First published online November 24, 2004; 10.1104/pp.104.050799
Plant Physiology 136:3990-3998 (2004)
© 2004 American Society of Plant Biologists
Green Fluorescent Protein-mTalin Causes Defects in Actin Organization and Cell Expansion in Arabidopsis and Inhibits Actin Depolymerizing Factor's Actin Depolymerizing Activity in Vitro1
Tijs Ketelaar2,
Richard G. Anthony and
Patrick J. Hussey*
The Integrative Cell Biology Laboratory, School of Biological and Biomedical Sciences, University of Durham, Durham, DH1 3LE, United Kingdom (T.K., P.J.H.); and School of Biological Sciences, Royal Holloway University of London, Egham, Surrey, TW20 0EX, United Kingdom (R.G.A.)
Expression of green fluorescent protein (GFP) linked to an actin binding domain is a commonly used method for live cell imaging of the actin cytoskeleton. One of these chimeric proteins is GFP-mTalin (GFP fused to the actin binding domain of mouse talin). Although it has been demonstrated that GFP-mTalin colocalizes with the actin cytoskeleton, its effect on actin dynamics and cell expansion has not been studied in detail. We created Arabidopsis (Arabidopsis thaliana) plants harboring alcohol inducible GFP-mTalin constructs to assess the effect of GFP-mTalin expression in vivo. We focused on the growing root hair as this is a model cell for studying cell expansion and root hair tip growth that requires a highly dynamic and polar actin cytoskeleton. We show that alcohol inducible expression of GFP-mTalin in root hairs causes severe defects in actin organization, resulting in either the termination of growth, cell death, and/or changes in cell shape. Fluorescence recovery after photobleaching experiments demonstrate that the interaction of GFP-mTalin and actin filaments is highly dynamic. To assess how GFP-mTalin affects actin dynamics we performed cosedimentation assays of GFP-mTalin with actin on its own or in the presence of the actin modulating protein, actin depolymerizing factor. We show that that GFP-mTalin does not affect actin polymerization but that it does inhibit the actin depolymerizing activity of actin depolymerizing factor. These observations demonstrate that GFP-mTalin can affect cell expansion, actin organization, and the interaction of actin binding proteins with actin.
1 This work was supported by the Biotechnology and Biological Sciences Research Council, UK (to T.K., R.G.A., and P.J.H.).
2 Present address: Tijs Ketelaar, Laboratory of Plant Cell Biology, Wageningen University, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.050799.
* Corresponding author; e-mail p.j.hussey{at}durham.ac.uk; fax 44–191–334–1201.
Received July 29, 2004;
returned for revision September 23, 2004;
accepted September 29, 2004.
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