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Comparative Study
. 2004 Dec;136(4):3968-78.
doi: 10.1104/pp.104.049411. Epub 2004 Nov 19.

A green fluorescent protein fusion to actin-binding domain 2 of Arabidopsis fimbrin highlights new features of a dynamic actin cytoskeleton in live plant cells

Michael B Sheahan  1 , Chris J Staiger, Ray J Rose, David W McCurdy
Affiliations
Comparative Study

A green fluorescent protein fusion to actin-binding domain 2 of Arabidopsis fimbrin highlights new features of a dynamic actin cytoskeleton in live plant cells

Michael B Sheahan et al. Plant Physiol. 2004 Dec.

Abstract

The actin cytoskeleton coordinates numerous cellular processes required for plant development. The functions of this network are intricately linked to its dynamic arrangement, and thus progress in understanding how actin orchestrates cellular processes relies on critical evaluation of actin organization and turnover. To investigate the dynamic nature of the actin cytoskeleton, we used a fusion protein between green fluorescent protein (GFP) and the second actin-binding domain (fABD2) of Arabidopsis (Arabidopsis thaliana) fimbrin, AtFIM1. The GFP-fABD2 fusion protein labeled highly dynamic and dense actin networks in diverse species and cell types, revealing structural detail not seen with alternative labeling methods, such as the commonly used mouse talin GFP fusion (GFP-mTalin). Further, we show that expression of the GFP-fABD2 fusion protein in Arabidopsis, unlike GFP-mTalin, has no detectable adverse effects on plant morphology or development. Time-lapse confocal microscopy and fluorescence recovery after photobleaching analyses of the actin cytoskeleton labeled with GFP-fABD2 revealed that lateral-filament migration and sliding of individual actin filaments or bundles are processes that contribute to the dynamic and continually reorganizing nature of the actin scaffold. These new observations of the dynamic actin cytoskeleton in plant cells using GFP-fABD2 reveal the value of this probe for future investigations of how actin filaments coordinate cellular processes required for plant development.

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Figures

Figure 1.
Figure 1.
Expression of GFP-fABD2 and GFP-mTalin in Arabidopsis. Confocal projections showing GFP-fABD2 (A–E) or GFP-mTalin (F–J) expression in roots (A and F), hypocotyls (B and G), leaf epidermis (C and H), trichomes (D and I), and stomata (E and J). A, Longitudinal cables in mature root epidermal cells and hairs; inset shows diffuse, irresolvable fluorescence in growing root hair tip. B, Longitudinal cables and transverse perinuclear arrays in hypocotyls that have ceased growth; inset shows similar arrangement of AFs in immature hypocotyls. C, Pavement epidermal cells show randomly arrayed transvacuolar bundles; inset shows expanding epidermal cells. D, Longitudinal bundles in young trichomes; inset shows diffuse tip fluorescence. E, Intricate cortical and simple perinuclear arrays in guard cells; inset shows transversely oriented AF bundles. F and I, Helical arrays of filaments in root hairs and trichomes; nucleoplasmic labeling is evident (arrows). G, H, and J, Randomized networks of short, curved filaments in hypocotyl, leaf epidermis, and stomata. G, Mature hypocotyl tissue; inset shows immature hypocotyl. All images are confocal projections composed of 85 (A and F), 63 (B and G), 54 (D and I), 35 (B inset and G inset), 15 (E, J, and E inset), or 10 (C, H, C inset, and D inset) optical sections. Scale bar = 10 μm (A–J) or 20 μm (B and G, inset).
Figure 2.
Figure 2.
GFP fusions to full-length AtFIM1, fABD1, or fABD2 and transient expression in N. tabacum leaf epidermal cells. A, Schematic diagram of AtFIM1 with two ABDs (fABD1 and fABD 2) and amino- or carboxy- terminal fusions o f GFP with full-length AtFIM1, fABD1, or fABD2. B, Low-level GFP-AtFIM1 expression showing filamentous labeling with overexpression and cytoplasmic fluorescence in localized regions (arrow). C and D, Cytoplasmic localization of GFP-fABD1 and fABD1-GFP, respectively. E, Visualization of filamentous structures with GFP-fABD2. Image in B is single-optical section with depth of 2.5 μm. Images in C to E are confocal projections composed of 45 (C and D) or 30 (E) optical sections. Scale bar = 20 μm.
Figure 3.
Figure 3.
Expression of GFP-mTalin affects plant growth and morphology. A, Root and hypocotyl elongation of 4-d-old, dark-grown seedlings; elongation is retarded in GFP-mTalin, but not GFP-fABD2 plants. B, Retarded growth in 7-d-old GFP-mTalin but not GFP-fABD2 plants. C, Altered silique morphology in GFP-mTalin plants. Error bars shown in A, se (n = 20); scale bar = 5 mm (B and C).
Figure 4.
Figure 4.
GFP-fABD2 labels rich AF networks in different cell types from diverse species. A and B, Dense cortical and perinuclear AF arrays in cultured N. tabacum BY-2 cells. C, Cortical AF arrays in M. truncatula suspension cultures. D, Cortical AF arrays in N. tabacum (L. esculentum; top inset) leaf epidermis, and chloroplast baskets in underlying mesophyll (bottom inset). Cells predicted to be entering division are indicated (*). All images are confocal projections composed of 75 (A), 71 (B), 15 (C and D top inset), or 4 (D and D bottom inset) optical sections. Scale bar = 20 μm (A and B) or 10 μm (C and D).
Figure 5.
Figure 5.
Expression of GFP-fABD2 reveals a dynamic framework of AFs. A, Recovery from LatB treatment in washed N. tabacum protoplast populations; rapid recovery of filamentous actin within 1.5 h of washing. Insets show half-cell (approximately 15 μm), confocal projections of protoplasts treated with 1 μm LatB or 0.1% DMSO for 24 h. B, Recovery from LatB treatment in washed Arabidopsis leaf epidermis; 8 h of LatB treatment (a) caused near complete depolymerization of the network, contrasting normal arrays in 0.1% DMSO-treated controls (b). Substantial recovery of the actin network 1.5 h after LatB washout in GFP-fABD2-expressing plants (c) but not in plants expressing GFP-mTalin (d). C, Fluorescence recovery along bleached AF bundles occurs rapidly and in a pattern resembling the prebleach image; images show typical fluorescence recovery in bleached bundle over 45 s, with left, center, and right boxes used in recovery analysis shown. Fluorescence recovers to 84% of prebleach intensity by 45 s in this example. D, Detailed analysis of fluorescence recovery following bleaching of bundled AFs; graph shows recovery ratio for one end compared with the center; arrows on graph correspond to time point for images displayed on right (n = 7). E, Plotted mean fluorescence intensity over time along bleach window in a GFP-fABD2-tagged transvacuolar actin bundle. F, Plotted mean fluorescence intensity over time along bleach window in an FDA-stained transvacuolar strand; fluorescence recovery is uniform over the bleach window with FDA, but is irregular and occurs fastest at the edges of the bleach window in GFP-fABD2-expressing cells. Insets in A are confocal projections (half-cell) and images in B are single-plane images with a z-depth of 2.5 μm. Scale bar = 10 μm (A–C).
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References

    1. Baluska F, Jasik J, Edelmann HG, Salajova T, Volkmann D (2001) Latrunculin B-induced plant dwarfism: plant cell elongation is F-actin-dependent. Dev Biol 231: 113–124 - PubMed
    1. Baluska F, Wojtaszek P, Volkmann D, Barlow P (2003) The architecture of polarized cell growth: the unique status of elongating plant cells. Bioessays 25: 569–576 - PubMed
    1. Blanchoin L, Pollard TD, Mullins RD (2000) Interactions of ADF/cofilin, Arp2/3 complex, capping protein and profilin in remodeling of branched actin filament networks. Curr Biol 10: 1273–1282 - PubMed
    1. Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735–743 - PubMed
    1. El-Assal SE, Le J, Basu D, Mallery EL, Szymanski DB (2004) DISTORTED encodes an ARPC2 subunit of the putative Arabidopsis ARP2/3 complex. Plant J 38: 526–538 - PubMed

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