Living markers for actin block myosin‐dependent motility of plant organelles and auxin

Abstract

Expression-based techniques using recombinant actin-binding proteins (ABPs) have been developed as advantageous means of visualising actin filaments. As actin function is linked to the movement of cellular cargoes, and overexpression of ABPs may compete with endogenous cytoskeletal proteins, such as myosins, secondary effects on cellular motility might be observed during actin visualisation. Cytoplasmic streaming and auxin transport were chosen as examples of cargo movement and investigated in two Arabidopsis thaliana lines stably transformed with fluorescently labelled talin (GFP-mTn) or fimbrin (GFP-FABD2). In both lines, the maximal streaming velocity of organelles was reduced to 80% in hypocotyl epidermal cells, where actin was broadly equally labelled by both ABPs. In contrast, observations of streaming and actin organisation during treatments with cytochalasin D (CD) suggested GFP-mTn-labelled actin to remain more stable. Furthermore, basipetal auxin transport was undisturbed in the GFP-FABD2 line but reduced by GFP-mTn. Remarkably, treatments with CD and 2,3-butanedione monoxime, which immobilizes myosin by impairing its ATPase, produced not only failures in organelle movement but also in basipetal auxin transport in the wild-type. These observations suggest that myosin is involved in processes of auxin translocation. In parallel, reduced motility in transgenic plants may be explained by a disturbed acto-myosin interplay, if overexpressed ABPs block the processive movement of myosin along actin filaments. This report shows that the use of live markers for actin visualisation may affect motility of cellular compounds and underlines the general need for critical investigation of actin-related processes in wild-type as well as transgenic plants prior to further interpretation.

Inhibition of Tobacco Mosaic Virus Movement by Expression of an Actin-Binding Protein

“…Collectively, these findings are consistent with the proposal that the intracellular transport of TMV occurs via the ER and that the adjacent actin cytoskeleton may exert control over ER-mediated transport via myosin bridges. The observation that transient ABD2:GFP expression interfered with myosin-mediated motility is in agreement with several other reports indicating that the expression of GFP fusions with actin-binding proteins can interfere with actin dynamics and organization, even though they are valuable tools for evaluating cytoskeletal functions (Kost et al, 1998;Mathur et al, 1999;Fu et al, 2001;Brandizzi et al, 2002;Ilgenfritz et al, 2003;Holweg et al, 2004;Ketelaar et al, 2004;Weerasinghe et al, 2005;Xu and Scheres, 2005;Holweg, 2007;Wang et al, 2008). The inhibition of myosin-based dynamics upon transient expression of ABD2:GFP in our N. benthamiana system is probably related to the ABD2:GFP expression level, which during the time of the experiment (1.5 and 2.5 dpa) was in excess of the level of ABD2:GFP in transgenic plants.…”

Critical role of actin bundling in plant cell morphogenesis

“…[4][5][6][7][8] Ketelaar et al (2004), using in vitro actin microfilament co-sedimentation assays, demonstrated that FP-mTn inhibits the actin depolymerization activity of actin depolymerization factor (ADF). 4 As ADF disfunction does not directly result in actin bundling, as shown by real-time in vitro visualization systems, 9 the molecular mechanisms of FP-mTn-mediated actin bundling appear to be more complex and related to other components, such as actin bundling proteins.…”

“…4 As ADF disfunction does not directly result in actin bundling, as shown by real-time in vitro visualization systems, 9 the molecular mechanisms of FP-mTn-mediated actin bundling appear to be more complex and related to other components, such as actin bundling proteins. 1,2 It has been recognized that FP-mTn overexpression tends to perturb cellular functions, including root hair tip growth, 4 organelle motility, 5 synchrony of cell division, 6 gravitropism 7 and stomatal opening. 8 Consequently, FP-mTn expression techniques have helped identify the relationships between actin bundling and various plant cellular events and the phenotypes of the overexpression lines.…”

Actin bundling in plants

“…Secondly, GFP-mTn triggers more abundant and severe side effects on actin cytoskeleton organization and cell growth than GFP-fABD2 [Ketelaar et al, 2004;Sheahan et al, 2004]. Holweg [2007] reported that, although a slight reduction in cellular motility occurred in both Arabidopsis GFP-FABD2 and GFP-mTn expressing lines, only the latter displayed actin cytoskeleton over-stabilization and a significantly reduced basipetal auxin transport. Although GFP-FABD2 and GFPmTn are the most commonly used actin markers, other ABP-derived markers yielded substantial results.…”

“…Indeed, fastest organelle movements were recorded along robust actin bundles located in the transvacuolar strands [e.g. Holweg, 2007]. In contrast, organelle velocity is much slower in cortical regions where finer bundles and single AFs predominate.…”