Green fluorescent protein fusions to Arabidopsis fimbrin 1 for spatio‐temporal imaging of F‐actin dynamics in roots

Abstract

The visualization of green fluorescent protein (GFP) fusions with microtubule or actin filament (F-actin) binding proteins has provided new insights into the function of the cytoskeleton during plant development. For studies on actin, GFP fusions to talin have been the most generally used reporters. Although GFP-Talin has allowed in vivo F-actin imaging in a variety of plant cells, its utility in monitoring F-actin in stably transformed plants is limited particularly in developing roots where interesting actin dependent cell processes are occurring. In this study, we created a variety of GFP fusions to Arabidopsis Fimbrin 1 (AtFim1) to explore their utility for in vivo F-actin imaging in root cells and to better understand the actin binding properties of AtFim1 in living plant cells. Translational fusions of GFP to full-length AtFim1 or to some truncated variants of AtFim1 showed filamentous labeling in transient expression assays. One truncated fimbrin-GFP fusion was capable of labeling distinct filaments in stably transformed Arabidopsis roots. The filaments decorated by this construct were highly dynamic in growing root hairs and elongating root cells and were sensitive to actin disrupting drugs. Therefore, the fimbrin-GFP reporters we describe in this study provide additional tools for studying the actin cytoskeleton during root cell development. Moreover, the localization of AtFim1-GFP offers insights into the regulation of actin organization in developing roots by this class of actin cross-linking proteins.

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“…However, our chemicals did not alter the plasma membrane localization of the styryl dye FM4-64. In addition, neither a ABD2-GFP construct that marks the actin cytoskeleton (26) (Fig. 7, which is published as supporting information on the PNAS web site) nor a chimeric gene that targeted GFP to plastids (27) displayed any apparent altered morphology (data not shown).…”

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“…7B, bottom row). We included transgenic GFP-MAP4 (Marc et al, 1998) and GFP-ABD2 (Sheahan et al, 2004;Wang et al, 2004) lines as controls for the MT and actin cytoskeleton, respectively. Oryzalin treatment efficiently disrupted MT networks but had no effect on the actin cytoskeleton.…”