5. [DOI] [PubMed] [Google Scholar]
  19. Perelroizen I., Didry D., Christensen H., Chua N. H., Carlier M. F. Role of nucleotide exchange and hydrolysis in the function of profilin in action assembly. J Biol Chem. 1996 May 24;271(21):12302–12309. doi: 10.1074/jbc.271.21.12302. [DOI] [PubMed] [Google Scholar]
  20. Pollard T. D. Myosin purification and characterization. Methods Cell Biol. 1982;24:333–371. doi: 10.1016/s0091-679x(08)60665-2. [DOI] [PubMed] [Google Scholar]
  21. Pollard T. D. Rate constants for the reactions of ATP- and ADP-actin with the ends of actin filaments. J Cell Biol. 1986 Dec;103(6 Pt 2):2747–2754. doi: 10.1083/jcb.103.6.2747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Rozycki M., Schutt C. E., Lindberg U. Affinity chromatography-based purification of profilin:actin. Methods Enzymol. 1991;196:100–118. doi: 10.1016/0076-6879(91)96012-g. [DOI] [PubMed] [Google Scholar]
  23. Rubenstein P. A. The functional importance of multiple actin isoforms. Bioessays. 1990 Jul;12(7):309–315. doi: 10.1002/bies.950120702. [DOI] [PubMed] [Google Scholar]
  24. Ruhlandt G., Lange U., Grolig F. Profilins purified from higher plants bind to actin from cardiac muscle and to actin from a green alga. Plant Cell Physiol. 1994 Jul;35(5):849–854. doi: 10.1093/oxfordjournals.pcp.a078668. [DOI] [PubMed] [Google Scholar]
  25. Sheterline P., Clayton J., Sparrow J. Actin. Protein Profile. 1995;2(1):1–103. [PubMed] [Google Scholar]
  26. Staiger C. J., Yuan M., Valenta R., Shaw P. J., Warn R. M., Lloyd C. W. Microinjected profilin affects cytoplasmic streaming in plant cells by rapidly depolymerizing actin microfilaments. Curr Biol. 1994 Mar 1;4(3):215–219. doi: 10.1016/s0960-9822(00)00050-6. [DOI] [PubMed] [Google Scholar]
  27. Vidali L., Hepler P. K. Characterization and localization of profilin in pollen grains and tubes of Lilium longiflorum. Cell Motil Cytoskeleton. 1997;36(4):323–338. doi: 10.1002/(SICI)1097-0169(1997)36:4<323::AID-CM3>3.0.CO;2-6. [DOI] [PubMed] [Google Scholar]
  28. Weeds A. G., Taylor R. S. Separation of subfragment-1 isoenzymes from rabbit skeletal muscle myosin. Nature. 1975 Sep 4;257(5521):54–56. doi: 10.1038/257054a0. [DOI] [PubMed] [Google Scholar]
  29. Xu P., Lloyd C. W., Staiger C. J., Drobak B. K. Association of Phosphatidylinositol 4-Kinase with the Plant Cytoskeleton. Plant Cell. 1992 Aug;4(8):941–951. doi: 10.1105/tpc.4.8.941. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Yen L. F., Liu X., Cai S. Polymerization of Actin from Maize Pollen. Plant Physiol. 1995 Jan;107(1):73–76. doi: 10.1104/pp.107.1.73. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Zechel K., Weber K. Actins from mammals, bird, fish and slime mold characterized by isoelectric focusing in polyacrylamide gels. Eur J Biochem. 1978 Aug 15;89(1):105–112. doi: 10.1111/j.1432-1033.1978.tb20901.x. [DOI] [PubMed] [Google Scholar]

Articles from The Plant Cell are provided here courtesy of Oxford University Press

RESOURCES