The Actin Cytoskeleton

by Elison B. Blancaflor

Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, Oklahoma, USA.

The actin cytoskeleton plays a critical role in plant development by regulating a number of fundamental cellular processes including cell division, cell expansion, organelle motility and vesicle trafficking [1,2]. To function properly, the dynamics and organization of the actin cytoskeleton is mediated by a multitude of accessory proteins that are responsible for specifying whether cellular actin exists in the filamentous (F-actin) or monomeric, globular form (G-actin). Furthermore these accessory proteins are involved in actin nucleation, cross-linking of individual actin filaments to form actin bundles and facilitating actin interactions with other cellular components [2,3,4]. It is partly through this tight spatial and temporal control of actin organization that plant cells are able to achieve their highly polarized growth patterns and diverse shapes.
Fluorescent methods to visualize actin organization have contributed substantially to understanding actin function and organization during plant development. Prior to the discovery of fluorescent proteins (FPs), imaging of actin in plant cells at the light microscopic level has relied on fluorescently labeled phalloidin, a toxin from the basiodiomycete fungus Amanita phalloides that binds to F-actin and indirect immunofluorescence using actin specific antibodies. Although improvements in fixation and sample preparation methods continue to provide invaluable tools for studies on the plant actin cytoskeleton [5,6], these approaches are limited in terms of their ability to depict the intricate dynamics characteristic of actin in living cells. As with other cellular structures, studies on actin in plants has benefited significantly from the introduction of FPs. The first reported use of FPs to visualize actin organization in living plants was in 1998 where GFP was fused to the actin binding domain of mouse talin (GFP-mTalin; [7]). In addition to talin, GFP fusions to plant actin regulatory proteins have been demonstrated to bind F-actin in vivo. For example, translational GFP fusions to the headpiece of Arabidopsis villin decorated filamentous structures in suspension cells and seedlings [8]. Moreover, GFP linked to actin depolymerizing factor (ADF) localized to distinct actin cables in pollen tubes [9]. Despite the availability of GFP-ADF and GFP-Villin, the GFP-mTalin construct was the most widely used live reporter for documenting F-actin organization in plants at least until GFP fusions to the actin binding domain 2 (ABD2) of Arabidopsis fimbrin 1 were introduced (GFP-ABD2; [10,11,12,13]. In addition to Arabidopsis, GFP-ABD2 was used to image changes in F-actin organization in root hairs [14] and in response to colonization by mycorrhizal fungi in Medicago truncatula hairy roots [15]. Moreover, stable expression of GFP-ABD2 revealed F-actin-dependent vacuolar dynamics and the existence of novel mitotic structures in tobacco Bright Yellow-2 cells [16,17]. Recently, a new variant of GFP-ABD2 was described that exhibited a dramatic increase in fluorescence when stably expressed in Arabidopsis plants. This modification to the GFP-ABD2 probe involved the simple addition of GFP molecules to both the C and N termini of ABD2 [18].
Although, live F-actin reporters based on FPs have rapidly gained popularity, it is important to be mindful of potential problems and artifacts resulting from the over-expression of these side binding cytoskeletal reporters. For instance, in a study of pollen tube actin organization, GFP-Talin, GFP-ADF and GFP-ABD2 were compared with rapidly frozen pollen tubes and each of the FP reporters produced different actin labeling patterns from that of optimally fixed material [19]. Furthermore, Arabidopsis plants harboring the GFP-Talin construct exhibited defects in plant morphology possibly through interfering with the activity of other actin regulatory proteins [20] while expression of ABD2 with GFP tags on both the N and C termini (GFP-ABD2-GFP) exhibited subtle defects in cell morphology [18]. Recently, GFP-Talin expressing lines affected the efficiency of auxin transport while organelle motility in some GFP-ABD2 lines were found to be slightly inhibited [21]. Given the pivotal role that the cytoskeleton plays in defining plant morphology, the problems outlined above are not that surprising. However such observations highlight the need to develop additional fluorescent actin reporters and to use the currently available actin FP reporters with caution.

Recently a green to red photo-convertible probe, mEosFp::FABD-mTn has been reported [26]. This new addition introduces a high degree of precision in labelling F-actin locally for following actin dynamics and interactions with microtubules and other organelles.

Fluorescent protein probes for the actin cytoskeleton
 FP probes for Actin



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18. Wang Y-S, Yoo C-M, Blancaflor EB (2007) Improved imaging of actin filaments in transgenic Arabidopsis plants expressing a green fluorescent protein fusion to the C and N termini of the fimbrin actin binding domain 2. New Phytologist (in press)

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25. Liu, J.-Z., Blancaflor, E.B. and Nelson, R.S. (2005) The tobacco mosaic virus 126 kD protein, a constituent of the virus replication complex, alone or within the complex aligns with and traffics along microfilaments. Plant Physiol. 138, 1853-1865.

26. Schenkel, M. , Sinclair, A.M. , Johnstone, D. , Bewley, J.D.  and Mathur, J. (2008) Visualizing the actin cytoskeleton in living plant cells using a photo-convertible mEos::FABD-mTn fluorescent fusion protein. Plant Methods. 4.21.